The 7th ASIAN CROP SCIENCE ASSOCIATION CONFERENCE
Improving food, energy and environment with better crops

ISBN: 978-602-17500-0-1

PROCEEDINGSThe 7th ASIAN CROP SCIENCE ASSOCIATION CONFERENCE
Improving food, energy, and environment with better crops

IPB International Convention Center
Bogor, Indonesia, 27-30 September 2011

Research Center for Bioresources and Biotechnology
Bogor Agricultural University
2013

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Proceedings of The 7 ACSA Conference 2011i

Copyright:
This proceedings published by Research Center for Bioresources and Biotechnology,
Bogor Agricultural University(Pusat Penelitian Sumberdaya Hayati dan Bioteknologi, Institut Pertanian Bogor)
Jln. Kamper, Gedung PAU Kampus IPB Darmaga, Bogor (16680), Indonesia.

iithProceedings of The 7 ACSA Conference 2011

ISBN: 978-602-17500-0-1

PROCEEDINGSThe 7th ASIAN CROP SCIENCE ASSOCIATION CONFERENCE
Improving food, energy, and environment with better crops

IPB International Convention Center
Bogor, Indonesia, 27-30 September 2011

Editors
Chief:
Suharsono(Research Center for Bioresources and Biotechnology, Bogor Agricultural University)

Members:
Hiroshi Ehara(Graduate School of Bioresources, Mie University, Japan)

Hayati Minarsih(Indonesian Biotechnology Research of Estate Crops)

Komang G Wiryawan(Faculty of Animal Sciences, Bogor Agricultural University)

Miftahuddin(Faculty of Mathematics and Natural Sciences, Bogor Agricultural University)

Muhamad Yunus(Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture)

Tri Muji Ermayanti(Research Center for Biotechnology, Indonesian Institute of Sciences)

Utut Widyastuti(Research Center for Bioresources and Biotechnology, Bogor Agricultural University)

Technical Editor:
Noor F Mardatin(Research Center for Bioresources and Biotechnology, Bogor Agricultural University)

Bogor, January 2013th

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Foreword from the Chairman of Organizing Committee

We are currently facing an increasing population, diminishing resources and also escalating
environmental problems. These inconvenient conditions could be solved by intensifying ourattention to develop efficient, stable, and sustainable crop production systems. To achieve this goal,
the cooperation of crop scientists working on different aspects from basic to applied sciences, anddifferent commodities from food crops as considered the basic need of human being to ornamental
crops considered as secondary need, transcending the boundaries of region and nations isnecessary.
With the intention to accommodate the communication among researchers on crop science,
the Asian Crop Science Association (ACSA) conducts regular international conference every three
years. During four days on September 27-30th, 2011, we had successfully organized the 7th
Conference of ACSA with the theme on Improving Food, Energy, and Environment with Better
Crops, in Bogor, Indonesia. This event had been attended by 240 participants coming from 17
different countries. During three days of the conference, 13 papers were presented by invited
speakers in the plenary sessions, 81 papers were in the parallel sessions, and 71 posters were
presented in the poster sessions. This conference was in conjunction to the 48th anniversary ofBogor Agricultural University, annual meeting of Crop Science Society of Japan (CSSJ), JapaneseSociety of Breeding (JSB) and Japanese Society of Tropical Agriculture (JSTA), and the kick offmeeting and workshop of Japan International Research Center for Agricultural Science (JIRCAS).
The scientific activities were composed of three seminars: (1) ACSA Conference with thetheme on ―Improving food, energy, and environment with better crops‖, (2) seminar of annual
meeting of CSSJ, JSB and JSTA with the theme on ―Improvement of crop performance forsustainable agricultural development in wetlands‖, and (3) workshop of JIRCAS with the theme on"Rice innovation for environmentally sustainable production systems‖.
On behalf of the editor and also the organizing committee, I would like to express ourhappiness that the scientific papers presented in the Conference can be finally compiled andpublished in the form of Proceedings of the 7th Asian Crop Science Association Conference.
We apologize for the delay to publish these proceedings. We sincerely hope that these
Proceedings will be useful and beneficial for all parties.

Chairman of OC and Chief Editors,

Prof. Dr. SONY SUHARSONO

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Foreword from the Rector of Bogor Agricultural University

I would like to express my happiness that The Proceedings of The 7th Conference of Asian
Crop Science Association (ACSA) can finally be published. Looking on the number of scientificpapers coming from different countries and aspects, it is certainly that many tasks had been done topublish these proceedings. My expression of appreciation and gratitude are addressed to theeditors who had been working very hard to finish the proceedings.
Crop is very important to human and there is no life without crop. From crop we obtain foodand feed. With the increasing of the world population, the requirement for crop is increasing
substantially, therefore, serious efforts must be undertaken to improve crop production to supply
sufficient food for human. In addition, the climate change forces us to adapt to this environmental
changes. From this point of view, the development of crop science is highly important, from basic toapplied sciences, and collaboration amongst scientists is necessary to accelerate the progress ofcrop development. Crop does not only provide food, but also provides a future energy sources
which is more friendly to the environment, therefore, the proceedings consisted of the scientificpapers of the 7th Conference of ACSA with the theme of ―Improving food, energy, and environment
with better crops” is highly relevant to the current situation.
ACSA is expected to play major role in crop science development and in coordinating thecollaboration amongst scientists, not only in Asia but also throughout the world. Therefore I
personally and as a Rector of Bogor Agricultural University strongly support ACSA as a medium forcollaboration to share knowledge and experiences in recent advances of crop science development.
Finally, I extend my gratitude to contributors, and sponsors for participating in the compilingall scientific papers in the proceedings. I believe that these proceedings will enrich our knowledge,
especially in crop sciences, and beneficial to mankind.

Rector of Bogor Agricultural University,

Prof. HERRY SUHARDIYANTO, PhD.

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MINISTRY OF AGRICULTURE
REPUBLIC OF INDONESIAKEYNOTE ADDRESSMINISTER OF AGRICULTURE REPUBLIC OF INDONESIA
AT THE OPENING CEREMONY OF THE 7th ACSA CONFERENCE
Bogor, 27-30 September 2011

Distinguished guests,

Rector of Bogor Agricultural University,
Chairman of Asian Crop Science Association (ACSA),
Chairman of Crop Science Society of Japan,
Director of Japan International Research Center for Agricultural Sciences (JIRCAS),
Ladies and Gentlemen,
Assalamu'alaikum warahmatullaahi wabarakaatuh,
It is a great pleasure for me to be here with you and warmly welcome you all in thisimportant gathering of ―The 7th ACSA conference‖. On behalf of the Government of the Republic ofIndonesia, it is also a great honor and privilege for me to extend our warm welcome to allparticipants.
It is a great pleasure also for me to congratulate for 48th anniversary of IPB and warm
welcome and success for kick off meeting of Crop Science Society and Japan International
Research Center for Agricultural Sciences (JIRCAS).
Ladies and Gentlemen,
The on going changes in the economy, resources competition from other sectors,
environmental changes, increasing commercialization of agricultural activities, and the importanceof international trade mean that the way of agricultural product will be produced in the future will be
substantially different.
Some traditional agriculture-growing areas may lose their comparative advantage whileothers may become new growth centers for agriculture product. Clearly, there is a need to develop
a new vision for future agriculture activities given these global trends and likely scenarios. Thisvision is needed to strategically position investment in agricultural research, technology delivery,
and the design of policy reforms.
Agriculture in the next future will not only need to produce enough food and energy for
population, but will also be the key to conserve environment for a sustainable food production.
FAO‘s latest forecast for world cereal production in 2011 stands at 2,302 million tonnes, 2.9 percent
up from the 2010 harvest. World cereal utilization in 2011/12 is forecast to increase by 1.2 percent
from 2010/11. Given the latest production forecast and the expectation for utilization in 2011/12,

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world cereal stocks at the close of crop season ending in 2012 are now estimated at about 486
million tonnes.
Except for rice, the inventories of which are forecast to increase, stocks of coarse grains
and wheat anticipated to decline. With total cereal production in 2011 below the anticipatedutilization, international price are likely to remain high, especially in the wheat and coarse grainmarket. In Asian countries, high food prices and the generally good rainfall situation have been
conducive to bumper food production. In the 2011, annual harvest for rice are expected to increasein China, India, Vietnam, Indonesia, Thailand and Philippines.
Up to 2050 the world‘s population will rise from 6 bn to 8 bn (33%) and about 50% of thetotal population are lives in Asia. Simultaneously, demand of food will increase by 50%, demand ofwater will increase 30%, and demand for energy will increase by 50%.
Considering those features, the approach need for adaptation are (1) public awareness toenhance participation, (2) improving institutional capacity building, (3) policy reform, (4) strong
enhancing research and study.
Ladies and Gentlemen,
In terms of energy, more than 40% of our populations engage in producing energy fromfossil fuel and wood to run their daily activities including agriculture. So there is a need for using asustainable source of fuel in the rural area in the future.
The Central Bureau of Statistics (BPS) reported that population growth in Indonesia during
the period of 2000 to 2010 was 1.49 percent per year, compared to 1.45 percent during the periodof 1990 to 2000. The 0.04 percent increase for the current population (237 million people) resulted
in a very big increase in food demand. If we do not make a serious effort to control birth rate, thecountry's population may reach an alarming 400 million in the year 2060. In the case of rice, with
consumption rate of 134 kg/cap/year, the demand for hulled rice in 2015 will be 44,4 million tons. In
addition to the pressure on the demand, large population also represent a serious threat toenvironment that cause unsanitary living conditions, the depletion of resources, environmental
pollution, and poverty, which are the root of environmental problems.
The second problem is diminishing natural resources, particularly land and water
resources. Irrigation water is an important production factor in rice production systems but it is nolonger available in unlimited quantities in rice-growing areas due the increase of competition fromdomestic use. Meanwhile, conversion of rice field to non-agriculture use is estimated about 100.000
ha annually. These two will significantly limit food production capacity in the country.
The third problem is the impact of global warming. Indonesia is already a significant emitterof greenhouse gases due to deforestation and land-use change, estimated at 2 million hectares per
year and accounts for 85 percent of the country‘s annual greenhouse gas emissions. The effects
will be felt more acutely by the poorest people, who are living in the most marginal areas that arevulnerable to drought, floods and landslides. International Food Policy Institute estimates that riceproduction in developing countries, will be reduced as much as fifteen percent (15%), and the priceof rice will increase to twelve percent (12%) in 2050.
Ladies and Gentlemen,
Opportunities to cope with fore mentioned problems are by using environmentally friendly
technologies. Luckily, our researchers have generated innovative technology for better crops that
increase productivity with environmentally friendly manners. Among of them are more aerobic
cultural practices, include intermittent irrigation and aerobic rice varieties, specific site nutrient
management, integrated pest management, and higher yield rice varieties.

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Ladies and Gentlemen,
I believe that through the 7th Asian Crop Science Association Conference, we are all will
share knowledge and experience; therefore, I hope this conference will come up withrecommendation to cope all those problems.
Finally, with Bismillahirrahmanirrohim, I declare The 7th ACSA Conference officiallyopen. Have a pleasant meeting and please enjoy your stay in Indonesia, especially in Bogor.
Wassalaamu‘alaikum warahmatullaahi wabarakaatuh.

Minister of Agriculture,SUSWONOth

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Table of Content
page
Foreword from the Chairman of Organizing Committee ………………………………………….

v

Foreword from the Rector of Bogor Agricultural University .......................................................

vi

Keynote Address ……………………………………………………………………………………….

vii

Table of Content ……………………………………………………………………………………….

xi

Invited speakers:
1.

Improving Photosynthesis to Increase the Productivity of Crops (Abstract)(Akiho Yokota) ………………………………………………………………………………

1

Impacts of Climate Change and Climate Variability on Productivity of Food Grain
Crops (Abstract)(PV Vara Prasad) …………………………………………………………………………...

2

The Plant Improvement of Tolerance to Oxidative Stresses (Abstract)(Shigeru Shigeoka) ………………………………………………………………………..

3

Salt Resistance Mechanism of Metroxylon sagu, Starch-producing Palm
(Hiroshi Ehara, Wikanya Prathumyot, Hitoshi Naito) …………………………………..

5

How to Increase Food Crop Productivity in Asia (Abstract)(Narasimham Upadyayula) ……………………………………………………………….

11

Biosynthesis of Nicotine as an Anti-insect Defense in Plants (Abstract)(Takashi Hashimoto) ………………………………………………………………………

12

Food Security and Agriculture Biotechnology Progress in Indonesia (Abstract)(Tantono Subagyo, Fadlilla Dewi Rakhmawaty) ………………………………………..

13

14-3-3 Proteins Act as Intracellular Receptors for Rice Hd3a Florigen (Abstract)(Hiroyuki Tsuji, Ken-ichiro Taoka, Izuru Ohki, Kyoko Furuita, Kokoro Hayashi,
Tomoko Yanase, Midori Yamaguchi, Chika Nakashima, Yekti Asih Purwestri, Shojiro
Tamaki, Yuka Ogaki, Chihiro Shimada, Atsushi Nakagawa, Chojiro Kojima, Ko
Shimamoto) ……………………………………...…………………………………………...

14

Food Security and Sustainable Agriculture (Abstract)(Paul PS Teng) ………………………………………………………………………………

15

10. Improvement of Oil Palm Productivity (Abstract)(Tony Liwang) ………………………………………………….….………………………..

17

2.

3.

4.

5.

6.

7.

8.

9.

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Concurrent Session and Poster Presentations:
Rice
1.

Characterization of Water Availability, Management Practices and Grain Yield for
Deepwater Rice in Northwest Cambodia(Nguyen T.B. Yen, Akihiko Kamoshita, Yuji Araki, Makara Ouk) ………………….

19

Submergence Escape in Oryza glaberrima Steud.
(Jun-Ichi Sakagami, Chiharu Sone, Naoyoshi Kawano) ……………………………

24

Toposequnential Variation in Soil Fertility and Limiting Nutrient for Rice Growth in
the White Volta Floodplain of Northern Ghana(Yasuhiro Tsujimoto, Yukiyo Yamamoto, Keiichi Hayashi, Alhassan I Zakaria,
Yahaya Inusah, Tamao Hatta, Mathias Fosu, Jun-Ichi Sakagami) …………………

29

Varietal Differences of Rice (Oryza sativa L.) Genotypes for Aleurone Traits
Contributing to Lipid Content(Ohn Mar Khin, Masao Sato, Tong Li-Tao, Yuji Matsue, Atsushi Yoshimura,Toshihiro Mochizuki) …………………………………………………………………...…

34

Development of New Cytoplasmic Male Sterile Lines with Good Flowering
Behavior for Hybrid Rice Breeding(Indrastuti A Rumanti, Bambang S Purwoko, Iswari S Dewi, H Aswidinnoor) ……

39

Genetic Analysis of Superior Double Haploid Rice Lines Developed from Anther
Culture(Muhammad Syafii, Bambang S Purwoko, Iswari S Dewi) ……………………….…

45

QTLs on Chromosome 12 Responsible for Expressing Root Plasticity underTransient Soil Moisture Fluctuation Stress in Rice(Jonathan M Niones, Yoshiaki Inukai, Akira Yamauchi) ……………………………

51

Yield and Related Traits in Two Japonica Rice Lines Carrying Ur1 Gene
(Stanis Malangen, Takumi Iwakura, Toru Hata, Takeshi Akaoka, Amol Dahal,
Masayuki Murai) …………….....................................................................................

56

Oxidative Stress and its Relation to Radiation Use Efficiency in Rice Growing
under Rainfed Condition in Northeast Thailand(Kohtaro Iseki, Koki Homma, Boonrat Jongdee) ....................................................

61

10. The Growth Characteristics of Some Varieties of Aceh‘s Local Rice (Oriza sativa
L.) on Acid Soils(Elly Kesumawati, Erita Hayati, Muhafaz Zulusfitri) ................................................

66

2.

3.

4.

5.

6.

7.

8.

9.

11. Effect of NPK Fertilizer and Biochar Applications on Growth and Yield of Irrigation
Rice(Zaitun, Khairun Nisa, Sufardi, Chairunas, Anischan Gani, Peter Slavich, Malem
McLeod) ....................................................................................................................

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12. Effects of New Foliar-Application Fertilizer Containing 5-Aminolevlinic Acid on
Yield Increase of Direct-Sowing Rice Plants(Ryuji Yoshida, Keitaro Watanabe) .........................................................................

76

13. Yield Stability Evaluation of Upland Rice Lines Obtained from Anther Culture(Deni D Sulaeman, Bambang S Purwoko, Iswari S Dewi, Muhamad Syukur,
Desta Wirnas, Heni Safitri) .......................................................................................

80

14. Yield and Blast Resistance Evaluation of Upland Rice Lines with New Plant Type
Characters(Jose MA Ornai , Bambang S Purwoko, Desta Wirnas, Iswari S Dewi, Heni
Safitri) .......................................................................................................................

85

15. Functional Analysis of Drought-Induced OsLEA3 Promoter Isolated from Batutegi
Rice Cultivar(Agus Rachmat, Satya Nugroho, Bernadetta Rina Hastilestari, Kinasih Prayuni,
Sudarsono) ………………………………………………………………………………...

91

16. Evaluation of the Nutritional Environment for Rice in Cianjur, Indonesia, forDevelopment of an Advanced Basin Model for Asia
(Koki Homma, Gunardi Sigit, Handarto, Masayasu Maki, Chiharu Hongo, Koshi
Yoshida, Kazuo Oki, Hiroaki Shirakawa, Tatsuhiko Shiraiwa, Ritsuko Hara, Mitsuo
Kambayashi, Yoshihiro Hirooka, Hiroki Iwamoto) ……………………………………..

96

17 The Nutritional Environment in Nonfertilized Rice Production and Its Effect on the
Nutritional Quality of Brown Rice
(Ritsuko Hara, Koki Homma, Yoshihiro Hirooka, Mitsuo Kuwada, Tatsuhiko
Shiraiwa) …………………………………………………………………………………...

101

18 Effect of Slow Release Fertilizer on Yield and Yield Components in Chinese HighYielding Rice Cultivars(Toshiaki Kokubo, Akira Miyazaki, Tetsushi Yoshida, Yoshinori Yamamoto, Jing
Ju, Yulong Wang) ………………………………………...……………………………….

107

19 Drought Resistance of NERICA Compared with Asian Rice, African Rice andMillets in the Field with Different Fertilization Levels(Michihiko Fujii, Seiji Ishihara, Ryohta Shinohara) …………………………………..

113

20 Social Experiment of Volumetric Irrigation Fee Scheme: Case of Gravity Irrigation
System in Bohol, the Philippines(Shigeki Yokoyama, Kei Kajisa, Tatsuro Miyazaki) ………………………………….

119

21 Ultraviolet-induced Fluorescence of Rice Leaf as Influenced by Nitrogen
Application and Cultivars(Mikio Sekinuma, Takehiro Natori, Futoshi Sasaki, Futoshi Kato, Naoto Inoue) …

125

22 Locus for Malate Secretion in Rice Chromosome 3(Miftahudin, Irfan Martiansyah, Dewi Indriyani Roslim, Tatik Chikmawati) ............

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Maize1.

Responses of Nutrient Efficient Maize Genotypes to Bio-fertilizer at Low Chemical
Fertilizer Doses(Marlin Sefrila, Munandar, Renih Hayati, Sabaruddin) ……………………………….

137

Cassava
1. Kinetin and Calcium Pantothenate Effects on Shoot Multiplication in In Vitro
Cultured Cassava Var. Adira 2 and Adira 4(Nurul Khumaida, Sintho Wahyuning Ardie, Candra Catur Nugroho, Suwarto) .....

141

Wheat
1.

Wet Injury of Wheat in Upland Field Converted from Paddy Field in Japan(Kentaro Kawaguchi) ..............................................................................................

147

Roles of Root System Development and Function in the Growth and Yield under
Waterlogged Condition in Common Wheat(Tomohito Hayashi, Tomofumi Yoshida, Kiyoshi Fujii, Takako Tsuji, Yurie Okada,
Eriko Hayashi, Akira Yamauchi) ...............................................................................

153

Growth and Physiological Responses of Sago Palm Against Aluminum Stress in
Acidic Conditions(Ornprapa Anugoolprasert, Hiroshi Ehara, Shina kinoshita, Hitoshi Naito,
Somchai Chakhatrakan) ...........................................................................................

159

Transplanted Sucker Stem Growth in Sago Palm (Metroxylon sagu Rottb.) before
Trunk Formation(Satoshi Nakamura, Keita Nabeya, Mutsumi Akama, Teiji Nakamura, Youji Nitta,
Manabu Watanabe, Yusuke Goto) ...........................................................................

165

Soybean
1. Secondary Aerenchyma Formation and Root Growth Response of Soybean(Glycine max) Seedlings under Flooded Conditions(Toshihiro Mochizuki, Satomi Sakazono, Sayuri Kajihara, Satoshi Shimamura) ..

169

2.

Sago
1.

2.

2.

3.

4.

5.

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Agronomical Performances of Soybean Cultivated under Saturated Soil Culture
on Tidal Swamps(M Ghulamahdi, M Melati, S A Aziz, A Junaedi, Sahuri, Y Puspitasari, D Sagala) .

175

Agronomic Performance of F7 Large Seed Soybean Breeding Lines in Medium
Plains(Darman M Arsyad, Asadi) ......................................................................................

180

Organically Production of Soybean Supported by Fertilizers Residue under
Saturated Soil Culture(Maya Melati, Kalimatul Jumro) ...............................................................................

185

Factors Causing the Soybean Yield Gaps between Japan and USA(Keisuke Katsura, Koki Homma, Yohei Kawasaki, Larry C Purcell, Randall L
Nelson, Taiki Yoshihira, Tatsuhiko Shiraiwa) …………………………………………

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6.

Photosynthetic Acclimation to Elevated CO2 in Soybean(Mochamad Arief Soleh, Hai Yan, Tomohiro Konno, Makie Kokubun, Ryoji
Sameshima) ……………………………………………………………………………….

196

The Effect of Organic Materials and Decomposer on Soybean Growth andProduction(Sandra Arifin Aziz, Sri Ayu Dwi Lestari) ................................................................

200

Yield and Dry Matter Production of Japanese and US Soybean Cultivars underDrought Stress(Yohei Kawasaki, Yu Tanaka, Keisuke Katsura, Tatsuhiko Shiraiwa) ....................

205

Selection of F4, F5 and F6 Soybean Breeding Lines for High Yield and Large
Sees Size(Darman M Arsyad, Asadi) ……………………………………………………………..

209

10. Effect of Hydrogen Peroxide Spraying on Drought Stress in Soybean Plant(Yushi Ishibashi, Haruka Yamaguchi, Takashi Yuasa, Mari Iwaya-Inoue, Susumu
Arima, Shao-Hui Zheng) …………………………………….……………………………

215

11. Productivity of Several Lines of Soybean in Majalengka, West Java, Indonesia(Alia Astuti, Ence Darmo Jaya Supena, Suharsono) ……………………………….

220

7.

8.

9.

Beans
1.

Paclobutrazol Application Effectiveness on Growth of Two Peanut (Arachis
hypogaea L.) Varieties(Iskandar Lubis, Aries Kusumawati, Munif Ghulamahdi, Heni Purnamawati,
Yudiwanti WE Kusumo, Ahmad Ghozi Mansyuri, Sri Astuti Rais) ............................

225

Effect of Source or Sink Restriction on Flowering, Podding, and Yield
Performance in Field-Grown Adzuki Bean
(Shinya Kasajima, Hirotake Itoh, Yasuo Nakamaru, Hozumi Yoshida, HitoshiSato) ………………………………………………………………………………………..

230

Bio-oil
1. Analysis of Genes Expressed during the Early Maturation of Sesame Seeds(Motonobu Yoshida, Akihiro Kishikawa, Takayuki Nakano, Eiji Tanesaka) ………

235

2.

2.

Effects of Different Seeding Dates on Pattern of Internode Length in Sorghum
Variety ‗Kazetachi‘(Akihiro Fujii, Satoshi Nakamura, Mitsuo Saito, Yusuke Goto) ..............................

239

Pranajiwa as Raw Material for Bio-Oil(Endang Yuniastuti, Susanti Indriya Wati) ……………………………………………

244

Vegetables
1. Selection and Evaluation Characteristics of Six Candidate Varieties of Cucumber(Cucumis sativus) in the Dry Season Planting(ST Rahayu, U Sumpena, Ali Asgar) ......................................................................

249

3.

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2.

Blue Light Induced the Stem Growth in Vegetable Water Spinach(Futoshi Sasaki, Mikio Sekinuma, Futoshi Kato, Naoto Inoue) ..............................

252

Effects of Genotypes and Storage Time on Quality Parameters of Chinese
Flowering Cabbage (Caisim) Planted in Subang(ST Rahayu, R Kirana) …………………………………………………………………..

256

Influence of Organic Fertilizer on Growth and Vitamin E Content of Traditional
Vegetable, Codonopsis lanceolata(Futoshi Kato, Naoto Inoue, Seishi Yoshida) …………………………………………

263

Potato and Tuber
1. Review on the Effect of Postharvest Treatment on Potato Quality(Ali Asgar) ..............................................................................................................

269

Perception, Attitude and Factors Influencing Household‘s Acceptance toTransgenic Late Blight Resistant Potatoes(Asma Sembiring, Eri Sofiari) .................................................................................

274

Relations Between the Amyloplast Sedimentation in Tubers and theMorphogenesis of Tubers in Yams
(Michio Kawasaki, Seito Hosokawa, Hayuru Yoshida, Kyoko Takahashi, Hiroki
Suto) .........................................................................................................................

279

Optimalising Potato Productivity in Sembalun Highlands, Nusa Tenggara Barat –Indonesia(Baiq Nurul Hidayah, Sudjudi, Lia Hadiawati, Dwi Praptomo Sudjatmiko, Peter
Dawson, Terry Hill, Julie M. Warren) ........................................................................

282

Fruits and Flowers
1. Transformation and Transient Ekspresion Analysis of L-HBsAg DNA in Fruits ofMusa acuminata Colla cultivar ‗Ambon Lumut‘ and ‗Mas‘ using Agrobacterium
tumefaciens(Sony Suhandono, Lailatul B, Ernawati Giri R, Putri DU, Dina RR, Ima MZ) ..........

287

3.

4.

2.

3.

4.

2.

3.

4.

5.

6.

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The Study and Early Evaluation of Resistance of Banana Accessions for Wilt
Disease Caused by Fusarium oxyporum f.sp. cubense VCG 01213/16 (TR4)(Agus Sutanto, Sudarsono, Dewi Sukma, Catur Hermanto) ...................................

291

Response of Plant Roots to Drought Stress(Atsushi Kato, Saki Hoshiyasu, Akiho Yokota, Kinya Akashi) ................................

296

Growth and Development Characteristics of Hoya multiflora Blume(Sri Rahayu) ………………………………………………………………………………

300

Effects of High Water Table and Waterlogging on Sunflower Growth, Yield andSeed Quality(Satoko Yasumoto, Yukari Terakado, Morio Matsuzaki) ……………………………

305

Population Genetics of Hoya multiflora at Sukamantri of Gunung Salak, West
Java, Indonesia Based on Isozyme Analysis(Sri Rahayu, Rochadi Abdulhadi) ……………………………………………………….

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Spices
1. Endophytic Bacteria as an Alternative Agent for the Biological Control of Plant
Parasitic Nematodes on Black Pepper(Abdul Munif, Rita Harni, Siswanto) ........................................................................
2.

315

The Effect of Organic and Inorganic Fertilizers on Growth and Yield of Red Ginger(Zingiber officinale Rosc.)(Rudiyanto, Tri Muji Ermayanti) …………………………………….…………………

319

Growth Analysis of Superior Clones of Temulawak (Curcuma xanthorrhiza Roxb.)
Grown with Organic Fertilizers(Bambang Pujiasmanto, Samanhudi) ....................................................................

324

Forage1. Effect of Drought and Inoculation of Arbuscular Mycorrhizal Fungi in Enhancing
Productivity and Tolerance Mechanism of Grasses(PDMH Karti, L Abdullah, J Nulik, Nurhayati, OR Nahak, S Nofyangtri) .................

331

3.

2.

The Cooling Effect of Forage Crop, Kudzu (Pueraria lobata) Vine Covering overLivestock Buildings(Takuya Koyama, Mika Yoshinaga, Kei-ichiro Maeda, Akira Yamauchi .................

335

Effect of Weed Control Management on Herbage Yield and Quality in the
Established Dwarf Napiergrass (Pennisetum purpureum Schumach)(Renny Fatmyah Utamy, Yasuyuki Ishii, Sachiko Idota, Lizah Khairani) ................

340

Effect of Density of Mother Plants on Efficiency of Nursery Production in Dwarf
Napiergrass (Pennisetum purpureum Schumach)(Asuka Yamano, Yasuyuki Ishii, Koutarou Mori, Renny F Utamy, Sachiko Idota) .

345

Analysis of the Major Seed Storage Protein, 13S Globulin, in Common Buckwheat(Fagopyrum esculentum Moench)(Tomoyuki Katsube-Tanaka, Nadar Khan, Yusuke Takahashi, Mariko
Nakagawa) ..............................................................................................................

349

Estate and Forest species
1. Isolation of soc-tuf Gene Encoding Chloroplast Elongation Factor Tu (EF - Tu)
Protein from Sugarcane (Saccharum officinarum)(Dhika Amanda, Sony Suhandono) .........................................................................

355

3.

4.

5.

2.

3.

4.

Endophyte Microbes from Oil Palm (Elaeis guineensis) Tissues and its Potential
as a Biocontrol for Ganoderma boninense in vitro(Wisnu Adi Wicaksono, Rika Fithri Buana, Elizabeth Caroline Situmorang) .........

360

Chitinase Activities of Oil Palm Root at Early Infection of Arbuscular Mycorrhizal
Fungi(Happy Widiastuti) ..................................................................................................

366

In vitro Test of Rhizosphere Chitinolytic Bacteria as a Biocontrol for Ganoderma
boninense(Rika Fithri Buana, Wisnu Adi Wicaksono, Elizabeth C Situmorang) ....................

372

th

Proceedings of The 7 ACSA Conference 2011

xvii

5.

The Effect of Mulch, Trichoderma, and Arbuscular Mycorrhizal Fungi (AMF)
Biofertilizer on the Growth of Oil Palm Seedlings Inoculated with Ganoderma(Happy Widiastuti) ................................................................................…………....

377

The Use of RAPD Marker on Gambier‘s Breeding Program(Hamda Fauza, Jamsari, Istino Ferita, Ade Noverta, Azmi Dhalimi, Ahmad
Denian, Murdaningsih H Karmana) ..........................................................................

382

Shorea leprosula: the Most Commercial Trees to Improve: ―Production-Natural
Forest‖ Productivity(Wahyudi, Indrawan A, Irdika Mansur, Prijanto Pamoengkas) ……………………

387

Patogenity Test of Two Isolates of Ganoderma on Sengon Seedlings(Elis N Herliyana, Irfan K Putra, Darmono Taniwiryono, Hayati Minarsih) ..............

391

Pathogen
1. A PCR-based Technique for Detection Cylindrocarpon destructans, the Causal
Agent of Grapevine Black Foot Disease(Raed A Haleem, Samir K Abdullah, Jaladet MS Jubrael) ...................................

397

Photosynthesis
1. Photosynthetic Light Reactions in C4 Photosynthesi(Naoya Nakamura, Yuri Nakajima Munekage, Akiho Yokota) …………………….

403

Environmental
1. Heavy Metals Concentration in Irrigation Water, Soils and Fruit Vegetables in
Coastal Area, Kota Bharu, Kelantan, Malaysia(ER Aweng, M Karimah, Osuhaini) ..........................................................................

407

6.

7.

8.

2.

The Contribution of Agriculture in a Local Greenhouse Gas Turnover(Arief Sabdo Yuwono) ………………………………………………………….………

412

The Influence of Global Climate Indices on Rainfall Distribution Pattern and Its
Impact on Crop Yield in Gunung Kidul, Yogyakarta, Indonesia(Bayu D A Nugroho, Hiromitsu Kanno, Ryoji Sameshima, Hiroshi Fujii, Larry
C.M. Lopez) ………………………………………………………………………………..

418

Economy1. An Input-Output Analysis of the Economic Impact for Sustainable Rural
Development in Wonogiri District, Indonesia(Shintaro Kobayashi, Shigeki Yokoyama, Satoshi Uchida, Yoshiko Iizumi) ……..

423

3.

2.

xviii

Permutation Test in Evaluating the Significance of Plants in PLS-DA Model ofJamu Ingredients(Farit Mochamad Afendi, Md. Altaf-Ul-Amin, Shigehiko Kanaya) …………………

th

429

Proceedings of The 7 ACSA Conference 2011

Index of Authors ......................................................................................................................

435

List of Organizing Committee ..................................................................................................

439

List of Participants ...................................................................................................................

441

List of Organizers ....................................................................................................................

453

Acknowledgements .................................................................................................................

455

th

Proceedings of The 7 ACSA Conference 2011

xix

xx

th

Proceedings of The 7 ACSA Conference 2011

Improving Photosynthesis to Increase the Productivity of Crops
Akiho Yokota
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan
yokota@bs.naist.jp

Abstract
Environmentally friendly energy-producing systems that utilize natural sources of energy, such as
solar, wind, land heat, and sea waves, have been developed around the world. These new energy
systems are expected to replace fossil fuels for energy production. However, they do not assimilate
CO2 liberated into the atmosphere artificially, nor do they produce food or feed for heterotrophs.
Thus, it would be prudent to utilize plant systems to synthesize organic matter from inorganic CO2
with solar energy. However, there are intrinsic restrictions on plant production, one of which is theability of the key photosynthetic enzyme, ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase(RuBisCO), to fix CO2 via photosynthesis. Photosynthesis converts CO2 into sugars and lipids. This
process relies on biochemical energy in the forms of reduced nicotinamide adenine dinucleotide
phosphate and adenosine triphosphate, which are produced from solar energy. As a catalyst,
RuBisCO has disadvantages. It has a low reaction rate, a low affinity for its substrate, CO2, and it
catalyzes the oxidation and evolution of carbons in sugars as CO2. In addition to RuBisCO, activitiesof two phosphatases have been reported to be too low to give other enzymes full play. Here, I
would like to address our successes in improving photosynthetic performance of tobacco andpotato.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 20111

Impacts of Climate Change and Climate Variability on Productivity ofFood Grain Crops
P V Vara Prasad
Department of Agronomy, Kansas State University, Manhattan, Kansas 66506, USA
2004 Throckmorton Plant Sciences Center, Department of Agronomy,
Kansas State University, Kansas 66506, USA
Tel.: +1 785 532 3746; Fax: +1 785 532 6094
vara@ksu.edu

Abstract
Crop production is highly sensitive to changing environmental conditions. In recent years long-termclimate change and year-to-year climate variability has become a major challenge to crop
productivity. Current knowledge on effects of season-long high temperatures and elevated carbon
dioxide concentrations; and effects of short periods of high temperature stress on various
physiological, growth and yield processes will be presented and discussed. Main focus will be on
major grain crops (dry bean, groundnut, soybean, rice, wheat and sorghum). Results indicate thatabove optimum temperatures will have negative impacts on reproductive processes (such as pollen
production, pollen germination, fertilization, seed numbers and individual seed weight) resulting inlower seed yield. The beneficial effects of elevated carbon dioxide mediated through increased
photosynthesis will be negated by rising temperatures resulting in lower seed yields. Grain crops
are most sensitive to high temperature stress during micro-sporogenesis and flowering. Hightemperature stress during these stages leads to loss of pollen fertility, poor pollination anddecreased fertilization, resulting in fewer seed numbers. Development high temperature tolerant
cultivars will be of prime importance for adaptation to climate change and climate variability.
However, there are several challenges to develop stress tolerance genotypes, some of theseinclude (a) availability of genetic variability in trait of interest; (b) development and use of efficientand reliable screening method; (c) identification of stress tolerant genotypes; (d) understanding
physiological and biochemical mechanism(s) associated with tolerance or susceptibility; and (e)heritability of identified trait(s) and incorporation of tolerant traits into existing high yielding
genotypes. Genetic variability exists in high temperature tolerance in few grain crops. Some
physiological traits that may contribute to high temperature tolerance include increased membrane
thermostability, increased green leaf duration, higher reproductive fertility, canopy temperature
depression, increased thermal tolerance and high temperature avoidance during flowering by timeof the day of flowering. New emerging biochemical and molecular tools provides some opportunities
for screening and phenotyping. Continued collaboration between physiologists, breeders, molecularbiologists and agronomists is essential for developing strategies to combat effects of climatechange and climate variability on crop production.
Keywords: temperature stress, carbon dioxide, pollen viability, seed yield, gen-- back to Table of Content --2

thProceedings of The 7 ACSA Conference 2011

The Plant Improvement of Tolerance to Oxidative Stresses
Shigeru Shigeoka
Dept. Advanced Bioscience, Faculty of Agriculture, Kinki University
Nakamachi, Nara 631-8505, Japan
Tel: +81 742 43 8083
shigeoka@nara.kindai.ac.jp

Summary
The explosive increase in world population and in environmental deterioration poses a serious
threat to agricultural production and food supplies. To improve food production efficiency, crops thatmature earlier with enhanced environmental stress tolerance and higher yield must be developed.
When plants are exposed to a variety of environmental stresses, excess amount of reactive oxygen
species (ROS) is generated, resulting in oxidative damage at the cellular level (J. Exp. Bot.
53,1305-1319,2002;Plant Cell Physiol. 45,1586-1594,2004). Despite their potential for causing
harmful oxidations, ROS are also powerful signaling molecules that are involved in the control ofplant growth and development as well as priming acclimatory responses to stress stimuli (Plant Cell
Physiol.51,190-200,2010; Plant Physiol.155,93-100, 2011). A wide range of strategies, including
molecular genetics and genetic engineering approaches, have been used to enhance the toleranceof plants to abiotic stress. Many attempts have been made over the last two decades to protect
photosynthesis against stress-induced inhibition by manipulation of component antioxidant
enzymes, and an extensive literature exists showing that enhancing the capacity of the water-water
cycle through genetic engineering, including the overexpression of superoxide dismutase,glutathione reductase, and dehydroascorbate reductase, can improve the tolerance of plants toabiotic stress. Overall, the enhancement of chloroplast antioxidant defenses has proved to be oneof the most effective ways of protecting plant cells from abiotic stress (Biosci. Biotechnol. Biochem.
72,1143-1154, 2008, Plant Physiol. 155,93-100,2011). Transgenic tobacco plants overexpressing
thylakoid membrane-bound ascorbate peroxidase (APX) in the chloroplasts exhibited enhanced
tolerance to high-light (HL) and paraquat (PQ) treatment, a combination that is designed tomaximize photooxidative stress (Plant J. 32, 915-925, 2002). Similarly, transgenic tobacco andtomato plants overexpressing KatE, a gene encoding an Escherichia coli catalase (CAT) in thestroma, resulted in a greatly enhanced tolerance to photooxidative stress (Plant Cell
Physiol.41,311-320,2000; Plant Cell Environ. 26,2037-2046,2003; Plant Physiol.145,258-265,2007).
In this case, the enhanced tolerance was explained in terms of compensation for the inactivation ofchloroplastic APXs by KatE. Ectopic expression of useful genes from cyanobacteria or
Chlamydomonas (Plant J 37,12-33, 2004) into the chloroplasts of higher plants has proved to beeffective at enhancing tolerance to abiotic stress. The GPX-like proteins of Synechocystis PCC
6803 are able to reduce unsaturated fatty acid hydroperoxides using NADPH as an electron donor,
but not GSH and TRX (Plant Physiol., 136, 2855-2861, 2004). The expression of Synechocystis
GPX in Arabidopsis chloroplasts resulted in a suppression of photoinhibition and an increased
tolerance to photooxidative stress (Physiol. Plant 128, 251-262, 2006). Recently we have isolated
various HL and heat-shock (HS) stress-inducible genes, including a heat shock transcription factor
A2 (HsfA2), by suppression-subtractive hybridization from Arabidopsis plants. HsfA2overexpressing Arabidopsis (Pro35S:HsfA2) plants showed increased tolerance to combined stressconditions, HL+ HS plus PQ treatments due to the increased expression of 46 genes, including alarge number of heat-shock proteins, APX 2 and galactinol synthase 1 and 2 (GolS1 and GolS2)

thProceedings of The 7 ACSA Conference 20113

(Plant J. 48, 535–547, 2006). Our findings indicated that HsfA2 is a key regulator in the induction of
the defense system under several types of environmental stress (Plant Cell Physiol. 51,486–496,
2010; 52,933-945, 2011). Furthermore, mutant pqr-216 from an Arabidopsis activation-tagged line
showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μM PQ. Basedon the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was
clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X)
hydrolase gene, AtNUDX2 encoding ADP-ribose pyrophosphatase, was responsible for thetolerance, resulting from maintenance of NAD+ and ATP levels by nucleotide recycling from free
ADP-ribose molecules under stressful conditions (J. Biol. Chem. 280, 25277-25283, 2005; Plant
Cell Physiol 48,1438-1449,2007; Plant Physiol.148,1412-1424,2008; 151,741-754, 2009; 152,
2000-2012, 2010; Plant J. 57,289–301,2009; Plant Signaling Behavior 5,839-841, 2010). The
evidence described above confirms the importance of the antioxidative enzymes and the value ofgenetically enhanced antioxidants and other defense compounds as a mechanism for stabilizing
photosynthesis in stress situations. Other metabolites can serve protective functions in chloroplasts.
Oligosaccharides such as galactinol and raffinose, which often accumulate to high concentrations,can act as antioxidants as well as osmoprotectants in plant cells. Transgenic Arabidopsis plants
overexpressing GolS1 and GolS2, key enzymes in the synthesis of galactinol and raffinose, had
increased levels of these oligosaccharides and enhanced tolerance to enhanced oxidation causedby exposure to paraquat, chilling, and osmotic stress (Plant Physiol. 147, 1251-1263, 2008). Thesefindings suggest that carbohydrates including raffinose family oligosaccharides and sugar alcohols
are present at high levels under normal and/or stressfull conditions and act as antioxidants toprotect plant cells from oxidative damage and maintain redox homeostasis (Plant Signaling &Behavior 3, 1016-1018, 2008).

-- back to Table of Content --4

thProceedings of The 7 ACSA Conference 2011

Salt Resistance Mechanism of Metroxylon sagu,
Starch-producing Palm
Hiroshi Ehara a,*, Wikanya Prathumyot a,b, Hitoshi Naito caGraduate School of Bioresources, Mie University, Tsu, Mie, Japan
Faculty of Agricultural Technology, Rambhai Barni Rajabhat University, Muang,
Chantaburi, ThailandcCollege of Life Sciences, Kurachiki University of Science and the Arts, Kurashiki, Okayama, Japan* Corresponding author: Graduate School of Bioresources, Mie University, Kurimamachiya-cho 1577, Tsu,
Mie 514-8507, Japan.
Tel.: +81 59 231 9495; fax: +81 59 231 9495.
ehara@bio.mie-u.ac.jpbAbstract
Sago palm (Metroxylon sagu Rottb.) distributed in Southeast Asia and Melanesia grows in swampy,
peaty, and alluvial soils in areas with both fresh and brackish water where almost no other crops
grow without drainage and/or soil improvement. This palm species stores a large amount of starchin the trunk. The starch of sago palm is processed into various basic raw materials and is receiving
attention as a sustainable energy resource as well as for use in the production of bioethanol due tothe current situation, in which competition between biofuel and food production is taking place.
However, sago palm is harvested primarily from natural forests and is considered to be an
unexploited plant. Thus, the Na+ and some other ion concentrations in different plant parts as wellas the physiological and morphological features under NaCl treatments (86 to 342mM NaCl for 1 or
4 months in a hydroponic system) were investigated to study the salt resistance mechanism of sago
palm to develop a sustainable method of cultivation that is essential for the improvement of sago
palm as an economic plant. (1) Sago palm maintained a low Na+ concentration in the leaflets, which
may be attributed to Na+ storage mainly in the roots. K+ absorption and distribution to the leaflets
may not be affected by changes in the Na+ concentration in the roots and petioles in sago palm. (2)Based on X-ray micro-analysis, a dense distribution of Na was observed around the endodermis of
the adventitious roots. Salt resistance of sago palm might be due to salt avoidance to mechanically
restrict an excess of Na distribution in plant tissues as well as maintain the water status in theleaves by restricting the transpiration. (3) The development of Casparian strips in the endodermis
can be considered as an important mechanical factor relating to the avoidance mechanism forpreventing the excess influx of Na+ into the stele and its translocation from root to shoot in sagopalm. (4) The factor limiting the photosynthetic rate under NaCl stress was the reduction in stomatal
conductance that resulted from a trade-off with the decrease in the transpiration rate to maintain thewater status in the leaves. (5) Although chlorophyll production was depressed, the absorption ofmacronutrients was not inhibited by salt stress and there was no lack of materials, such as N and
Mg for chlorophyll production. The chlorophyll concentration could increase up to high levels over acomparatively long time. These factors may account for the resistance of sago palm to salt stressand its ability to grow even with a reduction of the growth rate.
Keywords: Casparian strip, Na+ absorption, sago palm, salt avoidance, transpiration rateth

Proceedings of The 7 ACSA Conference 20115

Introduction
Sago palm (Metroxylon sagu Rottb.) that is distributed in Southeast Asia and South Pacificareas grows in swampy, alluvial and peaty soils where almost no other crops can grow without
drainage or soil improvement (Flach, 1977). It supplied carbohydrates and, like banana and taro,
has long been cultivated (Barrau, 1959). This rare palm species stores a large amount of starch in
the trunk, approximately 300kg (dry wt.) per tree (Ehara 2006). The importance of sago palm as astaple food is well recognized and the palm is still being in some areas of Southeast Asia and South
Pacific (Ehara et al., 2000). Its carbohydrate can be further processed into various basic raw
materials for food, animal feed and for industrial uses. Sago palm is one of the most important
crops for sustainable agriculture and for rural development in swampy areas of the tropics.
Since, sago palm is distributed not only in fresh water areas but also in brackish water
areas near the coast, it is considered to be salt-resistant. Flach (1977) reported that saline water
treatment up to EC values of 6 to 7 mmho/cm did not affect leaf emergence in sago palm.
However, there are few studies on the mechanisms of salt tolerance in sago palm. Thus, weinvestigated the Na+ and the other ions concentrations of different plant parts with some otherphysiological features under NaCl treatment to study the absorption and distribution of Na+ in sago
palm.

Materials and Methods
Expt 1. Seedlings at the 8th leaf stage (mean plant length: 50cm) were treated with theculture solution lacking or containing NaCl concentrations of the rates of 86, 171 and 342mM NaCl
(0.5, 1.0 and 2.0%) in a pot filled with vermiculite for 30 days (n=3) under a 25klx at 30ºC and
RH75%. Kimura B culture solutions (Baba and Takahashi, 1958) containing the different NaCl
concentrations (pH5.5) were supplied every day according to the amount of solution consumed andthen they were renewed once a week. The Na+ and K+ concentrations in the leaflets, petioles androots determined using an ion chromatograph with a conductivity detector (Shimadzu CDD-6A, ICC2, Japan).
Expt 2. The seedlings at the 8th or 9th leaf stage were used for the NaCl treatments. The
culture solution containing 342mM NaCl was used from July 9 to August 9, 2004 in the phytotron
under natural sunlight. Ion concentration in the plant tissue was measured. A portion in the region
above the extension zone of the large roots of the treated plants was soaked in liquid nitrogen after
sampling and kept at -70°C. The frozen root samples were freeze-dried and prepared astransverse sections. The sections were coated with gold ion and used for SEM observation and Xray micro-analysis (Horiba EMAX-5770W, Japan).
Expt 3. At ten months after germination, the longest adventitious root was taken as
sample and preserved in 70% ethanol solution. The transverse sections of the root were preparedas 1st position: root tip, 2nd position: 10 mm from root tip, 3rd position: 20 mm from root tip, 4th
position: 30 mm from root tip, 5th position: 40 mm from the root tip, 6th position: between the firstand second lateral roots, and 7th position: above the second lateral root. The sections were stained
according to Brundrett et al. (1988) and observed with a fluorescence microscope (Axio Imager A1;
Carl Zeiss, Germany).
Expt 4. Four spiny seedlings were selected and transferred individually to a 7.3 L plastic
pot filled with vermiculite. The mean value of plant length of all the plant materials was 79.3 cm.
Two treated pots were connected individually to the first peristaltic pump that was used to supply
Kimura B culture solution containing NaCl 224 mM (1.3% NaCl) (pH 5.5) to the treated pots for 6h
and the 2nd pump that was used to supply the culture solution without NaCl for another 6h by turns
twice a day. The 4th and 5th leaves from the top were used for the measurement of thephotosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Cs) by a portable
6thProceedings of The 7 ACSA Conference 2011

photosynthetic meter (Analytical Development Company limited LCA-4, England) under 900 µmol
m-2 s-1 PAR.

Results and Discussion
+

+

400

250
200
150
100
50
0

B
C
D
c

bc

ab

a

-1

A

350
300

K＋concentration (µmol g )-1

Na＋ concentration (µmol g )Na and K concentrations in different plant parts
There were no significant differences in the Na+ concentrations in the leaflets between thethree NaCl concentrations in the culture solution, the values of which ranged from 22 to 44 µmol/g(Figure 1). In the petioles, Na+ concentrations under the 342mM NaCl treatment were significantly
higher than those in the absence of NaCl treatment (one and half times), and the differences
between the absence of NaCl treatment and the 86mM NaCl treatment, 86mM and 171mM NaCl
treatments, and 171mM and 342mM NaCl treatments were not significant. The Na+ concentrationsin the petioles tended to be higher at higher NaCl concentrations, but with a maximum increase of
1.5 times. The Na+ concentrations in the roots were significantly higher at higher NaCl
concentrations, increasing 1.5, 2.2 and 3.1 times under the 86, 171 and 342mM NaCl treatments,
respectively. Two types of salt tolerance include an avoidance mechanism and a resistancemechanism (Yeo and Flowers, 1983).
Through the avoidance mechanism, a low Na+
concentrations maintained in the leaf blade. Sago palm can be considered to display an avoidancemechanism to maintain a low Na+ concentration in the leaflets by storing Na+ in the roots andpetioles. The K+ concentrations in the leaflets, petioles and roots did not show any significant
differences at three NaCl concentrations in the culture solution and in the absence of NaCl
treatment, which seems very important to understand the mechanism of salt tolerance in sago palm
(Figure 1). K+ accumulation may be associated with osmotic adjustment in sago palm.

800
700
600
500
400
300
200
100
0

L P R

L P R

L P R

L P R

L P R

L P R

L P R

L P R

0

86

171

342

0

86

171

342

Different letters in the figure indicate significant differences (0.05 probability level, Tukey-Kramer test).
++

Figure 1. Na and K concentrations in leaflets (L), petioles (P) and roots (R) in different NaCl treatments (0, 86,
171 and 342mM).

Na+ concentration in different parts of the roots
The Na+ concentration in the roots increased with the NaCl treatment (Figure 1). In theadventitious roots, the Na+ concentration was lower in the stele than in the cortex (Figure 2). Figure
3 shows the Na distribution from the cortex to the stele in the adventitious roots of the treated plants
based on X-ray micro-analysis. The amount of Na was larger in the cortex than in the stele. A
dense distribution of Na was observed in the inner region of the cortex near the endodermis. Basedon our present findings, it appeared that the region with the endodermis was able to trap some ofthe excess influx of Na into the large roots. This mechanism must be very important to restrict thetranslocation of Na+ from root to shoot under salt stress.

th

Proceedings of The 7 ACSA Conference 20117

Control plants+-1

Na concentration (μmol g )

600

300µm

Treated plants
a
*

700
S

C

C

S

500
400
b

300

*

b*

C: cortex
S: stele△: indicates theoutline ofendodermis
En: endodermis

200
100
11 Na Kα
Full scale [cps]5000Cortex Stele Small
Cortex Stele Lateralrootof Adventitious root
root

Cortex Stele Small
Cortex Stele Lateralrootof Adventitious root
root

Different letters in the figure indicate significant differences indifferent parts (0.05 probability level, Tukey-Kramer test). Asterisks
indicate significant difference in each part (0.05 probability level, Ttest).
+

Figure 2. Na concentration in different parts of the roots.
En
C

S

En

C

Figure 3. SEM of adventitious root and Na distribution based on X-ray micro-analysis.

C
Coo

En

Co

P
Phh
En
X
Xyy

Xy
P
Pee

5500 μμmm

50 μm

Figure 4. The structure and component of adventitious root at 10 mm from the root tip under visible light (left)
and UV (right) microscope. Arrowheads indicate Casparian strips in the radial cell walls ofendodermis. Co: cortex, En: endodermis, Pe: pericycle, Ph: phloem, Xy: xylem.

Casparian strip in the root
We found that the Casparian strip was located in the endodermal cell wall of bothadventitious roots of sago palm (Figure 4). Taking into account the previous findings of Ehara et al.
(2008) that a dense distribution of Na was observed around the endodermis in the extension zoneof the adventitious root, therefore, the Casparian strip develops in the endodermis of which can beconsidered as one of the important mechanical factors relating to functional role of the avoidancemechanism for preventing the excess influx of Na+ through an apoplastic pathway into the stele andits translocation from root to shoot in sago palm.

8thProceedings of The 7 ACSA Conference 2011

SPAD value .

Photosynthetic rate and related characteristics
After the 4th week of the treatment, the SPAD value of the control plants started toincrease and became stable after the 6th week (Figure 5). Due to the slow rate of increase in the
SPAD value of the treated plants, the difference in the SPAD value between the control and treatedplants became more appreciable in the 5th to 7th week and gradually decreased after the 7th week.
Finally, the SPAD value of the treated plants reached nearly the same level as that of the control
plants. The SPAD value is a parameter positively related to the chlorophyll concentration in plantleaf or chlorophyll content per unit leaf area. Therefore, it can be suggested that the rate of increasein the chlorophyll concentration of the treated plants also may be low. Based on this result, it can besuggested that sago palm is able to produce chlorophyll even under NaCl stress, although the rateof chlorophyll production was low. The important structural components of chlorophyll aremagnesium and nitrogen that are located in the centre of chlorophyll (Lack and Evans, 2001). We
found that the total N and Mg2+ concentrations of the leaflets did not display any distinct differences
between the control and treated plants, and it was therefore clear that the deficiency of N and Mg
was not the cause of the low chlorophyll production.
80

60

40

20
14th leaf
0
0

2

4

6

8
10
12
Duration (week)

14

16

18

Figure 5. Changes in SPAD value of the leaflets at the 14th leaf of the control plants (C1, C2) and treated plants(T1, T2) during the NaCl treatment. Open and close symbols indicate the control and treated plants,
respectively. Circle and triangle symbols indicate plant replication No. 1 and 2 of the control andtreatment.

The photosynthetic rate, transpiration rate and stomatal conductance in both leaves of thetreated plants decreased by 40%, compared to the value of the control plants (Figure 6). Based on
the water content, which was not appreciably different between the control and NaCl treatment, it issuggested that a decrease in the stomatal conductance, which leads to the decrease in thetranspiration rate and also to the decrease in the photosynthetic rate is the mechanism that sago
palm used to avoid water loss and maintain the water status in the plant body under salineconditions. The decrease in the photosynthetic rate can be interpreted as a trade off with thedecrease in the transpiration rate. The decrease of the photosynthetic rate might be caused by 2
main factors, i.e. reduction in the stomatal conductance and low chlorophyll production in theleaves.
In conclusion, sago palm growing under NaCl treatment can maintain a low Na+
concentration in the leaflets by storing Na+ mainly in the roots and petioles at lower leaf positions
with a fair reduction of the growth rate. This accounts for the slow morphogenesis and the decreasein the stomatal conductance and results from the maintenance of the water status in the plant body.

thProceedings of The 7 ACSA Conference 20119

-2 -1Pn (μmol m s )

10
8
6
4
2

-2 -1

Tr (mmol m s )

0
1.5

1.0

0.5

-2 -1

Cs (mol m s )

0.0
0.08
0.06
0.04
0.02
0.00

C1 C2

T1 T2

4th leaf

C1 C2

T1 T2

5th leaf

Figure 6. Photosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Cs) of the 4th and 5thleaves from the top of the control plants (C1, C2) and treated plants (T1, T2) at 900 PAR in October
2007.

References
Baba, I. and Y. Takahashi 1958. Solution Culture. In: Sakumotus Shiken Ho (Togari, Y. ed.) Nogyo Gijutsu
Kyokai, Tokyo. 327–343.
Barrau, J. 1959. The sago palm and other food plants of Marsh Dwellers in the South Pacific Islands. Econ. Bot.
13: 151–159.
+
Ehara H, H Shibata, W Prathumyota, H Naito, H Miyake. 2008. Absorption and distribution of Na , Cl and someothers ions and physiological features of sago palm under salt stress. Trop.Agr.Develop. 52:7-16.
Ehara, H. 2006. Geographical distribution and specification of Metroxylon palms. Jpn J Trop. Agr. 50: 229–233.
Ehara, H., S. Susanto, C. Mizota, S. Hirose and T. Matsuno 2000. Sago palm (Metroxylon sagu, Arecaceae)
production in the eastern archipelago of Indonesia: Variation in morphological characteristics and pithdry matter yield. Econ. Bot. 54: 197–206.
Flach, M. 1977. Yield potential of the sago palm and its realization. In: Sago-‘76: The Equatorial Swamp as a
Natural Resource (Tan, K. ed) Papers of the 1st International Sago Symposium, Kuala Lumpur. 157–
177.
Lack, A.J. and D.E. Evans 2001. Instant notes in plant biology. BIOS Scientific Publishers, London. Pp.332.
Yeo A. R. and T. J. Flowers 1983. Varietal differences in the toxicity of sodium ions in rice leaves. Physiol. Plant.
59: 189–195.

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thProceedings of The 7 ACSA Conference 2011

How to Increase Food Crop Productivity in Asia
Narasim Upadyayula
Monsanto Singapore Co (Pte) Ltd
Singapore. Tel.: +65 97220310; fax: +65 64885610
narasimham.upadyayula@monsanto.com

Abstract
At Monsanto we believe that by improving agriculture, we improve people‘s lives. In the hands offarmers, better seeds along with good agronomic practices can help meet the needs of our rapidly
growing population. We believe that we need to increase productivity while protecting the Earth‘snatural resources. Monsanto‘s technologies are striving to make agriculture truly sustainable bygetting more from each acre, each raindrop and each seed. In this presentation I will talk about the
3 main pillars that we think we can help us increase the productivity by using less resources: (1)Breeding, (2) Agronomic Practices, and (3) Biotechnology.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201111

Biosynthesis of Nicotine as an Anti-insect Defense in Plants
Takashi Hashimoto
Graduate School of Biological Sciences, Nara Institute of Science and Technology,
630-0192 Nara, Japan
hasimoto@bs.naist.jp

Abstract
Nicotine and related pyridine alkaloids are synthesized in the root and then transported to the aerial
parts of tobacco plants. Expression of biosynthetic genes for these alkaloids is enhanced 3-4-foldupon insect herbivory to the leaf. Current model suggests that jasmonate acts as a transmissible
signal from the damaged leaf to the underground part, where it activates structural genes of nicotine
biosynthesis via the conserved COI1-JAZ-MYC2 pathway. In Arabidopsis, the MYC2-family basichelix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive
genes, and their transcriptional activities are suppressed by physical interactions with JasmonateZIM domain (JAZ) suppressors. Regulatory NIC loci that positively regulate nicotine biosynthesis
have been genetically identified and their mutant alleles have been used to breed low-nicotine
tobacco varieties. The NIC2 locus comprises tandemly arrayed transcription factor genes of an
Ethylene Response Factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes aredeleted altogether. Overexpression, suppression, and dominant-repression experiments usingtransgenic tobacco roots showed functional redundancy and divergence among the NIC2-locus
ERF genes. These transcription factors recognized a GCC-box element in the promoters of nicotine
pathway genes, and specifically activated all known structural genes in the pathway. We also
demonstrate that tobaccoMYC2 controls nicotine biosynthesis genes in two combinatorial ways, bydirectly binding G-box in the target promoters, as well as by up-regulating the NIC2-locus ERF
genes.

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thProceedings of The 7 ACSA Conference 2011

Food Security and Agriculture Biotechnology Progress in Indonesia
Tantono Subagyo, Fadlilla Dewi Rakhmawaty
Syngenta Indonesia, Perkantoran Hijau Arkadia, Tower C, 8FL Jakarta

Abstract
Food security in Indonesia become serious problems because land conversion from agriculture ishigh and almost uncontrollable and the population growth rate is still high, the conditions worsenedby climate uncertainty. One of the answer to this problems is to increase yield and it can beachieved by increasing seed quality using available technology. Although it is not panacea,
biotechnology can provide tools to increase seed quality. Biotechnology research in Indonesia isdone since 1980 but so far little products come to the market due to the regulations. Recent
improvements in the system and regulations make the product pipeline moving, and it is hoped thatthe first launch of transgenic plants happen in 2012. However some improvements is still needed inorder to make regulations in Indonesia facilitating biotechnology development.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201113

14-3-3 Proteins Act as Intracellular Receptors for Rice Hd3a Florigen
Hiroyuki Tsuji1, Ken-ichiro Taoka1, Izuru Ohki2, , Kyoko Furuita2 , Kokoro
Hayashi2, Tomoko Yanase1, Midori Yamaguchi1, Chika Nakashima1, Yekti Asih
Purwestri1, †, Shojiro Tamaki1, Yuka Ogaki1, Chihiro Shimada1, Atsushi
Nakagawa3, Chojiro Kojima2,3 and Ko Shimamoto1
1

Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192, Japan
2
Laboratory of Biophysics, Graduate School of Biological Sciences, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192, Japan
3
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan†Present address: Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

Abstract‗Florigen‘ was proposed 75 years ago to be synthesized in the leaf and transported to the shootapex, where it induces flowering. Only recently have genetic and biochemical studies establishedthat florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved inhigher plants. Nonetheless, the exact function of florigen during floral induction remains poorly
understood and receptors for florigen have not been identified. We show here that the rice FT
homologue Hd3a interacts with 14-3-3 proteins in shoot apical cells, yielding a complex thattranslocates to the nucleus and binds to OsFD1, a rice homologue of Arabidopsis FD. The resultant
ternary 'florigen activation complex' (FAC) induces transcription of OsMADS15, a homologue ofArabidopsis APETALA1 (AP1), which leads to flowering. We determined the 2.4-Å crystal structureof a rice FAC. The modelled FAC structure provides a mechanistic basis for florigen function inflowering. Our results suggest that 14-3-3 proteins act as intracellular receptors for florigen in shootapical cells and offer new approaches to manipulate flowering in various crops and trees.

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thProceedings of The 7 ACSA Conference 2011

Food Security and Sustainable Agriculture
Paul P.S. Teng
S Rajaratnam School of International Studies
Centre for non-Traditional Security Studies (RSIS/CNTS)
andNational Institute of Education, Nanyang Technological University, Singapore 637616
paul.teng@nie.edu.sg

Abstract
Food security is a complex interplay of forces and is more than just an issue of supply and demand.
Various definitions have been proposed but in general most consider access to safe food which isnutritious at all times to all people. Four dimensions of food security have been proposed in aconceptual model by RSIS/CNTS, namely food availability, physical access, economic access andutilization. While each dimension is necessary for overall food security, they likely have different
weightings in a rural setting as compared with an urban setting and also across countries withdifferent incomes and net food trade balances. Food availability, the first dimension of food security,
addresses the ‗supply side‘ and is determined by the level of food production, stock levels, food aid
and net trade. Raising farm productivity is a core issue; whether by accessing or increasing inputs,
improving seed varieties, or employing better farm management practices. Herein also lays theinterplay with sustainable agriculture, as raising farm productivity has the potential to clash withsustainable agriculture goals. Food security implies surplus production over demand so that theexcess may be sold or traded with food deficit households. If Sustainable Agriculture is taken tomean agriculture which is ―environmentally friendly, economically sound and socially just‖, thenensuring food availability means ensuring that there is sustainable agriculture. A key debate is theuse of appropriate technology and the modality in which it is practiced, whether in a conventionalmanner or through organic and subsistence farming. The debate is further complicated by the fast
uptake of biotechnology crops. Food availability is often the focus of much of the debate on foodsecurity but raising farm productivity alone is not sufficient to ensure household food security. The
second dimension is the physical access to food. This means an adequate amount of food must bewithin the physical reach of vulnerable households, whether through their own production or throughthe marketplace, to assure food security. Common threats to physical access to food are war, civil
strife, poor infrastructure, inadequate logistics for food distribution and market imperfections. The
third dimension is economic access to food or the ability of the household to purchase the food it
requires. As the most recent food crisis demonstrated, urban households were among the hardest
hit as they saw their purchasing power decline drastically and they had very limited capacity to
produce their own food. Additional factors that will influence economic access include employment
and income security, macroeconomic policies and social security programmes. The fourth
dimension in food security is food utilization, typically reflected in the nutritional status of anindividual and is determined by the quantity and quality of dietary intake, food safety and humanhealth factors. Strategies to combat food insecurity arising from this dimension include biofortification of common staples through conventional or bio-technology. In the longer term, foodsecurity considered as a four-dimensional complex, is inextricably linked to sustainable agriculture,thProceedings of The 7 ACSA Conference 201115

which in an Asian context, must take into account the millions of smallholder farmers and themillions of urban poor.
Keywords: food security, sustainable agriculture-- back to Table of Content --16

thProceedings of The 7 ACSA Conference 2011

Improvement of Oil Palm Productivity
Tony Liwang
Plant Production & Biotechnology Division,th
PT SMART Tbk, BII Plaza, Tower 2, 10 Fl., Jalan M.H. Thamrin 51, Jakarta, Indonesia.
tony-liwang@smart-tbk.com

Abstract
Since 2009 Indonesia is the largest palm oil producer in the world. At the same time, Indonesia
became the largest oil palm seed producer as well as consumer in the world. However, some major
challenges facing in oil palm upstream industries in Indonesia are a narrow basic of genetic
material, a long period needed for breeding cycle, limited human resources and research findings,
and a limited genetic source for abiotic as well as biotic stress in some specific areas, and low
absorption efficiency of nutrition. This paper explains the main research activities to improve the oilpalm productivity. To enlarge and enrich the oil palm germplasm in Indonesia, a consortium of oilpalm companies has carried out some explorations, such as to Cameron, Angola, and others
countries. Moreover, the application of new gene resources has been studied for the phenotypiccharacters, genetic diversities and heterosities by using molecular markers. Elite varieties of oilpalm could be obtained faster by the application of micro satellite and Single Nucleotide
Polymorphism (SNP) markers from DNA genome sequences of selected genotype in early phase.
The application and commercialization of oil palm tissue culture were the big challenge for theindustry to improve the oil palm productivity at large scale, it might be included the clone of elite oilpalm and DxP bi clonal seedlings with 20-30% higher of productivity compared to conventional oil
palm. The originality of ramets could be done by using DNA fingerprinting. Genetic engineering
technology focused on water stress tolerant, resistance to Ganoderma sp. and oil synthesis.
Subsequently, the techniques of anther culture to produce dihaploid homozygous plant as a pureline will be used as a parental for F1 Tenera hybrid in order to produce oil palm with two to threetimes higher productivity compared to conventional oil palm.
Key words: oil palm, Elaeis guineensis Jacq, biotechnology, tissue culture, productivity-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011

17

18

th

Proceedings of The 7 ACSA Conference 2011

Characterization of Water Availability, Management Practices and Grain
Yield for Deepwater Rice in Northwest Cambodia
Nguyen T.B. Yena, Akihiko Kamoshitaa,*, Yuji Arakia,b, Makara OukcaAsian Natural Environmental Science Center, The University of Tokyo, Japanb
Faculty of Education, Saitama University, Saitama, Japan
Cambodian Agricultural and Development Institute, Phnom Penh, Cambodia*Corresponding author: Asian Natural Environmental Science Center,
The University of Tokyo, 1-1-1 Midoricho, Nishitokyo, 188-0002 Japan.
Tel/fax.: +81-42-463-1696.
akamoshita@anesc.u-tokyo.ac.jpcAbstract
This study aimed to characterize rice area from the shallower (lower toposequence of) rainfed lowlands side to
the deeper floating rice side in Northwest Cambodia during wet season rice (WSR) production in 2009 and 2010
for the yearly and spatial differences in field water conditions, management practices and grain yield. This area
considered as deepwater rice (DWR) area was divided into three groups with (1) upper fields located near the
National Road Number 5 (referred as ULR); (2) middle fields (referred as MLR and MFR where lowland rice (LR)and floating rice (FR) were planted, respectively); and (3) lower fields located near to the Lake (referred as LFR).
Water came to the paddy fields from both the inundation from Tonle Sap Lake and rainfall in WSR 2009, but
only from rainfall in WSR 2010. Water depths increased from upper to lower fields in 2009, while they wereshallower and relatively similar between the field locations in 2010. Broadcasting time started earlier andharvesting time finished later in FR fields than in LR fields. The overall average grain yield for both years of 2009
and 2010 was low. The lowest yields were observed in MLR and MFR in 2009 as well as they were in MFR and
LFR in 2010. Lower yield in WSR 2009 was mainly due to the water shortage at heading stage for both FR and
LR, improper application of N fertilizer and insufficient weed management for LR, and late sowing for FR. Lower
yield in WSR 2010 was mainly due to low water level for FR, low N fertilizer rate and insufficient weed
management. This study identified important spatial and yearly variation in rice management and grain yield forfarmers to cope with unpredictable flooding environments in DWR area.
Keywords: Cambodia, deepwater rice, flooding pattern, rice type

Introduction
In Cambodia, deepwater rice (DWR) areas are located in the provinces near to the Tonle
Sap Lake, the Mekong River, and Tonle Bassac River. In 1960s, the DWR area occupied up to 16%
of Cambodia‘s rice land (about 400,000 ha) (Javier, 1997; Seng et al, 1988). However, as thediscouragement of growing DWR during Pol Pot regime, DWR area decreased sharply and it was
only 120,000 ha in 1988 (Seng et al., 1988). DWR presented 3.9% of the cultivated area in 2006
(MAFF, 2006). Cambodian‘s Tonle Sap Lake (TSL) floodplain is well known for its unique dynamic
flooding pattern between dry and rainy season. Volume of the Lake ranges from about 1.3 to 75
km3, its surface area varies from 2,500 to about 15,000 km2, and its water level increases from 1.4
to 10.3 m above sea level, between dry and rainy season (MRC, 2010a). DWR is important sourceof livelihood to many poor villages in the TSL floodplain that do not have access to better
agricultural land higher up. There is a need to further increase the productivity of DWR in order toimprove the local farmers‘ livelihood and to conserve the DWR area. However, necessary
information for yield improvement in DWR area such as flooding pattern, land use pattern,management practices in Cambodia are not sufficient. We conducted a study to quantify the yearlyand spatially difference in field water condition in DWR area in Northwest Cambodia, and to assess
rice management practices and grain yield in the area.
th

Proceedings of The 7 ACSA Conference 201119

Materials and Methods
The study was carried out at a DWR area inside a flood plain area of TSL located in
Kampong Preah village, Kampong Preah commune, Sangke district, Battambang province,
Northwest Cambodia in 2009 and 2010. Eighty five fields (91.2 ha in planting area) were selectedwhich were located continuously along a transect line from a toposequentially upper zone nearby
National Road Number 5 toward TSL. These 85 fields were divided into three groups according totheir field locations : (1) upper fields located closer to the National Road Number 5 where only LR
was grown (19 fields; 11.4 ha) and referred as ULR; (2) middle fields where both LR and FR weregrown (19 fields; 31.6 ha) and referred as either MLR or MFR; and (3) lower fields located near to
the Lake where only FR was grown (37 fields; 48.2 ha) and referred as LFR. Variety, field size andtype of rice were determined for the 85 fields in WSR 2009 and 2010. Thirty fields within the 85
fields were selected from each field group; sowing time, mid-season tillage practice, fertilizer input,
pest control and grain yield were determined by the interviews to the owners in ending time of each
WSR.

Results and Discussion
Field water environment
Field water regime of the studied area in WSR 2009 was completely different from that in
WSR 2010 (Figure 1). In WSR 2009, water came to the fields from both the inundation from the
Tonle Sap Lake and rainfall. Flood started earliest in lower fields in September, then middle andupper fields afterward. Flood started receding to the Lake in late October and rice fields became
non-flooded conditions in middle fields and upper fields in late November and in lower fields in early
to mid December. There was large difference in water depth between the 3 field locations in 2009.
Rice ecosystems of the three locations could be classified as DWR for middle and lower fields andmedium-deep rainfed lowland rice for upper fields (Mackill et al., 1996). In WSR 2010, water cameto the fields only from the rainfall; field water regime was relatively similar to all the 3 field locations
with the average maximum water depths less than 30 cm in mid October. These water conditions in2010 were more favorable for rainfed lowland rice than floating rice. Low water level in Tonle Sap
Lake leading to shrink floodplain area in 2010 was probably due to extremely low water level in
Mekong River (IRIN news, 2010) which was caused likely by a combination of an early end to the2009 wet season, low monsoon rainfall and very low rainfall in the dry season in upper Mekong
Basin (MRC, 2010b).
Management practices
Crop calendar
Planting season for FR fields is earlier than that for LR fields. For FR fields, farmers startland preparation from early March to late April (Figure 1). Dry seeds with rate of about 100 kg ha-1
are broadcasted mainly from early April to early May. Farmers sometimes have to broadcast seeds
for the second time in late May or early June if rice establishment failed due to drought. For LR
fields, land preparation is often from mid April to mid May. Dry seeds with higher rate in comparison
with FR (150 kg ha-1) are broadcasted from mid May to mid June. Rice is harvested from early
December to early February in FR fields and from late November to early January in LR fields, both
depending on maturity type of rice varieties.
Distribution of rice type and varieties
LR and FR were both grown in the studied area in the both WSR 2009 and 2010. In
general, early medium varieties of LR were only planted in upper fields while medium varieties ofLR were planted in both upper and middle fields. Early varieties of FR were only planted in middle
20thProceedings of The 7 ACSA Conference 2011

fields while medium and late varieties of FR were planted in both middle and lower fields. In
comparison between WSR 2009 and 2010, there was a small change in the area ratio of LR and FR
in middle area. Area of LR decreased from 38% in WSR 2009 to 30% in WSR 2010 but area of FR
increased from 62% in WSR 2009 to 70% in WSR 2010. Among the varieties planted in the studiedarea, early varieties of FR in the middle area had the largest increase in area percentage, from 22%
in WSR 2009 to 47% in WSR 2010.
250

120
upper (LR)middle ( LR & FR)lower (FR)(a)

Rainfall: 1,218 mm, 74 days
100
80

150
60
100
40
50

20

0

0

250

120

(b)

Rainfall: 1,356 mm, 89 days
100

200
Water depth (cm)Rainfall (mm)rainfall

80
150
60
100

Rainfall (mm)Water depth (cm)

200

40
50

20

0

0

(c)

Land preparation
SowingFR

Heading
Harvesting
Land preparation
SowingLR

Heading
Harvesting
Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sept

Oct

Nov

Dec

(a) and WSR 2010 (b), and cropping calendar for FR and LR (c) in the studied area. The error bars indicate standard deviations.

Figure 1. Rainfall distribution and water depth in the 3 field locations (upper, middle and lower) in WSR 2009.

Relationship between grain yield, environmental and management factors
In WSR 2009, grain yields of ULR and LFR (120 and 164 g m-2, respectively) weresignificantly higher than those of MLR and MFR (50 and 56 g m-2, respectively). However, grain
yields of ULR and MLR (both 262 g m-2) were significantly higher than those of MFR and LFR (87and 111 g m-2, respectively) in WSR 2010.
Average grain yield of both WSR 2009 and 2010 was low with only 180 g m-2 for LR and-2
110 g m for FR. The lowest yields were observed in MLR and MFR in WSR 2009 which was due
to risky water environments in middle fields. With maximum water depth of 85 cm and absence ofstanding water after late November (Figure 1), the water condition in this area was too deep for LR
for a possible submergence damages and insufficient for medium and late maturing FR to maintain
good grain filling. This was also supported by the positive correlation between maximum water
depth and grain yield for FR and negative correlation between those for LR (Table 1). Farmers
responded to this risky water condition by changing rice type/varieties from year to year based onyield obtained from previous year. The increase in area of early variety of FR in WSR 2010 was dueto that farmers observed that yields of early variety of FR in middle fields were higher than othervarieties of LR and FR in this area (data not shown).
Low yield in WSR 2009 was mainly due to (1) the water shortage at heading stage for both
FR and LR, (2) non- or late application of N fertilizer for LR, (3) insufficient weed management forthProceedings of The 7 ACSA Conference 201121

LR and (4) late sowing for FR (Table 1). (1) Due to low rainfall occurring at pre-flood period (June toAugust; Figure 1a) in WSR 2009, rice plants suffered drought stress leading to delayed heading,mainly from late November to mid December while there was also low rainfall in the late season.
This caused the shortage of water at heading stage of 10 out of total 29 fields. Those fields where
rice plants did not suffer drought at late stage were mainly located in lower part, or bunded with highlevees, or pumped water from the water source nearby. This indicated that well-water management,
especially at heading stage, is crucial to improve yield in DWR area, particularly in middle andupper fields. (2) In WSR 2009, LR plants were likely submerged during high flood (40 cm in upper
fields and 85 cm in middle fields), growing situation of rice plants before flood commence was very
crucial for gaining high grain yield. Taller and more vigorous rice plants, which could be improved bybasal application of N fertilizer (before flood commence), were able to withstand submergence andhence gave higher yield and this was also indicated in Puckridge (1991). It was suggested that rice
plant should be able to uptake more than 20 kg N ha-1 before onset of flooding in order to reduceyield loss due to submergence (Puckridge, 1991) while Sharma and Gosh (1998) reported that
optimum basal fertilizer rate for semi-deep water environment was 30 kg N ha-1. The average N
fertilizer applied in LR field in WSR 2009 was less than half of this recommendation rate (4-15 kg N
ha-1; data not shown). (3) Our study showed that conducting midseason tillage practice, a weed
control method, helped to improve yield of LR in WSR 2009. All of LR fields were conducted thepractice in WSR 2010 while about half of LR field number were not conducted in WSR 2009, which
was perhaps due to water constraint (drought or flood at the time farmers wanted to do thepractice). (4) Late sowing significantly decreased yield of LFR. High rainfall occurred mainly fromlate April to mid May while only few rains with small amount occurred in June and July (Figure 1a).
This might be the reason leading the poor establishment of crop with late sowing. When the flood
arrived, this late sowing rice was likely more susceptible to the rapidly rising water in September.
The importance of sowing in time was also mentioned by Catling (1983), Javier (1997) and Sing etal. (2004).
Table 1. Correlation between rice yield and water condition, management factors of LR and FR in WSR 2009and WSR 2010

*P<0.1; **P<0.05; ***P<0.01
avalues for flooded at heading stage and those for other water conditions at heading were 1 and 0, respectively, as a dummy
variablebvalues for inorganic fertilizer applied before flood commence and those for not applied or after flood commence were 1 and 0,
respectively, as a dummy variablecvalues for conducting mid-season tillage and those without the practice were 1 and 0, respectively, as a dummy variabledvalues for conducting mid-season tillage by plowing and those for conducting the practice by harrowing were 1 and 0,
respectively, as a dummy variable

22thProceedings of The 7 ACSA Conference 2011

Low yield of FR in WSR 2010 was mainly due to low water level as the discussion at
beginning of this section. Beside that insufficient weed control was also another reason. Higher
application rate of herbicide was applied in WSR 2010 in comparison with that in WSR 2009 andlow application rate of herbicide significantly reduced yield of FR in WSR 2010 (Table 1). This was
because that weed infestation was more severe throughout the crop season due to the low waterlevel in WSR 2010 while FR fields were not conducted midseason tillage for controlling weed likeLR. Different from WSR 2009, water condition in WSR 2010 was more favorable for growing LR likein rainfed lowland environment. Therefore, yield of LR in WSR 2010 could be further improved byconducting midseason tillage with plowing for controlling weed (Table 1) and increase application
rate of N fertilizer. The N fertilizer rate applied this WSR was only about one third of therecommendation rate for rainfed lowland rice in drought or submerged prone area with rate of 60 kg
ha-1 (Balasubramnian and Hill, 2002). However, it should be noticed that field water environment in
WSR 2010 was not representative for most years in the studied area.
In short, it is risky for farmers to attempt to increase planting higher yielding lowland rice inthe middle part of the floodplain of TSL, due to the occurrence of deep flood incidence, but farmers
could miss chances of higher yield if planting traditional floating rice varieties just to escape fromsubmergence damage. It is desirable if information on water situation in the area is informed tofarmers by a long-term weather forecast before the cropping season (February or March). So that
farmers will be able to make a right decision of selecting rice type/variety to grow in the area.
Beside that early maturity varieties with higher yield potential and tolerance to submergence(suitable for medium-deep water area) can be introduced in to the area in order to improve grain
yield in a sustainable manner.

References
Catling, H.D., Hobbs, P.R., Islam, Z. and Alam, B., 1983. Agronomic practices and yield assessments of
deepwater rice in Bangladesh. Field Crops Research, 6: 109-132.
IRIN news. 2010. Cambodia: Record low water levels threaten livelihoods. [Online]. Available at
http://www.irinnews.org/Report.aspx?ReportId=90291 (accessed 22 July 2011).
Javier, E.L., 1997. Rice ecosystems and varieties. In: H.J. Nesbitt (Editor), Rice production in Cambodia.
Cambodia-IRRI-Australia project, pp. 39-82.
Mackill, D.J., Coffman, W.R. and Garrity, D.P., 1996. Rainfed lowland rice improvement. International Rice
Research Institute, Manila, Philippines, 242 pp.
MAFF (Ministry of Agriculture, Forestry and Fisheries) 2006. Agriculture, Forestry and Fisheries statistic in
Cambodia 2005-2006. [Online]. Available at http://faorapapcas.org/cambodia/AgriStat-Cambodia2005/
index.htm (accessed on 15 Jan 2011)
Mekong River Commission (MRC). 2010a. Assessment of basin-wide development scenarios: Technical note
10. The Mekong River Commission, Vientiane, Lao PDR. 45 pp.
Mekong River Commission (MRC). 2010b. Preliminary report on low water level conditions in the Mekong
mainstream. Technical note 10. The Mekong River Commission, Vientiane, Lao PDR. 10 pp.
Puckridge, D.W. et al., 1991. Nitrogen uptake by deepwater rice during the preflood and flooded phases, in
relation to drymatter and grain production. Field Crops Research, 27: 315-336.
Seng, L.T., Puckridge, D.W. and Thongbai, P., 1988. Cultivation of deepwater and floating rice in Kampuchea,International Deepwater Rice Workshop. International Rice Research Institute, pp. 159-167.
Sharma, A.R. and Ghosh, A., 1998. Optimum seed rate and nitrogen fertilizer requirement of rice under semideepwater ecosystem. Journal of Agronomy and Crop Science-Zeitschrift Fur Acker Und Pflanzenbau,
181: 167-172.
Singh, V.P., Hong, N.V., Sharma, A.R. and Dhanapala, M.P., 2004. Challenges and strategies in rice crop
establishment for higher productivity in flood-prone environments. In: S.I. Bhuian, M.Z. Abedin, V.P.
Singh and B. Hardy (Editors), Proceedings of the international workshop on flood-prone rice systems.
Los Banos (Philipines): International Rice Research Institute, Gazipur, Bangladesh, 9-11 January 2001,
pp. 189-204.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201123

Submergence Escape in Oryza glaberrima Steud.
Jun-Ichi Sakagamia,*, Chiharu Sonea, Naoyoshi KawanobaCrop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences,
Tsukuba, Ibaraki, Japanb
Rural Development Division, Japan International Research Center for Agricultural Sciences,
Tsukuba, Ibaraki, Japan*Corresponding author: Crop, Livestock and Environment Division,
Japan International Research Center for Agricultural Sciences,
1-1, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
Tel.:+81 29 838 6368; Fax: +81 29 838 6355.
sakajun@affrc.go.jp

Abstract
Oryza glaberrima, an African monocarpic annual rice derived from Oryza barthii, is grown in traditional riceproducing wetland areas of West Africa. Oryza sativa, an Asian rice that varies from annual to perennial, isderived from Oryza rufipogon. Genotypes of O. glaberrima are inherently lower yielding than those of O. sativa
and are, therefore, cultivated in fewer areas. However, because they grow adequately in unstable environments
such as those with water stress, they appear to tolerate severe environmental stress. Cultivars of O. glaberrima
are roughly divisible into two ecotypes: upland and lowland. However, it might be that O. glaberrima is avaluable rice species for flooding conditions in all cases. To elucidate the physiological responses of young rice
plants to short-term submergence stress, so-called flash flooding, under rainfed conditions for O. glaberrima bycomparison with several genotypes for lowland adapted, deepwater adapted shoot elongated escape and Sub1of O. sativa, 30-day-old seedlings were submerged completely for 10 d at 45 cm water depth at 13 d after
transplantation in a lowland field. In fact, O. glaberrima showed higher shoot elongation ability during
submergence than any genotype of O. sativa that we tested. However, O. glaberrima lodged easily after the endof submergence because of longer and more rapid shoot elongation during submergence. Therefore, it triggereda decrease in its survival rate. On the other hand, various lines of 35 O. sativa and 27 O. glaberrima, including
some classified as short-term submergence tolerant, were compared for submergence tolerance in field and pot
experiments to long-term submergence tolerant varieties in other words, deepwater varieties. Submergencetolerant cultivars of O. sativa were unable to survive prolonged complete submergence for 31–37 d, whichindicated that the mechanism of suppressed leaf elongation that confered increased survival of short-term
submergence was inadequate for surviving long periods underwater. The superior tolerance of deepwater O.
sativa and O. glaberrima genotypes to prolonged complete submergence appeared to be attributable to theirgreater photosynthetic capacity developed by leaves that had newly emerged above the floodwater. Cultivars of
O. glaberrima adapt to long-term complete submergence. Cultivar ―Saligbeli‖ adapted to short to long term
submergence.
Keywords: glaberrima, lodging, photosynthesis, shoot elongation, submergence escapeIntroduction
Rice farming in Africa began approximately 3500 years ago (Porter, 1970). The rice variety
cultivated at the time was African rice (Oryza glaberrima Steud.), which is native to the continent(Chu and Oka, 1972). African rice is well adapted to the severe local climate, but it has had little
opportunity to be improved or selected by humans. It is, therefore, regarded as wild and of low
productivity. For that reason, Asian rice (O. sativa L.) accounts for a high proportion of the riceconsumed in Africa today. However, the development and promotion of a NEw RIce for AfriCA(NERICA) is attracting attention in Africa. Moreover, the properties of African rice, which was
employed as a parent strain in the production of NERICA, are now being reassessed. In thismanner, rice farming in Africa has been developed differently from that in Asia, and cultivation andcooking methods also differ in many ways. Farmers cultivate rice in irrigated lowlands in Asia,
24

thProceedings of The 7 ACSA Conference 2011

although rainfed uplands in Africa account for most rice fields. African farmers attach great
importance to the quantity immediately after cooking in many cases (Sakagami et al., 2008).
Although rice consumption in African countries has shown an increasing trend in recent years, theproduction rate is not sufficient to meet the demand. The volume of imported rice is increasing
annually, which is having a detrimental effect on African countries‘ economies.
In general, rice cultivation is vulnerable to natural disasters in West Africa. One reason is
the shortage of suitable irrigation systems. The area of irrigation is less than 20% of the total area ofrice cultivation, and most rice is planted in rainfed regions (Balasubramanian et al., 2007).
Therefore, rice cultivation in West Africa is strongly influenced by precipitation or overflow fromrivers. However, the status of damage to rice plants by flooding in West Africa has not been
understood well until now. Upland rice is severely influenced by rainfall because of the lack ofstanding water. Yields of upland rice are very low (around 1 t ha-1) compared with those of lowland
rice cultivation (around 2 t ha-1) (Norman and Otoo, 2003). Lowlands, therefore, offer greater
potential for raising rice production, and represent about 20–50 million hectares, depending on thedefinition used. At present, only about 10–20% of this area is under cultivation (Africa Rice Center,2004), thereby offering great potential for rice farming expansion. However, rice plants in lowland
areas are often damaged by floods caused by heavy rain. It is, therefore, important to study theeffects of submergence on rice plants to develop sustainable rice production in West Africa.
Genotypes of O. glaberrima are inherently lower yielding than those of O. sativa and are,therefore, cultivated in fewer areas (Linares, 2002). However, because they grow adequately in
unstable environments such as those with water stress, they appear to tolerate severe
environmental stress. Flooding imposes severe selection pressure on plants, principally because
excess water in the plant surroundings can deprive them of certain basic needs, notably of oxygenand of carbon dioxide and light for photosynthesis. It is a major abiotic influence on species‘
distribution and agricultural productivity world-wide. Based on our analysis, most O. glaberrima
varieties adapt well when floods are deeper and when they entail long-term submergence in Africa
because of their greater photosynthetic capacity developed by leaves that have newly emerged
above floodwaters through rapid shoot elongation.

Materials and Methods
Experiment 1: Responses to short-term submergence “flash flood”
To elucidate the physiological responses of young rice plants to short-term submergencestress, so-called flash flooding, under rainfed conditions for O. glaberrima by comparison withseveral genotypes for lowland adapted, deepwater adapted shoot elongated escape and Sub1 of O.
sativa, 30-day-old seedlings were submerged completely for 10 d at 45 cm water depth at 13 d after
transplantation in a lowland field of Guinea. Dry matter production, plant height, lodging andsurviving rate were compared.
Experiment 2: Responses to long-term submergence “deep water”
Various lines of 35 O. sativa and 27 O. glaberrima, including some classified as short-term
submergence tolerant, were compared for submergence tolerance in field and pot experiments tolong-term submergence tolerant varieties in other words, deep-water varieties. Plants were
submerged completely for 31 d in a field experiment, and partially or completely for 37 d in a pot
experiment in a growth chamber. Dry matter production, plant height, leaf area, and photosynthetic
rate were compared.
Experiment 3: Flash flood tolerance for cultivated species
Lodging, plant height, and dry matter accumulation for 99 cultivars in O. sativa, O.
glaberrima, and interspecific hybridization progenies (IHP) were measured when 12-day-oldthProceedings of The 7 ACSA Conference 201125

seedlings were submerged completely for 7 d in pots and in fields to make an evaluation of flash
flood tolerance.

Results and Discussion
There was a high positive correlation (P < 0.01, r =0.86) between shoot length elongation
during short term submergence and lodging score at 15 DAS (Day After Submergence) inexperiment 1. DRL (Deep-water to Rainfed Lowland genotype in O. glaberrima) showed higher
shoot elongation during submergence and a higher lodging score after desubmergence than othergenotypic groups. On the other hand, ST (Submergence Tolerance genotype) showed the opposite
features to DRL with lower shoot elongation and lodging score. RL (Rainfed Lowland genotype),
DW (Deep-Water genotype) and SE (Shoot Elongation genotype) showed intermediate traits inshoot length elongation and lodging score between DRL and ST. Figure 1 shows the relationshipbetween shoot length elongation during submergence and survival rate at 19 DAS. There was anegative correlation between shoot length elongation and survival rate (P < 0.05, r = -0.66).

Figure 1. Effect of shoot length elongation on survival rate. Survival rate was calculated for number of plants
before submergence divided by number of surviving plants at 19 day after desubmegence

ST of Sub1 showed the highest survival rate (93%) and the shortest shoot length
elongation (6.8 cm) as well as IR 62293- 2B-18-2-2-1-3-2-3 (86%, 11.5 cm) in the SE group. The
survival rate of DRL was lower compared with ST. O. glaberrima showed higher shoot elongation
ability during submergence than any genotype of O. sativa tested. However, O. glaberrima lodged
easily after desubmergence due to longer and rapid shoot elongation during submergence, andthus triggered a decrease in its survival rate. We suggested that O. glaberrima was susceptible toshort-term submergence while it may be adapted to prolonged flooding because of improved
restoration of aerial photosynthesis and survival rate through shoot elongation ability
All cultivars of O. sativa with submergence tolerance based on the quiescence strategy
failed to regain contact with the aerial environment and died during 31 d submergence in the field
experiment 2. In contrast, all O. glaberrima genotypes resurfaced and survived submergence. The
photosynthetic rates of the youngest fully expanded leaf of the main shoot of pot-grown plants were
measured in pot experiment 2. The non-submerged rate for ‗IR73020‘ (30.9 μmol m-2 s-1) 1
DBS(Day Before Submergence) was significantly above those for other genotypes. The rate for‗Yele1A‘ (6.74 μmol m-2 s-1) was the lowest. It was, however, significantly greater for Yele1A‘ thanfor other genotypes in non-submergence plots (29.2 μmol m-2 s-1 ), in partial submergence plots(30.3 μmol m-2 s-1) and in complete submergence plots (34.8 μmol m-2 s-1) 37 DAS. The
photosynthetic rate at 37 DAS in partial and complete submergence was closely related to the NAR(Net Assimilation Rate) during submergence in the pot experiment (Figure 2).

26thProceedings of The 7 ACSA Conference 2011

Symbols represent non-submerged plants, partially submerged plants and completely submerged plants as indicated. The
number next to each symbol indicate the cultivars: 1, ‗Banjoulou‘; 2, ‗IR71700‘; 3, ‗IR73020‘; 4, ‗Nylon‘; and 5, Yele1A‘

Figure 2. Relationship between net assimilation rate during submergence and photosynthetic rate after 37 d
submergence in a pot experiment.

The superior tolerance of deepwater O. sativa and O. glaberrima genotypes to prolonged
complete submergence appears to be due to their greater photosynthetic capacity developed byleaves newly emerged above the floodwater. Vigorous upward leaf elongation during prolonged
submergence is, therefore, critical for ensuring shoot emergence from water, leaf area extension
above the water surface and a subsequent strong increase in shoot biomass. Increase in shoot
DMA after desubmergence was negatively correlated with shoot elongation during submergence, r=−0.36 (P < 0.01) in experiment 3. ST were plotted in the area of short shoot elongation and highincrease in DMA (Dry Matter Accumulation), while O. glaberrima, except for Saligbeli, were plottedin the area opposite to that of ST. Some LS (Lowland Sativa genotype) and LI (Lowland
Interspecific progenies genotype) were plotted close to the ST for both shoot elongation and shoot
DMA. Increase in DMA after desubmergence of all US (Upland Sativa genotype) was less than thatof ST.
The first principal component explained 46.8%, and for the classification of cultivars
according to their physiological responses to flash floods. The results of the cluster analysis werecompared to the principal component analysis results (Figure 3). Principal component analysis was
performed with the increase in DMA from 1 DAD to 14 DAD, and lodging at 1 DAD, shootelongation during submergence, and increased DMA during submergence. In Clusters I, III, and
VIII, the main genotypes belonging to each cluster group were classified on the principal component
analysis. Cluster I, including ST cultivars, and Cluster VIII, including O. glaberrima, were positioned
in opposite regions except for Saligbeli. Saligbeli exhibited enhanced shoot elongation with theincrease in DMA during submergence. These features seemed to be a unique way to cope withsubmergence.

th

Proceedings of The 7 ACSA Conference 2011

27

I

Axis I is the first principal component (y =−0.403942 x1 + 0.434866 x2 + 0.329416 x3 −0.271996 x4). Axis II is the second
principal component (y =−0.068947 x1 −0.080874 x2 + 0.618871 x3 +0.722613 x4), x1, x2, x3 and x4 represent increase in
DMA after desubmergence, lodging score, shoot elongation and increase in DMA during submergence respectively. All data arestandardized. (●) Upland Sativa, (○) Lowland Sativa, (▲) Upland Glaberrima (△) Lowland Glaberrima, (■) Upland IHP, (□)
Lowland IHP, (×) Submergence tolerance.

Figure 3. Principal component analysis of the physiological traits linked to submergence (shoot elongation during
submergence, increase in dry matter accumulation during submergence, lodging score and increase
in dry matter accumulation after desubmergence.

References
Africa Rice Center. 2004. Going beyond the upland NERICA: another African Rice is born. In Annual Report
2003–2004. Cotonu, Benin.
Balasubramanian, V., Sie, M., Hijmans, RJ. & Otsuka, K. 2007. Increasing rice production in Sub-SaharanAfrica: Challenges and opportunities. Advances in Agronomy 94:55-133.
Chu, YE. & Oka, HI. 1972. The distribution and effects of genes causing F1 weakness in Oryza breviligulata andO. glaberrima. Genetics 70:163-173.
Linares, O.F. (2002). African rice (Oryza glaberrima): history and future. Proceedings of the National Academyof Sciences of the United States of America, 99:16360–16365.
Norman, JC. & Otoo, E. 2003. Rice development strategies for food security in Africa In: Proceedings of the 20th
Session of the International Rice Commission. Food and Agricultural Organization of the United Nations.
Porteres, R. 1970. Primary cradles of agriculture in the Africa continent. In: Fage, J., Oliver, R. (eds.). African
Prehistory. Cambridge University Press, Cambridge, UK, pp. 43-58.
Sakagami, J-I., Hatta, T., Kamidozono, A., Masunaga, T., Umemoto, T. & Uchida, S. 2008. The actual conditionof traditional rice culture in inland valley delta of Niger River. JIRCAS Working Report. In: Sakagami, J-I.
Ito, O.(eds). pp. 37-52.

-- back to Table of Content --28

thProceedings of The 7 ACSA Conference 2011

Toposequnential Variation in Soil Fertility and Limiting Nutrient for Rice
Growth in the White Volta Floodplain of Northern Ghana
Yasuhiro Tsujimotoa*, Yukiyo Yamamotoa, Keiichi Hayashia, Alhassan I. Zakariab,
Yahaya Inusahb, Tamao Hattaa, Mathias Fosub, Jun-Ichi SakagamiaabJapan International Research Center for Agricultural Sciences, Japan
Savanna Agricultural Research Institute, Council for Scientific and Industrial Research, Ghana*Corresponding author: Japan International Research Center for Agricultural Sciences,
1-1 Ohwashi Tsukuba, Ibaraki 305-8686, Japan
Tel.: +81 29 838 6368; fax: +81 29 838 6355.
tsjmt@affrc.go.jp

Abstract
Integrated floodplain resource management for rice cultivation is imperative to satisfy the growing demand ofrice in West Africa. Irrigated pot experiments were conducted with different fertilizer treatments to identify
toposequential variation in soil fertility and limiting nutrient for rice growth within the White Volta floodplains in
Ghana. Eighteen experimental soils were examined across a riverside to upland gradient, ranging from 898 to4200 m in distance from the main riverside, from 73 to 106 m in elevation, and from 0.7 to 2338.1 m in distancefrom water sources. The soil analysis revealed close correlation between N-mineralization rates and carbon
contents of the soils, which were exponentially decayed with distance from water sources. In the non-fertilized
treatments, plant N uptakes at maturity also decreased along the same transect from the water sources.
However, the dry matter production was little relevant to this toposequential factor. Various fertilizer treatments
identified remarkable effect of sulfur on rice growth, which was more significant on soils closer to wastersources. NPK application without S increased only N concentration and N: S ratio in plant tissues but notbiomass production. The results indicated that sulfur is the primary limiting element for rice growth, and its
supplementation would be more beneficial as closer to water sources so as to effectively utilize greater Nsupplying capacity of this agro-environmental soils.
Keywords: rice, sulfur deficiency, floodplain, soil carbon, water source

Introduction
River floodplains, consisting of wide and flat plain of alluvium bordering rivers, areexpected to support a large expansion in rice cultivation area and production in Africa, of which the
major share is currently unexploited. Major constraints to expand agricultural activities into riverfloodplains include difficulty in water control, risk of complete submergence of plants, occurrence ofwater-borne diseases, and disadvantage in access to the road and market (Balasubramanian et al.
2007). On the other hand, this geographical environment can provide water resources as well as
relatively fertile alluvial soils compared to uplands (Buri et al. 1999). Moreover, the use of riverfloodplains for rice cultivation should not cause spatial competition with the other crops due to therisk of periodic flooding.
In the traditional lowlands in Asia, large variance in cultivation conditions and riceproductivity are commonly recognized within relatively small areas, and farmers‘ management
practices are adapted to this variance according to the sequential changes in soil fertility and water
availability (Fukai et al. 1998). However, either quantitative data or farmers‘ practical adoption on
the toposeqential distribution of environmental resources are scarce for rice cultivation within riverfloodplains in West Africa.
Primary objectives of this study were, therefore (1) to quantify the rice productivity of soils
in relation to small-scale toposequence and (2) to identify deficient nutrient for rice growth, whichthProceedings of The 7 ACSA Conference 201129

results can help effective use of soil nutrient resources and development of toposequence-specificfertilizer management practices within a target environment. To achieve these objectives,
phytometry experiments with rice were conducted under various fertilizer treatments by using
multiple soils collected within a Whiter Volta floodplain in the northern Ghana. In addition to themajor macronutrients of N, P and K, target nutrients included Zn, Si and S which deficiency havebeen previously reported in the region and in the similar hydromorphic soils in the other parts of the
sub-Sahara Africa (Buri et al. 2000; Tsujimoto et al. 2010).

Materials and Methods
Sampling of experimental soils
Experimental soils were collected from the top 0-15 cm at 18 points across a riverside to
upland gradient in the White Volta floodplains of northern Ghana at the beginning of rainy season in
June, 2010. The sampling points ranged from 898.7 to 4200.0 m in distance from the main Volta
riverside, from 73 to 106 m in elevation (ASTER-GDEM), and from 0.7 to 2338.1 m in distance fromwater sources. The average slope angle was 0.86 % between the closest and furthest sampling
points from the riverside. Distance from water sources were calculated after extracting rivers andback-swamps by the image analysis of Quickbird imagery. Experimental soils were air-dried for aweek in a screen house at the Savanna Agricultural Research Institute (9˚ 26‘ N, 0˚ 59‘ W, 183 masl.). Thereafter, the soils at the air-dry equivalent weight (4.2 kg) were put into 7-l plastic pots
followed by flooding and puddling with different fertilizer treatments.
The chemical and physical properties of the experimental soils were analyzed after airdried and sieved. Total C and N were determined by automatic high sensitive NC analyzer,
Sumigraph NC-220F (SCAS, Japan). Mineralizable nitrogen was determined by a 4-week anaerobic
incubation at 30°C as the amount of NH4+-N extracted with 10% KCl solution. Available phosphorus
content was measured by Bray No.2 method. Extractable sulfate was determined by extraction with
KH2PO4 solution containing 500 mg l-1 P. CEC was measured by the ammonium acetate extract
method at pH 7.0. Exchangeable bases were determined by plant atomic emission spectrometer,
ICPE-9000 (Shimadzu, Japan). Soil texture was determined by sieving and pipetting method.
Experiment design and measurements
Two pot experiments were conducted. In the Experiment 1, rice was grown with nofertilizer inputs, using all the 18 experimental soils. In the Experiment 2, three of the 18
experimental soils were selected in a wide range of the distance from water sources (Bottom: 40 m;
Middle: 501 m; Top: 1870 m), for which ten different fertilizer treatments were established including
no fertilizer treatment in the Experiment 1: 1. Control (no fertilizer); 2. +N; 3. +P, 4. +K, 5. +NP; 6.
+NK; 7. +NPK, 8. +NPKSi, 9. +NPKZn, 10. +NPKS. The chemical forms and application rates foreach nutrient were 0.70 g N, 0.22 g P, 0.36 g K, 1.87 g Si, 0.05 g Zn, and 0.23 g S per pot as Urea,
NaH2PO4, KCl, SiO2, ZnCl2, and Na2SO4, respectively. Both experiments had three replicates,
followed by the same cultivation management and measurements. Two 21-day old seedlings of a
local cultivar, Jasmine85, were transplanted at a rate of one hill per pot on June 26 in 2010, andwere subsequently harvested on October 8 in 2010. The water level was kept above 2 cm
throughout the rice growing period. Weeds were removed manually. No specific pest management
was conducted.
The plants were harvested from each pot at maturity. The dry weights of the plant samples
were determined after oven drying at 80˚C to a constant weight. The plant N concentration wasdetermined by using automatic high sensitive NC analyzer, Sumigraph NC-220F (SCAS, Japan).
The plant nutrient concentrations of P, K, S, Mg, Ca, Zn, Fe, Mn, Cu, and B were analyzed by plant
atomic emission spectrometer, ICPE-9000 (Shimadzu, Japan), after digesting each sample with
HNO3 and H2O2 in microwave digestion system (MLS-1200 MEGA, Milestone Inc.). The plant30

thProceedings of The 7 ACSA Conference 2011

nutrient uptakes were calculated as the product of the dry weight and the nutrient concentration ofeach plant tissue. Statistic analysis was performed by using JMP 8 software (SAS Institute Inc.).

Results and Discussion
Toposequential variation in soil fertility and plant growth (Experiment 1)
The results of soil analysis revealed large variances in soil properties within the gently
sloping floodplain area (Table 1). The total carbon (TC) and total nitrogen (TN) contents ranged
from 3.48 to 30.15 g kg-1 and from 0.28 to 2.25 g kg-1, respectively. Although differing among thelocations, available P and extractable S were both below the critical deficiency levels shown by
Dobermann and Fairhurst (2000). This was in accordance with the previous studies conducted in
the same agro-ecological zones of West Africa (Buri et al. 1999, 2000).
Table 1. Soil properties and its correlation with the total carbon contents (n=18).

Mean

Max

Min

coefficiencyof variation(%)

correlation
coefficient toTotal C

pH 1:2.5 (H2O)

6.29

7.56

5.42

10.8

-0.43

Clay (%)

13.65

30.96

5.60

54.2

0.80***

Total C (g kg-1)

11.91

30.15

3.48

64.4

-

Total N (g kg-1)

0.89

2.25

0.28

65.3

0.91***

Mineralizable N (mg kg-1)

43.53

141.10

1.42

104.5

0.96***

Available P (mg kg-1)

11.26

31.85

1.98

79.5

0.58*

Extractable S (mg kg-1)

8.72

14.48

6.09

24.4

0.50*

CEC (cmol kg-1)

6.97

15.10

3.07

56.8

0.80***

Exchangeable cation (cmol kg-1)
Na

0.75

1.54

0.33

50.3

0.57*

K

0.35

0.47

0.24

23.3

0.64**

Mg

1.05

2.65

0.40

68.9

0.80***

Ca

1.89

4.21

0.80

59.6

0.78***The total carbon (TC) contents of the soils correlated to most of the other soil properties,
exponentially decreased as the distance from water sources (Figure 1a). This toposequential
gradient in the TC, SOC or clay contents of the soils was also observed in the other rice-growing
lowlands of Asia (Homma et al. 2003; Tsubo et al. 2006). The accumulated TC contents in the lower
parts of the toposequence were most likely attributable to greater deposition of clay minerals, and tolonger periods of submergence that alleviates the decomposition of organic substrates (Sahrawat2004).
The total N uptakes of rice plants also showed exponential reduction along the sametransect from the water sources (Figure 1b). This could be explained by large differences in soil
mineralizable N, that were highly correlated with the TC contents (Table 2). Soil mineralizable N
was regarded as a good index of soil N-supplying capacity, and sufficiently correlated with plant N
uptakes under non-N fertilized conditions in previous studies (Russell et al. 2006; Tsujimoto et al.
2010). However, top dry matter (TDM) yield was less relevant to this toposequential factor (Figure1b). The TDM production highly fluctuated within a 100 m radius of water sources, and one had arelatively high yield level at upper area of the floodplain. This discrepancy in responses with riceproduction to N-supplying capacity of the soils indicated that there existed other limiting elements
apart from N.

thProceedings of The 7 ACSA Conference 2011

31

(b)

50

0.4

40

r² = 0.54***

0.3

30

0.2

20

0.1

10

0
0

0
500 1000 1500 2000 2500

Distance from water sources (m)Soil total C contents (g kg-1)

0.5

Dry matter yield (g pot -1)

Plant N uptakes (g pot -1)

(a)

35
30
25
20
15
10
5
0

r² = 0.68***

0 500 1000 1500 2000 2500
Distance from water sources (m)Figure1. Relationship between distance from water sources and (a) soil TC contents and (b) plant N uptakes(closed circles: ●) and top dry matter yield (cross marks: X).

Effects of fertilizer treatment and soil type on rice growth (Experiment 2)
The results of ANOVA showed significant F-values for the main and interactive effects offertilizer treatment and soil type on the TDM production. The application of NPKS demonstrated
stunningly higher TDM yields compared to the Control plots (Table 2). The differences in TDM
yields between NPKS and Control plots were greater as closer to water sources (80.5; 40.7; and29.7 g pot-1 on the Bottom, Middle, and Top soils, respectively). On the other hand, the applicationof N, P, and K or combinations of these elements without S showed no effects on the TDM yields
for any of the experimental soils.
Table 2. Top dry matter yields and N: S concentration ratio at maturity as affected by different fertilizer
treatments for the three experimental soils.

Fertilizer

Top dry matter yield (g pot-1)
BottomMiddleTop

BottomMiddleTopC

14.7 fghijkl#

23.5 def

12.7 hijkl

29.2 f13.0 hi12.9 hiN

12.4 ghijklm

27.0 d10.1 jklm

51.6 ab25.4 fgh

47.9 abP

14.6

fghijkl

hijkl

fg

K

18.6 defghij

17.1 defghijk

11.5 hijklm

NP

10.7 ijklm

24.5 defNK

14.6 fghijkl
16.5

fghijkl

18.9

defghij19.5

defghi

NPKS

95.2

a

Soil type (S)df
2F-values
166.1***

df
2

F-values
98.7***

Fertilizer (F)9319.2***

9

159.8***

NPK
NPKSi
NPKZn

i

14.7 ghi

26.9 fgh

12.5 hi

14.9 ghi

9.3 ijklm

45.0 abcd

31.6 ef

50.4 ab20.3 defgh

7.9 klm

57.1 a34.0 cdef

50.8 ab

16.5

efghijk

lm

51.8

ab

43.8

bcde

47.1 abc

25.2

de

49.6

ab

33.6

def

48.3 ab12.0

hijklm

33.7

def

44.6

abcde

56.5 ab

71.2

b

13.4

hi

22.2

defg

N: S ratio

12.8

5.9
12.0

hijklm

3.2

m

42.4

c

27.8

11.3

6.6

i

7.1 i

24.4***
11.1***
SxF
18
18#Values within the next three columns followed by the same letter do not differ significantly at
P<0.05 by Tukey's HSD multiple range test. ***Significant at P<0.001.

The high N: S ratio, notably on the bottom soil, and the increased values by the N
application without S also implies the unbalanced supplies between N and S (Table 2). The ratios

32thProceedings of The 7 ACSA Conference 2011

reduced only by adding S as a result of the substantial increases in biomass production. In a
conventional study, Yoshida and Chaudhry (1979) suggested a critical N: S ratio for S deficiency at
14 in straw, and they demonstrated 50% of biomass reduction at the value of 40. The other study
also indicated the similar value as a critical N: S ratio, and slightly higher value for grains (Islam andPonnamperuma, 1982). Our results indicated that sulfur shortage severely hampers rice growth aswell as maximum efficiency of N from either inherent soils or fertilizers in the target floodplain. The
limiting S supply was also apparent in the S concentration values, that were all below the critical
deficiency level at 0.06% (Dobermann and Fairhurst, 2000), among the rice plants in theExperiment 1. The other macro- and micro- nutrient concentration values were above the critical
deficiency level except four of the 18 plots for the P level (data not shown).

Conclusions
This study revealed steep gradients in soil fertility within a gentle slope of a river floodplainin the northern Ghana. The phytometrical experiments suggested that the spatial variances in soil
fertility for rice growth were mainly represented by the N- and S- supplying capacity of the soils.
Once the limit of S is lifted, rice production is expected higher as closer to water sources due toexponentially greater amounts of N-supplying capacity of the soils. A further study requires
identifying spatial distributions in S-supplying capacity of the soils, so that efficient fertilizer
management practices can be developed on a basis of balancing N and S applications. The
development of toposequence-specific fertilizer management will certainly help effective extensionof rice production within the target floodplain environments.

References
Balasubramanian V, M Sie, RJ Hijmans, K Otsuka, 2007. Increasing rice production in sub-saharan Africa:
Challenges and opportunities. Adv. Agr. 94, 55-133.
Buri MM, T Masunaga, F Ishida, D Kubota, and T Wakatsuki, 1999. Soils of flood plains in West Africa: general
fertility status. Soil Sci. Plant Nutr. 45(1), 37-50.
Buri MM, T Masunaga, and T Wakatsuki, 2000. Sulfur and zinc levels as limiting factors to rice production inWest Africa lowlands. Geoderma 94, 23-42.
Dobermann A, and TH Fairhurst, 2000. Rice: Nutrient disorders and nutrient management. International Rice
Research Institute (IRRI), Los Banos, Philippines.
Fukai S, P Sittisuang, and M Chanphengsay, 1998. Increasing production of rainfed lowland rice in droughtprone environments-A case study in Thailand and Laos. Plant Prod. Sci. 1(1), 75-82.
Homma K, T Horie, T Shiraiwa, N Supapoj, N Matsumoto, N Kabaki, 2003. Toposequential variation in soil
fertility and rice productivity of rainfed lowland paddy fields in mini-watershed (Nong) in Northeast Thailand.
Plant Prod. Sci. 6(2), 147-153.
Islam MM and FN Ponnamperuma, 1982. Soil and plant tests for available sulfur in wetland rice soils. Plant Soil
68, 97-113.
Russell CA, BW Dunn, GD Batten, RL Williams, JF Angus, 2006. Soil tests to predict optimum fertilizer nitrogenrate for rice. Field Crops Res. 97, 286–301.
Sahrawat KL, 2004. Organic matter accumulation in submerged soils. Adv Agr. 81, 169-201.
Tsubo M, J Basnayake, S Fukai, V Sihathep, P Siyavong, Sipaseuth, M Chanphengsay, 2006. Toposequential
effects on water balance and productivity in rainfed lowland rice ecosystems in Southern Laos. Field Crops
Res. 97, 209-220.
Tsujimoto Y, K Homma, and T Shiraiwa, 2010. The effects of soil drying and rewetting on rice growth in lowland
aquatic Ferralsols in the southeastern forest region of Madagascar. Plant Soil 333, 219-232.
Yoshida S and MR Chaudhry, 1979. Sulfur nutrition of rice. Soil Sci. Plant Nutr. 25 (1), 121-134.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201133

Varietal Differences of Rice (Oryza sativa L.) Genotypes for Aleurone Traits
Contributing to Lipid Content
Ohn Mar Khina, Masao Satob, Tong Li-Taoa, Yuji Matsueb, Atsushi Yoshimurab, Toshihiro
Mochizukib*aGraduate school of Bioresource and Bioenvironmental sciences, Kyushu University, Fukuoka, Japanb
Faculty of Agriculture, Kyushu University, Fukuoka, Japan*Corresponding author: Experimental Farm, Kyushu University, Harumachi 111,
Kasuya-machi, Kasuya-gun, Fukuoka 811-2307, Japan
Tel.:+81 92 612 2865; Fax: + 81 92 612 2865.
mochizuki@farm.kyushu-u.ac.jp

Abstract
The increasing demand for oil is more than the increasing production of oil seed crops. Rice bran oil has beenused extensively in Japan, Korea, China, Taiwan, Thailand and Pakistan. Interest in rice oil is increasing
because it is identified as ―healthy oil‖ that reduces serum cholesterol. In rice seed, the lipid content deposited in
the aleurone layer and embryo has been reported from 17.5 to 21.7%. To explore the useful genetic resources
for aleurone traits closely related to the lipid content, 333 varieties were investigated. Brown rice seeds were cut
in cross around center with razor blade and the outermost region of the cut surface stained with oil red O were
measured with a fluorescence microscope. As a result, there were wide variations in stained area among
varieties and many varieties with large stained area were found in Japonica group. Twenty varieties wereselected according to their stained area to cover the whole range among varieties used and confirmed their
thickness of aleurone layer by using micro thin section that was made by Leica cryostat with slight modificationof the Kawamoto‘s film method (2008). The photos of sections were taken under a light microscope. The
aleurone traits were measured by using Image J software. The amount and content of triacylglycerol (TAG) of
brown rice without embryo were also measured. Both amount and content of TAG were significantly correlatedwith the area, the average thickness and the percentage of aleurone layer. These results suggest that thealeurone traits will be able to use as good indicators for selection of rice varieties with high TAG amount andcontent.
Keywords: aleurone layer, lipid content, rice bran oil, triacylglycerol, varietal differences.

Introduction
Rice (Oryza sativa L.) is one of the world's most important food crops, primarily in East andSoutheast Asia. Rice bran is a valuable by-product of rice milling that contains a high concentrationof nutritional compounds including edible lipids. Rice bran oil is used in foods, feed and industrial
applications. More recent efforts have emphasized the nutritional benefits of rice bran oil. It canaptly be concluded that rice bran oil is an important future source for edible and essential oils in allover the world.
Rice bran oil has been extensively used in Asian countries such as Japan, Korea, China,
Taiwan, Thailand and Pakistan (Kahlon, et al., 1992). The use of rice bran oil in Japan, where it is
the largest volume domestically produces vegetable oil, is as a frying oil (Orthoefer, 2005). In brown
rice, the lipid content is from 2.3 to 3.9% (Juliano, 1977, Fujino, 1978). Embryos and aleurone
layers in rice are major tissues to deposit lipids and the content in these tissues has been reported
from 17.5 to 21.7%. The content in rice embryos and aleurone layers is equivalent to that ofsoybean and cotton seeds. The physiological role played by the aleurone layer is to provide areserve site for minerals to deposit, which are crucial as an essential nutrient when seeds germinate

34

thProceedings of The 7 ACSA Conference 2011

Tanaka et al., 1973). In the cells, oil bodies and aleurone particles are most predominant and seemto play a more important physiological function than starchy endosperm.
Because rice is primarily used for food, starchy endosperm or milled rice is more important.
Little effort has been made to study about rice bran layer. There is some information about the
thickness of aleurone layer of some varieties. Genes for enlargement of embryos and aleurone
layers can contribute to an increase in the oil content of rice grains (Omura and Satoh, 1981). For
such reason, characterization of genetically broad rice germplasms for thickness of aleurone layer
is of special importance in identifying potential varieties. Therefore, the objectives of this study wereto evaluate the thickness of aleurone layer and to compare it with the storage lipid of the seed.

Materials and Methods
Half–seed staining method
All experiments were conducted at Kyushu University in 2010-2011. Three hundred andthirty three rice varieties including 176 of IRRI Core Collection, 59 of World Rice Collection and 96
of Core Collection stored at Kyushu Uniersity, were used. The brown kernels were cut in halfaround center with razor blade and then stained with oil red O for 5 -10 minutes and then rinsed
well in 70% ethanol for 3 - 4 times. The total cut surface area and the area of the stained region
were measured by using a fluorescence microscope (BZ 9000, Keyence Co. Ltd Osaka,Japan).
Then the percentage of the stained area and the average thickness of the stained region can becalculated by using the following formula.
the area of the stained region (μm2)
The percentage of the stained area (%) =x 100
the total cut surface area (μm2)
the area of the stained region (μm2)
The average thickness of the stained region (μm) =the circumference (μm)
Sectioning method
To confirm that the relationship between the thickness of the stained region and the real
thickness of the aleurone layer, we selected 20 varieties according to their stained area to cover thewhole range among varieties tested. The measurements were made by using micro thin section thatwas made by Leica cryostat with slight modification of the method of Kawamoto (2009). The
sections were stained with Hematoxylin and Eosin and examined under a light microscope. The
aleurone traits were measured using Image J software. Then the percentage of the aleurone layer
and the average thickness of aleurone layer were calculated by the same formulae in half-seed
staining method.
Measurement of Triacylglycerol (TAG)
The storage lipids (triacylglycerol: TAG) of selected varieties were analyzed. Hundred
brown kernels without embryos were milled (Degree of milling: 90%) in a mall-scale rice mill. The
lipids were extracted from the rice bran by the method of Folch et al. (1957). The TAG content wasmeasured using enzyme assay kits (Triglyceride E test; Wako Pure Chemicals Osaka, Japan).

Results and Discussion
In each trait, the varieties were significantly different. Varietal groups were alsosignificantly different in all traits except in percentage of stained area. Most of the indica varieties
have smaller cut surface area. The smallest one was found in wild rice and the largest one inthProceedings of The 7 ACSA Conference 201135

tropical japonica. The mean of total cut surface area was 4.01 mm2. There was wide variation from0.07 to 0.35 mm2 for area of the stained region and the mean was 0.18 mm2. There were manyvarieties with large stained area in japonica group (Figure1). The smallest stained area was found inwild rice. Some varieties with large stained area were also found in indica, intermediate hybrid andtropical japonica. Although the percentage of stained area (%) was significantly different among
varieties, the varietal groups were not significant and the mean was 4.45 %. In japonica group,
many varieties with large thickness of stained region were found (Figure1). But there were also
some indica varieties with large thickness of stained region. The mean of the thickness of stained
region was 20.15μm. The results revealed that the varieties with smallest ones were found in indica
and wild groups.

indicaTropical japonica

japonica

Intermediate hybrid

Wild

Figure 1. Frequency distribution of aleurone traits of different varietal groups.

y = 14.956x + 1.3617
r= 0.78

6.5
Total cut surface area (mm2)

6
5.5
5

4.5
4
3.5

GEMJYAJYANAM

PAKHE DHAN
MA HAE KHAO KAP
LO1050XANG
FUKUHIBIKIYELAIK MEEDONAICHI ASAHI
TAICHUNG 65LO1050
KINMAZE
KOSHIHIKARI
Mizuhochikara
NIPPONBARE
PTB25
PAI-KAN-TAO
TA HUNG KU
MARAYA1
IR64

3

IR24

2.5
TAL 214

2
0.05

0.1

0.15

0.2

0.25

0.3

0.35

Stained area (mm2)▲ indicates the selected varieties to check the aleurone layer, *** indicates the significance at 0.001 level.
22

Figure 2. The relationship between the stained area (mm ) and total cut surface area (mm ) of 333 rice varieties.

We selected 20 varieties according to their value of stained area to cover the whole rangeof varieties tested (Figure 2). The total cut surface area was nearly the same in half seed staining2
method and sectioning (Table.1). The mean of aleurone area was 0.27 mm and the range was2
0.12 - 0.42 mm (Table 1). It was larger than the area of stained region. This may be due to the fact

36

thProceedings of The 7 ACSA Conference 2011

that the stained region may become thinner than the real one depending on the time taken to washthe seed in 70% ethanol after staining. The correlation between these two parameters was
significantly high (r=0.81***) (Figure 3). We can, therefore, use the area of stained region as a good
indicator for selecting varieties for thickness of aleurone layer. The range of the thickness ofaleurone layer was 22.88 - 48.78 μm (Table 1). The thickness of aleurone layer was larger than andsignificantly correlated with that of stained region (r=0.59**, data not shown). The percentage ofaleurone layer was higher than that of the area of stained region and the correlation between thesetwo parameters was not significant (r=0.24, data not shown).

*** indicates significance at 0.001 level.
22

Figure.3. The relationship between the stained area (mm ) and the aleurone area (mm ) of 20 rice varieties.
Table 1. Mean with the standard deviation of aleurone traits and Triacylglycerol (TAG) content of 20 rice
varieties

2

Total cut surface area (mm )Sectioning method

Half-seed staining method

4.19 ± 0.99
(1.93 - 5.60)

4.24 ± 0.86
(1.97 - 6.00)

Aleurone area (mm )0.27 ± 0.07
(0.12 - 0.42)

Percentage of aleurone area (%)

6.45 ± 0.95
(0.20 - 9.00)

Thickness of aleurone layer (mm)35.26 ± 6.12
(22.88 - 48.78)

Amount of TAG (mg) in 100 seed

18.78 ± 7.91
(6.89 - 41.1)

2

Content of TAG (mg /g of seed)

0.18 ± 0.05
(0.07 - 0.32)
4.14 ±0.83(2.30 - 6.49)
19.23 ± 4.28
(10.00 - 29.14)

8.19 ± 2.3(5.02 - 14.72)

The values in parentheses indicate the range of each parameter.

The amount of TAG increased with increasing aleurone area. The range of amount of TAG
without embryo in 100 brown seed ranged from 6.89 to 41.1 mg among 20 varieties tested. It was
highly correlated with the area (r=0.71***) and thickness of aleurone layer (r=0.67**) (Figure 4).
However, the percentage of aleurone area was not correlated with the amount of TAG. The content
of TAG also increased with increasing the aleurone area. The range of content of TAG without
embryo in 1g of brown kernel was from 5.02 to 14.72 mg/g among 20 varieties tested. It was highlythProceedings of The 7 ACSA Conference 201137

correlated with the area (r=0.61**), percentage (r=0.58**) and average thickness of aleurone layer(r=0.68**) (Figure 4).

** and *** indicate significant level at 0.01 and 0.001 respectively.

Figure 4. Relationship between TAG and aleurone traits of 20 rice varieties.

Conclusions
There was wide variation in aleurone traits among the genetically diverse rice varieties.
The correlation between the aleurone area by sectioning method and the stained area by half-seed
staining method was significantly high. Therefore, thestained area could be used as a good
indicator for selecting varieties for thickness of aleurone layer. Furthermore, there were highcorrelation between aleurone area and aleurone thickness, and TAG. So, selection of varieties withhigh lipid content could be done without measuring directly lipid content of the varieties. A great
potential was obtained to produce rice bran oil and it did not require any special cultivation since it isa by-product of the rice milling process.

References
Fujino,Y. 1978. Rice lipids. Cereal Chem. 55: 559 - 571.
Juliano, B.O.1977. Rice lipid, IRRI. Los Banos,Phillippines: 21pp.
Kahlon T et al., 1992. Cereal Chem. 69: 485 - 489.
Kawamoto.T. 2009. Kawamoto‘s Film method 2008. Medical Technology. 72: 76 - 83.
Omura, T. and H. satoh. 1981. Possibility of chemical breeding in rice. Current Advances in breeding(Ikushugaku saikan no shinpo) 22:10 -19. (J)thOrthoefer F.T. 2005. Rice Bran Oil. In Bailey‘s Industrial Oil and Fat Products, 6 Edition, volume 6. John Wiley& Sons, Inc., Hoboken, New Jersey. Pp 465 - 489.
TanakaK, T Yoshida, K Asada and Z Kasai. 1973. Subcellular particles isolated from aleurone layer of riceseeds. Arch. Biochem. Biophys. 155: 136 -143.

-- back to Table of Content --38

thProceedings of The 7 ACSA Conference 2011

Development of New Cytoplasmic Male Sterile Lines with
Good Flowering Behavior for Hybrid Rice Breeding
Indrastuti A Rumantia, Bambang S Purwokob*, Iswari S Dewic, H AswidinnoorbaIndonesian Center for Rice Research, Ph.D. Student at Graduate School, Bogor Agricultural Universityb
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural UniversitycIndonesian Center of Agricultural Biotechnology and Genetic Resources Research and Development*Corresponding author: Jalan Meranti, Darmaga, Bogor 16680 Indonesia.
Tel.: +62 251 8629353.
bambangpurwoko@gmail.com

Abstract
Intensive use of single source of male sterile cytoplasm (i.e. wild abortive or WA) in developing hybrid rice mightlead to genetic vulnerability associated with susceptibility to pests and diseases. The research aim was todevelop new cytoplasmic genetic male sterile lines (CMS) from 3 different cytoplasmic sources (WA, Gambiaca
and Kalinga). Anther culture and successive backcrosses were conducted to develop new CMS. The screeningof bacterial leaf blight to each CMS was done through artificial inoculation using three isolates i.e. pathotype III,IV and VIII. The results showed that combination of anther culture and backcrosses obtained 6 CMS lines of WA
type, 3 CMS lines of Kalinga type, and 1 CMS line of Gambiaca type. The lines showed complete and stablesterility from first to fifth backcross generations. Some of them also showed resistance to certain Xanthomonas
oryzae pathotypes. The new CMS lines with WA, Kalinga and Gambiaca cytoplasm were early in maturity withflowering behavior better than IR58025A (check CMS), such as larger stigma, higher stigma exertion, wider
angle and longer duration of glume opening during anthesis. The accumulation of good flowering behavior
increased seed set of the new CMS lines yielded from outcrossings. It ranged from 4.75–25.9%, meanwhile
IR58025A seed set only reached 2.98%. Corellation analysis showed high positive and significant value
between seed set of the new CMS lines and stigma width (r = 0.44*), stigma exertion (r = 0.54*) and angle ofglume opening during anthesis (r = 0.42*), respectively. The seed set was also affected by some flower
characters of the maintainer lines such as filament length and angle of glume opening during anthesis. The
results indicated that the new CMS lines with diferent cytoplasm sources could be used in hybrid rice breeding.
Keywords: rice, cytoplasmic male sterility, flowering behavior, resistance, bacterial leaf blight

Introduction
Hybrid rice is able to increase rice productivity about 15-20% higher than the bestcommercial inbred varieties. Currently, hybrid rice technology is used in a large scale cultivation inseveral Asian countries. Hybrid rice covers more than 50% of rice total area in China (You et al.,2006). Three line system is generally used in Indonesia. This system needs three parental lines:
cytoplasmic male sterile line (CMS/A line), maintainer line (B line) and restorer line (R line). Hybrid
is the first filial generation of a cross between cytoplasmic male sterile line as female parent andrestorer lines as male parent. The hybrid often exhibits heterosis or hybrid vigor, where hybrid
progeny shows superior growth characteristics relative to one of the parental lines (Eckards, 2006).
The commercial hybrid refers to a superior F1, which not only performs better than its parents but
also shows significant yield superiority over the best high yielding inbred variety of similar growthduration (Virmani & Kumar, 2004). Based on those, superior parental lines i.e. CMS needs to be
developed.
CMS as female parent is a key factor for hybrid rice breeding. CMS is a condition underwhich a plant is unable to produce functional pollen (Eckardt, 2006). This line provides a valuable
tool in production of hybrid seed in self-pollinating crop species including rice. Development of CMS
in three line systems of hybrid rice breeding faces problems among others instability of pollenthProceedings of The 7 ACSA Conference 201139

sterility, flower behaviour not fully supporting outcrossing rate, and susceptibility to pests anddiseases. Unstable pollen sterility causes difficulty in achieving high heterosis of the hybrid rice andits seed production. The low seed yield has been implicated in higher price of the hybrid seed. Wild
Abortive (WA) CMS is mainly used in hybrid rice breeding in Indonesia (Suwarno et al., 2003) andelsewhere. This might lead to genetic vulnerability associated with susceptibility to pests anddiseases (Li et al., 2007). Therefore, breeding of new CMS having stable pollen sterility derived
from diverse cytoplasm sources and resistant to pests and diseases are warranted.
Commercial CMS should have good outcrossing rate, complete and stable sterile pollen,easy restorability, good and wide combining ability with any restorer lines, short plant, good
exsertion of panicle, more than 70% of stigma exsertion, good quality, resistant to pests anddiseases and adapted to target environment (Yuan et al., 2003). Dewi et al. (2007) identified 19
doubled haploid of maintainer line candidates through anther culture from F1 of crossing between
introduced CMS and donor lines resistant to bacterial leaf blight. The doubled haploid lines arepotential maintainers to be converted to CMS because these lines were homozigous and having
male sterile gene with normal cytoplasm. Rumanti et al. (2009) tested 48 F1s, derived from testcrossbetween cytoplasmic male sterile sources with those doubled-haploid maintainer lines, and 14 F1s
were completely sterile (100 % pollen sterility). The objectives of this research were to develop
stable and completely sterile CMS lines from three different cytoplasmic sources, to evaluate
resistance of the new CMS to bacterial leaf blight and study of flower behavior of the new CMS that
support outcrossing ability.

Materials and Methods
Development of cytoplasmic male sterile lines through succesive backcross
Genetic material in this research were backcross population (F1BCn) between CMS
candidates with their maintainer doubled haploid lines. CMS from International Rice ResearchInstitute (IRRI) were used as cytoplasm donors, i.e. 3 CMS of wild abortive type (IR58025A,
IR62829A, dan IR68897A), one Gambiaca type (IR80154A) and one Kalinga type (IR80156A).
Succesive backcrosses were done 5 times. Each CMS candidate or female parent (F1BCn) was
planted in 2 rows with 12 plants each, while male parent or their maintainer were planted in 3 rows
with 12 plants each. Flowering syncronization was achieved by staggered planting of male parents
in a 3-day intervals. Selection was based on pollen sterility in each F1BCn. Observation were doneon each F1BCn generation for pollen sterility (%) (IRRI, 2002).
Resistance evaluation of the new cytoplamic male sterile lines to bacterial leaf blight
Ten new CMS‘ were planted in the field in 2 rows with spacing of 20 x 20 cm. Plants were
inoculated with 108 cfu concentration of Xanthomonas oryzae pathotype using artificial method (leafcutting/clipping). Disease severity was observed following modified IRRI method (IRRI, 2002) whensusceptible check achieved 90% severity. Score of disease severity is shown in Table 1.
Table 1. Score of disease severity
Scale
1
3
5
7
9

40

Ratio of lesio length and leaf length (%)
1-5
>5-12
>12-25>25-50>50-100

Reaction
R (Resistant)
MR (Moderate Resistant)
MS (Moderate Susceptible)
S (Susceptible)
VS (Very Susceptible)thProceedings of The 7 ACSA Conference 2011

Evaluation of flowering behaviour which supports outcrossing ability between new CMS andtheir maintainer
Five new CMS lines (BI485A, BI599A, BI619A, BI639A dan BI665A) and their maintainers
selected from the first experiment were evaluated for flowering behaviour. IR58025A was used as
check line. The field experiment was conducted using randomized complete block design with three
replicates. The days to flower, panicle exertion, stigma exertion, duration of glume opening during
anthesis, angle of glume opening and seed set were observed based on SES (IRRI, 2002).
Statistical analysis was conducted using SAS 9.0.

Results and Discussion
Development of cytoplasmic male sterile lines through succesive backcross
The successful use of hybrid vigor in rice depends on the availability of local cytoplasmic
male sterile (CMS) and restorer lines. Cytoplasmic male sterile line is a rice genotype having
abnormal anther. CMS anther has no pollen or has pollen, but easy to rupture, then there is no seed
set yielded by self fertilization (Yuan et al., 2003). Male sterile character facilitates natural crossing
thus ease the hybrid seed production.
Table 2 shows pollen sterility character of ten CMS from first backcross generation (F1BC1)
to fifth generation (F1BC5). From the research, we identified six CMS candidates of Wild Abortive,
three candidates of Kalinga and one candidate of Gambica types. Backcrosses formed segregating
population and gave chance for breeder to select based on spesific target, in this case pollen
sterility. The three cytoplasm types gave different pattern of pollen sterility in the five generations ofbackcrosses. In the WA type, there were unique pattern shown by BI497A and BI703A lines, inwhich both lines were completely sterile (100%) in the first and second generation of backcrosses,
but reversed to be partial fertile on third and fourth generation. This phenomenon indicated that bothlines showed unstable pollen sterility. Backcross should be done in several generations todetermine pollen stability of both lines. The other lines, i.e. BI485A, BI543A, BI571A and BI599A
showed positive increase in sterility percentage from first to fifth generation of backcrosses. The
CMS with Gambiaca and Kalinga cytoplasm sources also increased pollen sterility as backcrossprogressing.
Table 2. Pollen sterility character of new cytoplasmic male sterile lines on F1BC1 - F1BC5 generation

Note: WA= wild abortive; value after ± was standard deviationthProceedings of The 7 ACSA Conference 201141

All completely sterile CMS‘ with WA cytoplasm source were achieved in the fifth generation(F1BC5). In the fourth backcross generation, four lines showed 100% pollen sterility (completetysterile), while other lines ranged from 99.8 to 99.9%. Different patterns is shown by CMS of Kalinga
and Gambiaca types. The pollen stability of both CMS types were achieved earlier. All of CMS with
Kalinga and Gambiaca cytoplasm had more stable pollen sterility than that of Wild Abortive. A
commercially usable CMS line needs to have complete and stable male sterility, independent fromenvironmental changes specially temperature (Yuan & Fu, 1995), adaptive to tropical rice growing
conditions and possess good outcrossing potential to affect an economically viable hybrid seedproduction (Virmani & Kumar, 2004).
Resistance evaluation of the new cytoplamic male sterile lines to bacterial leaf blight
Male sterile lines should have good adaptation to their rice growing conditions andresistant to rice pest and disease. The three type of CMS were converted from dihaploid maintainer(DH2) lines. The maintainers were obtained from anther culture of F1 derived from crossing between
elite maintainer lines and donor lines having high resistance to bacterial leaf blight. Both ofmaintainer lines and donor lines were elite breeding lines originated from Indonesia. Therefore, inthe fifth backcross, the new CMS already had cytoplasm from introduced lines, but having
98.4375% nucleus genes from their male parents (recurrent parent). The new CMS‘ are expected
be more adapted to Indonesian environment.
Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is one of the mostimportant disease of rice which cause substantial yield losses (Lee et al., 2000). We tested 10 new
CMS lines for reaction to three Indonesian pathotypes. The new CMS shows variation in resistanceto bacterial leaf blight (Table 3). The new CMS of wild abortive cytoplasm and Kalinga cytoplasm
showed to be resistant to moderate resistant to pathotype III, while Gambiaca cytoplasm wasresistant to pathotype III. In Indonesia, the pathotype IV and VIII of bacterial leaf blight were morevirulence than pathotype III. The results showed variation in the reaction of all CMS from threecytoplasm sources to different pathotypes. One CMS of Kalinga type (BI665A) was resistant to bothof pathotypes. The BI485A, BI497A, BI599A and BI855A showed moderate resistance to bacterial
leaf blight pathotype IV. Reaction of CMS to patotype VIII also varied. BI543A and 571A wereresistant to the pathotype VIII, while BI703A and BI669A were moderate resistant to the pathotype
VIII.
Table 3.

Reaction of cytoplasmic male sterile lines to three Indonesian pathotype of bacterial leaf blight

Cytoplasmic Male SterileLines
CMS WA type:
BI485A
BI497A
BI543A
BI571A
BI599A
BI703ACMS Kalinga type:
BI639A
BI665A
BI669ACMS Gambiaca type:
BI855A

Resistance to Bacterial Leaf Blight (BLB)
Pathotype III
Pathotype IV

Pathotype VIII

3
3
3
3
3
1

3
3
7
5
3
5

5
5
1
1
5
3

3
3
1

7
1
5

5
1
3

1

3

5

Note: 1 Resistant, 3 Moderate Resistant, 5 Moderate Susceptible, 7 Susceptible, 9 Very Susceptible

42thProceedings of The 7 ACSA Conference 2011

Sudir & Suprihanto (2006) reported that virulence of the bacterial Xanthomonas oryzae pv.
oryzae (Xoo) was significant in affecting disease severity of BLB. The elite line donor having xa5
gene which has responsibility to disease resistance. The xa5 gene is recessive and constitutively
expressed at the same level as the dominant susceptible allele, Xa5, and neither allele is induced or
suppresed in response to Xoo (Bart et al., 2006). Availability of CMS lines stable in sterility, good inagronomic characters, and resistant to disease will increase the adaptability of the hybrid developed
from those CMS lines.
Evaluation of flowering behaviour that support the outcrossing ability between new CMS andtheir maintainer
Table 4 shows the observation of several flower behavior of five CMS lines. IR58025A wasused as check line because most rice hybrids in Indonesia were derived from this line. New CMS
lines were earlier to flower than IR58025A, except BI665A. The maintainers flowered 1–4 days
before their respective CMS pairs. The different days to flower between CMS and their respective
maintainers were in the threshold and important for flowering syncronization when produced CMS
seed (Virmani et al. 1997).
Table 4. Flowering behavior of five new CMS and their maintainersNote:

A: cytoplasmic male sterile lines, B: maintainers; PE: panicle exsertion, SE: stigma exsertion, GOD: duration of glume
opening during anthesis, GOA: angle of glume opening during anthesis; seed set formed in CMS (A) was yielded bynatural outcrossing, pollen came from their respective maintainers; seed set of each maintainer (B) was obtained byselfing

All new CMS‘ showed good panicle exsertion (more than75%), but BI485A (WA) andBI665 (Kalinga) were more enclosed than IR58025A. Duration of glume opening during anthesis of
BI485A and BI599A (WA type) was significantly longer than IR58025A. The other new CMS lines
also showed longer duration of glume opening than that of IR58025A but not statistically significant.
BI599A, BI855A and BI639A showed wider angle of glume opening than that of IR58025A i.e. 39.8;
36.0 and 40.6 degrees, respectively. The better duration and angle of glume opening would
increase the opportunity of new CMS stigmas to receive pollen from maintainers.
Stigma exsertion of the three types of CMS was higher than IR58025A. It ranged from
67.7 to 74.7% compared to that of IR58025A (43.3%). The better flowering behavior caused higher
seed set of the new CMS than IR58025A. The new lines set seed at about 4.8–25.9%, while
IR58025A achieved 2.9% of seed set. Corellation analysis showed positive high and significant
value between seed set of new CMS lines with stigma width (r=0.44*), stigma exertion (r=0.54*) andangle of glume opening during anthesis (r=0.42*). The seed set was also affected by some flower
characters of maintainer lines such as filament length and angle of glume opening during anthesis(data not shown). The results indicated that the new CMS lines with different cytoplasm source
could be used in hybrid rice breeding.

thProceedings of The 7 ACSA Conference 201143

Conclusions
Combination of anther culture and backcrosses obtained 6 CMS lines of WA type, 3 CMS
lines of Kalinga type, and 1 CMS line of Gambiaca type. The lines had stable sterility andresistance to 1-3 Xanthomonas oryzae pathotypes. The new CMS showed better flowering behavior
which increase the seed set.

References
Bart, R., P. Ronald, and S. Hake. 2006. Fertility versus disease resistance, a hard choice. Gene andDevelopment (20): 1215-1217.
Dewi, I.S., A. Apriana, A. Sisharmini, and I.H. Somantri. 2007. Resistance evaluation of doubled haploid ofparental lines candidate to bacterial leaf blight and brown planthopper. Bull. Agron 35(1):15-21.
Eckardt, N.A. 2006. Cytoplasmic male sterility and fertility restoration. The Plant Cell 18:515-517.
Gavino, R.B., Y. Pi, and C.C. Abon Jr. 2008. Application of gibberellic acid (GA3) in dosage for three hybrid riceseed production in the Philippines. Journal of Agricultural Technology 4(1): 183-192.
[IRRI] International Rice Research Institute. 2002. Standarization Evaluation System for Rice. 66p
Rumanti I.A., Dewi I.S., Purwoko B.S. and Aswidinnoor H. 2008. Evaluation of anther culture-derived doubled
haploid maintainer lines for developing rice male sterile lines. Jurnal Agronomy Indonesia 37 (1): 1 – 7(In Indonesian).
Takai, T., S. Matsura, T. Nishio, A. Ohsumi, T. Shiraiwa, and T. Horie. 2006. Rice yield potential is closely
related to crop growth rate during late reproductive periode. Field Crops Research 96: 328-335.
Li, J., S. Cai, J. Feng, Li, and G. Cheng. 2007. Comparisons on genetic diversity among the isonuclearallopasmic male sterile lines and their maintainer lines in rice. Rice Science 14(2): 94-100.
Peng, S., G.S. Khush, P. Virk, Q. Tang, and Y. Zou. 2008. Progress in ideotype breeding in increase rice yieldpotential. Field Crop Research 108: 32-38.
Sudir and Suprihanto. 2006. Change in virulence of strain Xanthomonas oryzae pv. oryzae, a causal pathogen
of bacterial leaf blight in rice. Jurnal Penelitian Pertanian Tanaman Pangan 25 (2): 100-107. (InIndonesian).
Suwarno, N.W. Nuswantoro, Y.P. Munarso, and M. Diredja. 2003. Hybrid rice research in Indonesia..In: S.S.
Virmani, C.X. Mao, and B. Hardy, eds, Hybrid Rice for Food Security, Poverty Alleviation, andthEnvironmental Protection. Proceedings of the 4 International Symposium on Hybrid Rice, HanoiVietnam, 14-17 May 2002. IRRI, Philippines. pp. 287-296.
Virmani, S.S., and I. Kumar. 2004. Development and use of hybrid rice technology to increase rice productivity
in the tropics. Internationasl Rice Research Notes 29 (1):10-19.
Virmani, S.S., B.C. Viraktamath, C.L. Casal, R.S. Toledo, M.T. Lopez, and J.O. Manalo. 1998. Hybrid Rice
Breeding Manual. IRRI, Philippines.
You, A., X. Lu, H. Jin, X. Ren, K. Liu, G. Yang, H. Yang, L. Zhu, and G. He. 2006. Identification of quantitative
trait loci across recombinant inbred lines and testcross populations for traits of agronomic importance inrice. Genetics 172:1287-1300.
Yuan, L.P., X. Wu, L. Fuming, G. Ma, and Q. Xu. 2003. Hybrid Rice Technology. China Agriculture Press.

-- back to Table of Content --44

thProceedings of The 7 ACSA Conference 2011

Genetic Analysis of Superior Double Haploid Rice Lines Developed from
Anther Culture
Muhammad Syafiia,*, Bambang S. Purwokob, Iswari S. Dewica

Postgraduate of Plant Breeding and Biotechnology, Bogor Agricultural University, Bogor, Indonesiab
Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor, IndonesiacIndonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development,
Cimanggu, Bogor, Indonesia* Corresponding author: Postgraduate of Plant Breeding and Biotechnology,
Bogor Agricultural University, Bogor, 16680-Indonesia.
Tel.: +62 251 862 9353; fax: +62 251 862 9353.
syafii_m@yahoo.com

Abstract
Rice is the most important food commodity in Indonesia. The need for rice continues to increase with the
population increase. Plant breeding‘s objective to obtain high-yielding varieties is an attempt to increase
production plateu. Biotechnology is one of the breeding aproaches using tissue culture and genetic engineering
techniques. The availability and diversity of plant genetic resources are important factors to develop highyielding varieties with desirable traits. This study was aimed to investigate the agronomic characters of double
haploid lines developed from anther culture, evaluate genetic diversity, phenotypic variations and broad sense
heritability of double haploid lines developed from anther culture, and to obtain rice genotypes potential as
superior lines. Analysis of 18 genotypes showed there was diversity of agronomic characters among the rice
double haploid genotypes. Characters of dry weight-based grain (yield) per hectare and the number of empty
grain per panicle had high level of genetic variability, while other characters had rather low to low level of genetic
variability. Yield per hectare in KP44223 line was the highest (4537.8 kg / ha or 4.5 tons / ha) among the lines,
even higher than that of conrol varieties, Ciherang and Celebes. The characters of plant height, flowering age,
harvesting time, panicle length, number of empty grain/ panicle, 1000 grain weight and yield per hectarehad highbroad sense heritability, suggested that those characters can be used as selection characters to improve crop
performance
Keywords: anther culture, double haploid, rice

Introduction
Rice is the most important food commodity in Indonesia. In addition, rice is also a major
source of carbohydrate material for Indonesian people. The need for rice continues to increase withpopulation increase. (Prasetyo 2008), Plant breeding to obtain high-yielding varieties is one attempt
to overcome rice production plateu. Utilizing biotechnology through tissue culture technique andgenetic engineering could be one of the appraches in plant breeding (Somantri et al. 2003). The
availability and diversity of rice genetic resources is an important factor in developing high yielding
varieties with desirable traits. Development of new high yielding varieties of rice in Indonesia haslong been conducted since the early 1970s. After the 1980's breeding toward new high yielding
varieties (VUB) started to be intensified to obtain varieties that are responsive to fertilizer and highquality of rice, such as IR64, Membramo, Cisadane and Ciherang (Samaullah 2007). Conventional
breeding to develop new rice varieties require a long time (7-10 years), especially the time forselection process to obtain pure lines. Utilization of modern technologies (biotechnology) such as
anther culture is expected to shorten the selection process thus saving time, effort, and cost. Dewi
and Purwoko (2001), states that the researchers have applied the anther culture technique to obtainrice lines that are resistant to pests and diseases, quality of rice as well as tolerant of environmentalthProceedings of The 7 ACSA Conference 201145

stress. Kim (1986), also reported that anther culture-based breeding approach have also been
applied in Korea, Vietnam, China and India.

Materials and Methods
The study was conducted in January-May 2011 at IPB laboratory, Dramaga, Bogor. The
materials used were 18 rice double haploid lines developed from anther culture (KP1-3-1-2, KP3-72-1, KP3-18-1-1, KP3-18 -1-2, KP3-18-1-3, KP3-19-1-1, KP3-19-1-2, KP3-19-1-3, KP3-19-1-4, KP419-1 -3, KP4-19-2-3, KP4-42-2-1, KP4-42-2-2, KP4-42-2-3, KP4-43-1-2-43-KP4 1-4 , KP4-43-2-3,
KP4-43-2-4), and 2 control varieties (Ciherang and Celebes). The experiment was conducted at theExperimental station Babakan IPB, Darmaga, Bogor, using Randomized Block Design with four
replicates. The treatments used were 18 double haploid rice genotypes and two control rice
varieties. The 21 day old seedlings of each genotype were planted in field plots (0.8 x 5 m) withplant spacing of 20x20 cm having 2 seedlings/hole. Plants were fertilized with 200 kg/ha of Urea,
100 kg/ha of SP36 and 100 kg/ha of KCl applied three times at 10, 25 and 45 days after planting(DAP). Plants were maintained based on the paddy rice cultivation. The observations were
performed on parameters of plant height (cm), total number of tillers (stems), number of productive
tillers (stems), flowering date (days), harvesting date (days), panicle length (cm), number of filled
grain per panicle (grains), number of empty grain per panicle (grains), total grain number per
panicle (grains), weight of 1000 grains (g), and grain yield (kg/ha).
The data were analyzed using analysis of variance, Duncan multiple range test andanalysis variance components (Singh and Chaudhary 1979). Variance component and heritability
were determined based on Singh and Chaudhary (1979) as follow:

Vg =; V P = V g + Ve ; h 2 b s =; KVG =(√). 1 0 0 % ; K V P = (√). 1 0 0 %Which was:Vg: various genotypes; Vp: various phenotypes; r: replication; X: average common genotype; KVG:
coefficient of genetic diversity; KVP: coefficient of phenotypic diversity; KTg: sum square of
genotype; KTE: sum square error. Broad sense heritability values according to Stanfield (1983) ishigh (12:50 <h2 <1.00), moderate (12:20 <h2 <0.50) and low (h2 <0.20).

Results and Discussion
The results showed that the genotype effect was significant to all characters observed.
This suggested that there was variability among genotypes for all the observed characteristics.
Plant Height
KP44323 and KP44324 lines were genotypes having highest average plant height (112.6
and 112.3 cm, respectively), while KP31812 and KP31813 lines had shortest average of the plantheight (83.6 and 84.95 cm, respectively). Ciherang was classified as medium plant height (103 cm),while Celebes was classified as relatively short plant height (82.8 cm). From anther culture, thecharacters inheriteged from parent was the plant height, both on descent Ciherang and Celebes.
This indiated that the plant height was dominant.

46thProceedings of The 7 ACSA Conference 2011

Total Productive Tillers and Tillers
Although not signicantly different among genotype, number of tillers and productive tillers
per plant were varied among genotypes ranging from 14.1 to 19.55 and 11.55 to 15.25,
respectively. KP31913 and KP44223 were the genotypes producing the highest and the lowest
tillers. While KP1312 and KP44314 were the genotypes having the highest and the lowest
productive tillers. The genotypes tested showed moderate number of tillers.
Flowering and Harvested
Flowering KP31812 of genotype lines and varieties Cilebes (control) i.e 63.25 consecutive
days and 57 days compared to genotype lines KP41913, KP44312, KP44221, and KP44324 theconsecutive 76 days and 75.75 days. This indicated that the lines have descended from varieties
Ciherang age slower than lines flowering varieties descent Celebes. To determine the general
harvesting age, the age of the rice crop can be classified into three groups calculated from the day
after the scatterplot (HSS), namely age genjah (90-104 HSS), age was 105-120 HSS and age in atmore than 120 HSS) (BB-padi 2009a). The average age of crop genotypes tested genjah belonging
to moderate, with harvest age genjah of lines KP31811 and KP31813 was 91.75 day and mostvarieties genjah in Celebes (control) to 89.75 days old. While the maximum harvest age on
genotype KP41913 and KP41923 a consecutive were 106 days and 105.75 days, respectively.
Panicle length
Panicle length characters in genotypes tested was quite short to medium. Longest panicle
was found at KP44323 which was 24.96 cm. This was slightly shorter than that of varieties Ciherangat 25.82 cm, while the shortest panicle of KP44222 was 22.82 and 21.82 cm resulted from Celebes
varieties (Table 3). DMRT test showed that all genotypes were not significantly different between
the lines tested, except in the control varieties and Celebes Ciherang.
Number of Grain per Panicle
Line KP3721 had highest total amount of grain per panicle of 178.85 points, while thelowest was found in line KP31914 and KP31813 at 121.55 points and 121.88 points, respectively.
Thus these genotypes were ssified as moderate (Table 4). All the genotypes tested were notsignificantly different with the second control test DMRT at 5%, except in line KP3721 and
KP31914. Lines KP44312 and KP44324 had high in grain content / panicle at 100.35 points,
respectively and 100.1 points higher than the control plants respectively 90.45 and 90.1 for the
Celebes and Ciherang, while the lowest was found in line KP41923 had 56.45 points lower than thecontrol varieties. KP41923 line had the most empty grain number/panicle at 106.3 points, while thelowest in line was KP31914 had 33.75 points. Although the line had a number of grain hollow
KP31914/panicle, this line produced the lowest total amount of grain. That affected the yield per
hectare. Calculation of the percentage (%) content of grain/panicle to total grain/panicle produced inline KP31914 was the highest at 72.23%, this value was higher than that of the varieties of Celebes(control) atf 70.06%. The lowest percentage was found at lines KP41923 and KP44314 which were
34.6 and 46.04%, respectively.
1000 Grain Weight
Weight of 1000 grains of rice indicated the size of grains of rice and beans level. The
results showed that the highest weight of 1000 grains of rice was found at KP1312 line at 39 925 g.
This value was higher than both the control varieties and Celebes Ciherang consecutive which were
36 025 and 30,575 g, respectively. The weight of 1000 grains of line KP3721 was the lowest at 26
325 g. This value was lower than the control Celebes varieties which was 30 575 g. KP3721 line
had the lowest weight of 1000 grains allegedly due to low resistance to various diseases in the field,
either by bacteria or viruses. Value of 1000 grain weight assessed with DMRT test at 5% level isshown in Table 3.
thProceedings of The 7 ACSA Conference 201147

Grain Yield (kg /ha)Grain yield per plot can be used to measure the amount of the plant production. Dry grain
yield per plot was then converted to a hectare (kg/ha). The highest production was found at line
KP44223 (no.entry 14) and KP44221 (no. entry 12) in a row having 4.5 and 4.3 t/ha, respectively,
this significantly different with both the control varieties and Celebes Ciherang at 3:50 and 3:43 t/ha,
respectively. The lowest dry grain was found at line KP41923 (no.entry 11) and KP31811 (no.entry
3) consecutive at 1:03 and 1.82 tonnes/ha, respectively. This differed markedly lower than thecontrol based on test DMRT 5%. DMRT test at 5% for the grain yield/ha showed that all lines tested
had distinct intangible, except in line KP44223 (highest) and line KP41923 (lowest) (Table 3).
Variety Component
Various components and heritability estimation is performed to determine the proportion ofdiversity caused by genetic factors and environmental factors. Heritability determines the success of
selection for the appropriate environment, because it indicates the heritability of a trait is influenced
by genetic or environmental factors. High heritability indicates the relative importance of genetic
influences that can be used from the elders to their offspring as well as useful to determine the mostappropriate selection method for improving a plant characters (Falconer and Mackay 1996). The
value of plant genetic parameter estimation is shown in Table 3. Coefficient value of genetic
diversity (KVG) ranged between 3.25 and 27.31 and phenotypic diversity coefficient (KVP) ranged
between 4.51 and 36.83. Value KVG KVG 0.00-27.31% was the absolute value, that value wasdetermined from the relative value of KVG. Absolute value of 27.31% KVG KVG was a relativevalue of 100%. According Moedjiono and Mejaya (1994), KVG relative value of the following criteria:
low (0 <x <25%), somewhat lower (25%<x<50%), high (50%<x<75%) and high (75%x<100%).
Based on the above criteria, it can be determined KVG absolute criteria in this trial, namely low(0.00 <x <6.83%), somewhat lower (6.83% <x <13.66%), high (13.66% <x <20:48%), and high
(20:48% <x <27.31%). Murdaningsih et al. (1990) stated that the value was low and rather low KVG
classified as narrow genetinya character variability, while quite high and high KVG classified ascharacter mempynyai wide genetic variability.
Table 3. The value range of components and heritability estimates the value of agronomic characters and yieldin rice
Agronomic character

mean

Vg

Vp

2

KVG (%)KVP (%)

h bs (%)

Plant hight

93.79

87.61

103.04

9.98

10.82

85.03

Productive tillers

13.55

0.24

5.44

3.62

17.22

4.42

Total tiller

16.43

0.37

7.54

3.70

16.71

4.89

Flowering

69.59

30.54

31.56

7.94

8.07

96.79

Harvested

98.00

18.59

19.53

4.40

4.51

95.17

Panicle lenght

23.79

0.60

1.19

3.25

4.59

50.13

Number of grain/panicle

85.53

116.29255.69

12.61

18.70

45.48

number of empty grain/ panicle

55.37

228.61415.72

27.31

36.83

54.99

Total grain/panicle

140.90165.78372.94

9.14

13.71

44.45

1000 grain weight

34.43

9.46

14.01

8.93

10.87

67.53

Yield (ton/ha)

2.94

5.64

11.24

25.58

36.12

50.17

Remarks: Vg=varian genotype, Vp=varian phenotype, KVG=coefisien varian genotype, KVP=coefisien varian phenotype, h2bs=
boad sense heritability

48thProceedings of The 7 ACSA Conference 2011

Based on the criteria of absolute KVG, there were two characters having relatively highKVG ie grain yield / ha and the number of empty grain per panicle, five characters have a rather low
KVG, namely plant height, flowering age, number of grain content per panicle, total grain number
per panicle and 1000 grain weight, and the four characters have low KVG ie the number of
productive tillers, total number of tillers, harvesting age, and length of panicle. Thus there are two
characters having a broad genetic variability, and there are nine characters that have a narrowvariability. Nugraha Lestari (2007) mentioned the opportunities for improvement of grain yield canbe done with the selection of the character of the number of grain per panicle and grain yield dry.
Broad sense heritability value of the characters observed was between 04:42 and 96.79% (Table
3). According to Stanfield (1983), broad sense heritability values can be grouped into three highheritability (12:50 <h2 <1.00), the heritability of moderate (0.2 <h 2 <0.50) and low heritability (h2<0.20). Based on the above criteria that broad sense heritability values for characters: plant height,
flowering age, age of harvest, panicle length, number of empty grain / panicle, 1000 grain weightand dry grain yield per hectare was high, whereas the character of grain number per panicle andthe number of content total grains per panicle classified as being; then to the character of
productive tillers and total number of tillers per hill had a low broad sense heritability values.
Therefore, the improvment of grain yield can be done through the selection of plant height, floweringage, age of harvest, panicle length, number of empty grain / panicle, 1000 grain weight and grain
yield per hectare dry.

Conclusions
Analysis of agronomic characters of 18 genotypes indicated that there was diversity inagronomic characters and character among the double haploid genotypes of rice anther culture
results. Dry grain yield per hectare and the number of empty grain per panicle had a high level ofgenetic variability, agronomic characters and yield while others had a rather low level of genetic
variability to low. Dry grain yield per hectare obtained from line KP44223 was the highest of 4.5
ton/ha than varieties Ciherang and Celebes. High broad sense heritability value generated on thecharacters were plant height, flowering age, age of harvest, panicle length, number of empty
grain/panicle, 1000 grain weight and grain yield per hectare dry Therefore, the characters can beused as benchmarks for improving the character of the next generation of this crop.

References
Alluri K.1986. Screening rice varieties in acid upland soil. Progress in Upland Rice Research. IRRI. Los banos.
Philippines. pp 263-270.
Bhojwani SS, Razdan MK, 1996. Plant Tissue Culture. Theory and Practice, a revised edition. Elsevier Science.
Amsterdam. The Netherlands. 767p
Chang TT, Bardenas EA. 1965. The morphology and variety characteristics of the rice plant. Technical Bulletin
4. IRRI. Laguna. Philippines. 40p.
Chung GS. 1992. Anther culture for rice improvement in Korea. In: K. Zheng, T. Murashige (eds). Anther Culture
for Rice Breeder. Seminar and Training for Rice Anther Culture at Hangzhou. China. P8-37.
Dewi IS, Purwoko BS. 2001. Kultur antera untuk mendukung pemuliaan padi. Bul. Agron. 29(2):59-63
Donald CM. 1968. The breeding of crops ideotypes. Euphytica 17:385-403
Falconer DS, Mackay TFC. 1996. Introduction to Quantitative Genetics. Fourth edition. Longman. London
Fehr WR. 1987. Principles of Cultivar Development. Vol 1. McGraw-Hill. Inc. New YorkHu H. 1998. In-vitro induced haploids in higher plant. Di dalam : Genetic Manipulation in Crops. Proceeding ofrd
the International symposium on Genetic Manipulation in crops: the 3 International Symposium on
haploid. Beijing. Oktober 1984. IRRI. And Sinica Academy. p8-12International Rice Research. 2004. Rice Knowledge Bank. CD ROM Version 3.1 (15 April 2004).
Kush GS. 1995. Breaking the yield frontier of rice. Geo Journal 35: 329-332.
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Kim K. 1986. Ststus of Korean biotechnology and its role in improving crop production. In: Workshop on
Biotechnology for crop Improvement, potentials, and limitations. 13-17 oktober 1986. IRRI. Philipines
35p.
Moedjiono dan Mejaya MJ. 1994. Variabilitas genetik beberapa karakter plasma nutfah jagung koleksi Balittas
Malang. Zuriat 5 (2):27-32
Murdaningsih HK, Baihaki A, Satari G, Danakusuma T, Permadi AH. 1990. Variasi genetik sifat-sifat tanaman
bawang di Indonesia. Zuriat 1(1): 32-36
Prasetyo YT. 2008. Bertanam Padi Gogo Tanpa Olah Tanah. Bibliografi. hal 69.
Samaullah Y M. 2007. Pengembangan varietas unggul dan komersialisasi benih sumber padi. Apresiasi Hasil
Penelitian Padi. Balitpa.Sukamandi
Singh RK, Chaudhary BD. 1979. Biometrical Method in Quantitative Genetic Analysis. Kalyani Publ. New Delhi.
304p.
Soemantri IH, Ambarwati AD, Apriana A.2003. Perbaikan Varietas padi melalui kultur antera. Prosiding Hasil
Penelitian Rintisan dan Bioteknologi Tanaman. hal. 2008-214rd
Stanfield WD. 1983. Theory and Problems of Genetics, 2 edition. Schain‘s Outline Series. Mc. Graw Hill Book
Co. New Delhi.
Yoshida. 1981. Science of The Rice Plant. Food and Agricul;ture Policy Center. Tokyo. Japan. p21-25-- back to Table of Content --50

thProceedings of The 7 ACSA Conference 2011

QTLs on Chromosome 12 Responsible for Expressing Root Plasticity underTransient Soil Moisture Fluctuation Stress in Rice
Jonathan M. Niones, Yoshiaki Inukai, Akira Yamauchi*
Graduate School of Bioagricultural Sciences, Nagoya University*Corresponding author: Nagoya University, Chikusa, Nagoya 464-8601 Japan
Tel : +81-52-789-4021; Fax : +81-52-789-4022
ayama@agr.nagoya-u.ac.jp

Abstract
Soil moisture fluctuations (i.e. transient waterlogged to drought) frequently occur in rainfed rice fields due toerratic rainfall pattern, and irrigated fields due to wetting and drying system practices and inefficient irrigation
system. Such conditions are stressful for growth and development of rice. With the utilization of chromosome
segment substitution line (CSSL), we previously demonstrated the significant roles of root plasticity as
expressed by higher lateral root production in response to soil moisture fluctuation stress. Moreover, ourmolecular mapping analysis showed the presence of QTL that is associated with root plasticity, at short arm ofchromosome 12 region. This QTL was found to control the productions of L-type lateral roots with the increase
effect from Kasalath allele. In this study, we attempted to quantify the significant functions of QTL (qLLRn-12) on
chromosome 12 in relation to the shoot dry matter productions and root system development. CSSL genotypes
with QTL or substituted segment of Kasalath allele on chromosome 12 were evaluated and subjected to 38 daysof transient soil moisture fluctuation stress. Results revealed that CSSL genotype with qLLRn-12 showed greaterroot system development as expressed its greater total root length than those without qLLRn-12 in response tomoisture fluctuation stress. Longer total root length was attributed to higher production of L-type lateral roots,
which can effectively maintain water and nutrient uptake in the soil. Such plastic root responses resulted inincreased stomatal conductance and photosynthesis, eventually increased shoot dry matter production by 37%
compared to its Nipponbare parent. Comparison of the location of the QTL across rice cultivars showed that
there is no QTL reported in this region related to lateral root production. This may possibly be a putative QTL
and potentially be used in the marker-aided breeding program to improve adaptation under fluctuating soil
moisture environment.
Keywords: chromosome segment substitution lines (CSSL), lateral root, rice, root plasticity, soil moisturefluctuation

Introduction
With the scarcity of water for irrigation and continuing increase of world population, future
increase in rice production will more heavily rely on rainfed rice ecosystem. Rainfed lowland
conditions are characterized by frequent fluctuation in soil moisture at varying degrees throughout
the cropping season, which have stressful effects on the growth and development of rice (Niones etal., 2009). Plant roots play an important role in water and nutrient acquisition. Both constitutive andadaptive root growth have been implicated in the improvement of plant performance under rainfed
conditions (Kamoshita et al., 2002). A plant has an ability to change its phenotype, in response tosoil‘s heterogeneous environment (Yamauchi et al., 1996), which is termed as plasticity, and root
plasticity is a key trait for plant adaptation to stressful conditions. Moreover, we reported that traits
associated with developmental root plasticity triggered by mild drought stress played significant
roles in water uptake and dry matter production in rice (Kano et al., 2011).
With the use of chromosome segment substitution lines (CSSL), we previously identified
one line CSSL47 that consistently showed more plastic root growth than Nipponbare in terms ofbranching of lateral roots and aerenchyma formation at early vegetative stage (Suralta et al., 2008,

th

Proceedings of The 7 ACSA Conference 201151

2010) and until maturity (Niones et al., 2009) under transient soil moisture fluctuations. This line
contains 10 substituted segments from Kasalath allele with Nipponbare genetic background. Niones
et al. (2010) identified the location of the QTL at short arm of chromosome 12 region, which areresponsible for controlling root developmental plasticity specifically the L-type lateral root
production. The expression of such root plasticity QTL and its effect on dry matter production androot system development when rice plants are grown under transient soil moisture fluctuation
conditions have not yet been further verified.
In this study, we examined the significant function of the QTL or substituted segment from
Kasalath allele on chromosome 12 in relation to the shoot dry matter production at vegetative stage.
Furthermore, we tested the variation in root plasticity expression of parents and CSSL genotype
with QTL or substituted segment of Kasalath allele on chromosome 12 under 38 days of transientsoil moisture fluctuation stress.

Materials and Methods
Plant materials
Four genotypes (Nipponbare, CSSL47, CSSL27, CSSL52) were used in this study.
Nipponbare, an irrigated lowland japonica variety and recurrent parent of the 54 chromosome
substituted segment line (CSSL) (Nipponbare/ Kasalath cross) population genotypes. CSSL47
genotype has ten substitute segments across genome from Kasalath allele with a uniquecharacteristics of having greater plastic responses in root development under soil moisturefluctuations than the other CSSLs (Suralta et al., 2010; Niones et al. (2009). The CSSL27 andCSSL52 genotypes were selected from the 54 CSSL population. CSSL27 genotype contained no
substituted segment of Kasalath in short arm of chromosome 12 region, and thus is hereon referred
as - SL, while CSSL52 (referred as + SL) has substituted segment of Kasalath in short arm ofchromosome 12 region only. The seeds of CSSL population was provided by the Rice Genome
Research Center of the National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Treatment conditions
Seedlings were grown in soil-filled plastic root box and exposed to three different soil waterconditions; the well-watered (WW), transient waterlogged to drought (W-D) and transient drought towaterlogged (D-W) conditions. Plants were exposed to waterlogged conditions for 17 days andthereafter the water was allowed to drain to the target soil moisture content (SMC) of 20%, which
was then maintained for 21 days by watering every two days. Plant samplings were done at 38
DAS.
Root phenotyping
Lateral roots (LRn) were manually counted. Each nodal root was cut into 5-cm segments
keeping the lateral roots intact. In this way, the number of each type of lateral root; L and S type oflateral roots in rice (Yamauchi et al., 1996) can be determined and expressed as linear frequency(number of lateral roots per unit length root axis; Ito et al., 2006). One of their major noticeable
differences is that L type is branching while S type is non-branching. For total root length (TRL)
measurements, root samples from the FAA were rinsed with running water and spread ontransparent sheet without overlapping. The digitized images were taken using Epson scanner(ES2200) at 300 dpi resolution. TRL was analyzed using a NIH image software (ver.1.60), a publicdomain released by the National Institute of Health, USA.
Statistical analysis
The experiments were laid out in split-plot randomized complete block design with three
replicates. The significance differences of four genotypes were compared at LSD > 0.05
significance level using the Cropstat software.

52

thProceedings of The 7 ACSA Conference 2011

Results and Discussion
Plastic root development was mainly attributed to enhanced lateral root production
particularly the L-type lateral (Bañoc et al., 2002; Suralta et al., 2010), which is a key trait for theplant adaptation under transient soil moisture production. Lateral roots generally comprised thegreater proportion of the whole root system (Yamauchi et al., 1987), and thus promoted lateral root
development in response to moisture stress directly reflects the performance of the entire rootsystem. Enhanced lateral root development results in increased root surface area, and soil water
extraction and water use. Therefore, the maintenance of stomatal conductance and photosynthesis
could be a consequence of greater supply of water to leaves due to the enhanced lateral root
development as a result of their plastic responses.
QTL reported by Niones et al. (2010) was located at the short arm of chromosome 12
region, which is hereon referred as qLLRn-12. This was associated with root developmental
plasticity with the increase effect from Kasalath allele. The function of this QTL was to regulate theproduction of L-type lateral roots, which is associated with plastic root system development. It is
assumed that the possible location of the qLLRn-12 may be near TG156 marker locus with theapproximate distance of 17.6 cM between the adjacent markers RM6296.
In this study, greater lateral root development of + SL genotype over Nipponbare and – SL
genotypes was shown in higher linear frequency of lateral root number in response to transient soil
moisture fluctuation stress. This result showed the evidence the importance of QTL or Kasalath
segment that was present in chromosome 12 (Figure 1). Figure 2 shows the significant positive
correlation between total root lengths and the production of lateral root under both transient soil
moisture fluctuation stress treatments, with r-value of 0.624 for D-W and 0.646 for W-D. This result
indicates that under transient fluctuations of soil moisture, higher production of lateral root had asignificant contribution to greater root system development that eventually leads to enhanced waterand nutrient uptake in the soil.

Figure 1. The linear frequency of lateral root number per cm of the four genotypes grown under different soil
moisture conditions: i.e. WW, well-watered; W-D, transient waterlogging to drought; D-W, transient
drought to waterlogging. (☐)、
) - SL indicates the CSSL genotypes that do notcontain QTL or introgressed segment from Kasalath, ( ) + SL indicates the CSSL genotypes that do
not contain QTL or introgressed segment from Kasalath and ( 、CSSL47. Same letter means thatvalues are not significant at LSD > 5% level of significant level.

thProceedings of The 7 ACSA Conference 201153

Figure 2. Relationships of linear frequency of lateral root with total root length under different soil moisture
conditions; A, transient waterlogging to drought; B, transient drought to waterlogging. ( ), - SLO(CSSL28); ( ), CSSL47; ( ), + SL (CSSL52); ( ), Nipponbare.

To further demonstrate the effect of the existence of QTLs in response to transient soil
moisture fluctuation stress, + SL genotypes were evaluated for plastic root system development inrelation to shoot dry matter and root system development as shown in Figure3. The + SL genotype
showed significantly greater shoot dry matter production with the increase of 30% in W-D and 43%
in D-W than Nipponbare. The increase in shoot dry matter of + SL genotype was associated with a
10% (W-D) and 17% (D-W) increase in total root length compared to Nipponbare. On the other
hand, these parameters were not significantly different between – SL genotype and Nipponbare
parent. These facts clearly suggest that the function of QTLs at chromosome 12 was to regulate theproduction and branching of lateral roots (i.e., L-type lateral root), which resulted in greater plastic
root system development. This eventually led to the enhanced shoot dry matter production atvegetative stage under fluctuating soil moisture conditions.

Figure 3. Effect of the presence of QTL at short arm of chromosome 12 on the shoot dry matter production andtotal root length under different transient soil moisture fluctuation conditions. Same letter means thatvalues are not significant at LSD > 5% level of significant level.

We identified in short-arm of chromosome 12 region, the location of QTL that is
responsible for regulating the production of L-type lateral roots with the increase effect from
Kasalath allele. The use of the CSSL genotypes that contain substituted segment of Kasalath inchromosome 12 only further validated this QTL function. There have been reports on QTLs that areassociated with root length, root number, root dry weight and branching index traits (Price et al.,
2002; Horii et al., 2006; Gowda et al., 2011), however, with the best of our knowledge, there havebeen no reports on QTL in this region, which are related to lateral root production under soil
moisture fluctuation stress. Fine mapping is necessary to narrow down the distance of linked

54

thProceedings of The 7 ACSA Conference 2011

markers to the target trait. Then, this could be potentially used in the marker-aided breeding
program for enhanced plant adaptation under such environment.

References
Bañoc DM, A Yamauchi, A Kamoshita, LJ Wade, JR Pardales Jr 2000. Genotypic variations in response oflateral root development to fluctuating soil moisture in rice. Plant Prod Sci 3:335-343.
Gowda VRP, A Henry, A Yamauchi, HE Shashidhar, R Serraj 2011. Root biology and genetic improvement fordrought avoidance in rice. Field Crops Res 122:1-13.
Horii H, K Nemoto, N Miyamoto, J Harada 2006. Quantitative trait loci for adventitious and lateral roots in rice.
Plant Breed 125:198-200.
Ito K, K Tanakamaru, S Morita, J Abe, S Inanaga 2006. Lateral root development, including responses to soil
drying, of maize (Zea mays) and wheat (Triticum aestivum) seminal roots. Physiol Plant 127: 260-267.
Kano M, Y Inukai, H Kitano, A Yamauchi 2011. Root plasticity as the key root trait for adaptation to various
intensities of drought stress in rice. Plant Soil 342:117-128 (DOI 10.1007/s11104-010-0675-9).
Niones JM, RR Suralta, Y Inukai, A Yamauchi 2009. Evaluation of functional roles of plastic responses of rootsystem in dry matter production and yield under continuous cycle of transient soil moisture stresses byusing chromosome segment substitution lines in rice under field conditions. Jpn. J. Crop Sci. 78 (Extra 1)
260-261.
Niones JM, Y Inukai, A Yamauchi 2010. Identification of linked QTLs for root plasticity in rice under continuoustransient soil moisture fluctuation stress in field. Jpn. J. Crop Sci. 79 (Extra 2) 268-269.
Price AH, KA Steele, BJ Moore, RGW Jones 2002. Upland rice grown in soil filled chambers and exposed tocontrasting water-deficit regimes II. Mapping quantitative trait loci for root morphology and distribution.
Field Crop Res 76:25-43.
Suralta RR, Y Inukai, A Yamauchi 2008. Utilizing chromosome segment substitution lines (CSSLs) for evaluationof root responses under transient moisture stresses in rice. Plant Prod Sci 11:457-465.
Suralta RR, Y Inukai, A Yamauchi 2010. Dry matter production in relation to root plastic development. Oxygen
transport, and water uptake of rice under transient soil moisture stresses. Plant Soil 332:87-104.
Yamauchi A, Y Kono, J Tatsumi 1987. Quantitative analysis on root system structure of upland rice and maize.
Jpn J Crop Sci 56:608-617.
Yamauchi A, JR Pardales Jr, Y Kono 1996. Root system structure and its relation to stress tolerance. In O. Ito,
K. Katayama, C. Johansen, J.V.D.K. Kumar Rao, J.J. Adu-Gyamfi and T.J. Rego eds., Roots and Nitrogenin Cropping Systems of the Semi-Arid Tropics. JIRCAS Publication. Tsukuba, Japan. 211-234.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201155

Yield and Related Traits in Two Japonica Rice Lines Carrying Ur1 Gene
Stanis Malangena, Takumi Iwakurab, Toru Hatab, Takeshi Akaokab, Amol Dahalb,
Masayuki Muraib, *a

United Graduate School of Ehime University, Matsuyama, Ehime, 790-8566, Japan
Faculty of Agriculture, Kochi University, Monobe, Nankoku, Kochi, 783-8502, Japan* Corresponding author: Faculty of Agriculture, Kochi University, Monobe, Nankoku, Kochi, 783-8502, Japan
Tel.: +81- 088-864-5120; Fax: +81- 088-864-5200 (common)
muraim@kochi-u.ac.jpbAbstract
Ur1 (Undulate rachis-1) is an incompletely dominant gene on chromosome 6 in rice, being characterized byundulation of primary and secondary rachis branches. Ur1 increases not only the number of secondary branches
per panicle but also spikelet number per single secondary branch, resulting in a large spikelet number per
panicle. This genic effect can increase grain yield by enlarging sink size. We examined the yielding abilities of
the two Ur1-carrying lines under field conditions (mid-April sowing and early-May transplanting in two
experimental years). The two Ur1-carrying lines (MR79 and MR53) were selected from 108 recombinant inbredlines (F8 generation) originating from 108 F2 plants of the cross of ‗Nishihikari‘ × an isogenic line of Taichung 65
carrying both Ur1 and sd1-d. ‗Hinohikari‘, a leading variety in southern Japan was the check variety in thepresent study, being denoted by ―Hi‖. Additionally, ‗Nishihikari‘ (―Ni‖), a high yielding variety adaptable to fertile
land, was employed. They were grown under three fertilizer levels (4, 8 and 16 of Ng/㎡ in total) and two fertilizer
levels (12 and 21 of Ng/㎡ in total), together with P and K elements, in 2003 and 2005, respectively．Yield andother traits were measured. The results at 21 of N g/㎡in 2005 are described as follows. In yield (brown rice,g/㎡), the lines-varieties were ranked in the order MR79 (728) > MR53 (723) > Ni (695) > Hi (558). MR79 andMR53 had 170 g/㎡ (30%) and 165 g/㎡ (30%) higher yields, respectively, than Hi. Regarding spikelet number
per panicle, they were in the order MR79 (132.7) > MR53 (116.0) > Hi (94.5) > Ni (90.5). As for panicle number
per ㎡, Ni and Hi were higher than MR79 and MR53, although the difference is smaller than that in the previous
trait. Regarding ripened-grain percentage, Ni (88.9%) and MR53 (85.0%) were higher than MR79 (79.8%) andHi (79.2%). Regarding sink size (single-grain weight × fertilized-spikelet number per ㎡), they were in the sameorder as in yield. The yield of MR79 (728 g/㎡) was outstandingly high as compared with yield levels of ordinary
japonica varieties in southern Japan. This high yield was due to its more spikelets per panicle caused by Ur1, its
ripened-grain percentage being maintained at almost 80%. Moreover, MR79 had significantly higher yield thanMR53, Ni and Hi at 12 of N g/㎡ in 2005 as well as at the three fertilizer levels in 2003. Hence, MR79 hadoutstanding high yields at the low to high fertilizer levels. MR79 was 14-18 days later in 80%-heading date than
the rather late variety Hi in all cultivated conditions in the two years. MR79 was higher-yielding than the rather–early line MR53 in every environmental conditions. The lateness of MR79 might be advantageous to attain highyield. MR79 has high eating quality comparable to that of Hi, one of high eating-quality varieties in Japan.
Consequently, MR79 could be a super-high yielding variety with exceptionally late maturity in southern Japan.
Keywords： rice, Ur1 gene, spikelet number per panicle, sink size, yield

Introduction
Ur1 (Undulate rachis-1), located on chromosome 6 (Sato & Shinjyo, 1991), is an
incompletely dominant gene of rice, being characterized by undulation of primary and secondaryrachis branches. Ur1 increases not only number of secondary branches per panicle but also
spikelet number per secondary branch, resulting in a large spikelet number per panicle (Nagao etal., 1958; Murai & Iizawa, 1994). This genic effect can increase grain yield by enlarging sink size

56

thProceedings of The 7 ACSA Conference 2011

(Murai & Iizawa, 1994; Murai et al., 2002 and 2005 b). However, Ur1 decreases both ripened-grain
percentage and 1000-grain weight.
Murai et al. (2005 a) preliminarily reported that a japonica Ur1-carrying line Murai 79(hereafter ―MR79‖) had a higher yield than the levels of ordinary japonica varieties in southern
Japan, due to its higher sink size caused by more spikelets per panicle, which was derived from thecross of a Japanese commercial variety and an isogenic line of Taichung 65 carrying Ur1. MR79
and another Ur1-carrying line ―MR53‖ were used in the present study, which possess extremely lateand rather early heading times, respectively. The two lines, a representative variety of southern
Japan ‗Hinohikari‘ and a japonica high-yielding variety ‗Nishihikari‘ were grown under three fertilizer
levels in 2003 and two fertilizer levels in 2005. Yield, its components and other traits were
measured for the lines-varieties. On the basis of two-year data, characteristics of the two Ur1carrying lines were examined. We discuss utility of the lines in southern Japan.

Materials and Methods
Development of two Ur1-carrying lines
The highest-yielding F1 in the yield tests for various F1 hybrids with the Ur1 / + genotype(Murai et al., 1997 and 2003) was used for developing recombinant inbred lines with and without
Ur1. Its maternal and paternal parents were ‗Nishihikari‘ and an isogenic line of Taichung 65
carrying both Ur1 and sd1-d (dee-geo-woo-gen dwarf), respectively. The F2 population was grownin 1992, and the generation was progressed to F8 generation without selection in glasshousecondition. In 1999, the 108 F9 lines originating from the respective 108 F2 plants were grown in apaddy field; the two most well-ripened lines carrying Ur1, viz. MR53 and MR79 were selected from
Ur1-carrying lines by field observation. The uniformity (non-segregation) of MR53 and MR79 was
confirmed from 2000 to 2003 (F14 generation).
Field experiments
Besides MR53 and MR79, ‗Nishihikari‘ and ‗Hinohikari‘, (denoted by ―Ni‖ and ―Hi‖
respectively) were used for the field experiments. Ni is a short-culm and panicle-number typevariety possessing the highest lodging tolerance in southern Japan (Nishiyama, 1982). Hi is aleading variety in southern Japan, which possesses rather-late heading, rather long culm, rather
many panicles.
The two Ur1-carrying lines, Ni and Hi were seeded in mid April and transplanted to apaddy field of the faculty of Agriculture, Kochi University, Japan in early May, in the two
experimental years (Table 1). Two seedlings per hill were transplanted at a spacing of 30.0 x 15.0
cm. They were grown under three fertilizer levels (4, 8 and 16 of N g/㎡ in total) and two fertilizer
levels (12 and 21 of N g/㎡ in total) together with P and K elements, in 2003 and 2005, respectively(Table 1). The randomized block design with three replications was adopted for all combinations of
the lines-varieties and fertilizer levels in each year.
Measurements of yield and other traits
All panicles of about 30 hills were sampled from each plot at maturity, and the panicle
weight of each hill was checked after cutting just at their panicle bases after air-drying. Out of nine
hills randomly selected from about 30 hills of each plot, five hills having intermediate panicle
weights were selected. The panicles in the five hills were threshed, and all spikelets in each hill
were counted. Grains after hulling (hereafter ―grain‖) were sieved at 1.7 mm to select ripened grainsby thickness. All ripened grains in each of the five hills were counted, and were weighed. The
percentage of ripened-grain weight to panicle weight in the five selected hills of each plot wasthProceedings of The 7 ACSA Conference 201157

calculated; then, the ripened-grain weight (yield) of 30 hills of each plot was estimated from thispercentage.
Table 1. Fertilizer application, dates of sowing and transplanting for MR78, MR53, Ni and Hi in the experiments
in 2003 and 2005
Year
2003

1)

2003

2)

Total amount of fertilizer
2 2)
applied (N g/m )
16.0
8.0
4.0
21.0
12.0

Basal dressing
2 2)(N g/m )
3)
8.0
4)
4.0
2.0
6)
7.0
4.0

Top dressing
2 2)(N g/m )
5)
8.0
5)
4.0
5)
2.0
7)
14.0
7)
8.0

1)

Dates of sowing and transplanting were April 16 and May 9 in 2003, and April 20 and May 8 in 2005.
P2O5 and K2O elements were applied at the same level as N element
The 6.0 g/m2 of each nutrient element was applied with LONG® 100 type, Chisso Asahi Fertilizer Co., Ltd. (about 7% of eachnutrient element is readily available) six days after transplanting. In additional to 2.0 g/m2 of each nutrient element applied withan ordinary chemical fertilizer before puddling.
4)
The 2.0 g/m2 of each nutrient element was applied with LONG® 100 type, in addition to 2.0 g/m2 of each nutrient element
applied before puddling.
5)
LONG® 100 type was applied on June 30 for MR53, July 27 for MR 79, July 14 for Ni (‗Nishihikari‘) and July 9 for Hi(‗Hinohikari‘).
6)
The 3.0 g/m2 of each nutrient element was applied with LONG® 100 type six days after transplanting. In additional to 4.0 g/m2
of each nutrient element applied with the ordinary chemical fertilizer before puddling.
7)
Another slow-release coated fertilizer, LONG® 100 type, Chisso Asahi Fertilizer Co., Ltd. (about 3% of each nutrient element isreadily available) was applied on May 20 for MR53, June 2 for MR79, June 9 for Ni and June 5 for Hi.
2)
3)

Results and Discussion
In yield, the lines-varieties were ranked in the order MR79 > MR53 > Ni > Hi at every
fertilizer level in the two years: MR79 and MR53 were 30 to 70% and 23 to 48%, respectively,
higher than Hi in the five environments (Table 2). Regarding spikelet number per panicle, they werein the order MR79 > (or ≒) MR53 > Hi > (or ≒) Ni at each of the fertilizer levels in the two years. In
this trait, MR79 and MR53 were 24 to 40% and 21 to 30%, respectively, higher than Hi in the fiveenvironments. As for panicle number per m2, MR79, MR53 and Ni were 95-110%, 92-100% and103-111%, respectively, of Hi in the two years, although the difference among the four linesvarieties in this trait was smaller than that in the previous trait in every environment. In ripened-grain
percentage, Hi was around 80% and the lowest among the lines-varieties in every environment.
MR53 was similar to or not so different from Ni in every environment. MR79 was not significantly
different from Ni in all environments except at 21 of N g/m 2 in 2005. In terms of 1000-grain weight,
Ni was heavier than the other lines-variety, and either MR53 or MR79 was similar to or not so
different from Hi in both years. Regarding sink size, they were in the same order as in yield. In culm
length, MR79 was similar to Hi. MR53 was rather longer than low-height Ni in both years. They had
panicle lengths within the ranges of 2.1 and 2.5 cm in 2003 and 2005, respectively, althoughvarietal difference was detected. MR53 and MR79 were 11-17 days earlier and 14-18 days later,
respectively, in 80% heading date than Hi in the two years.
The higher the fertilizer level, the higher the yield in each line/variety in each year (Table
2). Similar fertilizer responses were noticed in both spikelet number per panicle and panicle number
per m2, although the latter trait was more responsive than the former trait in each line/variety in both
years. All line-varieties except MR53 had the lowest ripened-grain percentage at 4 of N g/m2 in
2003, whereas they had higher values at 12 of N g/m2 than at 21 of N g/m2 in 2005. However, range
caused by fertilizer effect within each line/variety was less than 5.1% inclusive in each of the years.
Every line/variety had little higher 1000-grain weight at 16 of N g/m2 than at 4 of N g/m2 in 2003, butsuch consistent response was not detected in 2005.

58

thProceedings of The 7 ACSA Conference 2011

Regarding sink size (single-grain weight × fertilized-spikelet number per m2), they were in
the same order as in yield (Table 2). The yield of MR79 at 21 of N g/m2 in 2005 (728 g/m2) wasoutstandingly high as compared with yield levels of ordinary japonica varieties in southern Japan.
This high yield was due to its more spikelets per panicle caused by Ur1, and its ripened-grain
percentage being maintained at almost 80%. Furthermore, MR79 had significantly higher yield thanMR53, Ni and Hi at the other four environments. Hence, the high yielding ability of MR79 was stable
over the low to high fertilizer levels.
Table 2. Yield and other traits of MR79, MR53, Ni and Hi at the three fertilizer levels in 2003 and two fertilizer
level in 2005

Trait
Yield

year
2003

2005
Spikelets/
panicle2003

2005
Panicle/
m2

2003

2005
Ripenedgrain
percenttage

2003

1000grain
weight1)
(g)

2003

Sink
size3)(g/m2)

2003

2005

2005

2005

Fertilizer
Level(N g/m2)
16
8
4
21
12
16
8
4
21
12
16
8
4
21
12
16
8
4
21
12
16
8
4
21
12
16
8
4
21
12
16
21

MR79
a

673
626 b
553 c
728 a
662 c
103.8 ab101.4 bc
102.0 b132.7 a123.8 b
338 b
317 b
289 c
354 bcd
326 ef94.0 ab95.3 a92.4 b79.8 c84.9 b
20.4 cde
20.4 cde20.3 de19.5 d19.3 d
695 a
640 b
577 c
827 a
726 c
73.5 a77.0 aNi („Nishihikari‟)

MR53
(149)
(165)
(170)
(130)
(131)
(124)
(130)
(137)
(140)
(134)
(107)
(110)
(105)
(95)
(96)
(115)
(117)
(117)
(101)
(104)
(98)
(99)
(100)
(97)
(97)
(141)
(151)
(155)
(131)
(129)
(97)
(191)

b

612
549 a
481 a
723 a
626 a
107.4 a101.8 bc
96.9 c116.0 c111.2 c309 c
288 cd268 e360 bc312 f
91.6 b
93.0 ab92.4 b85.0 b88,9 a20.2 e20.2 e20.1 cde
20.4 b
20.3 bc
640 b
566 c
496 d
780 b
659 d
65.9 b66.1 b

(136)
(145)
(148)
(130)
(123)
(128)
(130)
(130)
(123)
(121)
(97)
(100)
(98)
(96)
(92)
(112)
(114)
(117)
(107)
(109)
(97)
(98)
(99)
(102)
(101)
(130)
(134)
(133)
(123)
(117)
(87)
(87)

2)

c

554
478 a
442 a
695 a
572 a
80.6 de76.5 ef
74.1 f90.5 de85.2 e
336 a
314 b
304 b
389 a
349 cd94.1 ab93.2 ab92.7 ab88.9 a85.5 b
21.7 a21.3 b
21.1 b
22.2 a22.5 a567 c
492 de458 ef733 c
618 e
63.6 c64.6 b

(123)
(126)
(136)
(124)
(113)
(96)
(98)
(100)
(96)
(92)
(106)
(109)
(111)
(104)
(103)
(115)
(114)
(117)
(112)
(105)
(105)
(103)
(104)
(111)
(112)
(115)
(117)
(123)
(116)
(110)
(84)
(85)

Hi („Hinohikari‟)
de

451
379 a
326 a
558 a
507 a
83.6 d78.3 ef
74.3 f94.5 d92.1 d317 b
287 cd274 de374 ab339 de82.0 c81.6 cd
78.9 d79.2 c81.3 cb
20.8 c20.6 cd
20.3 de20.0 c20.0 c492 de422 f
373 g
631 de562 f75.6 a75.9 a

LSD
37

32
5.2

6.3
14

20
2.7

2.4
0.4

0.4
38

32

Culm
2003
2.1
length
2005
2.4
(cm)
Panicle2003
16
20.3 ab(110)
19.7 b(107)
20.5 a(111)
18.4 c0.8
length2005
2122.8 a(112)
21.6 b(106)
20.5 c(101)
20.3 c0.9(cm)
80%2003
4-16
Aug.22
July 28/29
Aug.13
Aug.9
heading
2005
12-21
Aug.23/24
July 21Aug.11
Aug.7
date
1)
Brown rice weight after sieving at 1.7mm, being adjusted into 15% moisture. 2) Percentage to H in parentheses 3) Single-grain
weight x fertilizer-spikelet number per m2. Values followed by the same letter within each year in a trait are not significantly
different at the 0.05 level being determined by LSD in the table.

MR79 was 14-18 days later in 80% heading date than the rather late variety Hi in allenvironmental conditions in the two years. MR79 was higher-yielding than the rather–early line
MR53 in every environmental condition. The lateness of MR79 might be advantageous to attainthProceedings of The 7 ACSA Conference 201159

high yield. MR79 has high eating quality comparable to that of Hi, one of excellent-taste varieties inJapan (Murai, unpublished). Consequently, MR79 could be a super-high yielding variety withexceptionally late maturity in southern Japan.

References
Murai, M. and M. Iizawa.1994. Effects of major genes controlling morphology of panicle in rice. Breed. Sci. 44:
247-255.
Murai, M., K. Ise, S. Sato and I. Takamure. 1997. Utility of Ur-1 for developing F1 cultivars. In ―Proceedings ofthe International Symposium on Two-line System Heterosis Breeding in Crops‖ China National Hybrid
Rice Research and Development Center, Changsha, China. p. 146-151.
Murai, M., Y. Sato, A. Nagayama, N. Ishii, and S. Ihashi. 2002. Effects of a major gene Ur1 characterized byundulation of rachis branches on yield and its related traits in rice. Breed. Sci. 52: 299-307.
Murai, M., A. Nagayama, S. Sato, H. B. KC and K. Ise. 2003. High yielding F1 hybrid carrying Ur1 (Undulated
rachis-1) gene in japonica rice. Breed. Sci. 53: 263-269.
Murai, M., S. Malangen, K. Nakamura, Y. Fukumoto, and S. Matumura. 2005 a. High-yielding japonica rice lines
carrying Ur1 (Undulated rachis-1) gene, preliminary report. SABRAO Journal of Breeding and Genetics.
37: 51-54.
Murai, M., K. Nakamura, M. Saito, A. Nagayama and K. Ize. 2005 b. Yield increasing effect of a major gene,
Ur1(undulated rachis -1) on different genetic backgrounds in rice. Breed. Sci. 55: 279-285.
Nishiyama, H. 1982. A new rice variety ‗Nishihikari‘. J. Agric. Sci. (Nogyou gijutu) 37: 125-127 (in Japnese).
Sato, S. and C. Shinjyo (1991) Genetic studies on genetic male sterile mutant obtained through breeding ofisogenic translocation line of Taichung 65 in rice, Oryza sativa L. Jpn. J. Breed. 41: 451-459.

-- back to Table of Content --60

thProceedings of The 7 ACSA Conference 2011

Oxidative Stress and its Relation to Radiation Use Efficiency in
Rice Growing under Rainfed Condition in Northeast Thailand
Kohtaro Isekia,*, Koki Hommaa , Boonrat JongdeebaGraduate School of Agriculture, Kyoto University, Kyoto, Japanb
Ubon Rice Research Center, Ubon Ratchathani, Thailand* Corresponding author: Graduate School of Agriculture, Kyoto University, Sakyo-ku, 6068502, Kyoto, Japan
Tel.: +81 75 753 6042; fax: +81 75 753 6065.
kiseki@kais.kyoto-u.ac.jp

Abstract
In rainfed cultivation of rice, the scarcity of available water often inhibits plant growth. Under such conditions, thedrought induces oxidative stress which damages to photosynthetic cells such as protein degradation, lipid
peroxidation and enzyme inactivation. In this study, we evaluate oxidative damage for 10 rice genotypes underrainfed condition in relation to radiation use efficiency (RUE). Although the drought stress was relatively mild asshown in the slightly decreases of stomatal conductance and leaf water potential, RUE under rainfed condition
was significantly decreased from that under flooded condition. The oxidative damages evaluated by chlorophyll
fluorescence and lipid peroxidation were also increased under rainfed condition. The genotypic variation of RUE
in both water conditions was negatively correlated to the value of midday Fv/Fm and it was thought to beaffected by the canopy structure of each genotypes. Damage of leaf cells evaluated by membrane stability index
under rainfed condition was not significantly different from that under flooded condition, but the larger
photoinhibition and lipid peroxidation seems to cause severe cell damage when the water scarcity continues forlong term.
Keywords: rice, photoinhibition, chlorophyll fluorescence, drought, oxidative stress

Introduction
Plant oxidative stress is induced by various abiotic stresses and damages to leaf cells(Mittler, 2002). Under drought and high light conditions, decreasing of energy use for Calvin cycle
leads to over excitation of energy which causes generation of reactive oxygen species (ROS) andpromote oxidative damage to photosynthetic cells (Murata et al., 2007). Oxidation of cell membraneincreases water leakage and decreases leaf water potential. In addition, severe oxidative stress isthought to be a cause of metabolic limitation in Calvin cycle, inducing non-stomatal inhibition ofphotosynthesis (Lawlor, 2002). Drought is an increasingly important constraint of rice production:
the scarcity of water resource for agriculture provides the opportunity for rice to have water deficit
even for irrigated production as well as for rainfed production. However, the relation between
oxidative stress and drought stress has not been examined in terms of crop response under field
conditions with various environmental stresses.
To clarify the relationship between drought and oxidative stress under field condition, we
firstly focused oxidative stress of rice in relation to plant water status. Effect of the oxidative stresson plant growth was evaluated in relation to RUE.

Materials and Methods
Field growth conditions
Ten rice genotypes from Surin1 backcross introgression lines with IR68586-FA-CA-143(BC3) were used. 3 weeks seedlings were transplanted with the plant density of 25 plants m-2 inthProceedings of The 7 ACSA Conference 201161

rainfed and flooded field on 12th August 2010 at Ubon Rice Research Center, Northeast Thailand.
Fertilizer was applied with 3-3-1.5 g m-2 of N-P2O5-K2O for each field. The items below were
measured from 2 to 9 weeks after transplanting.
Radiation use efficiency
Total shoot dry weight was measured at 2 and 9 weeks after transplanting, and thedifference of dry weight was defined as total shoot growth. Radiation use efficiency (RUE) was
calculated as RUE (g MJ-1) = (total shoot growth)/(total intercepted solar radiation). To determine
canopy light absorbance rate (kc), photosynthetic active radiation (PAR) at the top and bottom of
the canopy in each plot was measured with a linear PAR intercept-meter (AccuPAR, Decagon,
USA) under diffused solar radiation conditions once every week. The weekly accumulation ofincident solar radiation was multiplied by the weekly value of kc, and summarized during the period
as total intercepted solar radiation..
Chlorophyll fluorescence, gas conductance (gs) and leaf water potential (LWP)
Maximum quantum yield (Fv/Fm) and effective quantum yield (ΦPS2) of photosystemⅡ was
measured by chlorophyll fluorometer (OS-30p, Opti Science, USA) in early morning and midday of aclear sunny day every week. The topmost fully expanded leaves were used for the measurement.
Simultaneously, gs and LWP of the topmost fully expanded leaves were also measured.
Membrane stability and lipid peroxidation
The topmost fully expanded leaves were sampled at 8 weeks after transplanting and used
for the following analyses. The leaf membrane stability index (MSI) was determined according to
Sairam and Saxena (2000). Leaf discs were soaked in distilled water at 40 °C for 60 min and its
electrical conductivity recorded (C1). Subsequently the same samples were kept in boiling water for
10 min and its electrical conductivity also recorded (C2). MSI was calculated as MSI (%) = [1–(C1/C2)] × 100.
The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content,a product of lipid peroxidation. Fresh leaf sample was homogenized in 20% trichloroacetic acid
(TCA). The homogenate was centrifuged at 12000 G for 10 min. The 0.5 ml aliquot of thesupernatant was added to 1.5 ml of 0.5% thiobarbituric acid in 20% TCA. The mixture was heatedat 95°C for 30 min and then quickly cooled in ice. After centrifugation at 3000 G for 10 min, theabsorbance of the supernatant was measured at 532 nm. The value for non-specific absorption at
600 nm was subtracted. The MDA content was calculated using its extinction coefficient of 155mM-1
cm-1 and expressed as nmol MDA g-1 fresh weight.

Results and Discussion
Most genotypes decreased their total shoot growth under rainfed condition from that underflooded condition. The smaller shoot growth under rainfed condition was mainly caused by the lower
RUE (Figure 1). The Surin1 which showed higher RUE under flooded condition also showed higher
RUE under rainfed condition. Another parent line, IR68586-FA-CA-143 showed lower RUE underboth water conditions (Table 1). However, except these two parent genotypes, the relationship of
RUE between rainfed and flooded condition was not obvious. It implies that the effect of water
limitation on RUE was different among genotypes. In the theory, RUE can be explained as thefunction of canopy photosynthesis (Sinclair and Horie, 1989, Reynolds et al., 2000). In terms of
single leaf, photosynthetic rate is closely related with stomatal conductance, which is governed by
LWP (Medrano et al., 2002). The slightly but significantly lower value of gs and LWP under rainfed
condition suggested that mild drought stress was occurred (Table 1). The drought stress might
decrease RUE.
62

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Proceedings of The 7 ACSA Conference 2011

Total shoot growth (g m -2)

600

500
RUE=2.0400

300

RUE=1.5
RUE=1.0

200
200

250

300

350

400

Total Intercepted radiation (MJ m -2)

Figure 1. Genotypic variation in total shoot growth, total intercepted solar radiation and RUE under rainfed andflooded conditions. Open circle and closed diamond represents the genotype grown under flooded andrainfed condition, respectively. Data are average of three replicates.

Table 1. Radiation use efficiency, stomatal conductance and leaf water potential of 10 genotypes under flooded
and rainfed conditions-1

RUE (g MJ )-2

gs (mmol m )LWP (Mpa)

FloodedRainedFloodedRainedFloodedRainedIR68586-FA-CA-143
Surin 1IRUBN030055-5-87
IRUBN030055-5-112
IRUBN030055-5-190
IRUBN030056-10-42
IRUBN030056-10-107
IRUBN030062-1-9
IRUBN030063-9-4
IRUBN030070-9-32

1.42
1.83
1.38
1.73
1.68
1.49
1.65
1.68
1.33
1.66

0.97
1.34
1.43
1.17
1.25
1.32
1.32
1.20
1.14
1.09

218
214
239
261
286
280
229
249
311
229

184
224
244
192
230
220
189
222
229
194

-0.796-0.797-0.808-0.808-0.819-0.789-0.822-0.806-0.847-0.861-0.836-0.864-0.906-0.856-0.853-0.850-0.847-0.864-0.847-0.861

Average

1.59

1.22

252

213

-0.803-0.858

Genotype (G)Water condition (E)
GxE

*
***
*

ns
***
ns

ns
***
ns

Data are average of three replicates. * and ** represent statistically significant at 0.05, 0.01 level, and ns represents not
significant.

The water limitation, even mild drought, decreases carbon fixation of rice and induces
oxidative damage (Zhou et al., 2007). Measurement of chlorophyll fluorescence can easily evaluate
photosynthetic capacity, and is widely applied for environmental stresses studies. The maximum
quantum yield of photosystemⅡ (Fv/Fm) is an indicator of photoinhibition which relate to oxidative
stress (Maxwell and Johnson, 2000). Generally, Fv/Fm decreases with increasing of exposed lightintensity at midday. Decrease in Fv/Fm by high PAR was larger under rainfed condition than that
under flooded condition (Figure 2). The same tendency was observed for the effective quantum
yield (ΦPS2) but the difference was not significant between rainfed and flooded conditions. It is
known that the carbon fixation rate is closely related to the value of ΦPS2 under the environment ofconstant CO2 concentration (Kato et al., 2003). Decrease of ΦPS2 induces over reduction in electrontransport chain and increases oxidative damage. The lower value of Fv/Fm under rainfed conditionis thought to be resulted by increased oxidative damage that derived from inhibition of carbon
fixation. The genotypic variation in Fv/Fm and ΦPS2 was not significant, because of large residual
variation in this study.

thProceedings of The 7 ACSA Conference 2011

63

0.85

0.6

0.4

0.75

ΦPS2

Fv/Fm

0.80

0.70

0.2

0.65
0.60

0.0
0

500

1000

1500

0

PAR (μmol m -2 s-1)

500

1000

1500

PAR (μmol m -2 s-1)

Open circle and closed diamond represents the genotype grown under flooded and rainfed condition, respectively. Data areaverage of four replicates.

Figure 2. Decrease of Fv/Fm and ΦPS2 with increasing of PAR in midday. Fv/Fm was measured in early morningand midday, and ΦPS2 was measured only in midday.

MDA content was higher under rainfed condition, indicating that damage of cell membraneby reactive oxygen species was larger under rainfed than that under flooded condition (Table 2). On
the other hands, difference of MSI, another indicator of membrane damage, was not significant
between the two water conditions. MDA is a product of lipid of peroxidation, while MSI indicateswater leakage from cell induced by oxidative damage to cell membrane (Mittler, 2002). Accordingly,
the result in this study may suggest that lipid peroxidation proceeded under rainfed condition but thedamage was alleviated by antioxidation mechanisms.
Table 2 Malondialdehyde content and membrane stability index of 10 genotypes under flooded and rainfed
conditions-1

IR68586-FA-CA-143
Surin 1IRUBN030055-5-87
IRUBN030055-5-112
IRUBN030055-5-190
IRUBN030056-10-42
IRUBN030056-10-107
IRUBN030062-1-9
IRUBN030063-9-4
IRUBN030070-9-32
Average
Genotype (G)Water condition (E)GxE

MDA (nmol g f.w.)
FloodedRainfed
88.4
107.3
88.3
93.5
67.7
151.9
70.2
78.4
85.5
109.3
73.1
57.2
75.3
58.0
44.2
102.0
88.1
97.8
89.6
114.5
77.0
97.0
***
***
***

MSI (%)
FloodedRainfed
81.7
76.1
81.5
76.2
79.7
78.6
77.8
75.7
75.7
77.1
79.6
76.0
78.5
79.0
82.3
80.7
83.1
78.1
76.5
80.0
79.7
77.7
ns
ns
ns

Data are average of three replicates. * and ** represent statistically significant at 0.05, 0.01 level, and ns represents not
significant.

Drought stress decreases RUE and Fv/Fm, implying the RUE was positively correlatedwith Fv/Fm. However, the relationships for each water condition showed negative correlations(Figure 3). This suggests that the genotypes which showed larger canopy photosynthesis were
suffered with larger photoinhibition. Because RUE is not determined only by single leaf

64thProceedings of The 7 ACSA Conference 2011

photosynthesis but also canopy structure, further study is necessary to reveal the effect ofphotoinhibition on plant production.
2.0

RUE (g MJ-1)

RUE (g MJ-1)

2.0

1.5

1.0

0.5

1.5

1.0

0.5
0.70

0.72
0.74
Fv/Fm

0.76

0

50
100
150
200
MDA content (nmol g -1 f.w.)

Open circle and closed diamond represents the genotype grown under flooded and rainfed condition, respectively. Fv/Fm is theaverage of three measurement days from sixth to eighth weeks after transplanting.

Figure 3. Relationships of RUE to Fv/Fm and to MDA content.

In this study, oxidative stress in rice was evaluated under field environment of rainfed
condition during wet season where drought stress was relatively mild. The drought stressdecreased RUE and, increased photoinhibition and lipid peroxidation. The results suggest that theoxidative stress was associated with dry matter production. To elucidate the relation between
drought and oxidative stress and to apply it for breeding program of drought tolerance, further
studies are necessary.

References
Kato MC, K Hikosaka, N Hirotsu, A Makino and T Hirose. 2003. The excess light energy that is neither utilized inphotosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation inphotosystem II. Plant Cell Physiol. 44:318-325.
Lawlor DW. 2002. Limitation to photosynthesis in water-stressed leaves: stomata vs. metabolism and the role ofATP. Ann. Bot. 89:871-885.
Maxwell K and GN Johnson. 2000. Chlorophyll fluorescence--a practical guide. J. Exp. Bot. 51:659-668.
Medrano H, JM Escalona, J Bota, J Gulias and J Flexas. 2002. Regulation of photosynthesis of C3 plants in
response to progressive drought: stomatal conductance as a reference parameter. Ann. Bot. 89:895905.
Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7:405-410.
Murata N, S Takahashi, Y Nishiyama, and SI Allakhverdievd. 2007. Photoinhibition of photosystem II underenvironmental stress. Biochim. Biophys. Acta 1767: 414-421.
Reynolds MP, M Ginkel and JM Ribaut. 2000. Avenues for genetic modification of radiation use efficiency inwheat. J. Exp. Bot. 51:459-473.
Sairam RK and DC Saxena. 2000. Oxidayive stress and antioxidant in wheat genotypes: possible mechanism ofwater stress tolerance. J. Agron. Crop Sci. 184:55-61.
Sinclair TR and T Horie. 1989. Leaf nitorogen, photosynthesis and crop radiation use efficiency: a review. Crop
Sci. 29:90-98
Zhou Y, HM Lam and J Zhang.et al. 2007. Inhibition of photosynthesis and energy dissipation induced by waterand high light stresses in rice. J. Exp. Bot. 58:1207-1217.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201165

The Growth Characteristics of Some Varieties of Aceh‟s Local Rice(Oriza sativa L.) on Acid Soils
Elly Kesumawatia,*, Erita Hayatia, Muhafaz Zulusfitria
Faculty of Agriculture Department of Agrotechnology, Syiah Kuala University
Banda Aceh, Aceh Province, Indonesia* Corresponding author: Faculty of Agriculture Jl.Tgk.Hasan Krueng Kalee, Kopelma Darussalam,Banda Aceh 23111, Aceh Province, Indonesia
Tel.: +0651-7552223; fax: +065-17555269
ekesumawati@yahoo.com

Abstract
To identify the agronomic and morphological characteristic of some local rice varieties in Aceh, to describe
based on the classifications of rice and to observe the varieties resistant to soil acidity, it was needed to conduct
a study of 18 Aceh‘s local rice varieties and one ‖Dupa‖ variety as a comparison. The results showed that there
were 6 local varieties of Aceh tolerant to Al toxicity and 12 were somewhat tolerant varieties, as well as varieties
had tolerant nature to ―Dupa‖. There were differences in flowering age, counted from planting rice to grewpanicle by 10%. This age difference could be grouped into three, namely: age of flowering under 100 days wasfound in 2 varieties, age of flowering ranged from 100 to 119 days was found in 12 varieties (including Dupa),
while flowering more than 120 days was found in 5 varieties. There were 15 varieties of rice belonging to theclass Indica and 4 varieties in Javanica group. The largest stem diameter by Sigupai Wangi variety and smallest
by Pade Cut Kresek variety. Longest panicle varieties belonged to Pade Rangan and shortest Sigupai Nagan
Raya. There were 14 varieties which did not possess grain hair-tip, while shortest grain hair-tip by 4 varieties
and 1 varieties of grain had long hair-tip. The widest seed variety was found in Dupa and the smallest was from
Pade Cut Kresek. The thick seed was Dupa variety and the thinnest was Ramos Tihion. Based on length, thelongest seed character was found in 17 varieties and other 2 variety was just long.
Keywords: characteristics, variety, local Aceh’s rice, acid soils

Introduction
Rice is a strategic commodity plays an important role in the economy and national foodsecurity and become a major base in the revitalization of agriculture in the future. About 95% ofnational rice production depends on the production of rice paddies and only 5% of upland rice.
Increased productivity of rice reached only 0.40% per year while the land use field reaches 0.77%
per year (Puslitbangtan, 2007).
Expansion of the current wetland is difficult because suitable land for new rice fields arevery limited, the cost of irrigation system development and printing of new rice fields are very
expensive. Alternative increase rice production can be done by the expansion of upland rice
cropping on dry land. Dry land area reached 54.4 million ha, but the 45.8 million ha of land of whichis acid (BPS, 2007), and 11 million hectares more of them has the potential to be developed as
upland rice cropping land (Puslitbangtan, 2007). However, the development of upland rice on dry
land faced with the problem of Al toxicity and nutrient deficiency phosphate appearing
simultaneously on acid soils of land (Kochian et al., 2004). Liming and P fertilization are used toovercome these problems, but this way is not economical for areas that are far from sources oflime, not a good means of transportation, the availability of P fertilizers is often not continuous andexpensive. It would require the development of varieties that grace Al and efficient in the use of P.
Aceh is a province in Indonesia rich in diversity of rice local varieties, which needs to bepreserved and utilized for sustainable development in order to improve the welfare of all people.

66

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Proceedings of The 7 ACSA Conference 2011

Characterization of genetic resources will provide added value in enriching the "gene pool", the newdiversity of local varieties is useful for the assembly of new varieties (Neereja et al., 2005). These
genetic resources continue to decline due to lack of attention, their utilization, and the changes in
traditional farming practices. Introduction of high yielding varieties called genjah to rice production
centers in various regions may cause genetic erosion of local varieties existing in certain areas(Mishra et al., 2009). The existence of genetic erosion due to modern farming practices led toincreasingly felt the importance of germplasm collection and conservation. To anticipate the erosionof plant genes, it is necessary to preserve the plant genetic material through exploration,
characterization, and documentation (Hanarida et al., 2005). Genetic diversity can be determinedthrough characterization and identification. Improved varieties can be done either throughconventional breeding or modern breeding program utilizing biotechnology assemblies germplasm
using seeds from existing genetic resources. Each variety has certain important characteristics
which can be used for crossing among varieties (Liu et al., 2007). Aceh local rice varieties are stillwidely used by farmers in many districts in Aceh, but in relatively small quantities. Until now, local
rice germplasm has not been characterized and evaluated, therefore, this requires the identification,conservation and classification which are useful for the in breeding programs and serve for theprotection of local plant varieties (Menkumham, 2000; Bhuyan et al. 2007; Silitonga, 2008).
This study aimed to identify the agronomic properties and morphology of 18 local rice
varieties in Aceh, based on the classification of rice varieties and to observe their resistant to soil
acidity.

Materials and Methods
The research was conducted at the Garden Experiments Faculty of Agriculture, University
of Syiah Kuala Banda Aceh from January to June 2010. Rice seeds were 18 local varieties(varieties Ramos Merah, Rom Mokot, Sigupai Wangi, Sigupai Nagan Raya, Sambei, Rasi Singke,
Kepala Gajah Kinco, Bo Padang, Rasi Kuneng, Pade Mangat Bu, Bo Santeut, Ramos Tihion, Pade
Cut Kresek, Pade Pineung, Cantek Puteh, Itam Tangke, Pade Rangan, Sigupai Blang Pidie),obtained from farmers from several districts of Aceh, and a control varieties of varieties of Incense
originally from stone Estuary Borneo leaf blast-resistant and tolerant to Al toxicity.
The research was conducted using a randomized block design (RGD), non-factorial with 3
replicates. Treatment of rice varieties (V) consisted of 19 varieties, and 57 units experiment. Eachexperimental unit consisted of four plants, so that overall there were 228 units observation. Soils
used were soil type ultisol (pH 4.35) at 950 kg obtained from Jantho Aceh Besar district. The soil
weight was 8 kg/bucket. Fertilizers used were Urea, KCl, and SP 36 at 2 kg of each. Seeds were
germination on paper soaked in water to thei field capacity, then left for 5 days. Each bucket
consisted of four seedlings of rice plants. A seeding was planted in each planting hole. Furadan at
0.5 g/bucket was applied to soil surface in each bucket. Urea, SP 36 and KCl at 1 g / bucket of eachwere given one day before planting. At 15 DAT urea at 1 g/bucket were applied, at 45 DAT Urea
and KCl were again applied at 1 g / bucket. At 55 DAT Urea applied at 1 g/bucket. Plant
maintenance is carried out by watering, weeding, as well as controlling pest and disease.
The characters observed were agronomic trait (plant height and number of tillers per hill at
the age of 30, 45 and 60 DAT, level of tolerance, and the age of flowering) and morphology(diameter of the stem segment below, panicle length, feather edge grain, seed width, seed
thickness, and length of seed). The data were described based on Characterization and Evaluation
System Guide Rice with scoring for each character observed (Deptan, 2003). Data were calculated
for the relative values (RV) to determine differences in plant height and number of tillers of the soil
acidity. Grouping level of tolerance to soil acidity was based on the criteria modified from Sarkarung(1986) then grouped to tolerant (when the value of RV was greater than 75%), somewhat tolerantthProceedings of The 7 ACSA Conference 201167

(when the value of RV was between 50 to 74%), sensitive (when the value of RV was less than50%).
The relative value (RV) (%) = Plant height or number of pups at acid conditions
x 100%
Plant height or number of pups at normal conditions

Results and Discussion
Agronomic characteristic
The results showed that rice varieties significantly affected the relative values for plantheight at 30 DAT, but not at 45 and 60 DAT (data not showed). The relative value of number oftillers per hill of rice plants was not significantly different at 30, 45, and 60 DAT (data not showed).
The Aceh local rice varieties Sigupai Nagan Raya (V4) and Cantek Manis (V15) were taller thanother local varieties such as Dupa (V19) variety. The shortest rice plant varieties was Red Ramos(V1). The plants high in rice plants had a positive and negative values on the cultivation system.
The tall rice plants can be used as an elder male in producing hybrid seed. Tall rice plants will be
more easily to spread its pollen to the shorter one (female elders). Hybrid rice production needa thestud taller than 20 cm for the elder females. However, tall rice plants are susceptible to fall down
(Samac and Tasfaye, 2003).
Grouping of local rice varieties tolerant to acid soil in Aceh was based on the relative value(RV). According to Sarkarung (1986), local rice in Aceh produces 6 varieties having a relative valueof more than 75% (tolerance type), and 12 varieties having a relative value between 50-74%(somewhat tolerant type) similar to the control Dupa tolerant nature (Table 1). Varieties tolerant tohigh acid soil may also tolerant to stress of Al. This was shown by the relative value of more than75%. Tolerant plants allegedly had the ability to suppress the adverse influence of Al by reducing
the Al3+ ion uptake by roots and neutralize toxic Al in the network, so that plant growth is not
disturbed (Watanabe and Osaki, 2002; Sopandie et al., 2003). Variations in the nature of resistance
18 local varieties of Aceh on Al toxicity in this study presumably because the genes resistant to Al
toxicity did not work together (Nguyen et al., 2002).
Age of flowering plants in Aceh local rice was varied. Sambei (V5) and Rom Mokot
varieties (V2) were flowering under 100 days. Varieties flowerring at 100-119 days were 11
varieties, including control variety Dupa (V19), varieties flowerring at more than 120 days were 5varieties (Table 1).
Morphological characteristic
The results showed that local rice varieties based on morphological characters of rice weregrouped in two, namely the Indica and Javanica groups. There were 15 varieties Indica type, and 4varieties of Javanica. Indica rice (paddy cere) had several characteristics of : having a lot of roots,
smooth leaf surfaces, long panicle, litle hair on the the surface of the grain, no tail on the end grain,
slender grain shape, grain size was small to moderate and harvest age was faster. Characteristicsof the group Javanica rice (paddy fur) were : the number of tillers was less, leaves had a roughsurface, long panicle, hairy grain surface, a tail on the end grain, medium to large grain size and theage old crop (Grubben and Partohardjono, 1996).
The largest diameter stem segment was found in Sigupai Wangi (V3), while the smallest
diameter of the rod segment was found in Pade Cut Kresek variety (V13) (Table 2). The longest
panicle variety was found in Pade Rangan (V17) and the shortest panicle variety was found in
Nagan Raya Sigupai (V4) (Table 2). According to the AAK (1990), panicle length is divided into
three different sizes which are short panicle (less than 20 cm), medium panicle (20-30 cm), andlong panicle (more than 30 cm).

68

thProceedings of The 7 ACSA Conference 2011

Table 1. Grouping of local rice varieties for tolerance to soil acidity based on the relative value (NL) and by ageof flowering

Varieties of rice
V1 (Ramos Merah)
V2 (Rom Mokot)
V3 (Sigupai Wangi)
V4 (Sigupai Nagan Raya)
V5 (Sambei)
V6 (Rasi Singke)
V7 (Kepala Gajah Kinco)
V8 (Bo Padang)
V9 (Rasi Kuneng)
V10 (Pade Mangat Bu)
V11 (Bo Santeut)
V12 (Ramos Tihion)
V13 (Pade Cut Kresek)
V14 (Pade Pineung)
V15 (Cantek Puteh)
V 16 (Itam Tangke)
V17 (Pade Rangan)
V18 (Sigupai Blang Pidie)
V 19 (Dupa)

Relative value
(%)
66,40
75,52
65,71
79,53
72,75
68,09
72,44
77,37
68,69
73,43
74,98
73,03
69,19
77,00
75,23
75,06
65,54
70,71
77,21

Flowersage

Criteria
somewhat tolerant
Tolerant
somewhat tolerant
Tolerant
somewhat tolerant
somewhat tolerant
somewhat tolerant
Tolerant
somewhat tolerant
somewhat tolerant
somewhat tolerant
somewhat tolerant
somewhat tolerant
Tolerant
Tolerant
Tolerant
somewhat tolerant
somewhat tolerant
Tolerant

113
98
112
112
96
120
130
130
130
120
101
112
115
110
101
119
115
116
100

Table 2. Classification of rice based on morphological characters, diameter of rod segment and panicle length ofrice plants

Varieties of riceGroup

Diameter of rod
segments (cm)Length ofpanicle (cm)V1 (Ramos Merah)
V2 (Rom Mokot)
V3 (Sigupai Wangi)
V4 (Sigupai Nagan Raya)
V5 (Sambei)
V6 (Rasi Singke)
V7 (Kepala Gajah Kinco)
V8 (Bo Padang)
V9 (Rasi Kuneng)
V10 (Pade Mangat Bu)
V11 (Bo Santeut)
V12 (Ramos Tihion)
V13 (Pade Cut Kresek)
V14 (Pade Pineung)
V15 (Cantek Puteh)
V 16 (Itam Tangke)
V17 (Pade Rangan)
V18 (Sigupai Blang Pidie)
V 19 (Dupa)

IndicaIndicaIndicaIndicaIndicaIndicaIndicaJavanica
Javanica
IndicaJavanica
IndicaIndicaIndicaIndicaJavanica
IndicaIndicaIndica

0.51
0.53
0.91
0.58
0.55
0.55
0.59
0.61
0.70
0.43
0.52
0.61
0.42
0.53
0.50
0.54
0.60
0.50
0.60

24.03
23.50
20.65
18.85
23.28
29.70
24.03
24.23
30.17
24.63
28.85
26.50
21.75
29.65
24.50
20.18
34.88
23.78
21.68

Grain hair-tip was not found in 13 local rice varieties and the control Dupa (V19). Fourvarieties had short grain hair-tip and some furry at Rasi Singke (V6), Bo Padang (V8), Rasi Kuneng(V9) and Itam Tangke (V16), while Bo santeut (V11) varieties had a long grain hair-tip and all furry(Table 3).
thProceedings of The 7 ACSA Conference 201169

Differences in width and thickness of seeds in each variety are presented in Table 3.
Control varieties (V19) had the widest seed, meanwhile the Pade Cut Kresek varieties (V13) was
the slimmest variety. Varieties of Dupa (V19) also had the thickest seeds and variety Ramos Tihion(V12) was a thin variety. Character of the length scale 1 (seed longer than 7:50 mm) was found at
17 varieties, including varieties of Dupa (V19). Character length scale 2, (seed length of 6.61-7.50mm) was found in Singke Rasi variety (V6) and Itam Tangke (V16).
A number of rice varieties characteristic were difficult to distinguish. Abdullah et al., (2006)
stated that the differences will affect the nature of plant varieties. Each plant has the characteristics
for special properties that may differ from each other, so it will show the diversity of characters.
According to Lesmana et al., (2004) morphological features often used to distinguish varieties ofrice are plant height, stem color, leaf color, leaf surfaces, grain shape, grain color and grainsurfaces. The character of grain morphology on the presence of feathers covering the tip of grain,
seed thickness, seed width and seed length. According Aldair et al., (1966) in Grist (1986), grain
shape consisting of three kinds, namely rounded, medium and lean. In addition, the inflorescencecharacters can distinguish rice varieties (Wet et al., 1986).
Table 3. The presence of feather tip of grain, seeds character width, thickness and length of seed grain Aceh
local

Varieties of rice

The presence of grainhair- tip

V1 (Ramos Merah)
V2 (Rom Mokot)
V3 (Sigupai Wangi)
V4 (Sigupai Nagan Raya)
V5 (Sambei)
V6 (Rasi Singke)
V7 (Kepala Gajah Kinco)
V8 (Bo Padang)
V9 (Rasi Kuneng)
V10 (Pade Mangat Bu)
V11 (Bo Santeut)
V12 (Ramos Tihion)
V13 (Pade Cut Kresek)
V14 (Pade Pineung)
V15 (Cantek Puteh)
V 16 (Itam Tangke)
V17 (Pade Rangan)
V18 (Sigupai Blang Pidie)
V 19 (Dupa)

Lint
Lint
Lint
Lint
Lint
Short and partly feathered
Lint
Short and partly feathered
Short and partly feathered
Lint
Length and all hairy
Lint
Lint
Lint
Lint
Short and partly feathered
Lint
Lint
Lint

Width seeds
(mm)
2.10
3.00
2.10
2.10
2.70
2.49
3.04
2.15
2.70
2.15
2.10
2.06
1.90
2.90
2.50
2.20
4.10
2.20
4.50

The thicknessof the seeds
(mm)
1.50
2.00
1.90
1.90
2.00
1.79
2.00
1.68
2.00
1.92
2.00
1.50
1.52
2.00
1.90
1.79
2.00
1.88
2.10

Length ofseeds
Very long
Very long
Very long
Very long
Very longLong
Very long
Very long
Very long
Very long
Very long
Very long
Very long
Very long
Very longLong
Very long
Very long
Very long

Acknowledgement
This work was supported by the Excellent Research of National Strategic (2009), the Ministry of
National Education Indonesia.

References
Abdullah B., R. Mudjisihono., Prajitno. 2006. Some rice genotypes towards improved quality of rice. Researcher
center for rice, Sukamandi. 5 page.
AAK . 1990. Rice cultivation. Kanisius. Yogyakarta. 172 page.

70

thProceedings of The 7 ACSA Conference 2011

[BPS] Central bureau of statistics. 2007. Statistics Indonesia 2005/2006. Biro Pusat Statistik, Jakarta.
Bhuyan N., N.Basanta., K.Borah., R.N.Sarma. 2007. Genetik diversity analysis in traditional lowland rice (Oryza
sativa L.) of assam using RAPD and ISSR markers. Current Science. 93 (7): 976-972.
[Deptan] Departemen Pertanian. 2003. Guidelines characteristics and evaluation system for rice. National
Commission Germplasm. Agency for Agricultural Research and Development. 57 page.
Grist D.H. 1986. Rice (Tropical agriculture series). Sixth Edition. London : Longman Inc.
Grubben G.J.H., S.Partohardjono. 1996. Plant resources of South-East Asia No. 10: Cereals. Bogor: Prosea.
Hanarida I.S., M.Hasanah., S.Adisoemarto., M.Thohari., A.Nurhadi., I.N.Orbani. 2005. Series know your foodplant germplasm. Germplasm National Commission, Bogor. 34 page.
Kochian L.V., O.A.Heokenga., M.A.Pineros. 2004. How do crop plants tolerate acid soils. Mechanisms ofaluminium tolerance and phosphorous efficiency. Annu Rev Plant Biol 55 : 459-493.
Liu Q.L., X.H.Xu., X.L.Ren., H.W.Fu., D.X.Wu., Q.Y.Shu. 2007. Generation and characterization of low phyticacid germplasm in rice (Oryza sativa L.). Theor. Appl. Genet. 114:803-814.
Lesmana O.S., H.M.Toha., I.Las., B.Suprihatno. 2004. Description of a new rice variety superior. Sukamandi,
Subang: Agency for Agricultural Research and Development - Research Institute for Rice.
[Menhumkam] Minister of Justice and Human Rights Republic of Indonesia 2000. Laws of the Republic ofIndonesia number 29 Year 2000 on the protection of plant varieties.
Mishra B., R.K.Sing., D.Senadhira. 2009. Enhancing genetic resources and breeding for problem soil.
http://www.idrc.ca/en/ev-85296-201-1-DO_Topic.html [14 Febuari 2009].
Neereja C.N., A.S.Hariprasad., S.Malathi., E.A.Siddiq. 2005. Characterization of tall landraces of rice (Oryza
sativa L.) using gene-derived simple sequence repeats. Current Science. 88 (1): 149-152.
Nguyen V.T., B.D.Nguyen., S.Sarkarung., C.Martinez., A.H.Paterson., H.T. Nguyen. 2002. Mapping of genes
controlling aluminum tolerance in rice: Comparison of different genetic backgrounds. Mol Genet
Genomics. 267:772 – 780.
[Puslitbangtan] Center for Food Crops Research and Development. 2007. Annual Report 2006 Food crops
research and development. Jakarta: National Agricultural Research and Developmen
Samac D.A., M.Tasfaye. 2003. Plan Improvement for tolerance to aluminium in acid soils. Plant Cell Tissue andOrgan Culture 75: 189-207
Silitonga T.S. 2008. Genetic resources conservation and development for rice farmers welfare. Paper presented
on the day of Rice Culture on Subang West Java.
Sarkarung S. 1986. Screening upland rice for aluminium tolerance and blast resistance. Proceedings of the1985 Jakarta Conference. IRRI. Philipphines. pp:271-281.
Sopandie, D., I.Marzuki., M. Jusuf. 2003. Alumunium tolerance in soybean: protein profiles and accumulation of
in roots. Hayati 10 (1):30-33.
Watanabe, T., M. Osaki. 2002. Mechanisms of adaptation to high alumunium conduction in native plant species
growing in acid soil. Communication Soil Science Plant Analysis 33:1247-1260.
Wet J.M.J.D., J.R.Harlan., D.E.Brink. 1986. Reality of infraspecific taxonomic units in domesticated cereals instyles, B. T. (ed). Infraspecific Classification of Wild and Cultivated Plants. New York: Oxford University
Press. pp: 210-222.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201171

Effect of NPK Fertilizer and Biochar Applications on
Growth and Yield of Irrigation Rice
Zaituna*, Khairun Nisab, Sufardia, Chairunasc, Anischan Ganid, Peter Slaviche, Malem
McLeodeaAgriculture Faculty, Syiah Kuala University, Banda Aceh, Indonesia
Agency Implementation of Extension and Food Security of West Aceh, IndonesiacProvincial Agricultural Service (BPTP NAD), Banda Aceh, IndonesiadIndonesian Centre for Rice Research, Sukamandi, West Java, Indonesiae
New South Wales Primary Industries, Australia* Corresponding author: Agriculture Faculty, Syiah Kuala University, Darussalam,Banda Aceh, Indonesia 23111.
Tel.: +62 81360301372; fax: +62 651 7555214
zaitundara@gmail.comb

ABSTRACTThe objective of experiment was to investigate the effect of NPK and Biochar applications on paddy rice growthand its production. The research was conducted at Empetrieng village, Aceh Besar district, Aceh Province,
Indonesia. The experimental was arranged in a randomized complete block design with two factors and four
-1
replicates. First factor was NPK at ratio of 15:15:15 as fertilizer application (0; 60; and 120 kg ha , respectively)-1
and second factor was Biochar application at 0 and and 10 ton ha ). The results showed that the application of
Biochar affected significanly on plant height at 28 and 90 DAP (day after planting), percentage of unfilled grain
per peanicle; whereas aplication of NPK affected significanly on plant height at 45 and 90 DAP, number of tiller
45 DAP, number of peanicle per clump, number of total grain per peanicle, number of filled grain per peanicle,
and yield ton per ha, respectively.
Keywords : NPK fertilizer, Biochar, paddy rice

Introduction
Rice is the main food of Indonesian people. The rice field has dominant contribution inyield of paddy rice because generally rice paddy is planted in wet land. Irrigation is very important
for the rice field paddy because this influences the yield of paddy rice productivity (Adiratma, 2004).
Biochar or charcoal represents one option to soil management. Biochar had been applied
traditionally by some farmers at many places. Several reports showed that biochar has the role in
improving fertility soil. Biochar has a lot of pores because it has large surface, so it has high water
holding capacity. Although biochar is not fertilizer, it can be used as mixed fertilizers (Gani, 2009).

Materials and Methods
The field experiment was established at Empetring Village, Darul Kamal Subdistrict, Aceh
Besar District, Aceh Province, Indonesia on December 2009 to May 2010.
This research was designed as experiment field continued in laboratory with the thefollowing step: (a) field trial was done by planting Ciherang varietas paddy rice. NPK fertilizer andbiochar treatment were applied as according to each plot combination treatment; (b) observation
was done by looking at the plant growth i.e. plant height, and number of tiller; (c) paddy rice growth
was measured by determining the plant height at 28, 35, 45 and 90 day after planting (DAP) andnumber of tiller 28, 35, and 45 day after planting (DAP). Measurement of final plant height andnumber of tiller were done, at 1-2 days before harvest paddy rice; and (d) estimation to yieldcomponent of paddy rice was done by determining the number of panicle per clump, number of total

72

thProceedings of The 7 ACSA Conference 2011

grain per panicle, percentage of unfilled grain per panicle, number of filled grain per panicle, weightof 1.000 grain at water content 14%, and yield ton per hectare at water content 14%.
The experiment was arranged in a randomized complete block design with two factors andfour replicates. First factor was NPK at 15:15:15 fertilizer application. F0 was without fertilizer;
F1was 60 kg ha-1 NPK which was equal to 150 g plot-1; and F2 was 120 kg ha-1 NPK which wasequal to 300 g plot-1. Second factor was Biochar application; B0 was without biochar; and B1 was-1-1
10 ton ha which was equal to 25 kg plot .

Results and Discussion
Plant Height and Number of Tiller of Paddy Rice Plant
Vegetative growth with parameter of plant height and tiller number were not significantly
affected by biochar and NPK treatments (Tables 1 and 2), eventhough, plant height and tiller
number tend to increase with biochar 10 ton ha-1 and NPK 120 kg ha-1 treatments. Biochar and
NPK applications can improve soil fertility by repairing soil chemical properties in the form of content
improvement and in the N, P, K nutrient availibility. Improvement of N, P, K nutrient availibility would
affect the nutrient availibility, so that can this would improve the growth of plant height and tiller
number.
Table 1. Average of plant height effect biochar and NPK treatment
Plant height (cm)Treatment
Combination

28 DAP

35 DAP

45 DAP

90 DAPB0 F042,70 a A

52,40 a A

66,45 a A

69,88 a A

B0 F1

44,45 a A

55,09 a A

71,30 a A

71,18 a A

B0 F2

43,68 a A

54,85 a A

71,73 a A

74,80 a A

B1 F045,23 a A

45,27 a A

65,63 a A

71,05 a A

B1 F1

46,28 a A

48,35 a A

72,80 a A

76,93 a A

B1 F2

46,75 a A

48,74 a A

76,88 a A

78,23 a A

BNT 0,05

3,206

15,341

4,627

5,700

Tabel 2. Average of tiller number effect biochar and NPK treatment
Treatment
Combination

28 DAP

35 DAP

45 DAP

B0 F0

14,08 a A

12,70 a A

11,98 a A

B0 F1

12,50 a A

13,05 a A

13,68 a A

B0 F2

14,35 a A

14,53 a A

16,23 a A

B1 F0

13,05 a A

13,05 a A

11,65 a A

B1 F1

13,70 a A

15,03 a A

14,83 a A

B1 F2

14,15 a A

14,83 a A

17,18 a A

BNT 0,05

1,802

2,269

1,937

th

Number of tiller (bar)Proceedings of The 7 ACSA Conference 201173Mori and Marjenah (1993) reported that biochar or husk chrocoal can be used as soilmaterial repairing with increasing air permeability as well as the water percolation. Purnomo (2009)
stated that paddy rice plant responded to single NPK and compound NPK. Compound NPK
fertization significantly improves paddy rice plant height at the primordial stage. Improvement of thisgrowth is caused of by soil chemical properties may increase the rate of N and P in soil.
Kaderi (2004) stated that number of tiller per clump was higher after the addition of NPK
compared to control plant. This showed that compound of NPK added the nutrient element for plant.
Growth of tiller number is related to N sufficiency and efficacy to primordial forming.
Yield of Paddy Rice Plant
Yield component of paddy rice plant did not significantly effected either by Biochar or NPK
treatments (Table 3), eventhough the yield component of paddy rice plant tend to increase with theapplication of Biochar at 10 ton ha-1 and NPK at 120 kg ha-1 compared to the control
treatmentwithout Biochar and without NPK.
Tabel 3. Average of yield component effect biochar and NPK treatment

Variable

Treatment combination

BNT
0,05

B0 F0

B0 F1

B0 F2

B1 F0

B1 F1

B1 F2

9,67
Aa

11,04
aA

12,54
aA

9,56
aA

10,96
aA

11,75
aA

1,249

146,72aA

130,75aA

117,09aA

145,14aA

126,61aA

122,65aA

20,425

20,37
aA

22,84
aA

23,14
aA

16,68
aA

19,11
aA

17,86
aA

4,569

Number of filled grain per panicle(grain)116,29aA

100,81
aA

89,78
aA

120,61aA

102,44aA

100,63aA

14,190Weight of 1.000 grain at watercontent 14% (gram)

24,78
aA

24,93
aA

24,74
aA

25,02
aA

25,28
aA

24,21
aA

1,576

4,32
aA

5,88
aA

6,20
aA

4,33
aA

5,89
aA

6,79
aA

0,748

Number of panicle per clump(panicle)
Number of total grain per panicle(grain)Percentage of unfilled grain per
panicle (%)

Yield ton per hectare at water-1
content 14% (ton ha )Biochar at 10 ton ha-1 tend to increase the yield component of paddy rice plant as iscompared to without Biochar treatment. This is because Biochar at 10 ton ha-1 gave the growth
media of soil microorganism. Consequently this may improve the amount of soil microorganism but
this was not followed with the increase of nutrient availibility in soil. Therefore, soil microorganism
exploit the nutrient source to energy that give in the emulation between soil microorganism with theroot plant in fulfilling nutrient. This may cause competition between soil microorganism and the root
plant.
Increase of NPK dose tend to to improve the yield component of paddy rice plantcompared to that without NPK treatment. This is caused by NPK fertilize that can directly gave thenutrient element required by plant. So that with the existence of nutrient element provided by NPK
can fulfill the nutrient required by the plant growth. This may also repaire soil chemical properties
with sufficiency of nutrient element so that this affected the increase of yield component of paddy
rice plant.

74

thProceedings of The 7 ACSA Conference 2011

Conclusions
Treatment of biochar affected significantly onthe plant height at 28 and 90 day after
planting (DAP), percentage of unfilled grain per panicle. Meanwhile, treatment of NPK affectedsignificantly on the plant height at 45 and 90 DAP, number of tiller 45 DAP, number of panicle perclump, number of total grain per panicle, number of filled grain per panicle, and yield ton per ha.

References
Adiratma, E.R. 2004. Stop Rice Plant. Penebar Swadaya. Jakarta.
Gani, A. 2009. Science and Technology of Main Crop Journal. 4 (1) :33-48.
Kaderi, H. 2004. Processing Technique of Pellet Fertilizer from Weed as Compound Fertilizer and Its Effect toPaddy. Buletin Teknik Pertanian. 9 (2).
Mori, S. and Marjenah. 1993. Mychorriza Incubation with Rice Husk Charcoal. Journal of Asosiasi Politeknik
Indonesia. Vol. I. No. 1. 2008. Samarinda.
Purnomo, J. 2009. Effect of Compound Fertilizer NPK to Yield of Ciherang Variety and Soil Chemical Properties
Inceptisol. Bogor .

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201175

Effects of New Foliar-Application Fertilizer Containing 5-Aminolevlinic Acid
on Yield Increase of Direct-Sowing Rice Plants
Ryuji Yoshidaa*，Keitaro WatanabebaToyama prefectural University, Toyama, Japanｂ
Cosmo Oil Co., Ltd., Japan
Corresponding author: Toyama Prefectural University, Imizu, 5180, 939-0398, Japan
Tel.: +81 766 56 7500; Fax: +81 766 56 0396
yoshida@pu-toyama.ac.jp*

Abstract
In Japan, the cultivation methods for rice plants (Oryza sativa L.) in paddy field can be mainly classified into two
types, i.e. mechanical transplanting cultivation and direct-sowing. However, in the case of direct-sowing
2
cultivation the grain number per m becomes often an excess of 30,000 grains. As the result, the decrease ofpercentage of ripened grains takes places in inferior grains than in superior ones and, as a consequence, thegrain yield decreases. In the present experiment, effects of foliar application fertilizer containing 5-aminolevulinicacid (5-ALA fertilizer: commercial name; Penta-keep) on the percentage of ripened grains in direct-sowing rice
plants were examined. This 5-ALA fertilizer was sprayed at the heading stage of cvs. Koshihikari andTenkomori, the promotive effect on the yield increase of grains was found in both the cultivars tested. It should
be noted that the high yielding effects were at least in part supported by the increasing of grain filling in inferiorgrains and superior, especially in inferior grains. On the other hand, the excellent effect of 5-ALA fertilizer was
found in the aerial spray by small automated helicopter. From all results, we emphasize that the application offoliar application fertilizer containing 5-ALA are very useful to achievehigh yield of direct-sowing rice plants.
Keywords: 5-aminolevulinic acid (5-ALA), direct-sowing of rice plants, new foliar-application fertilizer (-ALA
fertilizer; Penta-keep), yield-increase

Introduction
In Japan, the cultivation methods for rice plants (Oryza sativa L.) in paddy field can bemainly classified into two types, i.e. mechanical transplanting cultivation and direct-sowing. In recent
year, the growing area using the latter cultivation type progressively tends to the extension becauseof the establishment of labor-saving cultivation for rice. However, the grain number per m2 of rice
plants cultivated by direct-sowing becomes often an excess of 30,000 grains. As the result, thedecrease of percentage of ripened grains take places in inferior grains than in superior ones and, asa consequence, the grain yield decreases.
Yoshida et al. (1996) reviewed that 5-aminolevulinic acid (5-ALA) has often promoting
effects on yield increase of crops. Hotta et al. (1997) reported that ALA has plant growth regutating
properties at low concentration (<30ppm) and the foliar application of this compound enhanced thegrowth and yield of kidney beans, barley, potato and garlic. However, there is insufficient
information about the promotive effect of ALA on the grain filling of rice plants. The supply of large
amount for 5-ALA having high-quality and inexpensive price has already been successfully
achieved by Tanaka (1995). A new foliar-application fertilizer containing 5-aminolevlic acid using theextension in agriculture field was also developed by Cosmo Oil Research Group (Tanaka et al.
2006). Commercial name for this fertilizer is PENTAKEEP® super or 5-ALA fertilizer.
This experiment, therefore, was carried out to clarify whether the foliar application of 5-ALA
fertilizer was able to alter the percentage of ripened grains, especially in direct-sowing rice plants.
The utility for achieving high yielding grains of direct-sowing rice cultivation was discussed.

76thProceedings of The 7 ACSA Conference 2011

Materials and Methods
Experiment I (2009)
Rice cultivation. Rice cultivars used were Koshihikari (medium maturing variety) andTenkomori (late maturing variety) in Toyama, Japan. The coating pregerminated seeds with calcium
peroxide (CaO2 of 2-fold) were sown in paddy soil by using direct-shooting seeder. The sowing rateof both the cultivars was 2.5～2.8 kg per 0.1ha.The controlled availability fertilizer (LPs,
N:P:K=18:12:12 ) was basally dressed at rate of 30～35kg per 0.1ha. When the growth of rice
plants reached the full heading time, 5-ALA fertilizer was sprayed to the whole plant.
Spray of 5-ALA fertilizer. Fertilizer 5-ALA was composed of N at 8.0%, P2O5 at 5.0%, K2O
at 3.0%, MgO at 3.0%, Fe at 0.29%, Mn at 0.12% and other micro-elements, but the concentrationof 5-ALA was not only open. At the full heading time, 5-ALA fertilizer was sprayed weekly twice towhole plants. The concentration and application volume of 5-ALA fertilizer were 2000-fold solution
and 200 liter per 0.1ha, respectively. The percentage of ripened grains, such as superior andinferior gains of panicle, was determined at the harvesting time. Yield component of rice plants andquality of grains was also observed.
Experiment II (2010)
Cv. Koshihikari was grown in paddy field. The rates of basal-dressing fertilizer were thesame as those used in Experiment I. The application of 5-ALA fertilizer was sprayed by small
automated helicopter. In this case of aerial application, the concentration and application volume of
5-ALA fertilizer were 8-fold solution and 0.8 liter per 0.1ha, respectively. The effects of spraying 5ALA fertilizer on the percentage of ripened grains and yield component were evaluated at theharvesting stage.

Results and Discussion
In Japan, the yield decrease of direct-sowing rice plants is attributed to the excess of grain
number per m2 (more than 30,000 grains). This excess of unfulled grains per m2 takes place theincrease of sterile and imperfect grains in panicle. In practical producing field of direct sowing
cultivation, the percentage of ripened grains in cv. Koshihikari was well known to vary the range of
65～74%. For achieving high-yielding grains in direct-sowing rice cultivation, therefore,
understanding the effects of 5-ALA fertilizer was particularly important. In this experiment, theunfulled grains of panicle were divided into superior and inferior grains (Figure.1).

Superior grain

Inferior grain

Uppermost internodeFigure 1. Classification ofFig.1
superior
and inferiorgrains andin panicle.
Clssificationof superior
inferior
grains in panicle.

thProceedings of The 7 ACSA Conference 201177

All grains attached directly to primary branches and the terminal grains of secondarybranches were defined as superior grains and the grains on secondary branches, excepting theterminal one, were defined as inferior grains. The following data (Tables 1, 2, 3) were obtained from
Experiment I. Table 1 shows the effects of spraying 5-ALA fertilizer on yield component. In the caseof cv. Koshihikari, the rate of grain-straw was 1.1 in both the control plants (untreated-treated with
5-ALA fertilizer) and the plants treated with 5-ALA fertilizer. The unfulled grain number per m2 wasabout 33,000 in both the plants. In the case of cv.Tenkomori, the grain-straw ratio and unfulled
grain number were 0.9 and 30,000～33,000, respectively. The yield components in both thecultivars did not vary with the spraying 5-ALA fertilizer.
Table 1. Effects of 5-ALA fertilizer on yield component
Cultivar

Treatment
5-ALA
fertilizer

Koshihikari

Untreated(control)
Treated
Untreated(control)
TreatedTenkomori

Unfulledgrain
weight(g/hill)51

Strawweight(g/hill)

Unhulled
grain straw ratio

Panicalnumberper hill
22.9

Unhulled
grain
numberper hill
1.839

Unhulled
grain
number2
per m
33.470

48

1.1

52
45

49
48

1.1
0.9

23.0
21.2

1.806
1.654

32.86030.103

46

51

0.9

21.7

1.786

32.504

1. The density of direct-sowing rice plants is 21~24 per m2
2. The value show the means of 3 replications. The number of hills tested is 240.

The percentage of ripened grain in rice plant treated with 5-ALA fertilizer is shown in
Tables 2. As was expected, the 5-ALA fertilizer enhanced the percentage of ripened grains. This
increase was much more marked in the inferior grains than in the superior ones. The extent of theincrease in both the cultivars brought about by the spraying treatment amounted from 13.6 to 29.9%(cf. control) in the inferior grains. Thus, the spraying treatment of 5-ALAfertilizer significantly
affected the grain filling of inferior grains (p＜0.05).
Table 2. Effect of 5-ALA fertilizer on percentage of ripened gains

Cultivar
Koshihikari

Tenkomori

Treatment 5ALA fertilizer
Untreated(control)
Treated
Untreated(control)
Treated80.3+45

Ratio of grains per panicle(%)
Superior
Inferior grain
grains
77.9+0.7
22.1+0.7

Percentage of ripened grain(%)
Superior
Inferior grain
grains82.7+3.0a
54.9+2.1b78.5+5.5
78.0+7.0

82.8+2.3
71.8+2.6

17.2+2.3
28.2+2.6

88.8+2.8a
94.0+3.3a84.8+4.6a
79.8+4.4b

82.3+2.8

72.7+3.0

27.4+3.0

95.7+1.4a

93.4+1.5a

Unhulled
grain number
per panicle

1.Date are means+SE of 150 panicles.
2.Different letters within columus in each cultivar indicate significant differences by Tukey‘s multiple range test (p<0.05)

As for the 1,000 grain weight, the cv. Tenkomori showed higher value in inferior grains
than that in superior ones (Table 3). In the case of cv. Koshihikari, the fulled grain yield of the plants
treated with 5-ALA fertilizer was 627.5kg per 0.1ha, this value was higher than that of control plants(Table 4). The effect of spraying 5-ALA fertilizer also was much more marked in the case of cv.
Tenkomori. The degree of yield increased was 145.5kg per 0.1ha. These findings showed that 5ALA fertilizer acts as a regulator for yield increase of rice plants through a steady increase of grain
filling, especially in inferior grains. Regarding the physiological function of 5-ALA, Tanaka (1995)

78

thProceedings of The 7 ACSA Conference 2011

has been reported in radish plants that 5-ALA enhanced the activity of photosynthesis andsuppressed the rate of respiratory activity. Yoshida (1996) showed that the accumulation fractan in
rakkayo and shallot plants was increased by 5-ALA application. From the results obtained here andthese findings, the steady increase of the percentage of ripened grains by the application of 5-ALA
fertilizer seemed to be dependent on the activity of photosynthesis during the grain filling period.
However, the physiological functions of this fertilizer during maturation of rice grains are still almost
unknown at the present.
Table 3. Effect of 5-ALA fertilizer on yield and quality of gains (hulled grains)

Cultivar
Koshihikari

Tenkomori

Quality of grain (%)
AmiloseIndex ofcontent (%)
paratability
18.7
73

Treatment 5ALA fertilizer

1000 grain
weight (g)Grain yield(kg/0.1ha)

Untreated(control)
Treated
Untreated(control)
Treated22.2.0+0.5

506.6

Proteincontent (%)
6.1

22.0+0.5
22.3+0.3

627.5
583.3

5.9
6.0

18.7
18.9

74
74

23.7+0.5

728.8

6.0

19.0

75

1.means+SE
2.quality of grains is determined by Satake rice*grain paratability tester

Table 4. Effects of 5-ALA fertilizer sprayed by the helicopter on the percentage of ripened grains in cv.
Koshihikari
Treatment
5-ALA
fertilizer
Untreated (control)
Treated1000 grains weight(g)
21.2+0.3a
20.8+0.2a

Percentage of ripened grains (%)
Superior grains inferior grams
83.5+3.9a
68.7+5.1b86.2+5.0a
79.2+4.3aGrain yield(kg/0.1ha)
670.0
696.2

1.Data are means+SE of 150 panicles
2. Different letters within columns indicate significant differences by Tukey‘s multiple range test (p=0.05)

References
Hotta Y., T. Tanaka, H. Takaoka, Y. Takeuchi and M. Konnai. 1997. Promotive effects of 5-aminolevulinic acidon the yield of several crops. Plant Growth Regulation. 22:109-114.
Tanaka T.･1995.Application and production of 5-aminolevlinic acid by photosynthetic fungi, Rhodobacter
sphaeroides. Doctor thesis (Hroshima University) . 5-138.
ndYoshida R., Y. Hotta, T. Tanaka, Y. Takeuchi and M. Konnai, 1996. Agricultural use of 5-aminolevulinic acid 2International Crop Science Congress. Abstracts 392. New Delhi.
Yoshida R., T. Tanaka and Y. Hotta. 1996. Regulation of fractan accumulation in rakkyo (Allium Bakeri) andshallot (A. ascalonicum) by 5-aminolevulinic acid. Proceedings of Plant Growth Regulator Society ofAmerica. 177-182.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201179

Yield Stability Evaluation of Upland Rice Lines Obtained
from Anther Culture
Deni D Sulaemana, Bambang S Purwokob*, Iswari S Dewic, Muhamad Syukurb, Desta
Wirnasb, Heni SafitridaGraduate Student of Plant Breeding and Biotechnology Major, the Graduate School of Bogor Agriculturalb
University, Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural Universit,cIndonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor,dIndonesian Center for Rice Research, Bogor*Corresponding author: Jln Meranti, Darmaga, Bogor 16680 Indonesia.
Tel: +62 251 8629353.
E-mail address: bambangpurwoko@gmail.com

Abstract
The objective of this research was to study the adaptation and yield stability of upland rice lines obtained fromanther culture. Ten upland rice doubled-haploid (DH) lines were tested for their potential yield in eight different
locations (in Provinces of Lampung, West Java, Central Java, Yogyakarta, East Java - Indonesia) in the rainyseason of 2010/2011 along with two check varieties (Way Rarem and Batutegi). In each location, the design was
Randomized Complete Block Design with four replicates. The method of Francis & Kannenberg, Finlay &Wilkinson, Eberhart & RusseIl and AMMI (Additive Main Effect Multiplicative Interaction) were used to analyze
the adaptation and yield stability of the tested DH lines. The results indicated that the line showing the moststable yield in different environment was I5-10-1-1 followed by WI-44, and IG-38. I5-10-1-1 produced 4.01 tonsof dry grain per hectare. The line showing the highest yield was WI-44, and this line produced 4.72 tons of dry
grain per hectare. Visualization with AMMI showed that IW-56 and IW-67 lines were specifically adapted in
Purworejo, O18-b-1 is specifically adapted in Bogor, and IG-19 is specifically adapted in Malang, respectively.
Keywords: upland rice, DH lines, yield stability

Introduction
Efforts to increase the food production, especially rice, through the utilization of dry land
can be reached by breeding upland rice, since the high-yielding upland rice varieties may be
accepted by farmers. Anther culture has been reported to produce doubled-haploid (DH) plants orpure lines in a short time (Dewi et al., 1996). On the contrary, conventional breeding will require along period of time (more than 5 years) in obtaining pure lines as a result of combining the desirable
traits of different varieties or parents. Through anther culture of F1, several number of upland rice
DH lines were obtained from previous experiment, i.e. IW-56, IW-67, IG-19, IG-38, and GI-8 lines
tolerant to low light intensity or shade (Sasmita et al., 2006); III3-4-6-1 and I5-10-1-1 lines tolerant
to aluminum toxicity (Herathwati et al., 2009); while O18-b-1 and B13-2e lines tolerant to bothshade and Al toxicity (Purwoko, 2007). Those DH lines need to be evaluated further.
Subandi (1981) asserted that in the establishment of superior varieties breeders need to
pay attention to the yield stability in a systematic and continuous manner, starting from theformation of the basic population to test varieties. Adaptability evaluation in different growing
environments need to be done because of the diversity of land, soil, cultivation, cropping patterns
and planting season in Indonesia. The diversity of the growing environment will affect the yield perunit area, because the plant growth is a function of genotype and environment (Allard, 1960).
Appearance of plants depends on the genotype and environmental conditions where the plant grew,as well as the interaction between the two. Yield stability evaluation through a series of multi
location test is an important stage before the varieties can be released. Therefore, this research

80thProceedings of The 7 ACSA Conference 2011was aimed to study the adaptation and yield stability of upland rice DH lines obtained from antherculture in 8 different environments.

Materials and Methods
The yield stability test of upland rice was held at eight locations in the rainy season during
October 2010 until April 2011. Testing locations spread across Java and Sumatra, namely: theTaman Bogo - Lampung, Natar - Lampung, Cikarawang Bogor - West Java, Sukabumi - West Java,
Indramayu - West Java, Purworejo - Central Java, Wonosari - Gunung Kidul, and Malang - East
Java. A total of 12 genotypes were used as test material, which consisted of 10 upland rice DH lines
obtained from anther culture, i.e. III3-4-6-1, I5-10-1-1, WI-44, GI-7, O18-b-1, IW-67, IG-19, IG-38,
IW 56, and B13-2e, while Batutegi and Way Rarem.were two check varieties used for comparison.
The experimental design was Randomized Complete Block Design with four replicates. An
experimental unit consisted of 4 x 5 m2 plot. Each of the tested genotypes were planted with aspacing of 30 x 15 cm. Observations and data analysis were conducted on dry grain yield per
hectare. The method of Francis & Kannenberg, Finlay & Wilkinson, Eberhart & RusseIl and AMMI
(Additive Main Effect Multiplicative Interaction) were used to analyze the adaptation and yieldstability of the tested DH lines.

Results and Discussion
The variance analysis of pooled data at eight test locations showed the occurrence ofinteraction between lines (genotype) and environment (G × E) (Table 1). The existence of suchinteractions will cause each lines responded differently to the environment in which testing was
conducted. The response was mainly shown by the fluctuations in grain yield. A genotypes orvarieties though, will not always produce the same great results if planted in different environments.
This is caused by the large diversity of the macro geophysical environment that will provide thelarge environmental diversity for plant growth as well (Satoto et al, 2009).
Table 1. Analysis of variance of pooled data at 8 locations
Source of Variation

df

SS

MS

F value

Location

7

123.18

17.60

30.68

**

Rep/Location

24

66.29

2.76

4.82

**

Lines

11

123.48

11.23

19.57

**

Lines × Location

77

230.93

3.00

5.23

**

Error

264

151.41

0.57

Total

383

695.30Notes : ** very significantly different

The average of dry grain yield of WI-44 is able to outperform other lines and checkvarieties at two locations, namely Bogor and Malang (Table 2). The average dry grain yield showedthat only the WI-44 and IW-67 was able to outperform Batutegi varieties, while Way Rarem varieties
only surpassed by WI-44. The difference in average yields between WI-44 with Batutegi was 0.34
tons/ha, where WI-44 was about 7.67% higher than Batutegi. The difference in average yield
between IW-56 with Batutegi was 0.01 tons/ha or approximately IW-56 was only 0.2% higher thanthProceedings of The 7 ACSA Conference 201181

Batutegi. However, the difference in average yields between WI-44 with Way Rarem was 0.1
tons/ha or WI-44 approximately 17.2% higher than Way Rarem.
Tabel 2. The average dry grain yield (tons/ha) of upland rice DH lines in every test location

Wonosari
2.39

Purworejo
4.88

LOCATION
SukaBogor
bumi
2.59
1.52

Malang

III3-4-6-1

Indramayu
2.95

I5-10-1-1

2.88

3.44

4.62

4.70

4.81

WI-44

3.24

3.74

5.55

5.69

GI-7

2.72

2.60

2.53

O18-b-1

2.17

2.41

IW-67

3.14

IG-19

1.86

IG-38

DH Lines

Yield
Average(tons/ha)

3.11

Lampung
2.79

Taman
Bogo
3.36

4.11

2.99

4.51

4.01

5.40

5.98

4.15

4.02

4.72

3.73

5.16

5.10

2.68

3.69

3.52

3.19

3.45

5.04

3.58

2.68

2.52

3.13

4.35

6.94

5.08

4.14

3.90

3.76

3.85

4.39

3.61

2.66

3.40

3.79

4.32

2.26

4.48

3.30

2.06

3.38

3.28

3.70

2.89

4.51

2.31

4.13

3.28

IW-56

2.51

4.39

6.81

4.14

2.69

3.25

3.53

3.21

3.82

B13-2e

2.91

2.52

3.54

4.07

4.22

4.66

4.00

4.41

3.79

Batutegi

3.01

3.14

5.89

4.16

3.04

5.63

4.66

5.54

4.38

Way Rarem

3.79

3.87

4.35

6.95

3.46

5.33

4.99

4.24

4.62

Average

2.77

3.32

4.52

4.30

3.85

4.46

3.40

4.00

3.81

CV (%)

24.0

23.2

20.6

15.6

23.1

10.6

12.6

25.7

2.95

The parameters of stability of dry grain yield of upland rice DH lines from eight locations
are presented in Table 3. Lin et al.(1986) suggested three concepts of stability. A genotype isconsidered to be stable if: (1) the variance among environment is small, (2) the response to its
environment is parallel to the mean response of all genotypes in the trial, (3) residual mean square(MS) from the regression model on the environmental index is small.
Tabel 3. Parameters of stability of dry grain yield of upland rice DH lines from eight locations
2

δ2

Ri

*

1.04

0.31

ns

0.73

0.64

1.52

*

1.25

0.43

31.16

0.77

*

1.40

0.29

29.55

0.76

*

0.98

0.37

1.00

26.56

1.31

*

1.58

0.32

3.30

1.23

28.85

0.84

ns

1.05

0.37

IG-38

3.28

1.10

25.70

1.02

ns

0.82

0.47

IW-56

3.82

1.38

35.92

1.18

ns

2.19

0.20

B13-2e

3.79

1.04

19.71

0.78

*

0.65

0.67

Batutegi

4.22

1.20

27.98

1.17

ns

1.75

0.29

Way Rarem

4.62

1.15

24.42

0.90

ns

1.47

0.35

Rata-rata

3.81

DH lines

Yield (t/ha)

SDi

CVi

bi

III3-4-6-1

2.95

1.09

32.51

0.64

I5-10-1-1

4.01

0.74

19.74

1.10

WI-44

4.72

1.02

22.17

GI-7

3.53

1.21

O18-b-1

3.13

1.01

IW-67

4.39

IG-19

1.00

Notes : SDi= Standard deviation of genotype; CVi=Coeficient of variance of genotype;, bi= Coefficient of regression of
genotype, value of bi * (significantly different with 1), ns (not significantly different with 1); δ2=parameter of deviation;,
Ri2=Coefficient of determination.

82

thProceedings of The 7 ACSA Conference 2011

Francis & Kanennberg (1978)
Francis and Kanennberg (1978) measure the stability by using the coefficient of variability(% CVI) of each genotype tested in multiple environments. The small values of its coefficient ofgenotype diversity meant that the genotype is more stable. Moedjiono and Mejaya (1994)
categorized the value of the coefficient of genotype diversity in four groups, namely low (<25%),
rather low (25 -50%), rather high (50-75%), and high (75-100%). Therefore, based on those criteria
the DH lines tested in this study fell into of low and rather low groups. I5-10-1-1, WI-44, B13-2e, and
Way Rarem have a low coefficient of genotype diversity, thus classified as stable.
Finlay and Wilkinson (1963)
Stability analysis of Finlay and Wilkinson (1963) is a method of measurement stability thatis based on the regression coefficient (bi) between the average yields of a genotype with a general
average of all genotypes tested in all test environments. This analysis can explain the phenomenonof stability and adaptability of a genotype. Finlay and Wilkinson categorize standard bi values ofstability in the three groups, namely (1) the stability is below the average, if the value of bi > 1, (2)stability is equivalent the average, if the value of bi = 1, (3) stability is above the average, if the valueof bi <1.
Based on those criteria, Batutegi and Way Rarem and also three DH lines had bi values
not significantly different from 1, i.e. I5-10-1-1, IG-19, IG-38, and IW-56. Those DH lines werecategorized as a stable line, which meant the lines will be able to adapt to the large environment.
Lines with the stability below the average were WI-44 and IW-67 with the bi of 1.52 and 1.31, while
the lines with the stability above the average were III3-4-6-1, GI-7, O18-b-1, and B13-2e with bivalues of 0.64, 0.77, 0.76, and 0.78, respectively. Lines that have below-average stability aresensitive to environmental changes and adapt to particular environmental benefit (favorable). Thelines which have above average stability are generally able to adapt to marginal environments.
Eberhard dan Russel (1966)
Stability analysis of Eberhard and Russell (1966) is a measure of stability based on thedeviation from the regression of the average value of genotype on the environmental index. A
genotype is stable if residual mean square (MS) from the regression model on the environmental
index is small. Parameter stability seen from the deviation value (δ2) and coefficient of determination(Ri2) of the tested genotypes. Model stability of a genotype is good if it has little δ 2 value and large
Ri2 value (approaching 1).
Based on these parameters, two DH lines namely B13-2e and I5-10-1-1 had the smallest
value of δ2, i.e. 0.65 and 0.73 (Table 3). Coefficient of determination (Ri2) from these two lines also
had the largest value among lines and other varieties, i.e. 0.67 and 0.64. Therefore, the regression
model used to estimate the stability of those two lines are better than the other lines.
AMMI (Additive Main Effect Multiplicative Interaction)
Due to limited visualization of graphs that are only capable of displaying two-dimensionalgraph, then the model described in this paper is AMMI2. AMMI2 model can explain the diversity of
the interaction effect up to 70.98%. Bi-plot of interaction affects AMMI2 model for the dry grain yieldof upland rice DH lines are presented in Figure 1. The bi-plot of principal components 1 and 2 canexplain the main components of lines which are stable at all location test or specific to a particularlocation. Mattjik and Sumertajaya (2000) states that a line or genotype is said to be stable whenclose to the axis or point (0,0). The lines or genotypes that are far away from the axis but close to
the line location, then the lines were classified as specific location lines. Based on these, lines thatare stable at all location test are I5-10-1-1, WI-44, IG-38, and Way Rarem. IW-56 and IW-67 were
specific to the location of Purworejo, O18b-1 line-specific for the location of Bogor. IG-19 is specificto the location of Malang.

thProceedings of The 7 ACSA Conference 201183

Figure 1. Bi-plot of interaction effects AMMI2 model for the dry grain yield of upland rice DH lines derived fromanther culture

Conclusions
The DH line showing the most stable yield in different environment was I5-10-1-1 followed
by WI-44, and IG-38. Visualization with AMMI showed that IW-56 and IW-67 lines were specifically
adapted in Purworejo, O18-b-1 was specifically adapted in Bogor, and IG-19 was specifically
adapted in Malang.

References
Allard RW. 1960. Principles of Plant Breeding. J Wiley and Sons. New York. 485 p.
Dewi IS, Hanarida I, Rianawati S. 1996. Anther culture and its application for rice improvement program inIndonesia. Indon. Agric. Res. Dev. J. (18) : 51-56.
Eberhart SA, Russel WA. 1966. Stability parameters for comparing varieties. Crop Sci. 6:36-40.
Finlay KW, Wilkinson GN. 1963. The analysis of adaptation in a plant-breeding programme. Aust. J. Agric. Res
14:742-754.
Francis TR, Kannenberg LW. 1978. Yield stability studies in short-season maize. A descript method for grouping
genotypes. Can. J. Plant Sci. 58:1029-1034.
Herawati R, Purwoko BS, Dewi IS. 2009. Keragaman genetik dan karakter agronomi galur haploid ganda padi
gogo dengan sifat-sifat tipe baru hasil kultur antera. J.Agron. Indonesia 37(2) : 87-94.
Lin CS, Binns MR, Levkovitch LP. 1986. Stability analisis : Where do we stand?. Crop Science. Vol. 26 : 894900.
Mudjiono, Mejaya MJ. 1994. Variabilitas genetik beberapa karakter plasma nutfah jagung koleksi Balittas
Malang. Zuriat 5(2) : 27-32.
Purwoko BS. 2007. Aplikasi kultur antera dan marka RAPD dalam perakitan padi gogo toleran cekaman
lingkungan. Laporan Riset Unggulan Terpadu Bidang Pertanian dan Pangan.
Sasmita P, Purwoko BS, Sujiprihati S, Hanarida I, Dewi IS, Chozin MA. 2006. Evaluasi pertumbuhan dan
produksi padi gogo haploid ganda toleran naungan dalam sistem tumpang sari. Bul. Agron. (34) (2) 7986.
Satoto, Utomo STW, Widyastuti Y, Rumanti IA. 2009. Usulan Pelepasan Varietas Padi Hibrida H45, H47, H64,
H68 dan H78. Makalah pada Sidang Pelepasan Varietas, Desember 2009.
Subandi, 1981. Genotype x environment interactions in corn variety test. Food Crop 65:1-9.

-- back to Table of Content --84

thProceedings of The 7 ACSA Conference 2011

Yield and Blast Resistance Evaluation of Upland Rice Lines
with New Plant Type Characters
Jose MA Ornaia , Bambang S Purwokob*, Desta Wirnasb, Iswari S Dewic, Heni SafitridaGraduate Student, Plant Breeding and Biotechnology Major, the Graduate School,
Bogor Agricultural Universityb
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural UniversitycIndonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogord
Indonesian Center for Rice Research, Bogor* Corresponding author: Jln. Meranti, Darmaga, Bogor 16680 Indonesia.
Tel/ Fax: +62 251 8629353.
bambangpurwoko@gmail.com

Abstract
The present studies were conducted to determine agronomic characters, genetic variability, broad sense2
heritability (h bs) and blast resistance of doubled haploid rice lines and to obtain new plant type (NPT) of upland
rice with high yield potential and blast resistant. The research was conducted in Bogor and Sukabumi, Indonesia
started from November 2010 to June 2011. There were 14 genotypes evaluated namely the new lines of ricefrom anther culture (FG1-66-2-1, FG1R-108-1-1, FG1R-51-2-1, FM1-14-1-1, FM1-14-1-2, FM1-25-1-1, FM1-251-2, FM1-57-1-2, FM1R-32-1-1, FM1R-23-1-1, FG2-47-1-3, FM1R-19-2-4, FM2-12-1-1, FAT-4-1-2), as well as
Fatmawati, Limboto and Batutegi as check varieties. The experimental design used was randomized block
design (RBD). The results showed that there was variability on agronomic traits and blast resistance amonglines evaluated. FG2-47-1-3 did not have complete characters of NPT, but the line had superior characteristics
such as high percentage of filled grain, medium days to harvest, medium height, medium 1000-filled grain
weight, stay green when harvested and high resistance to blast disease. The results also indicated that the2tested population had high heritability (h bs) in leaf blast and neck blast resistance, plant height, filled grain per
panicle, days to harvest, number of grain per panicle and 1000-filled grain weight. They showed high variancecoefficient for leaf blast and neck blast resistance, flag leaf length and percentage of filled grain.
Keywords: new plant type, upland rice, blast resistance, heritability.

Introduction
Rice is staple food in Indonesia. Population growth will increase national rice consumption(Prasetyo 2008). In the year of 2000-2006, population growth rate was 1.36% with per capita
consumption of rice at 137 kg. It is estimated that in 2010, 2015 and 2020 consumption of rice anda population with declining population growth rate assumption of 0.03%, respectively was 32.13
million tons with a population of 235 million, 34.12 million tons with a population of 249 and 35.97
million tons with population of 263 million inhabitants (Puslitbangtan 2007).
Increase in national rice production must be continually pursued. One of the effort toincrease national rice production is the use of dry land to grow upland rice so as to support national
food security (Puslitbangtan 2007). Indonesia has 11.6 million ha of dry land (BPS 2005). Dry land
used for planting upland rice in 2010 reached 1.13 million ha (Deptan 2011), so there are still
opportunities to expand the area of upland rice cropping. Upland rice production reached 3.45
million tons with yield of 3.05 tons/ha (Deptan 2011). Contribution of upland rice to the national riceproduction is still low at 5% (Prasetyo 2008).
Increased productivity of upland rice can be done by developing a new plant type (NPT) of
upland rice lines. NPT ideotype characters desired, among others, are plant height <150 cm,
number of productive tillers > 6, number of grain per panicle > 150, short growth period (105-124
days), good filled grain (> 75%), 1000-filled grain weight more than 28 g (Herawati 2010).
thProceedings of The 7 ACSA Conference 201185

Based on previous research, rice lines were obtained from crosses of a new plat type oflowland rice Famawati with local upland rice varieties from the island of Buru namely Fulan Telo
Gawa (FTG) and Fulan Telo Mihat (FTM) through anther culture (Safitri 2010). Lines need to befurther evaluated both morphological and agronomic characters/ performance and resistance toblast disease.
Blast disease is caused by fungus Pyricularia grisea. The disease can reduce theproductivity of upland rice between 11-50% (Beker at al. 1997; Scardaci et al. 1997). Fungus
Pyricularia grisea infects leaves at vegetative stage, called leaf blast, while in the generative stage,
the pathogen also infects the panicle neck, called neck blast (BB Biogen 2009).
The present studies were conducted to obtain information on performance of agronomic
characters and resistance to leaf blast and neck blast in the new rice lines. In addition, this study
was also aimed to select a new plant type of upland rice lines with high yield and resistant to blast
disease.

Materials and Methods
The research was conducted in Babakan Experiment Station, University Farm, Bogor
Agricultural University and in the village of Bojong, Cikembar Sub-District, Sukabumi District, WestJawa, Indonesia (endemic blast region) from November 2010 to April 2011. The genetic materials
were 15 doubled haploid upland rice lines obtained from anther culture of F1, crosses between
Fatmawati and Fulan Telo Mihat or Fulan Telo Gawa (Safitri 2010). Fatmawati, Batutegi and
Limboto were used as a check varieties.
This study used a randomized blocked design with lines as treatment and consisted of 3
replicates. Experimental unit in Bogor was done in the form of a plot of 3 x 3.6 m. Three seeds
were planted per hill with spacing of 30 x 20 cm. In Sukabumi, each replicate was 10 x 2 m. One
seed was planted per hill with spacing of 20 x 10 cm. Fertilizer use were 10 tons of manure/ha, 200
kg of Urea/ ha (given: 20% age 1 week, 40% at age 4 weeks and 7 weeks), 100 kg/ha of KCl and
100 kg/ha of SP36. Maintenance was performed in accordance with the conditions and crop needsin the field.
Observations were made on 5 plants per plot on variables: plant height, number of
productive tillers, days to harvest, total grain per panicle, percentage of filled grain, 1000-filled grain
weight, productivity, the scale and intensity of leaf blast and neck blast disease. The data obtained
were analyzed by F test, if significantly different then followed by Dunnett test on a level ofsignificance 5%, range estimation and analysis component of the correlation between characters.
The intensity of leaf blast disease was calculated with the formula: I = Σ (n x v) / (N x V)
x100%, where I = intensity of disease, n = number of plants infected, v = scale of plant infected, N =total number of plant observed, V = the highest scale of blast disease (9) (IRRI 1996). Resistance toblast disease were classified into resistant, if I ≤ 10% and susceptible, if I> 10%. Observation of thepanicle neck blast was performed at harvest which includes the scale and intensity of the disease.
The intensity was calculated by: I = Σ n / N x 100%, where I = intensity of blast disease, n = numberof panicles infected, N = number of panicles observed. The scale of the disease was determinedbased on the evaluation system on panicle neck blast disease from IRRI (1996) as follows: 0 = nosymptom, 1 = infection of less than 5%, 3 = 5-10% infected, 5 = 11-25% infected, 7 = 26-50%
infected, 9 = more than 50% infected. Panicle neck blast resistance were classified into resistant(score 0-1), moderately resistant (score 3-4), moderately susceptible (score 5-6) and susceptible(score 7-9).

86thProceedings of The 7 ACSA Conference 2011

Results and Discussion
The variance analysis showed that the genotype effect was significant on all charactersobserved. This indicated that there were differences between doubled haploid lines of upland rice.
The coefficient of variability (CV) ranged between 2.5 and 43.6% (Table 1). Plant height ranged
from 73.8 to 124.0 cm (Table 1). Higher plant meets the criteria as a new plant type of upland ricelines. According to Herawati (2010) and Abdullah et al. (2005), height of new plant type of upland
rice is under 150 cm and for a new plant type of lowland rice is 80-100 cm. The height of some lines
tested (FG1R-108-1-1, FG1R-51-2-1, FM1-14-1-1, FM1-57-1-2-32-FM1R 1-1 FG2-47-1-3) weresignificantly different from and were higher than Fatmawati (Table 1). Nonetheless, plant height stillmeets the general criteria for the plant height of new types of upland rice lines.
The number of productive tillers of 14 lines tested ranged from 6.5-15.5. According to Peng
et al. (2008), the number of productive tillers are ideal for a new pant type of rice (NPT) is 10-15 forlowland rice, while according to Herawati (2010) the number of productive tillers for a new plant typeof upland rice lines is > 6. Fatmawati has the potential to produce 6-14 tillers. The number of tillers
Fatmawati in upland conditions conducted in this study was 13.
Table 1. Agronomic performance of doubled haploid upland rice lines
Lines
FG1-66-2-1
FG1R-108-1-1
FG1R-51-2-1
FM1-14-1-1
FM1-14-1-2
FM1-25-1-1
FM1-25-1-2
FM1-57-1-2
FM1R-32-1-1
FM1R-23-1-1
FG2-47-1-3
FM1R-19-2-4
FM2-12-1-1
FAT-4-1-2
Fatmawati(a)Batutegi (e)CV (%)

PH
91.2
a+
124.0
a+
116.6
a+
102.7
97.6
90.4
93.2
a+
112.1
a+
118.7
77.4
a+
110.3
73.8
94.0
79.1
84.6
118.6
6.6

NPT
11.8
a6.5
7.9
8.3
10.4
8.4
8.1
11.3
10.7
11.3
15.5
13.3
11.2
11.1
13.0
10.7
12.5

DH
127.7
130.3
130.0
126.7
128.3
a+
134.3
a+
134.7
a+
134.3
130.0
a+
131.3
128.0
128.3
122.0
128.3
123.3
126.0
2.5

TS
177.6
e137.4154.0
e114.7e100.5e122.3e115.1e133.6e128.7e115.8e118.4
e69.0
e61.3
e112.7
102.6
213.3
9.9

FG
59.4
e11.6
e7.5
e18.4
e13.0
e5.4
e15.0
e29.5
46.4
e2.1
80.1
e2.5
e22.3
40.0
11.5
114.2
43.6

PFG

B1000

33.3
17.1
e4.5
15.0
e11.7
e3.9
2.1
e8.3
37.1
e2.3
68.3
e32.8
34.8
34.8
10.0
53.8
40.0

e+

29.7
26.3
e+
29.3
e+
30.3
e+
29.3
e+
29.0
25.3
e+
31.0
e+
33.3
25.3
25.3
25.7
e+
23.3
28.3
25.7
26.3
3.4

YP
0.52
e0.24
e0.08
0.41
e0.26
e0.12
0.65
1.48
1.06
e0.08
2.00
e0.04
0.31
0.72
0.31
1.88
26.3

Note: Numbers followed by the letters a+ and a- column differ significantly higher and differ or lower with Fatmawati. Numbers
and e+ e- is significantly different higher or lower than Batutegi based on the Dunnett test at significant level of 5%. PH =plant height, NPT = Number of productive tillers, DH = days to harvest. TS = Total spikelet, FG = number of filled grain,
PFG = Percentage of filled grain, B1000 = 1000-filled grain weight, YP = Yield potential (tons/ha), CV = coefficient ofvariability.

According to Herawati (2010), the days to harvest of new plant type of upland rice ranged
from 105 to 124 days. Days to harvest of the lines tested ranged between 122-134.3 days. The linethat meet the criteria was FM2-12-1-1. Fatmawati variety showed 123.3 days to harvest. Some of
the lines tested (FM2-25-1-1, FM1-25-1-2, FM1-57-1-2, FM1R-23-1-1) had significantly differentdays to harvest and were longer than that of Fatmawati.
Batutegi was a variety with the highest number of spikelet per panicle (213.3 spikelets), but
only had percentage of filled grain of 53.8 percent. The number of spikelet per panicle of the lines
tested ranged between 61.3-177. Lines with the highest total number of spikelets was FG1-66-2-1(177), but only 59.4 filled grain per panicle, that was 33.3%. Despite of only having a total number of
spikelet 118.4, FG2-47-1-3 has the highest percentage of filled grain. According to Herawati (2010)
the number of spikelet per panicle of NPT upland rice is more than 150 per panicle. Based on thenumber of spikelet per panicle, only FM2-12-1-1 and FG1R-51-2-1 meets the criteria. However,they had low percentage of filled grain. Weight of 1000 grain of rice ranged from 23.3 to 33.3 g

thProceedings of The 7 ACSA Conference 2011

87

(Table 1). Variety has comparable weight of 1000 grain, ranging from 25.7 and 26.3 g. Of the 15lines tested, 8 lines have weight of 1000 grain had more than 28 g (Herawati 2010) (Table 1).
Yield of 14 lines tested ranged from 0.04 to 2.00 tons/ha. Yield of varieties were 0.31 and1.88 tons/ha. Batutegi had the highest yield among the check varieties (1.88 tons/ha). There were 3
lines having yield of more than one ton/ha i.e. FG2-47-1-3 (2.00 tons), FM1-57-1-2 (1.48 tons) andFM1R-32-1-1 (1.06 tons) (Table 1). The low yield of the lines and check varieties was due to blastand bacterial leaf blight and scarce rain during the reproductive period.
Based on the criteria of CGV, then there are two characters that have relatively high CGV,
namely plant height and intensity of neck blast disease; two characters have the medium CGV i.e.
the number of grain per panicle and the percentage of filled grain and number of filled grain per
panicle; and several other characters have low CGV. Murdaningsih et al. (1990) stated that low
CGV showed rather low genetic variability of the characters and was classified as narrow, while themedium and high value CGV were classed as broad genetic variability. Thus there were 4
characters that have broad genetic variability, and six have a narrow genetic variability.
Broad sense heritability value of the characters observed ranged from 33.4-91.2% (Table
2). According to Stanfield (1983), broad sense heritability values can be grouped into three highheritability (0.50 <h2 <1.00), medium heritability (0.20 < h2 ≤ 0.50) and low heritability (h2 <0.20).
Based on the heritability values there are two characters that have moderate heritability (20-50%),
i.e. the number of productive tillers and yield potential. another character has a heritability of 50%(Table 3). This means that agronomic characters were controlled by genetic factors more thanenvironmental factors. Selection will be effectively carried out for characters with high heritability
value.
Table 2. Variance components, heritability and genetic variability coefficient new plant type of upland rice lines
Characters
1. Plant height
2. Days to harvest
3. Number of productive tillers
4. Number of filled grain per panicle
5. Number of spikelet per panicle
6. Percentage of filled grain
7. The 1000-filled grain weight
8. Yield
9. Intensity of leaf blast disease
10. Intensity of neck blast disease

2

ζg
315.9
18.8
0.1
5.6
3.0
3.7
9.0
0.04
3.4
12.4

2

ζp
357.4
29.1
0.3
9.7
4.1
6.8
9.9
0.1
3.6
13.2

2

2

ζe
41.4
10.4
0.2
4.1
1.1
3.1
0.9
0.1
0.5
2.2

h bs(%)
88.4
64.4
44.3
57.5
73.0
55.0
91.2
33.4
95.7
94.4

CGV (%)
17.0
3.8
2.0
10.0
5.3
9.2
5.7
1.9
8.4
15.0

Description: ζ2 g =Genetic variance, ζ2 p =Phenotypic variance , h2 bs = broad sense heritability, CGV = Coefficient of genetic
variance .

Line FM2-12-1-2 was blast-resistant but only had yield potential of 0.31 ton per ha. Theline was not tolerant to drought. FG2-47-1-3 line was resistant to leaf blast and neck blast and had
the highest number of productive tillers than all other varieties and lines tested, thus the yield of
FG2-47-13 was also the highest (2 tons/ha). There were ten other lines having yield less than one
ton. Low yield tons was due to low resistance of the lines to leaf blast and neck blast and drought
during reproductive period (Table 3 Location in Bogor).
Based on Dunnett tests the intensity of leaf blast disease in the endemic area in
Sukabumi, of 15 lines tested, there were two lines showing intensity of leaf blast disease notsignificantly different from control resistant (Limboto), namely FG2-47-1-3 and FM2-33-1-1 (Table3). Panicle blast resistance of the 15 lines tested showed that the lines with intensity of neck blast
disease not different from controls resistant (Limboto) were FM1-25-1-1, FM1-25-1-2, FG2-47-1 -3,
FM2-12-1-1, and FAT-4-1-2 (Table 3). Line showing both resistance to leaf blast and neck blast was
FG2-47-13.

88

thProceedings of The 7 ACSA Conference 2011

Table 3. The scale and intensity of leaf blast and neck blast in lines of upland rice in Bogor and Sukabumi
Lines

Bogor
Leaf Blast
S
I (%)
aR
5
17.4
aR
5
12.6
aR
5
38.5
aR
7
57.8
aR
7
84.8
T
3
5.6
T
1
2.9
T
3
4.1
a
5
45.2
a
7
50.0
T
0
0.0
a
7
71.5
T
1
1.1
aR
5
20.0
T
0
0.0

FG1-66-2-1
FG1R-108-1-1
FG1R-51-2-1
FM1-14-1-1
FM1-14-1-2
FM1-25-1-1
FM1-25-1-2
FM1-57-1-2
FM1R-32-1-1
FM1R-23-1-1
FG2-47-1-3
FM1R-19-2-4
FM2-12-1-1
FAT-4-1-2
FM2-33-1-1
Limboto (a)Neck Blast
S
I (%)
aR
9
86.3
aR
9
63.3
aR
9
85.0
aR
9
97.7
aR
9
94.7
aR
9
53.0
aR
9
73.7
R
5
14.7
aR
9
64.7
aR
9
80.7
T
1
3.0
R
7
32.7
MT
3
6.0
R
7
49.7
T
1
3.7

Sukabumi
Leaf Blast
Neck Blast
S
I (%)
S
I (%)
aR
aR
7
38.0
9
74.1
aR
R
5
23.4
5
23.5
aR
aR
5
25.4
7
35.0
aR
aR
7
55.5
9
87.8
aR
aR
7
44.8
9
100.0
aR
R
5
16.2
5
16.8
aR
R
5
19.5
5
13.2
aR
aR
7
53.2
9
64.4
aR
R
5
28.7
7
28.9
aR
aR
5
29.6
9
88.4
T
MT
3
7.8
3
5.5
aR
aR
9
62.6
9
96.3
aR
R
9
66.8
5
18.4
aR
T
7
34.1
1
2.3
T
aR
3
7.5
9
77.5
T
T
3
4.8
1
0.0

Note: Figures followed by letter (a) showed higher intensity of blast disease than Limboto (resistant variety), based on the
Dunnett test on a significant level of 5%. I = intensity of the blast disease, T = resistant, R = Susceptible, MT =Moderate resistant, MR = Moderate susceptible.

Conclusions
There was variability on agronomic traits and blast resistance among the lines evaluated.
FG2-47-1-3 did not have complete characters of NPT, but the line had superior characteristics suchas high percentage of filled grain, medium days to harvest, medium height, medium 1000-filled
grain weight, stay green when harvested and high resistance to blast disease. The population had
high heritability (h2bs) in leaf blast and neck blast resistance, plant height, filled grain per panicle,
days to harvest, number of grain per panicle and 1000-filled grain weight. They showed highvariance coefficient for leaf blast and neck blast resistance, flag leaf length and percentage of filled
grain.

References
Abdullah B, Tjokrowidjojo S, Justianto B, Darajat AA. 2005. Pembentukan padi varietas unggul tipe baru.
Penelitian Pertanian 24(1): 1-7.
[BB Padi] Balai Besar Penelitian Tanaman Padi (2009) Blas Padi. [Tersedia Berkala].
http://bbpadi.litbang.deptan.go.id [November 2009]
Beker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP. 1997. Signalling in plant-microbe interactions. J Sci
276:726-733.
[BPS]. Biro Pusat Statistik. 2005. Statistik Indonesia. 4 hlm.
[Deptan RI] Basis Data Statistik Pertanian 2011. [Tersedia Berkala]. http://www.bps.go.id/tnmn_pgn.php [11Agustus 2011]. 1 hlm.
Herawati R. 2010. Pembentukan padi gogo tipe baru toleran aluminium dan tahan blas melalui kultur antera(Disertasi). 162 hlm.
IRRI. 1996. Standard Evaluation System for Rice. International Rice Research Institute, Los Banos, Philippines.
PO. Box. 933, Manila.
Moedjiono, Mejaya MJ. 1994. Variabilitas genetik beberapa karakter plasma nutfah jagung koleksi Balittas
Malang. Zuriat 5(2):27-32.
Murdaningsih HK, Baihaki A, Satari G, Danakusuma T, Permadi AH. 1990. Variasi genetik tanaman padi sawahdi Indonesia. Zuriat 1(1): 32-36.
Peng S, Khush GS, Virk P, Tang Q, Zou Y. 2008. Progress in idiotype breeding to increase rice yield potential.
Review. Field Crops Research 108:32-38.
Prasetyo YT. 2008. Bertanam Padi Gogo Tanpa Olah Tanah. Bibliografi. 69 hlm.

thProceedings of The 7 ACSA Conference 201189

Puslitbangtan. 2007. Peningkatan Produksi Padi Menuju 2020. Puslitbang Tanaman Pangan. 7 hlm.
Safitri H. 2010. Kultur antera dan evaluasi haploid ganda untuk mendapatkan padi gogo tipe baru (Tesis).
61hlm.
Scardaci SC et al. 1997. Rice Blast: a new disease in California. J Agr Fact Sheet Ser 1:2-5.
ndStanfield WD. 1983. Theory and Problems of Genetics, 2 edition. Schain‘s Outline Series. Mc. Grow Hill Book
Co. New Delhi. 304p.

-- back to Table of Content --90thProceedings of The 7 ACSA Conference 2011

Functional Analysis of Drought-Induced OsLEA3 Promoter
Isolated from Batutegi Rice Cultivar
Agus Rachmat1, Satya Nugroho1, Bernadetta Rina Hastilestari1,
Kinasih Prayuni1, Sudarsono2
1.

Research Center For Biotechnology, Indonesian Institute of Sciences
2
Faculty of Agriculture, Bogor Agricultural University
guess_btk@yahoo.com

Abstract
Drought is the most significant environmental stress in agriculture and many efforts have been made to improve
crop productivity under water-limiting conditions. Improving yield under drought is a major goal of plant breeding.
Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. It has
been found that LEA genes are a gene family and play important roles in the protection of water stress.
Expression of OsLEA3 is induced by abiotic stresses, including drought and high salinity. The promoter of
OsLEA3 was cloned from Batutegi, Rojolele and Nipponbare cultivars and its function was analyzed intransgenic Nipponbare plant by a GUS reporter gene. Transient assay was conducted in order to investigate
GUS expression driven by OsLEA3 promoter. OsLEA3 promoter was expressed in embryogenic callus
indicated by the appearance of blue spots. Differences in color produced in embryogenic callus showed different
expression level.
Key words: rice, drought, Batutegi, Rojolele, Nipponbare, GUS, OsLEA3 promoter

Introduction
Rice (Oryza sativa L.) is one of the major staple foods. As the population growth goes up,
the rice production must be increased. However, the field for rice cultivation has decreased becauseof the change of paddy field into housing and industry. Moreover, drought influences the riceproduction especially in North China and South Asian. Thus, rice becomes one of the target sourceof gene and genome sequence analysis project among cereal plants (Goff et al. 2002).
LEA proteins constitute a large group of proteins that speciﬁcally expressed during seeddevelopment. LEA proteins are located in seeds and pollen. Many vegetative organs can also
express some LEA proteins under conditions of dehydration, osmotic stress, cold or exogenous
ABA. First LEA proteins were identiﬁed during the desiccation phases of seed development. These
proteins were reported to protect speciﬁc cellular structures or ameliorate the effect of drought
stress by sequestering ions and maintaining minimum cellular water requirements (Hu, 2008).
Plant has physiological and molecular response mechanisms to tolerate drought. Thosemechanisms are osmotic accumulation, scavenging ROS, and osmoprotectant protein production
such as late embryogenesis abundant (LEA) and heat shock protein. LEA gene is identified as an
expresed gene in the late phase of seed growth. Research on LEA genes has showed that LEA 3
gene is responsible for drought tolerance. A major problem under severe dehydration is that theloss of water leads to crystallization of cellular components during the course of dehydration anddormancy, which damages cell structures (Spelundm et al 1996). Some of the LEA proteins could
essentially be considered compatible solutes that likely play the role similar to sugars in maintaining
the structure of the cytoplasm in the absence of water (Hu 2008). The goal of this research is to
study the function of LEA3 promoter isolated from rice cv. Batutegi.

thProceedings of The 7 ACSA Conference 201191

Materials and Methods
Promoter OsLEA3
Promoter sequence primers were designed based on sequence of genes LEA3 HVA-likegene promoter from rice cultivar IRAT 109 (GenBank Acc. DQ837728) and has been adapted to the
BAC sequence of chromosome No. 5 rice cultivar Nipponbare (GenBank Acc. AC104713). Primer
sequences are also designed to include the start codon (ATG) of mRNA sequences LEA3 gene
(GenBank Acc. DQ789359) and the addition sequence EcoRI (G↓ AATTC) and BglII (A↓GATCT) in
order to add EcoRI and BglII restriction sites in the primers. The predicted promoter was amplified
and isolated by PCR. The amplified fragments were sequenced to ensure fidelity.
Isolation of rice genomic DNA
Isolation of genomic DNA of rice cv. Batutegi was conducted by CTAB method (SaghaiMaroof et al., 1984) that has been optimized. Rice leaf samples were taken from 1-month-oldplants. A total of 2 g leaf samples were prepared using aluminum foil wrapping. Leaf powder was
put into polypropylene tubes containing 10 ml of buffer solution which has been has been preheatedin the incubator at 65oC for 30 minutes.
PCR analysis
All PCR reactions were done using 20 ng DNA template. PCR reaction mix contained 6.5
uL nuclease free water, 12.5 uL Green Go Taq 2x Master, 2.5 uL for each LEA F primer (0.1 μM)
and LEA R (0.1 μM) promoter LEA3, and 1 μl template DNA. PCR conditions were: initialdenaturation 95oC for 3 minutes, followed by 35 cycles of denaturation at of 95oC for 1 minute,annealing at 60°C for 1 minute, and initial extention at 72oC for 1 minute. The cycle was terminatedby final extention at 72oC for 10 minutes.
Vector Construction
1.

hpt

p35S

Left Border

2.

p35S

Left Border

Right Border

GUS

pOsLEA3 RJ
Right Border

NosT

hpt
Left Border

pOsLEA3 NB

NosT

hpt

3.

GUS

p35S

GUS

pOsLEA3 BT
Right Border

NosT

Figure 1. Vector Construction of OsLEA3 promoter::GUS.

Agrobacterium mediated transformation
Agrobacterium tumefaciens LBA4404 strain was transformed using recombinant plasmid
pC1305 containing hygromycin phosphotransferase gene (hpt) controlled by CAMV 35S promoter,and GUS gene controlled by OsLEA3 promoter.

92

thProceedings of The 7 ACSA Conference 2011

Histochemichal GUS assay
Water deficit treated and non treated plant tissues were vacuum infiltrated for 1 h in the
GUS reaction mixture containing 1 mM 5-bromo-4-chloro-3-indolyl- b-D-glucuronide (X-gluc) and 50
mM sodium phosphate buffer and incubated at 37oC overnight. The reaction was stopped by adding
75% ethanol, and the pigments and chlorophylls were removed by repeated ethanol treatment.

Results and Discussion
Amplification of OsLEA3 promoter
Isolation of 1.291 bp OsLEA3 gene promoter was conducted by amplification of DNA
genome using PCR with specific primers and visualized with 0.8% agarose gel (Figure 2). The
PCR fragment was then cloned into pGEM-T Easy and then used to construct pC1305 based
recombinant plasmid.

Figure 2. Amplification of OsLEA3 promoter.

Verification of the recombinant pGEM-T plasmid was conducted using EcoRI restriction
analysis . The result showed 3 fragments, which were fragment with size 3.001 bp (pGEM-T Easy
vector plasmid), 1.291 bp (gene OsLEA3 promoter), and 12 bp (nucleotides between EcoRI site of
OsLEA promotor gene and EcoRI site of pGEM-T Easy) fragments (Figure 3).

Figure 3. Electrophoresis of recombinant vector digested with EcoRI.

Rice Transformation using Agrobacterium
Recombinant vector containing OsLEA3promoter was successfully constructed andtrasformed to rice cv. Nipponbare. Selection of Transformed plants was selected on hygromisin
contained medium.

th

Proceedings of The 7 ACSA Conference 201193

Figure 4. Successfull transformed plant in the selection medium.

Some transformed calli survived in medium containing hygroimisin. Hygromisin resistantcelli were subcultured to regeneration medium. Calli with green spot grew into planlets. Table 1
showed low transformation efficiency. It needs to be increased by optimizing conditions of theculture. Factors that influence the low efficieny of transfomation are method of tranformation, type of
explants and condition of culture.
Table 1. Genetic transformation of OsLEA3 ::GUS to rice cv. Nipponbare
No

Recombinant vectors

Rice Varieties

1.
2.
3.

OsLEA3 NB:: GUSOsLEA3 Rj:: GUSOsLEA3 BT:: GUS

Nipponbare
Nipponbare
Nipponbare∑ infected calli

GUS
assay
Positive
Positive
Positive

600
600
600

Number of
Planlets
32
19
7

Histochemical Assay
Transient expression assaysof OsLEA3::GUS promoter of rice cv. Nipponbare, Rojolele
and Batutegi were successfully conducted. GUS gene expression was identified with blue spot on
the transformed calli .Figure 5 showed the difference intensity of blue spot in three cultivars. It
means that the expression level of GUS gene differ among different OsLEA3 promoters. Real Time
PCR will be conducted for further qualitative analysis.

Figure 5. Histochemical assay of transformed calli, NB= Niopponbare, RJ= Rojolele, BT= Batutegi.

Conclusion
1.
2.

94

Recombinant plasmid pCambia1305 of OsLEA3::GUS of Batutegi, Rojolele and Nipponbare
cultivars was succesfully constructed.
Promoters OsLEA3 of Batutegi, Rojolele and Nipponbare cultivars controlled GUS expression
differently among those cultivars

thProceedings of The 7 ACSA Conference 2011

References
Bray, E. A. 1993. Molecular response to water deficit. Plant Physiology 103: 1035-1040.
Gao, J-P., D-Y. Chao & H-X. Lin. 2007. Understanding abiotic stress tolerance mechanisms: Recent studies onstress response in rice. Journal of Integrative Plant Biology 49(6): 742--750.
Goff, S. A. et al. 2002. A draft sequence of rice genome (Oryza sativa L. ssp. Japonica). Science 296: 92--100.
Kalefetoğlu, T. & Y. Ekmekçi. 2005. The effects of drought on plants and tolerance mechanisms. Fen Bilimleri
Dergisi 18(4): 723--740.
Promega. 2007. Technical manual pGEM-T and pGEM-T Easy vector systems. Promega Corporation, Madison:
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Spelundm, S., Aeboelarssen, S., Hughes, D.W., Galau, G.A.,Larsen, F., and Jakobsen, K.S., 1992. Late Embryo
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Sambrook, J. & D.W. Russel. 2001. Molecular cloning: A laboratory manual, vol 1-3. 3rd ed. Cold Spring Harbor
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Hu T. Z. 2008. OsLEA3, a Late Embryogenesis Abundant Protein Gene from Rice, Confers Tolerance to Water
Deficit and Salt Stress to Transgenic Rice. Russian Journal of Plant Physiology, Vol. 55, No.4, pp. 530–
537.
Xiao, B., Y. Huang, N. Tang, & L. Xiong. 2007. Over-expression of a LEA gene in rice improves drought
resistance under the field condition. Theory Application Genetics 115: 35--46.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 201195

Evaluation of the Nutritional Environment for Rice in Cianjur, Indonesia
for Development of an Advanced Basin Model for Asia
Koki Homma a,*, Gunardi Sigit b, Handarto c, Masayasu Maki d, Chiharu Hongo e, Koshi
Yoshida f, Kazuo Oki g, Hiroaki Shirakawa h, Tatsuhiko Shiraiwa a, Ritsuko Hara a, Mitsuo
Kambayashi a, Yoshihiro Hirooka a, Hiroki Iwamoto aaGraduate School of Agriculture, Kyoto University, Kyoto, Japan
Regional Office of Food Crops Service West Jawa Province, Indonesiac
Human Resource Development, Universitas Padjadjaran, IndonesiadGraduate School of Global Environmental StudieseCenter for Environmental Remote Sensing, Chiba UniversityfCollage of Agriculture, Ibaraki UniversitygInstitute of Industrial Science, the University of TokyohGraduate School of Environmental Studies, Nagoya University* Corresponding author: Graduate School of Agriculture, Kyoto University, Sakyo-ku, 6068502, Kyoto, Japan.
Tel.: +81 75 753 6042; fax: +81 75 753 6065.
homma@kais.kyoto-u.ac.jpbAbstract
Food production systems in tropical Asia face the following problems: 1) increased demand for production; 2)
increasing environmental load; 3) water scarcity and 4) effects of climate change. To overcome these problems,
strategies for better management of crop and environmental resources are recommended. Here, we describe
our research in Cianjur, Indonesia under the project ―Development and Practice of Advanced Basin Model in
Asia – Toward Adaptation to Climate Change‖. The study area, Cianjur, is one of the main rice production areas
in Indonesia, and has water reservoirs that supply water to Jakarta. However, excess nutrients from agriculturalland cause environmental problems. In this situation, evaluation of the nutritional environment for rice growth isnecessary. We have begun to evaluate the nutrient concentrations in the irrigation water, soil and plants. The
relationships of these nutrient contents to rice growth were analyzed using a rice growth simulation model. The
simulation model and remote-sensing technologies were used in combination to evaluate the geographic
distribution of rice growth and nutritional environment. The research produced baseline information fordeveloping the Advanced Basin Model.
Keywords: environmental problem, irrigation water, remote-sensing, simulation model, soil solution

Introduction
Food production systems in tropical Asia face the following problems: 1) increased
demand for production; 2) increasing environmental load; 3) water scarcity and 4) effects of climatechange. To overcome these problems, strategies for better management of crops andenvironmental resources are recommended.
Since the green revolution, modern agricultural technology has increased land productivity
and supported population growth. However, this technology tends to institutionalize agricultural
management and reduce diversity. Previous studies reported that such institutionalized
management increased the risk of disasters. Tsuno (1995) analyzed cold summer damage to riceproduction in Japan in 1993 and concluded that the simplification of management in cultivars andcultivation methods increased the extent of damage. Shiraiwa et al. (2002) analyzed the variabilityof rice production in Thailand and concluded that the annual variation was larger in an intensive
high yielding area (the Central Plain) than in an extensive low-yielding area (the Northeast).
Yoshino et al. (2000) analyzed the relationship between the Southern Oscillation Index and riceproduction in Indonesia and reported that the negative effect was largest in Java, which was the96

thProceedings of The 7 ACSA Conference 2011

most productive area. These examples indicate that the present management is insufficient for
coping with unfavorable conditions. Management variability may thus be the key to attaining
production stability.
The high fertilizer input required by modern agriculture technology often exceeds anacceptable amount in the field and increases the environmental load. To optimize the input, theavailable nutrients in the soil and the requirements of the plant should first be evaluated. Because
water scarcity is predicted in the future, the available water supply should also be evaluated.

The project “Development and Practice of Advanced Basin Model in Asia – Toward
Adaptation to Climate Change”
As mentioned above, the present situation demands not only a production increase, butalso management optimization based on resource evaluation. Because the smallest water
management unit is a basin and nearly all excess nutrients move with the water stream in the basin,a strategy to overcome these problems must be developed at the basin scale. Here, we establishedthe project ―Development and Practice of Advanced Basin Model in Asia – Toward Adaptation to
Climate Change‖ supported by the Ministry of the Environment, Japan．The project includes theevaluation of environmental resources and crop production, proposals for better management andalso the establishment of an environmental conservation community. The Citarum River inIndonesia and the Num Ngum River in Laos were selected as test sites (Oki, 2011). This
manuscript describes one of the project activities in Indonesia, which were managed by the leadauthor. In the activities, the nutrient concentrations of the irrigation water, soil solution and plants
are evaluated their relationships to rice growth will be analyzed using a rice growth simulation
model. The relations in terms of rice growth would be analyzed by using rice growth simulation
model. The simulation model and remote-sensing technologies will be used in combination toevaluate the geographic distribution of rice growth and then nutritional environment.

Overview of research area and activities
The research area is in the hydrographic basin of the Citarum River in West Java,
Indonesia. The basin includes Bandung, the third largest city in Indonesia, and a total of 5 million
people live in the area (Fig. 1a). Because the river is heavily polluted by human sewage andagriculture, the Asian Development Bank has called it the world‘s dirtiest river and approved a loan
for clean-up in 2008. The river has 3 dams and one of 3 dams directly supplies water for Jakarta;
thus clean-up of the river is of urgent concern.
Field observations have been conducted in the Bojongpicung and Ciranjang districts,
Cianjur regency, West Java. The area is located between the Saguling and Cirata reservoirs and isdownstream of Bandung. We selected 40 paddy fields, of which 30 were planted with rice and 10
were planted with soybeans in August 2011 (Fig. 1b). In addition to the chemical properties of thesoil, the nitrogen and phosphate concentrations of soil solutions are periodically monitored.
Because the nutrient concentrations in irrigation water are also measured along 3 points in the
major channels, the nutritional environment for crop growth can be assessed in terms of nitrogenand phosphate levels.
Plant growth is nondestructively evaluated in terms of plant height and canopy cover using
a digital camera. The spectral reflectance of the canopy is measured once a week with areflectance-measuring instrument (MS-720, Eiko seiki Co. Ltd.). The plants are also destructively
sampled once per month to determine the leaf area index (LAI), above-ground dry matter and root
density. The grain yield and above-ground biomass will be measured at maturity.

thProceedings of The 7 ACSA Conference 2011

97

a)

b)

JakartaJatilful
Cianjurb)

CirataBandung
Sagling

We selected 30 rice fields from 6 locations () and 10 soybean fields from 2 locations (o). Irrigation water is periodicallymonitored at 6 locations for rice fields.

Figure 1. Maps of the hydrographic basin of the Citarum River (a) and the field observation site (b).

Strategies for analysis and evaluation
To develop strategies for managing crop production in the hydrographic basin, thegeophysical distribution of environmental resources and crop growth must be evaluated. However,the data we obtain are point-based. Accordingly, a method that scales up from point to area isneeded. Satellite-based remote-sensing can be used for this purpose, and one of the co-authors of
this paper had already developed a method that has been adapted in an agricultural area of Japan(Nuarsa et al. 2011). However, obtaining satellite images for analysis is difficult because the area
often obscured by clouds.
To compensate for the missing data, we employed a simulation model. The authors
developed the simulation model to predict rice growth and yield under rainfed conditions based on afield survey in Northeast Thailand, where the water and nutrients are quite limited (Fig. 2; Hommaand Horie, 2008). Because the model was modified from SImulation Model for RIce Weather
relations (SIMRIW), which has a good reputation for predicting the effects of climate change on riceproduction (Horie et al. 1995), the modified model may be able to handle climate change issues forthe advanced basin model. We are now developing a simulation model to use in combination withremote-sensing (Fig. 3; Maki and Homma, 2011). The combined model will be used to evaluate thenutritional environment and predict rice yield by correcting the simulated data with satellite-based
remote-sensing data. The combined model may also help to analyze the relationship between the
remote-sensing data and field observations. The analysis will first be used for rice production andmay be expanded to examine green soybean (edamame) production.

98

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The basic concept of the model is derived from SIMRIW (SImulation Model for RIce Weather relations, Horie et al. 1995), andmodified for rainfed rice production in Northeast Thailand (Homma and Horie, 2008).

Figure 2. Schematic illustration of the rice growth simulation model.

Figure 3. Examples of leaf area index (LAI) simulated by the rice growth simulation model and corrected byremote-sensing (Maki and Homma, 2011).

thProceedings of The 7 ACSA Conference 201199

Conclusions
Here, we describe one of our activities in Cianjur, Indonesia. To develop better
management practices for overcoming environmental problems and adapting to climate change,
evaluation of the nutritional environment and rice growth are necessary. For this purpose, we havebegun to evaluate the nutrient concentrations of irrigation water, soil solutions and plants. The
relationship between these nutrient concentration and rice growth will be analyzed using a rice
growth simulation model. The simulation model and remote sensing technologies will be used in
combination to evaluate the geographic distribution of nutritional environment and rice growth.

Acknowledgement
The project is supported by the Environment Research and Technology Development Fund, Ministryof the Environment, Japan (E1104).

References
Homma K and T Horie. 2008. The present situation and the future improvement of fertilizer applications by
farmers in rainfed rice culture in Northeast Thailand. In: LR Elsworth and Paley WO (Eds.) Fertilizers:
Properties, Applications and Effects. Nova Science Publishers, N.Y. 147-180.
Horie T, H Nakagawa, HGS Centeno and M Kropff 1995. The rice crop simulation model SIMRIW and its testing.
In Modelling the Impact of Climate Change on Rice in Asia (Matthews, R. B. et al. eds.), CAB
International, Oxon, UK. p.51-66.
Maki M and K Homma 2011. Development of Simulation Model to Evaluate Geographical Distributions of Rice
Growth and Yield in Relation to Weathers, Varieties and Fields. Proc. Annual Meeting of Society ofEnvironmental Science, Japan in 2011.
Nuarsa IW, F Nishio and C Hongo. 2011. Spectral characteristics and mapping of rice plants using multitemporal landsat data. J. Agric. Sci. 3: 54-67.
Oki K. 2011. Development and Practice of Advanced Basin Model in Asia. Proc. Annual Meeting of Society ofEnvironmental Science, Japan in 2011.
Shiraiwa T, H Nakagawa, T Horie, T Matsui and K Homma. 2001. Variability in rice production and precipitationin Thailand. Chikyukankyo 6: 207-215. (in Japanese)
Tsuno Y. 1995. Clarification of actual situation and cultivation management problem in cool summer damage on
rice production in Japan in 1993. Research Project Number 06304012. Research Report of Grants-in-Aid
for Scientific Research, MEXT and JSPS, Japan. Pp. 233. (in Japanese)
Yoshino M, K Urushibara,-Yoshino, W Suratman. (2000) Agricultural production and climate change inIndonesia. Global Environ. Res. 3: 187-197.

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100

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The Nutritional Environment in Nonfertilized Rice Production and Its Effecton the Nutritional Quality of Brown Rice
Ritsuko Hara a, Koki Homma a *, Yoshihiro Hirooka a, Mitsuo Kuwadaab and Tatsuhiko
ShiraiwaaaGraduate School of Agriculture, Kyoto University, Kyoto, Japan.
NPO Nonorganic, Nonchemical Crop Production Research Group, Kyoto, Japan.
* Corresponding author: Graduate School of Agriculture, Kyoto University, Sakyo-ku, 6068502, Kyoto, Japan.
Tel.: +81 75 753 6042; fax: +81 75 753 6065.
homma@kais.kyoto-u.ac.jpbAbstract
The nonfertilized-nonchemical crop production (so-called nature farming) proposed by Mr. Mokichi Okada is a
popular farming method in both Japan and worldwide. The farming method uses neither fertilizers nor chemicals
but obtains comparable yields to those produced by conventional farming. Although the method was designed toutilize natural resources, the details of the mechanism are still unknown. This study was aimed to evaluate thenutritional environment on the basis of the measurement of the nutrients in the soil solution and to analyze theeffect on the nutrient concentrations of brown rice. For this purpose, we selected 2 nonfertilized-nonchemical
paddy fields (NN fields) and 1 experimental paddy field (Expt field) as a reference. One of the NN fields wascultivated for 6 years without fertilizer (6-year NN), and another was cultivated in this way for 59 years (59-year+
NN). In the Expt field, 3 types of fertilizer treatments (None, Basal and Conventional) were conducted. The NH 4
concentrations in the 6-year NN fields was lower than those in the fertilizer-applied plots (Basal and+Conventional), but they were comparable to that in the None treatment. Conversely, NH 4 was not detectable inthe 59-year NN field. Other nutrients were extremely different between the NN fields and Expt field, namely, thefertilizer treatments (Basal and Conventional) had little effect on the nutrient concentration of the soil solution.
+
2+
2+
The P2O5 and K levels in the Expt fields were notably higher than those in the NN field, whereas Ca and Mg
showed an opposite trend. Significant interactions of the genotype by environment were observed for the++nutrient concentrations in brown rice. The relatively low NH 4 and K level in the soil solution in the NN fields
tended to result for brown rice in low concentrations of those nutrients, which Japanese people tend to prefer.
The relationship between the nutritional environment and the uptake and concentration of nutrients in brown rice
should be studied further.
Keywords: rice, nonfertilized-nonchemical crop production, soil solution, nutritional quality of rice

Introduction
Agrochemicals such as fertilizers and pesticides largely increase agricultural production andsupport the huge population of the world. However, excess inputs of agrochemicals leak into theenvironment and cause various kinds of problems that pose threats to human life (Reichenberger et
al. 2007). Organic farming or nature farming is derived from the reactions against such
agrochemical agriculture.
The nonfertilized-nonchemical crop production proposed by Mr. Mokichi Okada is popular inboth Japan and worldwide. This method of farming uses neither fertilizers nor chemicals but obtains
comparable yields to those produced by conventional farming (Okumura, 2002). Okumura reported
that the sources of some of the nitrogen were irrigation water and biological fixation but that the
majority comes from soil. Although the method of farming was designed to utilize natural resources,
the details of the mechanism are still unknown.
Because crop productivity is restricted by nutrients supplied through soil, an evaluation of
the nutritional environment in the soil is quite important when discussing productivity. In general, theavailability of nutrients for plants is evaluated by chemical analyses. However, the nutritionalthProceedings of The 7 ACSA Conference 2011101

environment is quite influenced by the temperature, soil moisture and plant uptake and exudates,
which suggests that periodic monitoring of the nutritional environment is required to analyze theplant response to nutrition and the nutrient availability (Boivin et al., 2002).
When plants take up nutrients from the soil, the procurement does not occur on the surfaceof the soil particles but through the liquid phase of the soil (soil solution). Nutrients in their ion forms,
such as ammonium and potassium, move with the soil solution (mass flow) or by diffusion in thesolution. Accordingly, nutritional analyses of the soil solution are suggested to evaluate thenutritional environment (Smethurst, 2000).
This study investigated nonfertilized-nonchemical fields and an experimental field as areference. The soil solutions were sampled once every 2 weeks for the nutritional evaluation. Six
representative rice cultivars were planted to evaluate any cultivar differences in nutrient uptake, drymatter production, yield and grain quality. This report focused on the changes in the nutrients in soil
solution and the effect on the nutrient content of brown rice.

Materials and Methods
The experiment was conducted in 2010. We used two types of paddy fields: one wasoperated under nonfertilized-nonchemical management by NPO Nonorganic, Nonchemical Crop
Production Research Group (NN fields; Ogura, Uji city, Kyoto Prefecture, 34° 54‘ N, 135° 46‘ E),
and the other was an experimental field in the Graduate School of Agriculture, Kyoto University(Expt field; Sakyo, Kyoto city, Kyoto Prefecture, 35° 02‘ N, 135° 47‘ E). The NN fields included 2
fields: one was cultivated for 6 years without fertilizer or agrochemicals (6-year NN), and the otherwas cultivated for 3 years without fertilizer or agrochemicals after the top-soil was converted using
the soil of a field where nonfertilized-nonchemical management was conducted for 56 years. Thus,
the soil of the converted field was used for a total of 59 years under nonfertilized-nonchemical
management (59-year NN). The experimental year was the 7th and 60th year of cultivation,
respectively. In the Expt field, 3 kinds of fertilizer treatments (None, Basal and Conventional) wereconducted under pesticide application. The none treatment was conducted without fertilizer,
whereas the Conventional and Basal treatments included the application of chemical fertilizer at a
rate of N-P2O5-K2O = 5-5-5 g m-2 as basal, and the Conventional treatment was top-dressed withfertilizer on the 22th of July and the 7th of August. The rate of each top-dress application was NP2O5-K2O = 2.5-2.5-2.5 g m-2. Thus, the total application rate for the Conventional treatment was NP2O5-K2O = 10-10-10 g m-2. The Basal treatment was designed to starve nutrients around theheading period.
Based on the results of a study conducted in the same field (Matsuyama et al., 2010), six
representative rice cultivars were selected. Beniasahi (Japan) is a traditional japonica cultivar andhas been self-seed-produced for the 59 years under the nonfertilized-nonchemical management,suggesting that the cultivar has adapted to these conditions (Okumura, 2002). Nipponbare (Japan)
and Kasalath (India) are standard cultivars of japonica and indica, respectively (Kojima et al., 2005).
Takanari (Japan) is a high-yielding indica cultivar and is promising for the production of rice flour
and as a feed crop. B6144F-MR-6-0-0 (B6144F) was bred for upland cultivation in Indonesia (Atlinet al., 2006). Bei Khe (Cambodia) is a cultivar that produced the largest amount of dry matter in theexperiment conducted by Matsuyama et al. (2010). The cultivars were sown on the 10th of May andtransplanted to the Expt field on the 3rd of June and to the NN fields on the 8th of June. The
transplanting density was 22.2 hill m-2, with 1 plant hill-1. The experiment was arranged in arandomized block design for each field.
A soil solution sampler (DIK-301B, Daiki Rika Kogyo Co., Ltd.) was placed at a 10 cm-depth
in every Nipponbare and B6144F plot. The soil solution was sampled by a syringe aspiration
method once every 2 weeks and the concentrations of NH4+, P, K, Mg and Ca in the solution were
measured. The soil chemical properties were measured by a standard method. The brown rice
102

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grains at maturity and the above-ground biomass and leaves at heading were sampled, and its N, K
and Mg concentrations were determined.

Results and Discussion
Evaluation of the nutritional environment determined on the basis of the soil solution
The three major soil solution nutrients, NH4+, P and K, were quite different among the fields
and treatments (Fig. 1). The NH4+ concentration under the Basal and Conventional treatments in the
Expt field was initially quite high due to the effect of the basal fertilizer, but it markedly decreased by
30 days after transplanting. The conventional treatment maintained a relatively higher NH4+
concentration due to the top-dress application of fertilizer, whereas the NH4+ concentration underthe Basal treatment exhibited a lower level (almost 0 ppm) than the None treatment. The Basaltreatment resulted in nitrogen starvation conditions, as was intended by the experimental design. In
the NN fields, the NH4+ concentration in the 6-year NN field peaked at 60 days after transplanting
and maintained levels of approximately 0.70 ppm and 0.25 ppm before and after the peak at 60days, respectively. The NH4+ concentration in the 59-year NN field maintained levels of
approximately 0.2 ppm and 0 ppm for 30 days after transplanting and thereafter, respectively.
6

4

P conc. (ppm)

NH4+ conc. (ppm)

5

3
2

4

2

1

0
0

40
80
120
Days after transplanting（NN fields）
12
6-yearNN
59-year NN（Expt. field）
8
None
Basal
Conventional4

Mg conc. (ppm)4080Days after transplanting

120

10
5

5

0

4
3

0

Ca conc. (ppm)

40
80
120
Days after transplanting

2
1

160

120
80
40
0

120

15

0

0

40
80
Days after transplanting

20

K conc. (ppm)

00NH4+ conc. (ppm)

0

0

40
80
120
Days after transplanting

0

40
80
120
Days after transplanting（NN fields）
6-year NN
59-year NN（Expt. field）
None
Basal
Conventional

Figure 1. Change of nutrients concentration in soil solution.

thProceedings of The 7 ACSA Conference 2011103

The P concentration in the Expt field increased from the transplanting up to 70 days after
transplanting, and differences among the fertilizer treatments were not obvious, suggesting that theannual P application accumulated P in the soil. Conversely, P was barely detectable in the NN
fields. The difference in the P concentrations between the Expt and NN fields was corresponded tothe soil chemical analysis (data not shown). Although the P concentration in the soil solution was
negligible, the plants took up P from the soil (data not shown). Moreover, although the P
concentration in the soil solution in the 6-year NN field was as low as that in the 59-year NN field,
the P uptake by the plants in the 6 year-NN field was 1.6 times as large as that in the 59-year NN
field. These data suggest that another indicator is required to evaluate the availability of P.
The K concentration was affected by the fertilizer application in the Expt field, but the effectwas short-lived: the baseline concentrations under the Basal and Conventional treatments weresimilar to that under the None treatment. The K concentration in NN fields was one half of the basal
concentration in the Expt field, and apparent differences were not observed between the 6-year and59-year NN fields.
The Ca and Mg concentrations were different at transplanting among the fertilizer treatmentsin the Expt field, but the difference disappeared by 30 days after transplanting. The concentration
gradually decreased with time, whereas the concentrations increased until 50 days after
transplanting in the NN fields. The increase was repeated in a flood incubation using the soil
sampled before transplanting (data not shown), suggesting that the trend shown in Fig. 1 is derived
from the characteristics of the soil or the forms of Ca and Mg in the soil.
The effect of nutrients in the soil solution on nutrient concentrations of brown rice
The N and K concentrations in brown rice almost reflected those in the soil solution: theconcentrations of the brown rice in the NN fields tended to be lower than those in the Expt field.
However, the Mg concentration of the brown rice in the 6-year NN field was lower than that under
the Conventional treatment in the Expt field; thus, the concentrations of the brown rice did notreflect those in the soil solution. The interaction effects of the cultivar and the environment weresignificant for all of the nutrients, indicating that the plant responses to the nutritional environment
were different among the cultivars.
Previous studies report that the N concentration in brown rice is affected by that in the flag
leaves at heading (Taira, 1997). The relationship in this study suggests that the indica cultivars are
more affected than the japonica cultivars (Fig. 3). As available data atre inadequate for K and Mg,
the relationship of the K and Mg concentrations of the brown rice was compared with that in theabove-ground biomass at the heading stage. The relationships of the K concentration between thebrown rice and the above-ground biomass were significant for the traditional varieties but notsignificant for the improved varieties. The relationships of the Mg concentration between the brownrice and the above-ground biomass were significant for the traditional indica cultivars but notsignificant for the other cultivars (data not shown). As shown in Fig. 3 these cultivar differences may
be one of the causes of the significant interaction of the cultivar and the environment on the nutrient
concentration in brown rice.
In terms of the nutrient concentration of brown rice, 1/N, Mg/K or Mg / (K N) were proposed
as grain palatability indexes for Japan (Nakagawa et al., 2000). As mentioned above, the N and K
concentrations of the brown rice in the NN fields tended to be lower than those in the Expt field,
increasing the indexes in NN fields higher than those in the Expt field. However, as nitrogenstarvation under the Basal treatment apparently decreased the N and K concentrations of the
Nipponbare brown rice, the index for Nipponbare was the highest under the Basal treatment.

104

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Proceedings of The 7 ACSA Conference 2011

0.5

K conc. (g 100g–1)

N conc. (g 100g–1)

1.5

1.0

Cultivar**
Environment**
C×E**

0.5

0
59-year 6-year None Basal

1.5

59-year 6-year None

NN fields

59-year 6-year None

NN fields

Cultivar**
Environment**
C×E**

0.1

0

Cultivar**
Environment**
C×E**

0.1

Basal

Conv.

Expt. Field

0.8

J Beniasahi
B Nipponbare
3 Takanari
1 B6144F
H Kasalath
F Bei Khe

0.2

0.2

Expt. field

Mg/K (g g–1)

0.3

0.3

0

Conv.

Basal

Conv.

Expt. Field

N conc. (g 100g–1)

Mg conc. (g 100g–1)

NN fields

0.4

1.0

0.6
0.4

0
0.5

Cultivar**
Environment✝
C×E✝

0.2
59-year 6-year None

NN fields

Basal

Conv.

Cultivar**

Expt.
field
Environment**

Mg/(K N) (g g–1 g–1 )

C×E**

1.0
0

0
59-year 6-year

0.7
5
0.5
0
0.2
5
0

None Basal

NN fields

Cultivar**
Environment**
C×Ens

59-year 6-year None

NN fields

Basal

Conv.

Conv.

Expt. field

J Beniasahi
B Nipponbare
3 Takanari
1 B6144F
H Kasalath
F Bei Khe

Expt. field

Results of ANOVA are shown in the figure: †, *, ** and ns indicates 10, 5, 1 % and non- significance, respectively.

Figure 2. Effect of cultivar and environment on nutrition concentration of brown rice (a, b and c), and grain
palatability index (d and e).

thProceedings of The 7 ACSA Conference 2011

105

0.5
Brown rice (g 100g–1)

Brown rice (g 100g–1)

0.5

Brown rice (g 100g–1)

1.6 a) N conc.
b) K conc.

0.4
0.3
0.2
0.1

0.5

1.2

0.8

0.4

1.0
0.5

1.51.0 2.0 1.5 2.5

2.03.0 2.5–1)Above ground biomass (g 100g
–1

Leaf (g 100g )b) K conc.

0.4
0.3
0.2
0.1

0.5

1.0

1.5

2.0

2.5

3.0

Above ground biomass (g 100g–1)

Slope
N conc. P conc.
J Beniasahi
0.30*
0.067**
B Nipponbare 0.18**
ns
3 Takanarinsns
1 B6144F
0.63**ns
H Kasalath
1.01** 0.068**
F Bei Khe
0.66** 0.057**

Slope
N conc. P conc.
J Beniasahi 0.30* 0.067**
B Nipponbare 0.18**
ns
3 Takanarinsns
1 B6144F
0.63**ns
H Kasalath
1.01** 0.068**
F Bei Khe
0.66** 0.057**

Fig. 3 (b)Figure 3. (a) Relationship between N concentration in leaf at heading and that in brown rice at maturity. (b)biomass at heading and that in brown rice at
Fig. 3 (a) Relationship between K concentration in above-groundFig.
3 (b)maturity.

References
Atlin GN, HR Lafitte, D Tao, M Laza, M Amante and B Courtois. 2006. Developing rice cultivars for high-fertility
upland systems in the Asian tropics. Field Crops Res. 97: 43-52.
Boivin P, F Favre, C Hammecker, JL Maeght, J Delarivière, JC Poussin and MCS Wopereis. 2002. Processes
driving soil solution chemistry in a flooded rice-cropped vertisol: analysis of long-time monitoring data.
Geoderma 110: 87-107.
Kojima Y, K Ebana, S Fukuoka, T Nagamine and M Kawase. 2005. Development of an RFLP-based ricediversity research set of germplasm. Breed. Sci. 55: 431-440.
Matsuyama H, K Homma, M Kuwada and T Shiraiwa. 2010. Genotypic variation in rice productivity under
nonfertilized-nonchemical management. Research Report in 2009. NPO Nonorganic, Nonechemical
Crop Production Research Group, Kyoto, Japan. P 6-11.
Nakagawa S, Y Tamura and Y Ogata. 2000. Comparison of rice grain qualities as influenced by organic andconventional farming systems. Jpn. J. Crop Sci. 69: 31-37.
Okumura T. 2002. Rice production in unfertilized paddy field -Mechanism of grain production as estimated from
nitrogen economy-. Plant Prod. Sci. 5:83-88.
Reichenberger S, M Bach, A Skitschak and HG Frede. 2007. Mitigation strategies to reduce pesticide inputs into
ground- and surface water and their effectiveness; A review. Sci. Total Environ. 384: 1-35.
Smethurst P. 2000. Soil solution and other soil analyses as indicators of nutrient supply: a review. Forest Ecol.
Manage. 138: 397-411.
Taira T. 1997. Estimation of nitrogen content of brown rice in rice cultivar Koshihikari at heading time. Jpn. J.
Crop Sci. 66: 706-707.

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106

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Proceedings of The 7 ACSA Conference 2011

Effect of Slow Release Fertilizer on Yield and Yield Components
in Chinese High-Yielding Rice Cultivars
Toshiaki Kokubo a, Akira Miyazaki b*, Tetsushi Yoshida a, Yoshinori Yamamotob,
Jing Ju c and Yulong Wang ca

The United Graduate School of Agricultural Sciences, Ehime University, Japanb
Faculty of Agriculture, Kochi University, JapancYangzhou University, China* Corresponding author: Fac. of Agr, Kochi Univ., 200 Monobe-Otsu, Nankoku-shi, Kochi, 7838502 Japan.
Tel & FAX.: +81 88 864 5123.
miyazaki@kochi-u.ac.jp

Abstract
The effect of slow release fertilizer on yield and yield components in Chinese high-yielding rice cultivars were
evaluated and analyzed to avoid over-fertilization in China. Two different types, linear (L) and sigmoid (S)fertilizer as a nitrogen (N) source were applied at basal dressing, in comparison with split applications ofammonium chloride (C type). The application of the slow release fertilizer increased the brown rice yieldsignificantly in Chinese high-yielding cultivars, Yangdao 4 (YD) and Wuyugen 3 (WY), and was more effective at-2
low N conditions than at high N conditions. Maximum yield was achieved in YD grown at 21 g N m by S type,-2
and in HH grown at 21 g N m by L type. This indicates that about 30 % of N fertilizer could be saved by theapplication of the slow release fertilizer. These effects on yield resulted from the increase in sink size (the-2-2number of spikelets m multiply one grain weight) due to the increase in the number of spikelets m . The effectof the S type slow release fertilizer was more effective on the increase in sink size and the number of spikelets-1
panicle than that of L type. Grain filling percentage in WY and HH decreased with the increase in sink size,
while it was slight in YD. Therefore, the increase in sink size was important for YD to improve the yieldproduction. These responses to the N supply were different between YD and WY, suggesting that the optimum
amount of N fertilizer applied should be varied with these cultivars.
Keywords: Chinese high-yielding cultivar, Nitrogen fertilizer application, rice, slow release fertilizer, yield

Introduction
In Jiangsu province in China, one of the representative rice-producing areas, high-yielding
rice cultivars are widely used for breeding and cultivation with applying a large amount of nitrogen(N) fertilizer more than 30 g m-2 to achieve high-yield (Peng et al. 2006). These cultivars have large
yield potentials (Amano et al. 1993; Wang et al. 1995; Yao et al. 2000) and require the applicationof large amounts of nutrients, especially nitrogen (N). However, an excess fertilizer application
causes not only an increase of costs but also an outflow of nutrients with an environmental burden.
This requires an appropriate management of the fertilizer application and an improvement of
fertilizer use efficiency (brown rice yield / the amount of N fertilizer application) in the rice cultivation.
The method of fertilizer application has been improved by changing the amounts and timings offertilizer application (Peng et al. 2006), and by using farmyard manure (Liu et al. 2008) andsupplemental nitrification inhibitor (Huang et al. 1996) to raise the fertilizer use efficiency. As one of
the effective solutions for high fertilizer recovery rate, a slow release fertilizer has been commonlyused in Japan for the rice cultivation. However, there are few studies in Chinese high-yielding
cultivars (Ju et al. 2006). As recent high-yielding cultivars can achieve high yield even in a small
quantity of fertilizer application (Hasegawa 2003; Taylaran et al. 2009), the optimum fertilizer
condition should be examined by using the slow release fertilizer. In this study, effects of differentthProceedings of The 7 ACSA Conference 2011107

types of the slow release fertilizer on yield in Chinese high-yielding rice cultivars were evaluatedand analyzed to determine effective methods and amounts of N fertilizer application.

Materials and Methods
Experiments were conducted in 2006 to 2009 (Exp.1) and 2010 (Exp.2) in a paddy field ofFaculty of Agriculture, Kochi University, Japan. Three treatments were designed by types of N
fertilizer applied. Ammonium chloride was applied as conventional N fertilizer (C type) for basal andtop-dressing at each growth stage (basal:20 days after transplanting : 20 days before heading : 10days before heading : heading = 2 :1:1:1:1). Two types of the slow release fertilizer, linear type (LPcoat 100, L type) and sigmoid type (LP coat SS 100, S type) were applied as basal dressing all at
once, except for the S type in 2006 and 2007, in which both 6 g m-2 of sigmoid type and 4 g m-2 ofammonium chloride were mixed at basal dressing and 2 g m-2 of ammonium chloride wasadditionally applied at 20 days after transplanting to promote early growth. In Exp.1, Chinese highyielding Indica cultivar, Yangdao 4 (YD), Chinese high-yielding Japonica cultivar, Wuyugen 3 (WY)and conventional Japonica cultivar, Hinohikari (HH) were grown at the rate of 12 g N m-2 for eachtypes of N fertilizer. In Exp.2, three different amounts of N, 12, 21, 30 g m-2 for each type of fertilizer
were applied in YD and HH. The same amounts of P2O5 and K2O as N in both experiments wereapplied at basal dressing and at each growth stage (basal : 20 days before heading : 10 days
before heading =4:1:1), respectively. All treatments were arranged with 2 replications by arandomized block design. Yield and yield components were determined by using 20 hills sampled atmaturity.

Results and Discussion
The effect of the slow release fertilizer on brown rice yield
Brown rice yield significantly increased with the application of slow release fertilizer in
Exp.1 (Table 1). The effect of the slow release fertilizer on yield did not differ significantly amongcultivars, but the average increasing percentage in YD and WY (6 to 15%) was higher than that in
HH (0 to 5%). This result agreed with reports by Sato et al. (1993; 1997) in which the yield
increased by 33 to 55% and 12 to 14% respectively.
The effect of the slow release fertilizer on yield was not significant in Exp.2 (Table 2).
However, the increasing percentage at 12 g N m-2 was higher than that at 30 g N m-2 in both
cultivars, showing that the slow release fertilizer was more effective on yield in low N fertilizer
conditions than in high N fertilizer conditions. In addition, this effect was significantly different
between cultivars, and the increasing percentage in YD was higher than that in HH. Maximum yield
was obtained in YD grown at 21 g N by S type (781 g m-2), and in HH grown at 21 g N by L type(539 g m-2), which were higher than those grown at 30 g N by C type. This result indicates that theslow release fertilizer saves about 30% of N fertilizer applied, compared with the conventional
fertilizer.

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Table 1. Yield and yield components of rice cultivars in Exp.1 (the average value of 2005-2009)

Note: Italic values indicate the increasing percentage by the slow release fertilizers.
Values with different small letters indicate significant differences at the 5% level among methods of the fertilizer applicationand cultivars.
Values with different capital letters indicate significant differences at the 5% level among varieties.
*, **, ***; Significant at 5, 1, 0.1% levels by ANOVA, respectively; ns is Not significant.

Analysis of yield components
These effects of the slow release fertilizer on yield resulted from the increase in sink size(the number of spikelets m-2 multiply one grain weight, Venkateswarlu and Visperas, 1987) due tothe increase in the number of spikelets m-2 in both experiments, although 1000 grain weightsignificantly decreased with the increase in the number of spikelets m-2 (Table 1, 2). The increase in-2-2
the number of spikelets m was attributed to the increase in both the number of panicles m and-1
the number of spikelets panicle . The effect of the S type slow release fertilizer was more effectiveon the increase in sink size and the number of spikelets panicle-1 than that of L type. Kamekawa(1990) reported that sink size increased with the application of the slow release fertilizer of linear
type mainly due to the increase in the number of panicles m-2 in Japanese cultivars. These results
show that there is a difference of the effect on the yield components between L and S types.
The increase in sink size resulted in the decrease in grain filling percentage in bothexperiments. However, Tanaka (1988) and Nakanishi et al. (1990) reported that grain filling
percentage increased despite the increase in the number of spikelets m-2 with applying the slow
release fertilizer at panicle neck-node differentiation stage. This result may be because of latefertilizer application in comparison with our study.
A negative relationship between sink size and grain filling percentage was observed in WY
and HH (P<0.10), but not observed in YD in Exp.1 (Figure 1A). A similar trend was observed inExp.2; the decrease in grain filling percentage in YD was less than that in HH when grown with highN fertilizer conditions (Figure 1B). Yang et al. (2002) reported that grain filling percentage in recent
Chinese cultivars did not always decrease with the increase of the number of spikelets panicle -1.
These results show that YD maintains high grain filling percentage with high sink size in response tohigh N conditions, resulting in high fertilizer use efficiency. However, Chinese high-yielding
Japonica cultivar, WY had a different trend with YD, and showed the significant decrease (P<0.10)
in grain filling percentage with the increase in sink size. This indicates that the optimum amount of
fertilizer application should be different between YD and WY.

thProceedings of The 7 ACSA Conference 2011109

Table 2. Yield and yield components of rice cultivars in Exp.2 (2010)

Note: Italic values indicate the increasing percentage by the slow release fertilizers.
Values with different small letters indicate significant differences at the 5% level among methods of the fertilizer
application and cultivars.
Values with different capital letters indicate significant differences at the 5% level among varieties.
*, **, ***; Significant at 5, 1, 0.1% levels by ANOVA, respectively; ns is Not significant.

(A)

80

Yield
900 g m-2

80

70
700 g m60

WY: y = -0.0258x + 98.3
r = -0.399 †HH: y = -0.032x + 100.0-2Grain filling percentage (%)

90
Grain filling percentage (%)(B)

YD: y = -0.0305x + 98.9
r = -0.754 ***YD: r = 0.167 n.s.

Yield
900 g m-2

60

700 g m-2

40
500 g m-2
300 g m-2

20
HH: y = -0.0854x + 124.8
r = -0.792 ***r = -0.366 †50

500 g m-2

500

700

900

1100

YD WY HHC ▲ ▲ △
L ■ ■ □
S ◆ ◆ ◇

100 g m-2

0
500

700

-2

900

1100

1300

1500

Sink size (g m-2)

Sink size (g m )***, †; Significant at 0.1 % and 10 % levels, respectively. n.s.; Not significant.

Figure 1. Relationship between sink size and grain filling percentage in Exp.1 (A) and Exp.2 (B).

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Yearly differences
The effect of the slow release fertilizer on yield was minimum in 2006 (-1%) and maximumin 2009 (12%) on average of cultivars in Exp.1. This yearly differences of the slow release fertilizer
was because of the decreasing rate in grain filling percentage and the increasing rate in sink sizedue to the increase in the number of spikelets panicle-1. That is, the remarkable decrease in grain
filling percentage and the slight increase in sink size were observed in 2006, while the opposite
trend was observed in 2009.

Conclusion
From the above results, the application of the slow release fertilizer increased the brownrice yield significantly, especially in YD and WY, and was more effective at low N fertilizer conditions
than at high N fertilizer conditions. Maximum yield was achieved in YD grown at 21 g N m -2 by S
type, and in HH grown at 21 g N m-2 by L type. Therefore, about 30% of N fertilizer could be savedby the application of the slow release fertilizer without any decrease in yield, in comparison with theconventional fertilizer. This resulted from the significant increase in sink size due to the increase in
the number of spikelets m-2. The slow release fertilizer of S type was more effective on increases insink size and the number of spikelets panicle-1 than that of L type. The increase in sink size resulted
in the decrease in grain filling percentage in WY and HH, while it was negligible or slight in YD,
which contributed to the increase in the yield production. These responses to the N supply weredifferent between YD and WY, both Chinese high-yielding cultivars. Therefore, the amount of N
fertilizer applied should be varied with these cultivars.

References
Amano T, Qingsen Z, Yulong W, Naoto I and Hidehiko T. 1993. Case studies on high yields of paddy rice in
Jiangsu province, China: I. Characteristics of grain production. Jpn. J. Crop Sci. 62: 267-274.
Hasegawa H. 2003. High-yielding rice cultivars perform best even at reduced nitrogen fertilizer rate. Crop Sci.
43: 921-926.
Huang W-Y, David S and Irwin TH. 1996. On-farm costs of reducing residual nitrogen on cropland vulnerable tonitrate leaching. Rev. Agric. Econ. 18: 325-339.
Liu J, Qiufa X, Qinghua S and Muying L. 2008. Rice uptake and recovery of nitrogen with different methods of
15
applying N-labeled chicken manure and ammonium sulfate. Plant Prod. Sci. 11: 271-277.
Ju J, Yoshinori Y, Akira M, Tetsushi Y and Yulong W. 2006. Effects of the amount and the kinds of fertilizer on
the bleeding rate and nitrogen absorption in a Chinese high-yielding cultivar, Yangdao 4. Jpn. J. Crop Sci.
75: 249-256.
Kamekawa K. 1990. LP fertilizer - characteristic of fertilizer effect and utilization- in Nougyou gijyutsu taikei 2-2,
Noubunkyo eds., Tokyo, Japan, Gi 522: 38-43.
Nagata K, Satoshi Y, Jun-ichi T and Tomio T. 2001. Effects of dry matter production, translocation of
nonstructural carbohydrates and nitrogen application on grain filling in rice cultivar Takanari, a cultivar
bearing a large number of spikelets. Plant Prod. Sci. 4: 173-183.
Nakanishi M, Nobuyuki T and Ho A. 1990. Effect of coated urea topdressing on growth and yield of rice plant.
Jpn. J. Crop sci. 59: 265-269.
Peng S, Roland JB, Jianliang H, Jianchang Y, Yingbin Z, Xuhua Z, Guanghuo W and Fusuo Z. 2006. Strategies
for overcoming low agronomic nitrogen use efficiency in irrigated rice systems in China. Field Crops
Research. 96: 37-47.
Sato T, Kyoichi S, Masagiko S and Tokuro A. 1993. Single basal application of total nitrogen fertilizer with
controlled-release coated urea on non-tilled rice culture. Jpn. J. Crop Sci. 62: 408-413.
Sato T, Kyoichi S and Masahiko S. 1997. A single basal application of controlled-release coated urea to ricecultures using pot seedlings in a cool region. Jpn. J. Crop Sci. 66: 11-16.
Tanaka N. 1988. Topdressing of coated urea to rice plant for saving of labor. Jpn. J. Soil Sci. Plant Nutr. 59:
500-503.

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Proceedings of The 7 ACSA Conference 2011111

Taylaran DR, Satomi O, Naoko M, Taiichiro O, Takashi M and Tadashi H. 2009. Performance of a high-yielding
modern rice cultivar Takanari and several old and new cultivars grown with and without chemical fertilizer
in a submerged paddy field. Plant Prod. Sci. 12: 365-380.
Venkateswarlu B and Visperas RM. 1987. Source-sink relationships in crop plants. IRRI Rice Research Paper
Series 125: 1-19.
Wang Y, Yoshinori Y and Youji N. 1995. Analysis of the factors of high yielding ability for a Japonica type rice
line, 9004, bred in China : I. Comparison of yielding ability with a Japanese rice variety under the samelevel of spikelets number per area. Jpn. J. Crop. Sci. 66: 1-10.
Yang J, Shaobing P, Zujian Z, Zhiqin W, Romeo MV and Qingsen Z. 2002. Grain and dry matter yields andpartitioning of assimilates in Japonica/Indica hybrid Rice. Crop Sci. 42: 776-772.
Yao Y, Yoshinori Y, Yulong W, Tetsushi Y, Akira M, Youji N, and Cai J. 2000. Macro-element absorption at
maturity in relation to grain Yield in high-yielding rice cultivars. Soil Sci. Plant Nutr. 46, 815-824.

-- back to Table of Content --

112

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Proceedings of The 7 ACSA Conference 2011

Drought Resistance of NERICA Compared with Asian Rice,
African Rice and Millets in the Field with Different Fertilization Levels
Michihiko Fujiia,*, Seiji Ishiharaa, Ryohta ShinoharaaaFaculty of Education, Shizuoka University, Shizuoka, Japan* Corresponding author: Faculty of Education, Shizuoka University, 836, Ohya, Shizuoka, 422-8529, Japan.
Tel.: +81 54 238 4680; fax: +81 54 238 4680.
eimfuji@ipc.shizuoka.ac.jp

Abstract
Recently NERICA (New Rice for Africa) was developed by a crossing between African rice (Oryza glaberrima
Steud.) with Asian rice (Oryza sativa L.) in West Africa, and is considered to be drought resistant, but it is notclarified enough about the difference in reactions under dry condition. In this research, NERICA, Asian rice,
African rice and millets were cultivated in the field under drought condition with different fertilization levels tocompare dry matter production, stomatal and leaf characteristics and water absorbing characteristics to comfirm
2
the characteristics of NERICA under drought condition. Fertilization levels were 6gN/m in standard fertilization2
level and 2gN/m in low fertilization level. Stomatal conductance was measured by porometer and soil watercontents at individual depths (0-60cm) were measured by TDR method. Leaf thickness was measured by
micrometer and and SPAD value was determined with SPAD meter. In standard fertilization level under drought
condition, top dry weight at harvest was high in Dular, drought resistant Asian rice cultivar. Dry weight at harvest
in low fertilization level tended to be higher than that grown in standard fertilization level. In low fertilization level
one of NERICA showed high dry weight. Dry weight of glaberrima parent of NERICA was high in both standard
and low fertilization levels, but proportion of dead leaf was high. In standard fertilization level, Dular showed
highest stomatal conductance followed by sativa parent of NERICA and one of NERICA. In low fertilization levelsativa parent of NERICA, Dular and one of NERICA showed high stomatal conductance and it tended to behigher than in standard fertilization level. Glaberrima parent of NERICA showed low stomatal conductance inboth fertilization levels. Average soil water content at 0-60cm depth before harvest in standard fertilization level
was low in Dular followed by glaberrima parent of NERICA. In low fertilization level soil water content wasmaintained highest in one of NERICA. Soil water content at 0-20 cm depth in standard fertilization level was low
in glaberrima parent of NERICA. There was large cultivar differences in soil water content at 40-60 cm depth
and soil water decreased significantly in Dular in standard fertilization level. On the contrary it was maintained
highest in one of NERICA in low fertilization level. Dry weight was high in Dular, but soil water content
decreased especially in the standard fertilization level. On the contrary, dry weight was high in one of NERICA
and soil water content was maintained high especially in low fertilization level and at deep soil layer (40-60 cm**
depth). There was a significant correlation (r=0.907 ) between average stomatal conductance and dry weight at
harvest except glaberrima parent of NERICA. In low fertilization level stomatal conductance and dry weight at**
harvest tended to be larger than in standard fertilization level. There was a significant correlation (r=0.892 )between average leaf thickness and dry weight at harvest except glaberrima parent of NERICA and cultivar with**thick leaf tended to maintain high dry matter production. There was a significant correlation (r=0.828 ) between
average leaf thickness and SPAD value in rice. In one of NERICA leaf thickness and SPAD value were higher in
low fertilization level than in standard fertilization level. SPAD value tended to be low in Dular. High leaf
thickness and SPAD values seem to be effective to maintain dry matter production under limited water condition
with high water use efficiency. NERICA seems to be appropriate for the cultivation under drought condition withthe effective use of limited water for the sustainable crop production especially in low fertilization level.
Keywords: drought resistance, fertilization levels, NERICA, soil water content, stomatal conductanceIntroduction
Global water shortages are getting worse and drought is the major constraint to the crop
production (Blum, 2009, Boyer, 2010, Serraj et al., 2011). Recently NERICA (New Rice for Africa)thProceedings of The 7 ACSA Conference 2011113

was developed by a crossing between African rice (Oryza glaberrima Steud.) with Asian rice (Oryza
sativa L.) in West Africa, and is considered to be drought resistant, but it is not clarified enoughabout the difference in reactions under dry condition. In this research, NERICA, Asian rice, African
rice and millets were cultivated in the field under drought condition with different fertilization levels tocompare dry matter production, stomatal and leaf characteristics and water absorbing
characteristics to confirm the characteristics of NERICA under drought condition.

Materials and Methods
Plant Materials and Cultivation
In this study two NERICA lines which showed superior drought resistant in our previous
reports (Fujii et.al, 2004, 2008) and their two parent cultivars were used. Cultivars WAB450-24-3P3-1-HB, WAB450-I-B-P-82-2-1, sativa parent: WAB56-104, glaberrima parent: CG14. Rice
cultivars of O. sativa L. (Koshihikari (japonica, lowland, Japan), Dular (indica, lowland-upland, India)
and IRAT13 (japonica, upland, Cote d‘Ivoire)) and common millet (Panicum miliaceum L.) were also
used. Plants were seeded in paper pots on May 31, 2007 and seedlings were planted at uplandfield in the vinyl house of Shizuoka University on June 21. Sides of vinyl house were kept open.
Irrigation was applied on June 21, 27 and July 10, and after that no irrigation was applied. Plots
were fertilized by compound fertilizer at the rate of 6 g/m2 as nitrogen in standard fertilization plotsand 2 g/m2 in low fertilization plots individually.
Measurements
Plants were sampled on June 25, July 31, September 13 and October 22 at the harvestingtime. After dividing into organs, dry weight was measured after desiccating in the drying oven.
Stomatal conductance was measured at the center of abaxial side of topmost three leaves bydynamic diffusion porometer (AP4, Delta-T Devices Ltd., Cambridge, UK) on sunny days during thedaytime. Measurements were made on August 7, 13 and 20. Soil water contents at individual
depths were measured by TRIME-T3 tube access probe system (IMKO micromodultechnik,
Ettlingen, Germany) by TDR method. Measurements were made at intervals of 10cm depth on
August 6, 17, 30 and September 11.
Leaf thickness was measured by micrometer (No.193-111, Mitutoyo, Kawasaki, Japan) at
the middle length of topmost fully extended leaves avoiding midrib on August 10, 17, 22, 30 and
September 12. SPAD value was measured by SPAD meter (SPAD502, Konica Minolta, Tokyo,
Japan) on August 10, 17, 22, 30 and September 10. Both measurements were replicated 10 times
in each plot.

Results and Discussion
Top Dry Weight
In standard fertilization level under drought condition, top dry weight at harvest was high in
Dular, drought resistant cultivar in Asian rice (Figure1). Dry weight at harvest in low fertilization level
tended to be higher than in standard fertilization level. In low fertilization level one of NERICA
showed high dry weight. Dry weight in glaberrima parent of NERICA was high in both standard andlow fertilization levels, but proportion of dead leaf was high and heading was not observed.

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Proceedings of The 7 ACSA Conference 2011

1400

Dry Weight at Harvest (Oct. 22)
Dead Leaf
Stem
Leaf
Ear

2

Dry Weight （g/m ）

1200
1000
800
600
400
200

L common millet

L WAB450-I-B-P-82-2-1

L glaberrima parent

L sativa parent

L WAB450-24-3-P3-1-HB

L Dular

L IRAT13

L Koshihikari

common millet

glaberrima parent

WAB450-I-B-P-82-2-1

sativa parent

WAB450-24-3-P3-1-HB

Dular

IRAT13

Koshihikari

0Bars show standard errors.
Figure1. Top dry weight of individual organs at harvest, Oct. 22.

160

2

Stomatal Conductance （mmol/m /s）

Stomatal ConductanceIn standard fertilization level Dular showed highest stomatal conductance followed bysativa parent of NERICA and one of NERICA (Figure 2). In low fertilization level sativa parent of
NERICA, Dular and one of NERICA showed high stomatal conductance and it tended to be higher
than in standard fertilization level. Glaberrima parent of NERICA showed low stomatal conductancein both fertilization levels.
120

80

40

L common millet

L glaberrima parent

WAB450-I-B-P-82-2-1

WAB450-24-3-P3-1-HB

L IRAT13

L sativa parent

L Dular

L Koshihikari

common millet

glaberrima parent

WAB450-I-B-P-82-2-1

sativa parent

WAB450-24-3-P3-1-HB

IRAT13

Dular

Koshihikari

0Bars show standard errors

Figure 2. Average stomatal conductance from August 7 to 20.

Soil water content
Average soil water content at 0-60 cm depth before harvest in standard fertilization level
was low in Dular followed by glaberrima parent of NERICA. In low fertilization level soil watercontent was maintained highest in one of NERICA. Soil water content at 0-20 cm depth in standard
fertilization level was low in glaberrima parent of NERICA (Figure 3). There was large cultivar
differences in soil water content at 40-60 cm depth and soil water decreased significantly in Dular instandard fertilization level (Figure 4). On the contrary it was maintained highest in one of NERICA in
low fertilization level.

thProceedings of The 7 ACSA Conference 2011115

Soil Water Content （%）

15

0-20cm depth on September 11105Evaporation Plot

L common millet

L glaberrima parent

L WAB450-I-B-P-82-2-1

L sativa parent

L WAB450-24-3-P3-1-HB

L Dular

L IRAT13

L Koshihikari

common millet

WAB450-I-B-P-82-2-1

glaberrima parent

sativa parent

WAB450-24-3-P3-1-HB

IRAT13

Koshihikari

Dular

0

Bars show standard errors

Figure 3. Soil water content at 0-20 cm depth on September 11.

40-60cm depth on September 11

3

Soil Water Content (g/cm ,%)

40

30

20

10

Evaporation Plot

L common millet

L glaberrima parent

L WAB450-I-B-P-82-2-1

L WAB450-24-3-P3-1-HB

L IRAT13

L sativa parent

L Dular

L Koshihikari

common millet

glaberrima parent

WAB450-I-B-P-82-2-1

WAB450-24-3-P3-1-HB

IRAT13sativa parent

Koshihikari

Dular

0

Bars show standard errors

Figure 4. Soil water content at 40-60cm depth on September 11.

Relationship between stomatal conductance and dry weight
There was a significant correlation (r=0.907**) between average stomatal conductance anddry weight at harvest except glaberrima parent of NERICA (Figure 5). In low fertilization levelstomatal conductance and dry weight at harvest tended to be larger than in standard fertilizationlevel.
Dry Weigt at Harvest (g/m2)

1400

Asian rice
NERICA
African rice
Common millet
L Asian rice
L NERICA
L African rice
L Common millet

1200
1000
800

Common millet

600
African rice

400

Asian rice, NERICA
r=0.907**

200
0
0

20

40

60

80

100

120

140

Average Stomatal Coductance (mmol/m2/s)**: significant at 1%

Figure 5. Relationship between average stomatal conductance from August 7 to 20 and top dry weight at
harvest on Oct. 22.

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Proceedings of The 7 ACSA Conference 2011

Relationship between leaf thickness and dry weight
There was a significant correlation (r=0.892**) between average leaf thickness and dryweight at harvest except glaberrima parent of NERICA and cultivar with thick leaf tended tomaintain high dry matter production (Figure 6).

Dry Weigt at Harvest (g/m2)

1600

Asian rice
NERICA
African rice
Common millet
L Asian rice
L NERICA
L African rice
L Common millet

Common millet

1200

800
African rice

Asian rice, NERICA
r=0.892**

400

0
0.12

0.16

0.2

0.24

Average Leaf Thickness (mm)**: significant at 1%

Figure 6. Relationship between average leaf thickness from August 10 to September 12 and top dry weight at
harvest on Oct. 22.

Relationship between leaf thickness and SPAD value
There was a significant correlation (r=0.828**) between average leaf thickness and SPAD
value in rice (Figure 7). In one of NERICA leaf thickness and SPAD value were higher in low
fertilization level than in standard fertilization level. SPAD value tended to be low in Dular.

Average SPAD Value

50

40

rice
r=0.828**

30

Common millet

20
0.12

0.16
0.2
Average Leaf Thickness (mm)Asian rice
NERICA
African rice
Common millet
L Asian rice
L NERICA
L African rice
L Common millet

0.24**: significant at 1%

Figure 7. Relationship between average leaf thickness from August 10 to September 12 and average SPAD
value from August 10 to September 10.

Conclusions
Dry weight was high in Dular, but soil water content decreased especially in the standard
fertilization level. On the contrary dry weight was high in one of NERICA and soil water content wasmaintained high especially in low fertilization level and at deep soil layer. High leaf thickness andthProceedings of The 7 ACSA Conference 2011

117

SPAD values seem to be effective to maintain dry matter production under limited water condition
with high water use efficiency. Condon et al. (2004) showed cultivar differences in water use
efficiency in wheat. In rice, NERICA seems to be appropriate for the cultivation under drought
condition with the effective use of limited water for the sustainable crop production especially in low
fertilization level.

Acknowledgements
We thank Emeritus Professor Horie and Professor Shiraiwa, Kyoto University, for kindly giving an
opportunity to use seeds of NERICA lines in this study.

References
Blum A. 2009. Effective use of water (EUW) and not water-use efficiency (WUE) is the target of crop yield
improvement under drought stress. Field Crops Res. 112:119-123.
Boyer JS. 2010. Drought decision-making. J. Exp. Bot. 61(13): 3493-3497.
Condon AG, RA Richards, GJ Rebetzke and GD Farquhar. 2004. Breeding for high water-use efficiency. J. Exp.
Bot. 55: 2447-2460.
Fujii M, Andoh C and Ishihara S. 2004. Drought resistance of NERICA (New Rice for Africa) compared with
Oryza sativa L. and millet evaluated by stomatal conductance and soil water content In Fischer T et al.
New directions for a diverse planet: Proceedings for the 4th International Crop Science Congress,
Brisbane, Australia, 26 September - 1 October 2004. www.cropscience.org.au/icsc2004 5pages.
Fujii M, Y Miyamoto and S Ishihara. 2008. Studies on drought resistance of Asian rice (Oryza sativa L.) andth
NERICA -Water use in the field- In Abstracts of the 5 International Crop Science Congress & Exhibition,
April 13-18, 2008, Jeju, Korea: 209.
Serraj R, KL McNally, I Slamet-Loedin, A Kohli, SM Haefele, G Atlin and A Kumar. 2011. Drought resistance
improvement and resource management strategy. Plant Prod. Sci. 14(1)-- back to Table of Content --

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Social Experiment of Volumetric Irrigation Fee Scheme:
Case of Gravity Irrigation System in Bohol, the Philippines
Shigeki Yokoyamaa*, Kei Kajisab, Tatsuro MiyazakicaJapan International ResearchCenter for Agricultural Sceinces, Tsukuba, Japanb
International Rice Research Institute, Los Banos, the PhilippinescTsukuba University, Tsukuba, Japan* Corresponding author: Japan International ResearchCenter for Agricultural Sceinces,
Ohwashi 1-1, Tsukuba, 305-8686, Japan.
Tel.: +81 29 838 6343; fax: +81 29 838 6342.
syokoyam@affrc.go.jp

Abstract
Increasing in water productivity in rice is crucial as irrigated rice production consume a substantial part of thetotal world‘s fresh water. To promote the adoption of water saving technologies, farmers are provided with atangible incentive to save water is necessary. The social experiment survey of volumetric pricing had been
conducted in Bohol, the Philippines, where double rice cropping has been started since 2008 under gravity
irrigation. The preliminary results showed that both economic incentive and technical training promoted theefficient water use.
Keywords: social experiment, volumetric pricing, AWD, gravity irrigation, the Philippines

Introduction
The current scheme of irrigation fee, in most of Asia, is charged according to the area
irrigated. This scheme may induce overuse of irrigation water as the payment amount is fixed
regardless of the actual water use. Area basis charge scheme has long been practiced due totechnical difficulty of measuring water intake at farm level, for gravity irrigation system in particular.
Fresh water has become increasingly scarce resource worldwide due to increasing in demand of
urban and industrial use. Global warming and climate change may worsen this situation.
Considering that irrigated rice production consume a substantial part of the total world‘sfresh water use, increasing in water productivity in rice is crucial (Shivakoti et al 2005). To promote
the adoption of water saving technologies, providing farmers with a tangible incentive to save wateris necessary. Charging irrigation fee according to the volume of actual water use is such an
incentive. The social experiment survey of volumetric pricing had been conducted in Bohol, thePhilippines, where double rice cropping has been started since 2008 under gravity irrigation.
Research questions were: 1) Whether volumetric incentive effectively induces water saving efforts
of water users group, and 2) Whether training of water saving technology (Alternate Wetting andDrying, AWD) contributes efficient water use.

Materials and Methods
Bayongan Dam System of Bohol Integrated Irrigation System, Stage II (BIS-II), wasselected as the study site (Map) (Figure 1). The system was constructed with the financial andtechnical support of JICA (Japan International Cooperation Agency). The system started operationin May 2008 covering 4,000 ha (designed area).The main canal of 6,662 m and 15 laterals, LateralA to O, are fully lined, and each TSA (Turnout Service Area, unit of water users) is equipped withthProceedings of The 7 ACSA Conference 2011119

spindle gate, by which farmers can control water intake by themselves, and a level gauge by which
the volume of water is measured by the third party (Nippon Koei et al. 2007).
The experiment design was as follows. The volume of water intake was estimated basedon the gauge reading three times a day (morning, noon and afternoon) at head gate of each TSA.
Irrigation fee was firstly collected by area basis as usual, then to be paid back to TSA according to
the percentage of saved water based on the required water volume for conventional continuous
flooding practice.
Randomized field experiment was applied to 67 TSAs of upper portion of the systemamong total 147 TSAs. The data collections were conducted for five crop seasons from November
2008 to May 2011. No intervention was applied in the season 0 for all samples as the base line.
Volumetric incentives had been applied to the half of TSAs randomly selected for four seasons fromthe season 1 to the season 4. Technical training of AWD was provided to the half of experimental
TSAs and the half of control TSAs before the season 3 (May-Sep 2010). These two interventions
resulted in the four groups (Table 1).

Study Site
(Bohol Island)

Figure 1. Map of the Philippines.

Table 1. Number of sample TSAs by treatment and season
Season0
Nov08-Mar09

1
May-Sep09

2
Dec09-Apr10

34

34

33

33

67

3
Jun-Nov10
16
18
20
13

4
Dec10- May11
16
18
20
13

Treatment
CC
CA
VC
VA

CC: No intervention
CA: AWD training only
VC: Volumetric only
VA: Volumetric and AWD training

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Results and Discussion
In this analysis only the data of the season 0 to 3 were used, as the data of season 4 wasstill being processed. Profile of the sample TSAs is presented in Table 2. The average size of TSA
was around 15 ha of irrigated paddy field with 25 members of farmers. Double rice cropping waspracticed in irrigated paddy, while rice-upland crop in rainfed paddy and cassava in upland werecommon cropping systems. As the designed irrigation area was larger than the actual one, landleveling was still in process and irrigated area was gradually expanding. Main Farm Ditch (MFD)
was the soil canal draws water from a lateral into TSA area. National Irrigation Administration (NIA)
was responsible to design and construct MFD, while after completion its maintenance was assigned
to each TSA. Efficient water use and equity distribution among the members were highly dependenton operation and maintenance of MFD. Almost all of the TSAs organize a regular monthly meetingand collective maintenance work as members obligation. In average, water intake reduced by 30%
from Season 0 as baseline to Season 3, the third session of the experiment. Though other factors
affecting water intake such as rainfall should be carefully took into account, the experiment seems
successful as a whole.
Table 2. Profile of sample TSAs
n=67

Average

STDV

24.7

14.4

650.5

495.2

Season 0 (Nov08-Mar09)

14.36

9.32

Season 3 (Jun10-Nov10)

16.44

9.02

Season 0 (Nov08-Mar09)

10,6005,493

Season 3 (Jun10-Nov10)

7,308

4,964

Number of members/TSA
Season 0 (Nov08-Mar09)
Length of Main Farm Ditch (m)Season 0 (Nov08-Mar09)
Area irrigated (ha)3

Water intake (m /ha)Source: TSA interview survey, 2009.

Table 3. Rice yield under rainfed and irrigation by Lateral
Rainfed (2007)

Irrigated (2009)

LateralIrrg/RainnAverage (t/ha)

STDV

n

Average (t/ha)

STDV

A

28

2.39

0.67

84

2.79

1.43

1.17

B

5

2.46

0.59

12

2.22

0.86

0.90

C

NA

NA

NA

10

2.62

0.72

NA

D

11

2.68

0.83

29

2.92

1.59

1.09

E

5

2.04

0.55

14

2.87

1.6

1.41

F

13

2.02

1.14

33

2.42

1.39

1.20

G

5

1.86

0.9

14

2.67

1.45

1.44

Note: Lateral, A to G, aligns from up to down along the main canal.

Before evaluating the impacts of the experiment in detail, the effects of irrigation
construction were briefly examined. The rice yield increased by 20 to 40% after irrigation, with some
exception (Table 3). It should be noted that the effect was more obvious in down portion, suggestingth

Proceedings of The 7 ACSA Conference 2011121

irrigation contributed to not only production increase as a whole but also to mitigation of spatial
disequilibrium.
Change in Locational Water Distribution
The distribution of water intake by TSA in Season 0 and 3 are shown in Figure 2. As
expected, there was a tendency that TSAs in upper portion used more water than those in lower
portion both at lateral level and within a lateral. This finding was consistent with the frequently cited―Upstream vs Downstream problem.‖ Taking the advantage of position in the system, farmers in
upstream likely to take water excessively under the condition of area basis water fee. This caused
water shortage in the downstream, often resulting in conflict between the up- and downstream
farmers. It is worth to point out that additional water use did not necessary resulted in higher yield(Table 3). This suggested that saving water in upper portion could contribute to increase in total riceproduction of the system level, provided saved water is to be effectively distributed down portion
which suffers insufficient and unstable water supply.
Comparing the two seasons, a down ward slope from right to left became slightly flattened
after experiencing three experiments. The upper portion (Lateral A-D) saved more water than thelower E-F, suggesting saved water was transferred to down portion (Lateral G and after), resulted in
more equitable water distribution in the system-wide.
(m3/ha)
30000

Season 0 (Nov08-Mar09)
25000

y = -248.54x + 14953
R² = 0.2259

20000

15000

TSA
10000

5000

A-1
A-3
A-6
A-7
A-9
A-10
A-11
B-3
B-4
C-1
C-2
C-4
D-1
D-2
D-3
D-4
D-6
D-7
D-8
D-9
E-1
E-2
E-3
E-4
E-5
F-1
F-4
F-5
F-6
F-7
F-8
F-9
F-10
F-11
F-13

0

30000

Season 3 (Jun10-Nov10)
25000

y = -201.85x + 9260.2
R² = 0.2189

20000

15000

10000

TSA
5000

A-1
A-3
A-6
A-7
A-9
A-10
A-11
B-3
B-4
C-1
C-2
C-4
D-1
D-2
D-3
D-4
D-6
D-7
D-8
D-9
E-1
E-2
E-3
E-4
E-5
F-1
F-4
F-5
F-6
F-7
F-8
F-9
F-10
F-11
F-13

0

Figure 2. Spatial distribution of water intake by TSA.

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Proceedings of The 7 ACSA Conference 2011

Effects of Experimental Intervention
For further examining farmers‘ response to experimental interventions, changes in water
intake by experimental group were compared. Average of water intake by experimental group ispresented in Figure 3. Corresponding to the difference of experimental interventions, CC and CA
were treated as one group, same as VC and VA, from Season 0 to 2. As the samples were selected
randomly, water intakes of the two groups were mostly identical at the baseline (Season 0). The
volume has been consistently declined including the control group (CC). The factors affecting
overall water intake other than the experiment are rainfall, amount of water release from the dam,
NIA‘s promotion of water saving technology. To evaluate the experiment effects separately fromthese factors, deviation of experimental groups from the control group should be focused. Economic
incentive for water saving was effective, and efficacy of water saving efforts seem to be intensified
as time goes. It seems that once farmers are convinced of no adverse effects on crop production(Season 1), they continue water saving efforts and learned effective ways by themselves (Season
2-3).
Providing technical training also showed its effectiveness, as the group of AWD training
only (CA) substantially declined their water use compared with the control group (Season 3). There
was anecdotal evidence from farmer interview, indicating ADW or intermittent irrigation may
enhance yield by increased number of tillers and mitigating damage of golden apple snail. During
the experiment period, farmers had chance to learn these information from their neighbors. Part ofthe sharp decline from Season 2 to 3 might be explained by this spillover effect. In addition, farmers
had also chance to attend trainings by NIA and other institutions such as extension offices andNGOs. The synergy effect of economic incentive and training was not obvious. The performance ofthe group of volumetric and training (VA) is not so striking compared with the group of volumetric
only (VC) and training only (CA) (Season 3).
(m3/ha)

CC
CA
VC
VA

11000

10000

9000

8000

7000

6000
0

1

2

3

Experiment Season
CC (n=16): No intervention;
VC (n=20): Volumetric only;

CA (n=18): AWD training only
VA (n=13): Volumetric and AWD training

Figure 3. Change in water intake by experimental group.

Conclusions
The preliminary results of social experiment for volumetric water pricing clearly show 1)
farmers well respond to economic incentive to save irrigation water use, 2) technical training was
also effective to reduce water intake independently of economic incentive, 3) saved water was
efficiently redistributed to down portion, enhancing system-wide equity. In this preliminary studysocial factors, which are highly expected to influence collective actions like irrigation management,thProceedings of The 7 ACSA Conference 2011123

were not considered. Reduced water may entail the risk of yield loss, while technical training andcollective actions for water saving may contribute to yield increase and stabilization. These were theremaining issues for further analyses.

References
Nippon Koei Co Ltd, PKII Engineers, Integrated Philconsult, Inc and Sustainable Development Solutions, Inc.
2007. Bohol Irrigation Project Phase II, Operation and Maintenance Manual for Bayongan & Capayas
System Vol. 3.
Shivakoti GP, DL Vermillion, WF Lam, E Ostrom, U Pradhan and R Yonder eds. 2005. Asian irrigation intransition: Responding to challenges. Sage Publication, New Delhi. pp 528.

-- back to Table of Content --

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Proceedings of The 7 ACSA Conference 2011

Ultraviolet-induced Fluorescence of Rice Leaf as Influenced
by Nitrogen Application and Cultivars
Mikio Sekinumaa,*, Takehiro Natorib, Futoshi Sasakia, Futoshi Katob, Naoto Inouea,baInterdisciplinary Graduate school of Science and technorogy, Shinshu University, Japanb
Faculty of Agriculture, Shinshu University, Japan* Corresponding author: Shinshu University, 8304 Minamiminowa, 399-4598 Nagano, Japan.
Tel.: +81 265 77 1409; fax: +81 265 77 1409.
sekinuma.cultivator@gmail.com<
Abstract
New measurement system of ultraviolet-induced fluorescence was developed for detecting the secondary
metabolic materials and chlorophyll fluorescence in order to evaluate the photosynthetic activity of crops. In thisstudy, 4 rice cultivars; Koshihikari, IR72, Banten (traditional javanica variety), and CH86 (traditional indica
variety), were grown in pots and nitrogen fertilizers were added in 5 different levels. Fluorescence of leaves wasmeasured before and after the heading stage. Leaf blades were excited by light of 370 nm wavelength, which
was derived by passing light from xenon lamp through a band-pass filter. The results showed that thefluorescence peaks were located in the region of 400-650 nm, 685 nm and 740 nm, and the spectrum patterns
varied among cultivars and different nitrogen levels. Fluorescence intensity (400-650 nm wavelength) observed
under high nitrogen condition were relatively low as compared with observed under low nitrogen conditions.
These results indicated that the fluorescence pattern of the leaf blade was affected from plant growth andnitrogen level of soil. Our findings show that spectrum analysis of ultraviolet-induced fluorescence is useful forevaluation of rice plant nutrition.
Keywords: ultraviolet-induced fluorescence, nondestructive, rice

Introduction
Development of plant nutrition diagnosis systems is required for sustainable foodproduction in order to conserve resources. Diagnosis of crop is able to provide good information
about plant conditions, especially the nutrition status of a plant. Moderate amounts of fertilization
make it possible to conserve resource, and cost and to half nutrient enrichment of the soil.
In rice cultivation, increase of yield was established by restricting nitrogen supply in the
middle of the growth stage (Matsushima et al., 1964; Matsushima et al., 1966), as excess nitrogencaused lodging. Nitrogen restriction was determined with basis on leaf color (Matsushima et al.,1970; Matsuzaki et al., 1974). This cultivation method was termed the V-shaped rice cultivation.
Using leaf color to enable diagnosis is delaminated with information only of the leaf surface.
However, recent studies have attempted express to express the condition of a plant throughanalysis of internal leaf conditions; many methods have succeeded more easily with indirect
estimation. Direct detection will be expected to estimate photosynthetic ability and function in the
future of diagnosis.
A useful diagnosis method is light detection by chlorophyll fluorescence. Fluorescence
detection has been used as a nondestructive, noncontact, and continuous detection method toevaluate photosynthetic ability (Zoran et al., 1999). Chlorophyll concentration and water stress aremonitored by calculating the ratio of chlorophyll fluorescence (Saito, 2005).
The choice of an excitation light source is important in chlorophyll fluorescence because it
determines the fluorescence information that can be obtained. Visible (blue) excitation for plants
emits both red (RF, 630–700 nm) and far-red fluorescence (FRF, 700–800 nm). Ultraviolet (UV)
excitation emits RF, FRF, and blue-green fluorescence (BGF, 400–630 nm). The BGF spectrumthProceedings of The 7 ACSA Conference 2011125

includes information on leaf materials and structure, and this information reveals plant cultivation
conditions that are related to chlorophyll a information.
In this study, we developed a new UV-induced fluorescence measurement system todetect the fluorescence of secondary metabolic materials and of chlorophyll in order to evaluate thephotosynthetic activity of crops. To achieve this purpose, we investigated whether our system wasable to distinguish rice cultivar and nitrogen fertilizer levels.

Materials and Methods
Plant material
Four rice cultivars, Koshihikari (Japonica), IR72, Banten (traditional javanica variety), andCH86 (traditional indica variety), were grown in pots and nitrogen fertilizers were added in 5
different amounts (Table 1). The rice was grown in the Shinshu University greenhouse in Nagano
Prefecture (35°51′N, 137°56′E, 740 m above sea level). Measurement of leaf fluorescence wascarried out twice, before and after the heading stage, in 2010. The fluorescence of the cut leaves
was measured after dark adaption for 30 min at 25°C. Room temperature was maintained at 25°C
with air conditioning.
Table 1. Plant material

Cultivar

Ecotype

Plant type

Notes

Koshihikari

Japonica

Standard

Japanese commercial var.

IR72

IndicaMulti tillering

BANTEN

Javanica

Large panicle

CH86

IndicaGrassyYellow leaf

Fluorescence measurements
Figure. 1 illustrates the new fluorescence measurement system (FMS) for detection of thefluorescence spectrum. In this system, leaf blades were excited by a 370 nm-wavelength lightderived by passing light from a xenon lamp (LAX101, Asahi Spectra Co., Ltd., Japan) through aband-pass filter at 370 nm (XBPA370, Asahi Spectra Co., Ltd., Japan). UV light was directed to thetarget leaf through a quartz fiber. The band-pass filter had a center wavelength of 370 nm with a 10
nm half-bandwidth. The detector unit was a Photonic Multichannel Analyzer (PMA-11, Hamamatsu
Photonics, Japan). A long-pass filter (XUL0400, Asahi Spectra Co., Ltd. Japan) was positioned in
front of the detection fiber and the reflection of light below a 400 nm wavelength was ensured. The
detected fluorescence wavelength was 400–850 nm. The distance between the edge of the fiber
and the leaf sample was adjusted to approximately 5 mm. The PMA was operated in 50 msec
durations for each leaf area (point) and this was repeated 1000 times in the same area to determine
fluorescence excitation. Each leaf sample consisted of 5 areas (point). The acquisition spectra were
determined as the highest peak of the spectrum that had a maximum intensity at 680 nm and 740
nm in the 1000 runs. The acquisition spectra were identical to the mean spectra of peak timing in
the Kautsky curve (Govindjee, 1995).

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Proceedings of The 7 ACSA Conference 2011

(360nm<λ＜380nm)
Bandpass
filterLongpass
filter(400nm<λ)Coaxialfiver

Exciting unit
(Xenon light)

Detectingunit(Photonic
Multichannel
Analyzer)
Date

Fluorescence
Leaf blade

PersonalcomputerFigure 1. Schematic diagram of fluorescence measurement system. Detected bandwidth of spectrum had
wavelengths of 400–850 nm.

Results and Discussion
Figure 2 indicates the fluorescence patterns of leaf blades at various nitrogen levels. The
fluorescence peaks were in the region of 400–650 nm, 685 nm, and 740 nm, and the spectrum
patterns varied with the different nitrogen levels. The intensity of BGF observed under high-nitrogenconditions was low compared with BGF intensity observed under low-nitrogen conditions.
600(A) Koshihikari

5Ng/m220Ng/m2

400
300
200
100
0
400

500

600

700

800

2

2
20Ng/m2
20 Ng/m

400
300
200
100

400

2

5Ng/m25 Ng/m

2

300
200
100

Fluorescene intensity (Counts)

0 Ng/m0Ng/m2

2
20Ng/m2
20 Ng/m

400

500

600

700

800

900

Wavelength (nm)

600

500

2

5Ng/m25 Ng/m

0

900

600

(C) Banten

0 Ng/m0Ng/m2500

Wavelength (nm)Fluorescene intensity (Counts)(B) IR72

0Ng/m2500

Fluorescene intensity (Counts)

Fluorescene intensity (Counts)

600

20 Ng/m0Ng/m2(D) CH86

5Ng/m25 Ng/m500

2

2
20Ng/m2
20 Ng/m

400
300
200
100
0

0
400

500

600

700

800

900

Wavelength (nm)

400

500

600

700

800

900

Wavelength (nm)(A) Koshihikari, (B) IR72, (C) BANTEN, (D) CH86.

Figure 2. Fluorescence spectra of rice leaf. The spectra by excitation at 370 nm were measured at the leaf apex
prior to the heading stage.

thProceedings of The 7 ACSA Conference 2011127

Figure 3 illustrates the fluorescence spectrum of each cultivar. Fluorescence intensity
varied with each cultivar, and high BGF intensity was obtained from the Koshihikari spectrum.
These results might describe the fluorescence information containing the condition of plant
metabolic material affected by nitrogen levels. The effect of nitrogen treatment was related to BGF
intensity. BGF is caused by excitation of ferulic acid derivatives, other phenylpropanoids, andNAD(P)H; RF and FRF are caused by excitation of chlorophyll a (Cerovic et al., 1999). BGF
enables monitoring of the influence of nitrogen on the degree of growth. We believe that this system
can function as a nitrogen level monitor at the same growth stages in rice.

Fluorescene intensity (Counts)

600

Koshihikari

500

IR72

400

CH86

BANTEN

300
200
100
0

400

500

600 700 800
Wavelength (nm)900

Figure 3. Fluorescence spectra for the cultivars. The spectra by excitation at 370 nm were measured at the leaf
2
apex prior to the heading stage. All cultivars were grown at 5 Ng/ m .

The fluorescence spectrum of each cultivar demonstrated different patterns. However, we
also need to consider leaf structure, rate of development, and the response to nutrients. In
fluorescence detection, it has been suggested that a balance of FRF and BGF enables estimationof wheat lamina growth (Meyer, 2003). In this study, the balance of FRF and BGF may haveenabled monitoring of the degree of growth for the cultivars. This result suggests that the detection
for cultivars requires further testing and analysis in combination with their growth rates andconditions.
In conclusion, the differences in these results indicate that soil nitrogen levels and different
cultivars influence the fluorescence patterns of leaf blades. Our findings show that spectrum
analysis of UV-induced fluorescence is useful for evaluation of rice plant nutrition. In the future, UV
laser-induced fluorescence methods will be developed if a new diagnosis system becomes
necessary.

References
Govindje E. 1995. Sixty-Three Years Since Kautsky: Chlorophyll a Fluorescence. AUST. J. PLANT PHYSIOL.
22: 131–160.
Matsushima S, Matsuzaki A and Tomita T.1964. Analysis of Yield-Determining Process and Its Application to
Yield-Prediction and Culture Improvoment of Lowland Rice : ⅥⅩⅦ. Studies on the principles for
maximizing yield and their demonstration (1). JPN. J. CROP. SCI. 32: 48-52.
Matsushima S, Wada G and Matsuzaki A.1966. Analysis of Yield-Determining Process and Its Application to
Yield-Prediction and Culture Improvoment of Lowland Rice : ⅦⅩⅣ. Studies on the principles for
maximizing yield and their demonstration (3). JPN. J. CROP. SCI. 34: 321-328.
Matsushima S, Matsuzaki A and Tomita T.1970. Analysis of Yield-Determining Process and Its Application to
Yield-Prediction and Culture Improvoment of Lowland Rice: ⅭⅠ. On a method for expressing the leafcolour of rice plants under field conditions (1). JPN. J. CROP. SCI. 39: 231-236.

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Matsuzaki A, Matsushima S, and Tomita T.1974. Analysis of Yield-Determining Process and Its Application to
Yield-Prediction and Culture Improvoment of Lowland Rice : ⅭⅩⅩ. Effects of the nitrogen restriction on
the grain yield juded from the frowth amount, the leaf color ant the percentage of stained length of leaf
sheath. JPN.J.CROP. SCI. 43: 167-173.
Meyer,S. A. Cartelat, I. Moya and Z. G. Cerovic. 2003. UV-induced blue-green and far-red fluorescence along
wheat leaves: a potential signature of leaf ageing. J. Exp. Bot. 54 (383): 757-769.
Saito Y, Matsubara T, Kobayashi F, kawahara T, and Nomura A. 2005. Laser-induced fluorescence
spectroscopy for in-vivo monitoring of plant activities. The 7thFruit, Nut and Vegetable Production
Engineering Symposium: 699-708
Zoran G. Cerovica, Guy Samsonb, Fermín Moralesc, Nicolas Tremblayd and Ismaël Moyaa. 1999. Ultravioletinduced fluorescence for plant monitoring: present state and prospects. AGRONOMIE. 19 (7):543 – 578.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011129

Locus for Malate Secretion in Rice Chromosome 3
Miftahudin1*, Irfan Martiansyah1, Dewi Indriyani Roslim2, Tatik Chikmawati1
1

Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Kampus
IPB Darmaga, Bogor-Indonesia
2
Department of Biology, Faculty of Mathematics and Natural Sciences, Universiy of Riau Pekanbaru, Riau
*Corresponding author: miftahudinm@yahoo.com

Abstract
Aluminum (Al) toxicity is a major limiting factor of rice production in acid soil. One of the tolerance mechanismsof plant to Al stress is the secretion of malate from plant root. The objective of this research is to identifiyquantitative triat loci (QTL) in chromosome 3 that controls malate secretion from rice root during the period of Al
stress. The research was conducted in laboratory and green house. An F2 population derived from the crossbetween rice genotype Hawara Bunar and cultivar IR64 was used in this experiment. Analysis malate secretion
was carried out based on enzymatic method. Rice simple sequence repeats from chromosome 3 were as
moelcular marker. The mapping and QTL anaysis was performed using Mapmaker 3 and Mapmaker/QTL. The
result showed that the malate secretion trait was normally distributed in the rice F2 population indicating that thetrait is polygenic trait. A QTL for malate secretion was identified in the short arm of rice chromosome 3 located in
between markers RM545 and RM517.
Keywords: Aluminum stress, Chromosome 3, Rice, QTL

Introduction
In general, aluminum tolerance in plant is genetically controlled, and the diversity of thistrait can be found inter and intra plant species, including the member of Gramineae (Aniol andGustafson 1990). Aluminum tolerance mechanism in plant can be divided into two models (Kochian
1995, Matsumoto 2000). First, internal detoxification mechanism, which is the mechanism of plant
cells that are able to detoxify Al in the cell through organic acid chelation (Ma et 1998), vacuolar
accumulation, protein detoxification, or reactive oxigen species reduction in the root cells (Ezaki etal 2000). Internal detoxification mechanism can also be achieved through activation of calmodulinindependent NAD+ kinase (Aniol, 1991).
In the second model, Al is excluded from root tip cells through excluding Al accors plasma
membrane, increasing rhizosfeer pH, producing exudate or secreting organic acid (Taylor 1991,
Kochian 1995). Among those external mechanisms, organic acid secretion from root cells, such
malate, citrate and oxalate, has been the most accepted mechanism. Organic acid secretion has
important role in Al tolerance mechanism in wheat, rye and maize (Ryan et al 1995, Li et al 2002,
Pellet et al 1995), however the similar role has not been elucidated in rice.
Several researchers have shown that organic acid secretion closely related to Al tolerance
mechanis. However, the main factor that controlled organic acid secretion from root cells during Al
stress still unclear. The recent finding showed that over expression of malate transporter gene thatisolate from wheat could increase Al tolerance in transgenic tobacco, but the gene could notincrease Al tolerance in rice (Sasaki et al 2004).
This paper reported our research in identifying locus for malate secretion trait in rice using
an F2 population derived from a cross between an Indonesian local rice genotype that tolerant to Al
and a rice variety IR64 that sensitive to Al. It is expected that the locus can be used to isolate thegene controlling malate secretion in rice.

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Materials and Method
Plant Materials
Rice genotype Hawara Bunar (Al-tolerant parent) and cultivar IR64 (Al-sensitive parent),
F1 plants and F2 population derived from a cross between Hawara Bunar and IR64 were used inthis research.
Nutrient culture technique for alumininum stress treatment
A simple hydrophonic technique was used to grow rice seedlings. Seeds were sterilized
and soak in destilate water for 24 hours, and followed by germination for 48 hours. The seedlings
with homogenous root length were put in sterofoamfloated on sterile minimal nutrient solution(Miftahudin et al. 2004) at pH 4.0 in 15 ml tube. A 15 ppm of Al in the form of AlCl3.6H2O wasadminitered fro 24 hours. The seedlings were grown at room temperature in growth chamber with
12 hours light photoperiod.
Determination of malate secretion
Malate secretion was determined from F1 plants, 400 individual F2 population and bothparents. Determination of malate secretion followed procedure as described by Delhaize et al.
(1993). A 1.35 ml nutrient solution was sampled from each tube and was added with 1.5 ml buffer
solution (0.4 M hydrazine and 0.5 M glycine, pH 9) and 0.1 ml 40 mM NAD. The reaction mixtures
were thenincubated at room temperaturefor 30-60 min. The absorbance of the mixture wasmeasured using spectrophotometer at λ =340 nm (absorbance 1). The mixture was then added with
5 μL Malate Dehidrogenase (5 mg/mL, Sigma, USA), and the absorbance 2 was thenmeasured.The difference between absorbance 1 and 2 indicated the amount of NADH produced
and was used to calculate malate content of the solution as the following equation:
VxMC=--------------------------------- x ▲ A
ε x d x v x 1000
Notes: C = Malate concentration (mg/l)
V = Volume Total (ml)v = Sample Volume (ml)M = Molecular Weight of Malate (g/mol)d = Cuvette width (cm)ε = Absorption coefficientof NADH at λ 340 nm = 6.3 (l/mmol.cm)▲A = the difference between absorbance 1 dan 2
Moelcular marker analysis
The polymorphic SSR markers from rice chromosome 3 were applied to 300 individuals F2
population. The primers from those markers were used to amplify DNA. A 50 ul PCR reaction
mixture consist of100 ng DNA, 100 mM Tris-Cl pH 8.0, 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs,
0.3-0.5 uM tiap primer, and 1 U Taq DNA polimerase (NEB, USA) was made for each individual
plant. PCR process were performed using thermocycler with the following condition: 1 cycle of
94oC for 5 minutes followed by temperature cycles of94oC (35 second), 55oC (35 second), and 72oC
(1 minutes, 45 second) for 35 cycles and finally one cycle of 72oC for 10 minutes . PCR products
were analyzed using 2.5 - 3.0% superfine agarose gel electrophoresis in 0.5 x TBE (Tris-BorateEDTA) buffer.

thProceedings of The 7 ACSA Conference 2011131

Genetic mapping and QTL identification
Segregation analysis of molecular marker in the F2 population was performed using Chi
Squaretest pada  = 0.05. Genetic linkage and QTL analyses usedMAPMAKERS/EXP ver 3.0
andMAPMAKERS/QTL ver 1.1, respectively.

Results and Discussion
Malate secretion in parent plants under Al stress
Malate secretion was analyzed from nutrient culture media that has bee used for growing
F1, F2 and both parent seedling under 72 hours Al stress. The result showed that the malate
secretion was higher when the seedlings were Al stressed and the secretion increse as the periodof stress increase (Figure 1 and 2). The Al stress at thelevel of 15 ppm could differentiate malate
secretion level from both parents. In rice cv IR64, although malate secretion increase as theincrease of stress period, there was no significant difference between malate scretion at 0 and 15
ppm of Al stress along the 72 hour period of stress. Conversely, there wassignificant difference of
malate secretion along the 72 hours stress period between 0 and 15 ppm Al stressin Hawara
Bunar.The significant increase of malate secretion occurred when the seedlings were Al-stressed at
15 ppm at 72 hours. Therefore, the Al stress level of 15 ppm for 72 hour stress duration was used
for phenotyping the F2 population. The result also showed that the average malate secretion from
IR64, Hawara Buanr, F1 and F2 plants were 2.2, 3.8, 3.1 and 4.1 ppm, respectively.

ab

Figure 1. Malate secretion from, rice root cv IR64 (a) and Hawara Bunar (b) along the 72 hour period of Al
stress.

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thProceedings of The 7 ACSA Conference 2011

Distribution of Malate Secretion in F2 Population
Analysis of malate secretion in 400 individual F2 population showed normal distribution(Figure 2), which indicated that malate secretion character was multigenic controlled. It can beseen from the Figure 2 that the distribution curve rather skewed to the right and the secretion classfalled to higher value than that both parents. This is suggested transgresive segregation
phenomenon in this population. The average malate secretion of F2 population was also higher
than that of Hawara Bunar.
When the malate secretion was grouped into two class of secretion based on the range ofeach parent malate secretion, malate scretion in F2 population followed monogenic inharitance.
For individual F2 that secreted malate < 3 ppm, it was grouped into Al-sensitive plants, otherwise
the plants were grouped into Al-tolerant plants. The Chi square test showed that the segregation fit
to 3:1 ratio for Al-tolerant to Al-sensitive plants (Table1).

Figure 2. Distribution of malate secretion among individual F2 population.

Table 1. Chi square test for monogenic inharitance pattern of malate secretion in F2 population
Malate Secretion Class(ppm)

Number of Observed
PlantsNumber of
Expected Plants
(3:1)

< 3.0

79

91

Chi test

0.15
≥ 3.0

285

273

Locus for malate secretion in rice chromosome 3
Analysis of genetic factor controlling malate secretion trait in rice was performed into two
approaches. First, malate secretion trait was treated as polygenic trait based on the normal
distribution of malate secretion in the F2 population, and second, malate secretion trait was treatedas monogenic trait. The result showed that among the three chromosome that habe been
analyzed, which were chromosome 1, 2 and 3, the locus for malate secretion trait was only possibly
found om chromosome 3. QTL analysis based on poygenic trait indicated the presence of the QTL
in the short arm of chromosome 3 in the region between markers RM%$% and RM517. However,the LOD score in that region maximum only 0.98, which was not enough to conclude that the QTL
present in that region.
When analysis was performed based on monogenic trait, it was found that a locus formalate secretion trait present in the region with LOD 3.02. Genetic map of the rice chromosome 3that harbored the locus for malate secretion trait contained five markers i.e. RM569, RM545,
RM517, RM251, and RM232 with the total distance 131.8 cM (Figure 3). The locus for malate
secretion trait was located in between marker RM545 and RM517 with the exact position at 4 cM
thProceedings of The 7 ACSA Conference 2011133

from RM517 toward RM545. The result of this research was similar to Nguyen et al (2001) who
found QTL position for Al tolerance loci in the short arm of rice chromosome 3, but the position israther shifted to other region of the short arm. This difference was due to the different ricebackground used in both experiments.
RM569
43.1
RM545
14.5
RM517

MALATE

38.1
RM251
36.1
RM232
Figure 3. Genetic map of malate secretion locus in rice chromosome 3.

Conclussion
Rice secreted malate during the period of aluminum stress. Al tolerant rice genotype
secreted malate more than that of Al-sensitive rice genotype. The secretion increased as theduration of stress increase. There was a locus controlling malate secretion located in the short armof rice chromosome 3.

Acknowledgement
This research is supported by Basic Research Insentive Grant from Indonesian Ministry of Research
and Technology Year 2009 granted to Dr. Miftahudin.

References
Aniol A (1991) Genetics of acid tolerant plant. In Wright RJ, Baligar VC, Murrmann RP (eds) Plant-Soil
Interactions at Low pH. Kluwer Acad Publ, Dordrecht, pp.1007-10017
Aniol A, Gustafson JP (1990) Genetics of aluminum tolerance in agronomic plants. In Shaw AJ (ed) Heavy
Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, Florida, pp.255-267
Delhaize E, Ryan PR, Randall PJ (1993b) Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminumstimulated excretion of malic acid from root apices. Plant Physiol 103:695-702
Ezaki B, Gardner RC, Ezaki Y, Matsumoto H (2000) Expression of aluminum-induced genes in transgenic
Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress. Plant Physiol 122:657-665
Kochian LV (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annu Rev Plant Physiol
Plant Mol Biol 46: 237-260
Li XF, Ma JF, Matsumoto H (2002) Aluminum-induced secretion of both citrate and malate in rye. Plant andSoil 242:235-243
Ma JF, Hiradate S, Matsumoto H (1998) High aluminum resistance in buckwheat. II. Oxalic acid detoxifies
aluminum internally. Plant Physiol 117:753-759
Matsumoto, H (2000) Cell biology of aluminum toxicity and tolerance in higher plants. Intl Rev Cytol 200:1-46

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Miftahudin, Scoles GJ, Gustafson JP (2004) Development of PCR-based codominant markers flanking the Alt3
gene ini rye. Genome 47:231-238
Nguyen VT, Burow MD, Nguyen HT, Le BT, Le TD, Peterson AH (2001) Molecular mapping of genes
conferring aluminum tolerance in rice (Oryza sativa L.). Theor Appl Genet 102:1002-1010
Pellet DM, Grunes DL, Kochian LV (1995) Organic acid exudation as an aluminum-tolerance mechanism inmaize (Zea mays L) Plant 196:788-795
Ryan PR, Delhaize E, Randall PJ (1995) Characterization of Al-stimulated efflux of malate from the apices of
Al-tolerant wheat roots. Planta 196:103-110
Taylor GJ (1991) Current views of the aluminum stress response: The physiological basis of tolerance. Curr
Topics Plant Biochem Physiol 10:57-93-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011

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Responses of Nutrient Efficient Maize Genotypes to Bio-fertilizer
at Low Chemical Fertilizer Doses
Marlin Sefrilaa, Munandara, Renih Hayatia,* , SabaruddinaaPlant Science Department, Graduate School, Sriwijaya University,
Jl Padang Selasa No 524, Bukit Besar, Palembang, 30139, Indonesia* Corresponding author: Tel.: +62 711 354222; fax: +62 711 317202
renih_hayati@yahoo.com

Abstract
High dose of chemical fertilizer is usually required to obtain high maize (Zea mays L.) yield. Bio-fertilizer
application in combination with nutrient efficient genotype was expected to reduce the chemical fertilizer needwithout reducing the yield. The objective of the research was to evaluate the responses of nutrient efficientgenotypes to bio-fertilizer at low chemical fertilizer doses. The field experiment was conducted at Agro Techno
Park (ATP), South Sumatra. The treatments were arranged in Split-Split-Plot Design with three replications. The
main plots were chemical fertilizer doses, which were 25, 50, and 100 % of standard dose at ATP (400, 100, and-1
50 kg ha Urea, SP-36, and KCl, respectively). The sub-plots were nutrient efficient genotypes (B41, L167, andS219), and hybrid B 816 as comparison. The sub-sub-plots consisted of treatments with or without bio-fertilizers.
-1
The bio-fertilizer (10 ml L ) was sprayed to the soil around the plants at 2, 4, 6, and 8 weeks after sowing. The-1yields of nutrient efficient genotypes (5.23 to 7.89 ton ha ) were comparable to the yield of B 816 (6.58 to 7.62
-1
ton ha ) at all chemical fertilizer doses without bio-fertilizer. The genotypes responded differently to the biofertilizer application. Supply of bio-fertilizer at chemical fertilizer doses of 50 % tended to increase the yield of B
41 up to 16 % of its yield at 100 % chemical fertilizer dose without bio–fertilizer. This suggested that thechemical fertilizer dose can be reduced by 50 % only for B 41.
Key words: bio-fertilizer, low input, maize, nutrient efficiency

Introduction
High dose of chemical fertilizer is usually required to obtain high maize yield, especially forSouth Sumatra soil with deficiency of macro elements problem due to low soil pH (< 5.6). However,poor farmers often cannot afford it. According to Djafar and Halimi (1998), the farmers in South
Sumatra only apply the chemical fertilizer with sub optimum rate for maize production (75, 50, and
50 kg ha-1 Urea, SP 36, and KCl, respectively). An excessive use of chemical fertilizer can also
cause an environmental problem. Nutrient efficient maize genotype in combination with bio-fertilizer
may offer an alternative to overcome the problem.
Nutrient efficient genotype has the ability to produce a higher yield than other genotypes
under low nutrient supply (Presterl et al., 2003; Worku et al., 2007). Department Agronomy,
Sriwijaya University is now in the process of developing the nutrient efficient open pollinated maize
genotypes and B 41, S 219, and L 164 used in this experiment are some of them (Munandar et al.,2010).
Bio-fertilizer usually contains microorganisms having specific function such as Azospirillum
to fix N2 and P solubilizing bacteria to solubilize P from the soil and fertilizer to be available to theplants (Saraswati & Sumarno, 2008). Several researchers had conducted the experiments toevaluate the responses of various plants such as young Robusta coffee (Junaedi et al., 1999),
soybean (Noor, 2003; Totok & Rahayu, 2007), and turfgrass (Guntoro et al., 2007) to the biofertilizer application, but the results were still inconsistent. Further research is still needed in this
area.

th

Proceedings of The 7 ACSA Conference 2011137

Bio-fertilizer application in combination with nutrient efficient genotypes in this research
was expected to reduce the chemical fertilizer supply up to 25% of the standard dose without
reducing yield. The objective was to evaluate the responses of several nutrient efficient genotypes
compared to the hybrid to bio-fertilizer application especially at low chemical fertilizer rate.

Materials and Methods
The experiment was conducted at Agro Techno Park (ATP), South Sumatra in January to
April 2011. The soil was dominated by sand (69.91%) with 4.4 pH, 28.3 g kg-1 C organic matter, 2.1
g kg-1 N, 59.1 mg kg-1 P-Bray, and 0.19 c mol kg-1 exchangeable K. The experimental design was
Split-split-Plot with three replications. The main plot, the chemical fertilizer doses, consisted of 25,
50, and 100 % of the standard rate at ATP (400, 100, and 50 kg ha-1 Urea, SP 36, and KCl,
respectively). The split plot was the genotypes which consisted of B 41, S 219, and L164 as thenutrient efficient genotypes and Hybrid B 816 as comparison. The split-split plots were treatmentswith and without bio fertilizers which consisted of two types, Subur (bio fertilizer 1), and EM 4 (biofertilizer 2). Seeds were sown manually with 70 cm x 25 cm row spacing and two seeds per hole.
Plants were thinned into one plant per hole at one week after sowing (WAS), so in each 2.8 m x 2.5m sub-sub plot contained 40 plants. Chemical fertilizers (1/3 of Urea, SP 36, and KCl) were appliedat sowing and the rest of Urea was applied at 4 WAS. Organic fertilizer (10 ton ha-1) was applied at
2 WAS. Bio-fertilizer (10 ml L-1) was sprayed to the soil at 2, 4, 6, and 8 WAS as recommended
application time. Water was sprayed to the soil around the plants for treatment without bio- fertilizer.
All ears of each sub-sub-plot were harvested at harvest maturity, counted, and air dried in
the drying room for two weeks, weighted .and converted into yield (ton ha-1). Ears from threesample plants (randomly chosen) per plot were weighted and the average was used to get the ear
weight per plant data.

Results and Discussion
The yield of nutrient efficient genotypes (B 41, S 219, and L 164) at all chemical fertilizer
doses without bio-fertilizer (5.23 to 7.89 ton ha-1) was comparable to the yield of hybrid B 816 (6.58
to 7.62 ton ha-1). Among the nutrient efficient genotypes, S 219 had the lowest yield followed by B
41 and L 164 genotypes, especially at 25 and 50% chemical doses (Table 1). This suggested that L
164 genotype in this experiment had the highest nutrient use efficiency compared to B 41 and S
219 genotypes. The result was consistent with the previous experiment (Munandar et al., 2010).
The yield or ear weight (ton ha-1) significantly correlated with ear weight plant-1 (r = 0.54*).
Bio-fertilizer application had no significant effect on yield (data not presented), but tended
to increase yield only for B 41 and S 219 genotypes especially for Bio-fertilizer 1 (Table 1). The
yield of B 41 at 50 % chemical fertilizer dose was 94 % of its own yield at 100 % chemical fertilizer
rate without bio-fertilizer. The yield of B 41 genotype increased only 16 % by the application of Biofertilizer 1 or 9 % by bio-fertilizer 2 (Table 1). The yield of S 219 genotype increased 8 to 11 % at 25
and 50 % chemical fertilizer dose, respectively, but the values after the addition of bio-fertilizers
were still lower than the yield at 100 % chemical fertilizer dose without bio-fertilizer. The datasuggests that the chemical fertilizer dose can be reduced by 50 % with the application of biofertilizer only for B 41 genotype. The effect of genotype on yield was higher than the bio-fertilizer(data not presented).
The L 164 genotype and hybrid B 816 did not give positive responses to both bio-fertilizers
probably because their yields at all chemical fertilizer doses without bio-fertilizer were higher than
the yields of B 41 and S 219. It indicated that L 164 genotype and hybrid B 816 had an ability to

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Proceedings of The 7 ACSA Conference 2011

take up or use nutrient more efficient than that of the B 41 and S 219 genotypes as suggested by
Worku et al.( 2007).
The bio-fertilizers used in this experiment contained microorganisms to fix N2 and tosolubilize P, but the P Bray of the soil was high. Therefore, small increase in yield of B 41 and S
219 genotypes by application of bio-fertilizer probably related to a better N use efficiency than P
efficiency of the genotypes.
In conclusion, genotypes responded differently to the bio-fertilizers. Supply of bio-fertilizer
at 50% of chemical fertilizer doses tended to increase the yield of B 41 genotype up to 16% of
100% chemical fertilizer dose without bio-fertilizer. This suggested that the chemical fertilizer dosecan be reduced by 50% without reducing yield only for B 41 genotype.
Table 1. Yield (ear weight) of the nutrient efficient and hybrid genotypes as affected by bio and chemicalfertilizers
Genotypes
B 41 without biofertilizer+ Bio fertilizer 1+ Bio fertilizer 2
S 219 without biofertilizer+ Bio fertilizer 1+ Bio fertilizer 2
L 164 without biofertilizer+ Bio fertilizer 1+ Bio fertilizer 2
Hybrid B 816 without bioertilizer+ Bio fertilizer 1+ Bio fertilizer 2

Chemical Fertilizer (% of standard rate) ton ha
25
50
100
6.35 (101)5.94 (94)6.32 (100)5.84 (92)
6.96 (110)6.89 (109)
6.40 (101)6.48 (103)
6.66 (105)5.23 (79)
5.49 (83)6.61 (100)5.74 (87)
6.22 (94)
6.58 (100)4.48 (68)5.92 (90)6.60 (100)
7.12
6.83
7.89
6.37
6.23
7.44
5.94
6.53
7.06
6.58
7.62
7.19
6.25
6.36
6.38
6.03
7.05
6.83

-1

mean
6.20
6.56
6.51
5.78
6.18
5.67
7.28
6.68
6.51
7.13
6.33
6.64

Note: Number in parentheses is the relative to its yield at 100 % without bio-fertilizer

References
Djafar, Z.R., and E.S. Halimi. 1998. Increasing the corn production in South Sumatra. J Tan Tropika. 1:128134.
Guntoro, D., B.S. Purwoko, and R.G. Hurriyah. 2007. Growth, nutrient uptake, and quality of turfgrass at somedosages of mycorrhiza application. Bul. Agron. 35:142-147.
Junaedi, A., A. Wachjar, and A. Rahman. 1999. The effect of biofertilizer on the growth of young coffee (Cofee
canephora Pierre ex Froehner) var robusta. Bul. Agron. 27:12-17.
Munandar, R. Hayati, H. Adam, Sabaruddin, and R.J. Anjani. 2010. Developing nutrient efficient maize
genotypes in marginal land using recurrent selection. Proceeding of National Seminar Polinela,
Lampung. Pp. 674-689.
Noor, A. 2003. The effect of rock phosphate and combination of phosphate-solubilizingBacteria and farm yard
manure on soil available P and growth of soybean on Ultisols. Bul. Agron. 31:100-106.
Saraswati, R. and Sumarno. 2008. Application of soil microorganisms as component of agriculture technology.
Iptek. Tan. Pangan 3:41.

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Kinetin and Calcium Pantothenate Effects on Shoot Multiplication in
In Vitro Cultured Cassava Var. Adira 2 and Adira 4
Nurul Khumaida a,*, Sintho Wahyuning Ardie a, Candra Catur Nugroho a, Suwarto aaDepartment of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University* Corresponding author: Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural University,
Jl. Meranti, Kampus IPB Darmaga, Bogor 16680, Indonesia.
Tel.: +62 251 862 9353; fax: +62 251 862 9353.
nkhumaida@yahoo.com

Abstract
Cassava (Manihot esculenta Crantz.) is one of largest carbohydrate sources in the world which also potentiallydeveloped as the source of bio-energy and raw materials for severals industries. As the demand for cassava isincreasing, it is very important to supply the true-to-type cassava seedling continuously. In vitro propagation is
one advanced technique that can be applied to meet the increasing demand of cassava seedling. The objectiveof the research was to study the effect of kinetin and Calcium Pantothenate (CaP) concentration on shootmultiplication of in vitro cultured cassava var. Adira 2 and Adira 4. This research was conducted in a completely
randomized design with two factors. The first factor was kinetin concentration (0; 1; 1.5; and 3 ppm) and thesecond factor was concentration of CaP (0; 1; and 2 ppm) used in combination. The results showed that MS
medium supplemented with 1 ppm CaP and 1.5 ppm kinetin promoted the growth of cassava explants of Adira 2variety until 4 Weeks After Treatment (WAT). However, for longer culture period MS medium containing 3 ppm
of BAP was better to support the explants growth. MS Medium containing 1 ppm of CaP and 3 ppm of kinetin
promoted the growth of Adira 4 variety until 4 WAT. However, for longer culture period MS medium containing 2
ppm of CaP and 3 ppm of kinetin was better to support the explants growth. The highest shoot multiplication rate
for 20 weeks old plantlets of Adira 2 and Adira 4 varieties was obtained at MS medium containing 3 ppm of BAP.
Keywords: calcium pantothenate, cassava, kinetin, shoot multiplication

Introduction
Cassava (Manihot esculenta Crantz) is the third largest source of carbohydrate for humanconsumption in the world. It is the principal carbohydrate source for more than 500 million people in
the tropical world. Cassava plays a famine prevention role wherever it is cultivated widely. Cassava
has adaptability to a range of climatic and edaphic conditions including tolerance to drought, tosome pests and diseases relative to other crops, and confers a comparative advantage on cassava
under conditions of famine against alternative crops.
More recently, cassava has gained importance as a possible fuel commodity not only inIndonesia but also in the Philippines, China, Thailand, and other countries which have moreadvanced national bio-fuel programs. In situations where water availability is limited (i.e. not enoughfor the cultivation of sugar cane), cassava is the preferred feedstock for ethanol production.
However, cassava-based industries are facing the main problem of cassava availability in amountand continuity (Suryana, 2009). Therefore, in order to meet the large demand of cassava, farmers
need large amount of good quality cassava seedling in a relaitvely short time.
In general, cassava is propagated by stem cutting. Eventhough stem cutting is easy to bedone and relatively cheap, it is not always available when needed and it is difficult to assure to havethe true to type seedling. In vitro technique is one method for mass propagation can be applied for
rapid mass propagation of cassava seedlings. Research in cassava shoot multiplication and theinduction of embryogenic callus had been done recently (Guohua, 1998; Sudarmonowati et al.,th

Proceedings of The 7 ACSA Conference 2011141

2002; Onuoch and Onwubiku, 2007). Previous results showed that cassava ‗Mentega‘ formed thehighest shoot formation when cultured in MS medium containing 2 ppm of BAP (Sudarmonowati etal., 2002). In other varieties, Fauzi (2010) found that standard MS medium containing 1.5 ppm of
BAP was the most effective medium for shoot multiplication. The objective of this research was tostudy the effect of Kinetin and Calcium panthothenate (Ca-P) addition in the shoot multiplication of
Adira 2 and Adira 4 cassava varieties.

Materials and Methods
The research was conducted from January 2010 to January 2011 at Plant Biotechnology
Laboratory, Department of Agronomy and Horticulture, Agriculture Faculty, Bogor Agricultural
University. This research was consisted of two separated experiments differed in the cassava
variety used as explants (Adira 2 and Adira 4 varieties). The experiment was arranged in
Randomized Block Design with two factors and ten replicates. The two factors were kinetin andCalcium panthothenate (Ca-P) concentrations. There were four levels of kinetin concentrations (0;1; 1.5; and 3 ppm), and three levels of Ca-P concentrations (0; 1; and 2 ppm). A control experiment
was made using standard MS medium with an addition of 3 ppm BAP. Each replicate consisted of
one bottle culture with one explants.
Plant material used was one node of axillary shoot from in vitro culture of Adira 2 and Adira 4
cassava varieties provided by Indonesian Center for Agricultural Biotechnology and Genetic
Resource Research and Development (ICABIOGRD). MS medium was used as basic medium of alltreatments. Surface sterilization was conducted by dipping the axillary shoots in Agrept (2 g L-1) andDithane (2 g L-1) solution for 2 h, and consecutively into Chloramfenicol (2 g L-1) solution overnight.
Sterilization by dipping in 10% NaClO solution for 5 min, then into 5% NaClO for 2 min was
conducted in the laminar air flow cabinet. Sterilized shoots were planted in the precondition medium
with 4 -5 explants in each bottle for 4 weeks. Explants used for experiment were micro cutting withsingle node of cassava (approximately 1-3 mm in length) cultured in the precondition medium.
Those explants were transferred into MS medium containing kinetin or Ca-P according thetreatments.
Planted explants were cultured in the dark for one week to induce shoot initiation. After that,
cultures were transferred into 24 h light condition with 21oC for 9 weeks. Observations wereconducted on in vitro culture and acclimatized plants. On the in vitro culture, the first shoot formed
and the time of callus formation were observed daily. Percentage of callus formation (%), number
and percentage explants with shoot (%), number of shoot per explants, callus diameter (mm), callus
color, structure of callus, length of shoot (mm), shoot morphology, number of leaves, number of
explants with root, number of roots, and number of nodes were observed weekly until 10 Weeks
After Planting (WAP). Data was test for normality then tested with F-test at α = 5%. Data withsignificant difference was tested with Duncan‘ Multiple Range Test.

Results and Discussion
The use of Ca-P in several concentrations only affected the callus diameter of Adira 2variety, while kinetin addition significantly affected number of shoot and callus diameter of Adira 2
and Adira 4 varieties. Interaction between Ca-P and kinetin concentration only affected the callus
diameter of Adira 4 variety. The contamination rate in this research was relatively low for Adira 2variety (2.12%) and Adira4 variety (1.77%), respectively. Most of the contamination in the culture
was caused by bacteria. Fungi contaminated the culture media in a lower level than bacteria.
As shown in Table 1, percentage of explants with shoots of Adira2 variety were higher
except for three combination showing lower percentage (less than 50%) that were 1.5 ppm of142thProceedings of The 7 ACSA Conference 2011

kinetin, 1 ppm of CaP, and 2 ppm CaP. All treatment combination did not significantly affect theshoot formation. In the other hand, medium containing 3 ppm of BAP resulted in 100% callus
formation. Some treatments that failed to induce callus formation were in medium without kinetin or
low kinetin concentration (less than 1.5 ppm). For Adira 4 variety, the highest percentage of
explants with shoots was resulted in medium containing 0.5 ppm of kinetin. In general, percentageof explants with shoot was relatively high (more than 50 %) for all treatments, except for medium
containing 3 ppm of kinetin (44.44 %). The highest number of callus formed was achieved in
medium containing high level of cytokinin (3 ppm BAP and 3 ppm kinetin). In contrary, the root
formation was inhibited with the presence of high level of cytokinin. Root formation was induced in
medium containing low concentration of cytokinin.
Table 1. Percentage of shoot forming explants, callused explants, and rooted explants of Cassava Adira2 andAdira 4 variety at 10 WAP
Adira 2Treatment

Ca-P
(ppm)

0

Shoot forming
explants

Kinetin(ppm)
02

Rootedexplants…….………………….%..........................

Callused
explants

Rootedexplants…….…………….%..........................

75.00

0.00

75.00

66.67

11.11

44.44

1

50.00

0.00

50.00

57.14

0.00

42.86

1.5

33.33

0.00

0.00

80.00

0.00

40.00

3

100.00

33.33

0.00

44.44

44.44

33.33

75.00

50.00

16.67

50.00

1

25.00
50.00

0.00

22.22

44.44

1.5

100.00

25.00
33.33

77.78
66.67

22.22

22.22

3

75.00

10.00

33.33

25.00
33.33

66.67

0

25.00
0.00

77.78
70.00

40.00

50.00

1

75.00

0.00

25.00

0.00

57.14

1.5

100.0050.00

100.00

71.43
66.67

11.11

33.33

3

100.00

33.33

33.33

60.00

60.00

20.00

100.00100.00

0.00

75.00

50.00

0.00

0
1

Callused
explants

Adira 4Shoot
forming
explants

3 ppm BAP

25.00
33.33

At the 1 WAP, shoot of cassava was initiated at the nodes. Number of shoot formed inevery treatment was compared to number of shoot formed in the control treatment (medium
containing 3 ppm BAP, B3) and tested by Dunnet test as shown in Table 2.
Table 2 indicates that every treatments affected number of node when they werecompared to the control medium B3, except for C0K3−B3 (9 WAP), C0K3−B3 and C1K3−B3 (10WAP). In contrast, for Adira 4 variety none of the treatment affected number of node whencompared to the control medium. Based on the regression analysis, kinetin addition could be
increased above 3 ppm. For Adira 2 variety, the regression equation for shoot formation and kinetin
concentration was linear (y = 0.401x – 0.095, R2 = 0.866). This equation means that every 1 ppm
increase of kinetin concentration would increase shoot formation 0.401 per explants. For Adira4
variety, the regression equation for shoot formation and kinetin concentration was also linear (y =0.278x + 0.224, R2 = 0.613). This equation means that every 1 ppm increase of kinetin
concentration would increase shoot formation 0.278 per explants.

thProceedings of The 7 ACSA Conference 2011143

Table 2. Number of cassava shoot of Adira2 and Adira4 variety in the in vitro culture at 9 and 10 WAP
Adira 2Treatment combination

Adira 4Age (WAP)

Age (WAP)

9

10

9

C0K0-B3−3.75*−3.75*

−0.71

ns

−0.71

ns

C0K1-B3−4.00*−4.00*

−0.88

ns

−0.80

ns

C0K1.5-B3−3.00*−4.17*

−0.58

ns

−0.58

ns

ns

ns

−0.93

ns

−0.93

ns

−4.25*

−0.75

ns

−0.88

ns

−0.60

ns

−0.60

ns

ns

−0.71

ns

ns

0.29

−0.48

ns

−0.58

ns

−0.66

ns
ns

C0K3-B3−2.17

C1K0-B3−4.25*−2.17−4.00*

C1K1-B310−4.00*

C1K1.5-B3−4.17*−3.17*−0.71

C1K3-B3−3.00*

−2.75

ns

0.53

−4.17*

C2K0-B3−4.17*

ns

C2K1-B3−3.75*−3.75*−0.60

ns

C2K1.5-B3−3.50*−3.50*

−0.71

ns

−0.71

ns

ns

−2.50**

C2K3-B3
F test−3.50*
*

1.33
*

1.23
*

Note : Variety was not compared to each other; C0, C1, C2 = 0, 1, 2 ppm CaP, respectively; K0, K1, K1.5, K3 = 0, 1, 1.5, 3 ppm
kinetin, respectively; B3 = MS + 3 ppm BAP; ns = not significant at P = 0.05; * = significantly different (P<0.05)

Table 3. Number of leaf of cassava Adira2 and Adira4 variety at 3, 7, and 10 WAP
Adira 2Treatment
CaP (ppm)

Adira 4Age (WAP)

Kinetin (ppm)37

Age (WAP)
10

3

7

10

0

1.25

2.00 b2.75

0.80 jkl

0.30

0.33

1

1.00

0.33 b0.00

0.70 jkl

0.33

0.43

1.5

0.50

0.25 b0.00

0.60 jkl

0.14

0.20

3

1.25

1.33 b

1.33

0.40 jkl

0.22

0.11

0

0.25

0.25 b

0.25

1.10

j

1.00

0.67

1

0.50

0.50 b1.25

1.00 jk

0.67

0.33

1.5

2.00

2.00 b

1.00

1.20

j

0.33

0.44

3

0.50

0.50 b

0.25

1.20

j

1.10

0.56

0

0.00

0.00 b

0.00

1.10

j

0.30

0.40

1

1.00

1.25 b

1.25

0.90 jk

0.33

0.57

1.5

1.00

1.67 b

2.50

0.60 jkl

0.20

0.22

3

0.75

1.33 b

1.67

0.00

l

0.70

2.00

3 ppm BAP

1.00

5.50 b

7.50

0.11 kl

0.50

1.00

F test

ns

*

ns

*

ns

ns

0

1

2

Note: Variety was not compared to each other; Numbers followed by the same letter in the same columns are not significantly
different based on DMRT at level α = 5%.

Leaf formation of cassava explants started at the 2 WAP. The leaf formation rate wasslower than the leaf senescence rate at the 4 WAP and resulted in the decreased of leaf number inevery observation point. The leaf senescence might be caused by the production of ethylene gas,
nutrient deficiency, and toxicity. Magdalita et al. (1997) found that the accumulation of ethylene
fasten the senescence of leaves in in vitro culture. Nutrient deficiency might caused by longer

144

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Proceedings of The 7 ACSA Conference 2011

culture, thus periodically subculture might be needed at 4 WAP. Leaf senescence might be also
caused by endogen auxin-cytokinin imbalance in the plants tissues (Lizawati et al., 2009). As shownin Table 3, the highest number of leaf for Adira 2 variety at 10 WAP resulted in medium containing 3
ppm of BAP. In contrast, medium containing 1 ppm of kinetin, 1.5 ppm of kinetin, and 2 ppm of CaP
resulted in lowest leaf number (0). The highest number of leaf for Adira 2 variety at 10 WAP
resulted in medium containing 2 ppm of CaP in combination with 3 ppm of kinetin, while medium
containing 1.5 ppm of kinetin resulted in the lowest number of leaf formed.
Shoot multiplication rate could be increased by periodically subculture (Hartmann and
Kester, 1983). In this research, the subculture was done twice. The first subculture was done at 10
WAP, while the second subculture was done 4 weeks after the first subculture. The multiplication
rate for Adira 4 variety was observed only at the first subculture. As shown in Table 4, the highest
multiplication rate was achieved in the medium containing 3 ppm of BAP for both varieties.
Table 4. Shoot multiplication rate of in vitro grown cassava Adira 2 and Adira 4 varieties

Replication∑ Initial
explants∑ Initial
shoots∑ Shoot
first subculture∑ Shootsecondsubculture

Total numberof Shoot

Treatment
Variety

Kinetin
(ppm)

CaP
(ppm)

0

0

1

1

1

5

2a

8

3

0

1

1

1

3

3b

7

3

0

3

1

5

3

−

Means + Standard deviation
Adira 2

1

1

1

1

1

5

3bc

9

1.5

1

1

1

1

2

−

3

3

2

1

1

1

1

2

4

3 ppm BAP

1

1

7

17

34

58

3 ppm BAP

2

1

2

2

−

Means + Standard deviation
1

0

3
3

1
1

4

1

1

4
31.00 ± 38.18

2

−

3
7

7

1

3

4

−

8

1

2

1

−

Means + Standard deviation
Adira 48
7.50 ± 0.71

3
5.00 ± 2.83

0

2

1

1

2

1

−

3

3

2

3

1

6

8

−

14

3

2

6

1

6

11

−

17

3

2

7

1

5

1

−

6

3

2

9

1

8

1

−

9

−

11.50 ± 4.93
33

3 ppm BAP

Means + Standard deviation
10
1
6

27

Note : Variety was not compared to each other; a = sub-cultured to MS0 + 3 ppm kinetin; b = sub-cultured to MS0 + 2 ppm CaP+ 3 ppm BAP; c = sub-cultured to MS0 + 2 ppm CaP + 3 ppm kinetin

Conclusions
The use of several kinetin concentrations (0; 1; 1.5; dan 3 ppm) gave a positive linear
response to number of shoots for both varieties (Adira 2 and Adira 4). In contrast, concentration of
kinetin had negative linear response to number of roots. Media composition with 1 ppm of CaP in
combination with 3 ppm of kinetin promoted the growth of cassava explants of Adira 4 variety until 4

thProceedings of The 7 ACSA Conference 2011145

WAT, but for longer culture period MS medium containing 2 ppm of CaP and 3 ppm of kinetin was
better to support the explants growth. The highest shoot multiplication rate was achieved in themedium containing 3 ppm of BAP for both varieties.

References
Fauzi, AR. 2010. Induksi Multiplikasi Tunas Ubi Kayu (Mannihot esculenta Crantz.) var. Adira 2 secara In Vitro.
Skripsi. Fakultas Pertanian. Institut Pertanian Bogor. Bogor. 67 hal.
Hartmann, H.T. DE. Kester. 1983. Plant propagation, principles, and practice, p. 223-280. In E. Cliffs (ed.).
Prentice-Hall inc. New Jersey.
Guohua, M. 1998. Effects of cytokinins and auxins on cassava shoot organogenesis and somatic
embryogenesis from somatic embryo explants. Plant Cell Tissue Organ Cult. 54:1-7.
Lizawati, T. Novita, R. Purnamaningsih. 2009. Induksi dan multiplikasi tunas jarak pagar (Jatropha curcas L.)
secara in vitro. J. Agron. Indonesia 37: 78-85
Magdalita, PM., DG. Ian, AD. Roderick, WA. Stephen. 1997. Effect of ethylene and culture environment on
development of papaya nodal culture. Plant Cell Tissue Organ Cult. 49:93-100.
Onuoch, CI., NIC Onwubiku. 2007. Micropropagation of cassava (Mannihot esculenta Crantz) using different
concentrations of benzylaminopurine (BAP). J. Engineering Appl. Sci. 2:1229-1231.
Sudarmonowati, E., R. Hartati, T. Taryana. 2002. Produksi Tunas, Regenerasi dan Evaluasi Hasil Ubi Kayu
(Mannihot esculenta) Indonesia Asal Kultur Jaringan di Lapang. Pusat Penelitian Bioteknologi. LIPI:
Cibinong.

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146

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Proceedings of The 7 ACSA Conference 2011

Wet Injury of Wheat in Upland Field Converted from Paddy Field in Japan
Kentaro Kawaguchi*
Wheat and Barley Research Divison, NARO Institute of Crop Science (NICS),National Agriculture and Food Research Organization (NARO), Japan*Corresponding author: Wheat and Barley Research Divison, NARO Institute of Crop Science (NICS),National Agriculture and Food Research Organization (NARO).
2-1-18, Kannondai, Tsukuba, Ibaraki 305-8518, Japan.
Tel. +81 29 838 8694; Fax. +81 29 838 8694
kentaro@affrc.go.jp

Abstract
Wheat and barley are very important crops used as staple food and feed. Growth delay or injury of wheat andbarley under waterlogged soils is one of agricultural constraints to be solved. The common symptoms are
germination failure, leaf color degradation, and wilting. Since there are different types of waterlogging conditions,the mechanisms of injury also varied. However, root sensitivity to low-oxygen atmosphere in soil is critical. Rice
plant has been widely cultivated in Japan. In recent years the agricultural policy enhanced rice-wheat-soybean
rotation using paddy fields for economic reasons. Crop production using paddy fields is thought to be an
important key for sustainable agriculture in eastern Asia but wet injury of wheat usually occurs in the uplandfields converted from paddy fields because of the poor drainage. Under a particular case, a phenomenon called
ground subsidence due to the earthquake induced wet injury in this year. The most popular measure to mitigate
the waterlogging damage on farm level is the field management to facilitate drainage. In addition, we have greathopes for establishing tolerant varieties; however, such commercial one is not yet obtained by traditional
breeding. We are now engaged on an innovative research project about root aerenchyma which is thought tofacilitate gas exchange between root and aerial parts of plants. We hypothesized its modification could change
the sensitivity of root to low-oxygen. The project plan was; i) to investigate anatomical and physiological basis of
aerenchyma formation in the recipient wheat cv. Bobwhite. ii) to establish transgenic wheat lines using several
candidate genes for root aerenchyma formation. iii) to analyze function of the genes using the transgenic
Bobwhite lines.
Keywords: aerenchyma, barley, subsidence, waterlogging, wheat

Introduction
The main production areas of wheat in Japan are in Hokkaido, Kanto and Kyushu district.
Except in Hokkaido, they have relatively high precipitation. It is over one thousand mm a year.
Sowing and harvest of wheat often coincide with the two rain periods, we call them ―Akisame‖(autumnal rain in September and November) and ―Baiu‖ (rainy season in June). Moreover, wheat is
widely grown in upland fields converted from rice paddy fields in Japan. The main reason tocultivate wheat instead of rice is due to decline of the amount of rice consumption. Full efficientutilization of rice paddy fields is one of the main policies of the Japanese government. Average of
wheat acreage in recent 10 years is 210 thousand ha and the over half of the acreage is the rice
fields. Oyanagi (2008) reported that about 20% of the wheat acreage is affected by waterlogging.
Wet injury usually occurs in the converted fields because of the poor drainage and it
causes severe damage on the grain yields as well as their quality. One of the causes of the wet
injury is shortage of oxygen in the low layer, where the wheat roots develope and obtain oxygen to
grow. The most effective way to prevent wet injury is the field management for well drainage, forexample, ditch making and raised bed farming, etc., however, the economic cost for such fieldmanagement is relatively high in the farm level. Therefore, we develop new lines of wheat having
high tolerance to wet soil.

thProceedings of The 7 ACSA Conference 2011147

To overcome the agricultural constraint, understanding of the mechanisms of wet injury
and tolerance in wheat is essential. In our study, first, we characterized wheat growth under wet
conditions in farmer‘s fields and in our experimental paddy fields. The results showed that i) there
were high correlations between yield and soil moisture in the farm level and ii) wheat growthsuffered from wet stress from the early development under the condition of paddy field. Second, we
focused on root aerenchyma. Aerenchyma is thought to be enhancing gas exchange between
subterranean part (root) and aerial part (shoot) of plant tissues. Therefore, root aerenchyma is oneof the important traits to contribute wet tolerance of plants. To investigate the basic nature of root
aerenchyma formation during early development of wheat, we examined when and where root
aerenchyma were formed in the wheat seminal root under waterlogging condition using a plant pot
system.Third, we recently studied the strategy to improve wheat using modern biotechnology.

Materials and Methods
Field experiment
Wheat (Triticum aestivum L.) cultivars. Bobwhite (Mexican cultivar), Norin 61 (Japanesecultivar), Iskra and NS-302 (Yugoslavian cultivars) were used in this experiment. The latter two
were selected as candidates for high tolerant cultivars (Dr Sato, unpublished data). Norin 61 is the
major cultivar in Japan except in Hokkaido region. Bobwhite is often used as a host for establishingfertile transgenic by means of particle bombardment in wheat. Experimental fields were located at
Yawara, Tsukuba-mirai city, Ibaraki, Japan. Five are area each of contiguous two upland fields
converted from paddy fields were used. One was used under non-tilling cultivation as control plot
and the other was used after soil puddling as wet treatment plot. Wheat seeds were sown with 70
cm rows on 30th October, 2009 and 10th November, 2010. Fertilizers (6, 9 and 6 kg/10a each as N,
P2O5 and K2O) was applied at ten days before sowing. Growth characters were monitored in 3
weeks interval. The soil water contents were measured by TDR-method (Time Domain
Feflectometry) using a HydroSense CD620 display and CS620 sensor with a 12-cm probe(Campbell Scientific Australia Pty. Ltd., Thuringowa Central, Australia) in the top 12 cm of the soil.
The soil‘s redox potentials at 10cm depth were recorded using an Eh meter (PRN-41, Fujiwara
Scientific Co.LTD., Tokyo, Japan) with a reference electrode.
Pot experiment for aerenchyma study
Wheat cv. Bobwhite line SH 98 26 was used. Three seeds were sown at a 3-cm depth ineach of 27 soil-filled pots (Fujiwara Scientific, Tokyo, Japan; 1/5000a deep type Wagner pot, 30 cm
height x 15.9 cm inner diameter). The wheat plants were grown in a greenhouse maintained at atemperature of approximately 20°C day/night, with natural light. Water treatments were imposed to
5 d old seedlings: (i) control, with a well-drained; (ii) T-15 treatment, the water level was maintained
at 15 cm below the soil surface, and (iii) T+3 treatment, the water level was maintained at 3 cm
above the soil surface. The O2 concentrations were recorded at a 14 cm depth in the soil using an
OXY-10 O2-sensor (PreSens Precision Sensing GmbH, Regensburg, Germany). Transverse crosssections were obtained from primary seminal roots using a D.S.K. Microslicer (DTK-1000, Dosaka
EM Co. Ltd., Kyoto, Japan). The sections were examined and photographed with a light microscope
equipped with a camera (BX51 microscope and DP72 camera, Olympus Co. Ltd., Tokyo, Japan) at
10 magnification.

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Proceedings of The 7 ACSA Conference 2011

Results and Discussion
Growth of 4 cultivars of wheat in the experimental fields
The soil conditions in the experimental fields were measured. Ground water levels were
almost less than 75 cm below the soil surface during the entire experiment period. Soil watercontents were higher in puddling plot than that in control plot until April 19. Redox potentials in thesoil were constantly over +500 mV and showed no significant difference between the plots. The
plant height and dry weight were significantly affected by puddling (wet) treatment (Figures 1 and 2
show the results of 2009 – 2010 season). Similar tendency was observed in the repeated testing in
the next cropping season of wheat. Puddling/Control ratio in dry weight was very low (20%) atyoung (on 17 February) and was gradually recovered (80%) at ripening (on 13 May). These results
indicated that wheat suffered from wet stress since their early stage of development and suggested
that high tolerance to wet conditions at the seedling stage is important for wheat production in
paddy fields in Japan.

Figure 1. Changes in the plant height of 4 cultivars of wheat under control condition (solid line) and puddling(wet) condition (dashed line) in 2009 – 2010 season.

Figure 2. Dry weight of wheat plants (aerial part only) under control condition (gray bar) and puddling (wet)condition (black bar) in 2009 -2010 season.

thProceedings of The 7 ACSA Conference 2011149

Growth and root aerenchyma formation of wheat in pot system
The soil conditions in the plant pot were measured by TDR-method. Under the control, T15, and T+3, the soil water contents were 12-14; 23-27; and 74-82%, respectively. The O2
concentrations after 72 h in T-15 soil decreased from the control soil 18.7 to 10.1%, whereas that in
T+3 soil decreased to 4.9%. While the redox potential in the control soil remained around +400 mV,
the values in the T-15 and T+3 soils were about +360 and +290 mV, respectively, after 72 htreatment. These results showed that the soil conditions in T-15 and T+3 became hypoxic in orderof water depth during waterlogging treatment.
The length of primary seminal roots and the position of aerenchyma were schematicallydrawn in Figure 3. The root length was reduced by 25% in the T+3 plants. The reduction of seminalroot dry mass was 12.5% in T-15 and 31% in T+3 plants, respectively. For the aerial parts, theplants did not show significant difference (data not shown). Wheat primary seminal roots showed noaerenchyma in our control condition. Aerenchyma was appeared after 48 h under T+3 and after 72
h under T-15 waterlogging conditions, respectively, at 2 to 5 cm behind the root tip. The
aerenchyma in T+3 plants extended to 2-10 cm behind the tip at 72 h waterlogging.
24 h waterlogging

48 h waterlogging

72 h waterlogging

Base

C

T-15

T+3

C

T-15

T+3

C

T-15

T+3

0 cm

0

0

0

0 cm

0

0

0

0 cm

0

0

0

0 cm

3 cm

0

0

0

3 cm

0

0

0

3 cm

0

0

0

3 cm

5 cm

0

0

0

5 cm

0

0

0

5 cm

0

0

5

5 cm

5 cm

0

0

0

10 cm

0

0

7

15 cm

0

0

9

10 cm

2 cm

Root
length

0

0

0

5 cm

0

0

9

10 cm

0

7

9

5 cm

Tip

Tip

Tip

2 cm

0

1

9

5 cm

0

9

9

2 cm

Tip

Tip

Tip

2 cm

0

9

Tip

Tip

Tip

16
±0.7
cm

15
±0.7
cm

14
±0.8
cm

18
±0.7
cm

17
±0.8
cm

15
±0.8
cm

23
±0.4
cm

23
±0.4
cm

17
±0.8
cm

n=9

n=9

n=9

n=9

n=9

n=9

n=9

n=9

n=9

Numbers of root sections with developing aerenchyma are indicated at the sampling position. Roots (n=9) were tested. Blackbackground indicates aerenchyma formation in more than half number of the nine sections.

Figure 3. Aerenchyma formation in the seminal root of wheat seedlings under waterlogging conditions.

Our results provide basic information on the aerenchyma formation process in seminalroots of wheat. To our knowledge, this is the first report to describe the time of the aerenchyma
formation in wheat seminal roots under hypoxic conditions (Haque et al. 2010). The development
nature of aerenchyma, such as the formation timing, might be similar in part among several uplandcrops. Root aerenchyma can be seen within 48 h after the initiation of hypoxia in the seminal (ourstudy) and adventitious roots (Malik et al. 2003) in wheat. In maize, aerenchyma formation in theseminal (Gunawardena et al. 2001) and adventitious roots (Drew et al. 1981) is induced by hypoxia
within a few days. Thus, wheat requires several days to form aerenchyma by waterlogging stress,
while rice plant constitutively forms well developed aerenchyma in their roots. Such different ability
to form aerenchyma among plant species may be one of the important keys to determine theiradaptive nature to water.
Wheat plants grown in our pot system can be used for analyzing the molecular andphysiological mechanisms of aerenchyma formation. Proteome analysis using the wheat seminalroot showed that 10 candidates of proteins would be associated with the metabolism during root

150

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Proceedings of The 7 ACSA Conference 2011

development under hypoxia (Haque et al. 2011). Elucidation of the protein function during
aerenchyma formation is a next step of this study.
Future direction
Wheat cv. Bobwhite line SH 98 26 is known to be one of the most useful recipients for
producing transgenic wheat lines (Pellegrineschi et al. 2002). Therefore, the information obtained
from this study will be useful not only for understanding the basic nature of aerenchyma formation,
but also for efforts to improve aerenchyma formation, including the creation of transgenic plants. In
fact, the transgene approach in our experiment showed the ability satisfactorily on the investigationof seed dormancy of wheat and the related gene (Nakamura et al. 2011). Introducing genes can be
widely selected from various plant species, including rice and teosinte, etc., which have highcapacities to form aerenchyma (Nakazono et al. 2011 in this session, Mano and Omori 2009).

Acknowledgements
The author wish to thank the International Maize and Wheat Improvement Center (CIMMYT), Mexico
for providing seeds of wheat cv. Bobwhite SH 98 26 and the National Institute of Agrobiological Sciences (NIAS)
Genebank for supplying seeds of wheat cv. Iskra and NS-302. I would like to express my thanks to Drs. M.
Nakazono, Y. Mano and S. Shimamura for their valuable suggestions and critical comments to this study. I also
thank Mr. S Hamada for his skillful management of the field experiment. All of the achievements reported here
were produced through the collaborative investigation with my colleagues, Drs A. Oyanagi, F. Abe, Md Emdadul
Haque and M. Mori in NICS. The research was supported by a grant-in-aid from the Bio-oriented Technology
Research Advancement Institution (Promotion of Basic Research Activities for Innovative Biosciences, No. H20 /seeds-01-01).

References
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formation in adventitious roots of Zea mays L. subjected to exogenous ethylene or to oxygen deficiency.
Planta 153: 217-224.
Gunawardena AHLAN, DM Pearce, MB Jackson, CR Hawes and DE Evans. 2001. Characterisation ofprogrammed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize(Zea mays L.). Planta 212: 205-214.
Haque ME, F Abe and K Kawaguchi. 2010. Formation and extension of lysigenous aerenchyma in seminal root
cortex of spring wheat (Triticum aestivum cv. Bobwhite line SH 98 26) seedlings under different
strengths of waterlogging. Plant Root 4: 31-39.
Haque ME, K Kawaguchi and S Komatsu. 2011. Analysis of proteins in aerenchymatous seminal roots of wheatgrown in hypoxic soils under waterlogged conditions. Protein and Peptide Letters 18: 912-924.
Malik AI, TD Colmer, H Lambers and M Schortemeyer. 2003. Aerenchyma formation and radial O 2 loss along
adventitious roots of wheat with only the apical root portion exposed to O2 deficiency. Plant Cell Environ.
26: 1713-1722.
Mano Y and F Omori. 2009. High-density linkage map around the root aerenchyma locus Qaer1.06 in the
backcross populations of maize Mi29 × teosinte ―Zea nicaraguensis‖. Breed. Sci. 59: 427-433.
Nakamura S, F Abe, H Kawahigashi, K Nakazono, A Tagiri, T Matsumoto, S Utsugi, T Ogawa, H Handa, H
Ishida, M Mori, K Kawaura, Y Ogihara and H Miura. 2011. Wheat homolog of MOTHER OF FT AND
TFL1 acts in the regulation of germination. Plant Cell online publication, DOI 10.1105/tpc.111.088492.
Nakazono M, T Yamauchi, R. Imene, H. Takahashi, T Abiko, S Nishiuchi, S Yamazaki, K Shiono. 2011. Studies
on molecular mechanism of formations of aerenchyma and a barrier to radial oxygen loss in crops underwaterlogged conditions. In this proceedings.
Oyanagi A. 2008. Relationship of growth variability, with ground level and soil water content in a large paddy
field in Inashiki-city, Ibaraki-prefecture in 2007-Wet injury of wheat. Jpn. J. Crop. Sci. 77: 511-515. (inJapanese with English abstract)thProceedings of The 7 ACSA Conference 2011151

Pellegrineschi A, LM Noguera, B Skovmand, RM Brito, L Velazquez, MM Salgado, R Hernandez, M Warburton,
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152

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Roles of Root System Development and Function in the Growth and Yield
under Waterlogged Condition in Common Wheat
Tomohito Hayashia, Tomofumi Yoshidab, Kiyoshi Fujiibc, Takako Tsujib,
Yurie Okadaa, Eriko Hayashia, Akira Yamauchia＊aGraduate School of Bioagricultural Sciences, Nagoya University, Japanb
Aichi Prefecture Agricultural Research Center, Aichi, Japanc
Agricultural Production Division, Aichi Prefectural Government, Aichi, Japan*Corresponding author: Graduate School of Bioagricultural Science, Nagoya University,
Nagoya 464-8601, Japanayama@agr.nagoya-u.ac.jp

Abstract
Waterlogging stress substantially reduces the productivity of common wheat. Unavailability of oxygen to theroots is the major growth-limiting factor for plants exposed to waterlogging stress. The development and functionof roots play significant roles in expression of waterlogging tolerance of wheat under such conditions. However,
the mechanism of root function in the waterlogging tolerance is not clear yet. In this study, we, therefore, aimed
to evaluate the roles of root system development and function in the growth and yield of wheat plants that were
exposed to waterlogged condition at jointing stage. This study was conducted in 2009 at Aichi Agricultural
Research Center experimental field. Plants were grown under two different conditions; control for rainfed uplandconditions while in waterlogging treatment (WL), water level was maintained around 5 cm below the soil surface
from jointing stage till maturity. Soil type of this field was andsol. We previously examined a set of germplasm(144 cultivars) collection, and selected Nishikazekomugi (high waterlogging tolerance), Iwainodaichi (moderate)and UNICULM (low). Grain yield of Nishikazekomugi in WL was 64% of control, while that of Iwainodaichi and
UNICULM was 38 and 4%, respectively. The cultivars of greater watelogging tolerance further showed the ability
to maintain higher stomatal conductance in WL, which should reflect higher root ability of soil water uptake. In
fact, they showed higher root development as evaluated in total root length. Increase in nodal root porosity of
Nishikazekomugi and Iwainodaichi in response to exposure to WL then contributed to the significant increase intotal root length. Higher root porosity indicates greater root aerenchyma formation in response to waterlogging
stress. These results indicated that the tolerant cultivars had higher ability of aerenchyma formation, which
promoted root system development, and eventually contributed to the increase in shoot dry matter and grain
yield under waterlogged conditions.
Keywords: aerenchyma, common wheat, hydraulic conductivity, porosity, waterlogging tolerance

Introduction
Common wheat is known to be susceptible to waterlogging, and the grain yield
substantially decreases by excess moisture (Oyanagi et al, 2001) in most of the wheat producing
countries such as UK (Belford and Cannel, 1979), Australia (Setter et al., 2009). In Japan, 27% of
wheat-planted field (approximately 57,000 ha) is affected by excess moisture stress (Sakagami etal., 2010).
When the water table rises to or above the soil surface, roots surrounded by thewaterlogged soil suffer from O2 deficiency (Jackson et al., 1999; Malik et al., 2003). As one ofmechanisms for waterlogging tolerance, aerenchyma in root provides a low-resistance internal
diffusion pathway to supply O2 to the root apex (Arikado, 1975; Colmer, 2003; Armstrong andArmstrong, 2005). Therefore, root aerenchyma formation under waterlogged condition may play
important roles in waterlogging tolerance in wheat (Setter and Waters, 2003). Although wheat plants
generally have low efficiency of root aerenchyma formation (Watkin et al., 1998), genotypicthProceedings of The 7 ACSA Conference 2011153

variations in its functional roles in the maintenance of waterlogging tolerance under waterlogged
condition is not yet clear.
In this study, we examined if genotypes with high ability of root aerenchyma formation androot system development under waterlogging produce high dry matter and yield by evaluating theimpacts of root aerenchyma formation on root development, stomatal conductance, photosynthetic
rate and grain yield under waterlogged conditions, and compared with those under rainfed uplandconditions in the field. Such hypothesis of this study was summarized in Figure 1. The objective of
this study was, therefore, to examine the roles of root aerenchyma formation in the maintenance ofwaterlogging tolerance under waterlogged condition in wheat.

Figure 1. Schematic diagram of hypothetical mechanism of waterlogging tolerance in wheat.

Materials and Methods
Experiment design and Plant materials
A field experiment was conducted at Aichi Agricultural Research Center. Plants were grownunder two different conditions e.i control for rainfed upland conditions while in waterlogging
treatment (WL), water level was maintained around 5 cm below the soil surface from jointing stage
till maturity. Soil type of this field was andsol.
The seeds were sown on December 3, 2008. The waterlogging treatment lasted for 50 days
from April 13 to June 1, 2009. The plants were harvested on June 9, 2009 (188 days after sowing).
Based on the results of our previous study (Hayashi et al., 2008) that examined a set of 144
cultivars collection, we selected three cultivars e.i. Nishikazekomugi (high waterlogging tolerance),
Iwainodaichi (moderate) and UNICULM (low).
Grain Yield
Plants were harvested in six replicates for each cultivar. For shoots, after air-drying forabout three weeks, leaves and stems were oven-dried and weighed, and the grains were threshed.
After the grain moisture content was measured with a grain moisture tester, grains were weighed,and the yield components were determined.
Root Development
To determine total root length, after the harvest of shoots, roots were sampled in six
replicates for each cultivar. At the end of waterlogging treatment, the roots were sampled with theround monolith method (Kang et al. 1994) by using stainless cylinder of 15 cm in diameter, and thesoil cores of 20 cm in depth were taken. Soil samples were carefully washed to collect roots. Afterremoving the debris, the roots were stained and arranged on water in a transparent plastic tray.
They were then scanned to convert to a digitized image, and total root length was determined withthe use of NIH Image ver. 1.62 and Root Length ver. 1.54 (Kimura, 1999).

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Leaf Water Potential
We measured leaf water potential of the flag leaves in six replicates in each cultivar at -4,
4, 11, 16, 19, 28 and 34 days after the onset of waterlogging treatment. Leaf water potential wasmeasured with thermocouple psychrometry using sample chambers (Model C-52, Wescor) and a
microvoltmeter (Model HR-33T, Wescor). The sample chambers had been calibrated with NaClsolution of a known molarity in advance.
Stomatal Conductance and Photosynthetic Rate
We measured stomatal conductance and photosynthetic rate of the flag leaves in three
replicates for each cultivar at -4, 4, 11, 16, 19, 28 and 34 days after the onset of waterlogging
treatment. The measurements were conducted from 800 to 1300 on sunny days at PPFD of 1400
μmol m-2 s-1 with a portable type apparatus for photosynthesis and transpiration measurements (LI6400, LI-COR, Lincoln, NE, USA). Leaf temperature and CO2 concentrations surrounding leaf was
adjusted to be 28±1.7℃ and 400 μmol mol-1, respectively. Measurement duration average wasabout 40 to 60 s. The leaf areas enclosed by chamber were from 3.0 to 6.0 cm2.
Root Porosity
We measured porosity of the nodal root in six replicates for each cultivar. Nodal root
porosity was measured 50 days after the onset of waterlogging treatment.The nodal root axis wasused for root porosity measurement following the microbalance method (Visser and Bögenmann,
2003; Suralta et al., 2010).
A 1-cm segment from the middle portion of each root was cut. From each plant (replicate),
sixty 1-cm segments were further sampled. Each 1-cm root segment was cut with a sharp razor
blade and gently blotted by rolling it with a small brush on tissue paper for about 2 s to remove
adherent water. Then, to prevent weight loss by evaporation, the segments were transferred into a
capsule with cover that had been tared on a microbalance. After closing the capsule, the segments
were weighed (w1 in µg), transferred to a holder with small vials filled with water, and stored for 30
min. Up to 60 samples were weighed before they were infiltrated with tap water twice under vacuumfor 30 min. After water infiltration, the root segments were blotted again on tissue paper for about 2s and weighed in a capsule (w2 in µg). Using the specific weight (SW) obtained from larger
samples, the porosity was calculated using the formula: Porosity (%; v : v) = 100 × (w2 – w1) ×
SW/w2

Results and Discussion
In this study, we evaluated the roles of root system development and function in the growthand yield of wheat plants that were exposed to waterlogged condition at jointing stage. Grain yieldof Nishikazekomugi in WL was highest (64% of control), followed by Iwainodaichi (38%), and
UNICULM (4%) (Figure 2). Nodal root porosity of Nishikazekomugi in WL was 284% of control,
while that of Iwainodaichi and UNUCULM was 113% and 44%, respectively (Figure 3). Total root
length of Nishikazekomugi in WL was 104% of control, while that of Iwainodaichi and UNICULM
was 70 and 38%, respectively (Figure 4). These results showed that the cultivar with high porosity
in response to exposure to WL tended to have greater total root length. Specifically, increase innodal root porosity of Nishikazekomugi and Iwainodaichi contributed to the significant increase intotal root length. Higher root porosity indicates greater root aerenchyma formation in response towaterlogging stress.

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Proceedings of The 7 ACSA Conference 2011155

Data are means of 6 replicates. Vertical bar show standard errors (n=6). □ and ■ represents control and waterlogging conditions,
respectively.

Figure 2. The effect of waterlogging stress on grain yield under rainfed upland (control) and waterlogged
conditions.

Data are means of 60 replicates. Vertical bar show standard errors (n=60). □ and ■ represents control and waterlogging
conditions, respectively.

Figure 3. The effect of waterlogging stress on nodal root porosity under rainfed upland (control) and waterlogged
conditions.

Data are means of 6 replicates. Vertical bar show standard errors (n=6). □ and ■ represents control and waterlogging conditions,
respectively.

Figure 4. The effect of waterlogging stress on root length under rainfed upland (control) and waterlogged
conditions.

We also examined the effect of waterlogging stress on leaf water potential, stomatal
conductance and photosynthetic rate. Leaf water potential of Nishikazekomugi maintained to thelevel of control for 16 days after waterlogging treatment onset, for 11 days in Iwainodaichi and for 4days in UNICULM. Stomatal conductance of Nishikazekomugi maintained to the level of control for
16 days, for 16 days in Iwainodaichi and for 11 days in UNICULM. Likewise, photosynthetic rate of
Nishikazekomugi maintained to the level of control for 16 days, for 11 days in Iwainodaichi and for
11 days in UNICULM. As stated above, the cultivars of greater root system development (greater
total root length in WL) further showed the ability to maintain high leaf water potential and stomatal
conductance, which maintained higher photosynthetic rate in WL.
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thProceedings of The 7 ACSA Conference 2011

These results indicated that the tolerant cultivars have higher ability of aerenchyma
formation, which promotes root system development, and probably root hydraulic conductance,
both of which, through high ability to provide water to shoot, eventually contributed to the increasein shoot dry matter and grain yield under waterlogged conditions.

References
Arikado H. 1975. Waterlogging tolerance of aerenchyma and crops. Oriental print. Mie. Pp149.
Armstrong J and W Armstrong. 2005. Rice: sulfide-induced barriers to radial oxygen loss, Fe+ and water uptake,and lateral root emergence. Ann Bot. 96: 625-638.
Belford RK, RQ Cannel. 1979. Effects of waterlogging of winter-wheat prior to emergence on crop establishment
and yield. J Sci Food Agr. 30: 340-340.
Colmer TD. 2003. Aerenchyma and an inducible barrier to radial oxygen loss facilitate root aeration in upland,
paddy and deep-water rice (Oryza sativa L.). Ann Bot. 91: 301-309.
Jackson MB, W Armstrong. 1999. Formation of aerenchyma and the process of plant ventilation in relation tosoil flooding and submergence. Plant Biol. 1: 274-287.
Kang SY, S Morita, K Yamazaki. 1994. Root growth and distribution in some japonica-indica hybrid and japonica
type rice cultivars under field conditions. Jpn J Crop Sci. 63: 118-124
Kimura K, S Kikuchi and S Yamasaki. 1999. Accurate root length measurement by image analysis. Plant Soil.
216: 117-127.
Malik AI, TD Colmer, H Lambers and M Schortemeyer. 2003. Aerenchyma formation and radial O2 loss along
adventitious roots of wheat with only the apical root portion exposed to O2 deﬁciency. Plant Cell Environ.
26: 1713-1722.
Oyanagi A, C Kiribuchi-Otobe, T Yanagisawa, S Miura, H Kobayashi and S Muranaka. 2001.Selection of wheat
experimental lines with deep and shallow root systems based on the growth angle of seminal roots. Jpn J
Crop Sci. 70: 400-407.
Sakagami J, M. Nakazono, S. Shimamura, O. Ito, K. Ishizawa. Wetland environment and crops. Youkendo.
Tokyo. Pp139-150.
Setter and Waters. 2003. Review of prospects for germplasm improvement for waterlogging tolerance in wheat,barley and oats. Plant Soil. 253: 1-34.
Setter T.L, I. Waters, S. K. Sharma, K. N. Singh, N. Kulshreshtha, N. P. S. Yaduvanshi, P. C. Ram, B. N. Singh,
J. Rane, G. MCDonald, H. Khabaz-Saberi, T. B. Biddulph, R. Wilson, I. Barclay, R. MCLean, M. Cakir.
2009. Review of wheat improvement for waterlogging tolerance in Australia and India: the importance ofanaerobiosois and element toxicities associated with different soils. Ann Bot. 103: 221-235.
Suralta RR, Y Inukai, A Yamauchi. 2010. Dry matter production in relation to root plastic development, oxygen
transport, and water uptake of rice under transient soil moisture stresses. Plant Soil. 332: 87-104.
Hayashi T, T Yoshida, K Fujii, T Tsuji and A Yamauchi. 2008. Relationship between root system ability, andphotosynthesis and grain yield under excess soil moisture conditions in wheat. Jpn J Crop Sci. 77 (Extraissue 2): 254-255.
Visser EJW and GM Bögenmann. 2003. Measurement of porosity in very small samples of plant tissue. Plant
Soil. 253: 81-90
Watkin EJ, C Thomson and Hankgreenway. 1998. Root development and aerenchyma formation in two wheat
cultivars and one triticale cultivar grown in stagnant agar and aerated nutrient solution. Ann Bot. 81: 349354.

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Growth and Physiological Responses of Sago Palm against AluminumStress in Acidic Conditions
Ornprapa Anugoolpraserta, Hiroshi Eharaa,*, Shina kinoshitaa, Hitoshi Naitob, Somchai
ChakhatrakancaGraduate School of Bioresources, Mie University, Mie, Japan
College of Life Science, Kurashiki University of Science and the Arts, Okayama, Japanc
Department of Agricultural and Technology, Thammasat University, Pathumthani, Thailand*Corresponding author: Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu,
Mie 514-8507, Japan.
Tel.: +81 59 231 9495; fax: +81 59 231 9495.
ehara@bio.mie-u.ac.jpbAbstract
Sago palm (Metroxylon sagu Rottb.) grows in natural peat swamps, which are poorly drained and has highacidity and generally contain highly exchangeable Al. It is, therefore, considered to be acid- and Al-resistant. In
this study, the growth, physiological characteristics and nutrient concentrations in the plant tissues of sago palmgrown under a hydroponic system were investigated for 4.5 months. When sago palm seedlings were cultured at
pH 5.7, pH 4.5 and pH 3.6, the leaf morphogenesis, nutrient uptake and dry matter production were maintained
regardless of a small decrease in the photosynthetic rate through the decrease of stomatal conductance. In thecase in which seedlings were grown at pH 3.6 with different levels of AlCl 3·6H2O corresponding to 0, 10, 20, 100
and 200 ppm Al, the plant length and dry matter production increased with a mild Al concentration in the growth
media, such as 10 ppm Al. This result was attributed to the increase in the P and N uptake. In contrast, all thegrowth parameters significantly decreased under the 200 ppm Al treatment. The critical value to inhibit thegrowth of sago palm was considered to be around 200 ppm Al in the growth media. In addition, sago palm
3+
maintained a low Al concentration in all of the plant parts. Therefore, it could be concluded that sago palm has
high resistance to Al with mechanical restriction of the excess Al based on the Al exclusion ability under the acidcondition.
Keywords: acid resistance, aluminum resistance, dry matter growth, photosynthetic rate, stomatal conductanceIntroduction
Competition between biofuel production and food production has occurred in recent years
in the context of the current social background regarding the exhaustion of fossil energy and thegrowing world population. Various plants are receiving attention as sustainable energy resources forthe production of bioethanol and biodiesel. However, worldwide arable lands are limited. Thus, thedevelopment and/or improvement of new plant resources and their utilization are needed as a
strategy to secure a sufficient amount of biomass for producing foods and biofuel sources (Ehara,2009).
Sago palm (Metroxylon sagu Rottb.) stores large quantities of starch in its trunk. The total
starch storage in one trunk is approximately 300 kg dry weight (Ehara, 2005). As a staple food,sago palm continues to be important in parts of Southeast Asia and in areas inhabited by the
Melanesian people (Ehara et al., 2000). The carbohydrate or starch can be further processed into
various basic raw materials for human and animal consumption as well as an industrial energy
source, such as ethanol. In addition, sago palm grows in swampy, alluvial and peaty soils where
almost no other major crops can grow without drainage or soil improvement (Sato et al., 1979; Jong
& Flach, 1995). Nevertheless, the deep peat soils in swampy areas are usually characterized by low
pH values, a deficiency in mineral elements and a high rate of exchangeable Al (Sato et al., 1979).
Some former field studies on the growth of sago palm reported that sago palm grew under acidth

Proceedings of The 7 ACSA Conference 2011159

conditions (Purwanto et al., 2002; Osaki et al., 2003). According to Foy & Fleming (1978), there wasa possitive correlation between Al-resistant plants in nutrient solution and resistance to low pH
conditions. It is, therefore, assumed that sago palm is resistant to acidic pH and Al. However, few
studies have compared the growth characteristics of sago palm at widely different pH levels as wellas the Al-induced changes on sago palm growth. Thus, the objective of the present study was tocompare the physiological features and growth characteristics of young seedlings of sago palm at
different levels of pH and Al concentration under low pH condition in a hydroponic system toelucidate the acid- and Al-resistance.

Materials and Methods
Plant materials and treatments
Sago palm fruits were collected in the swampy areas of Rattapum, Songkhla, Thailand, on
1 August, 2006. Fertilized and well-developed fruits were selected and treated physically to remove
seed coat tissues. The cleaned seeds were placed in a plastic tray filled with tap water and then put
them in a dark temperature-controlled room at 30ºC in Thammasat University, Thailand. The
germinated seeds were brought to Mie University, Japan and each of them was transplanted to a
1/5000a Wagner pot filled with vermiculite and Kimura B culture solution containing (µM) 36.5
(NH4)2SO4, 9.1 K2SO4, 54.7 MgSO4, 18.3 KNO3, 36.5 Ca (NO3)2, 18.2 KH2PO4 and 3.9 FeO3 (Baba& Takahashi, 1958). The initial pH of the culture solution was adjusted to 5.5 using 1.0N HCl before
irrigation into pots. The pots were placed in a greenhouse under natural sunlight and maintained atover 15ºC, even at night, at Mie University. Daily additions were made to the culture solution,according to the amount of solution consumed, and the culture solution was renewed twice weekly.
In Experiment 1, three seedlings at the 7th leaf stage, with the mean plant length of all plantmaterial at 39 cm, were selected and treated with Kimura B culture solution at three different levelsof pH, 5.7, 4.5 and 3.6. In Experiment 2, three seedlings of the same size as those in Experiment 1
were treated with Kimura B culture solution without Al (referred to as control hereafter) or containing
different levels of AlCl3·6H2O corresponding to 10, 20, 100 and 200 ppm Al at pH 3.6. The pH of theculture solution was adjusted with 1.0N HCl as required. The pots were placed in the same
greenhouse under natural sunlight. An air pump was connected to the pots to provide air to theroots. The culture solutions in each pH and Al treatment were supplemented and renewed every
other day from 23 May to 9 October, 2007.
Photosynthetic rate, transpiration rate, and stomatal conductance
The leaflets of the most active leaves or the 4th leaf position from the top of the treatedplants (18 weeks after the treatments) were selected for measuring the net photosynthetic rate,transpiration rate, and stomatal conductance using a potable photosynthetic meter (Analytical
Development Co., Ltd., LCA-4, England) at saturation irradiance with incident photosynthetically
active radiation (PAR) of 800-1,000 μmol m-2s-1.
Sampling and nutrient concentrations in plants
The treated plants were separated into three parts: leaflets, petioles including rachis, androots. The leaflet areas were measured using an automatic area meter (Hayashi-Denko AAM-9,
Japan). The separated samples were dried in an oven at 80ºC for 72 h to measure the dry weight
and then ground into powder in order to analyze the ion concentrations. The ground samples werereduced to ash in a furnace and extracted with 1.0N HNO3, and the K+, Ca2+ and Mg2+
concentrations were determined using a high-performance liquid chromatography (HPLC) methodwith a conductivity detector (Shimadzu CDD-6A, IC-C3, Japan). The concentration of P was
evaluated by atomic absorption spectrophotometry. The total N concentration was determined by

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the semi-micro Kjeldahl method, while the Al3+ concentration was determined calorimetrically by thealuminon method.
The statistical difference of the data was determined using NCSS 2001 (Number Cruncher
Statistical Systems). The effects of treatments were determined by one-way ANOVA (analysis ofvariance), and the differences among the mean values of treatment were determined using the
Tukey-Kramer test.

Results and Discussion
During the experiment, approximately 7 leaves emerged in each pH treatment. The
number of dead leaves during the experiment was 4, 4 and 3 at pH 5.7, 4.5 and 3.6, respectively.
There were no significant differences in the number of emerged, dead and living green leaves
among the three pH treatments, which means that the low pH conditions had no effect on new leaf
emergence and senescence. Plant growth in weekly increment of length and total leaflet area did
not change with the pH treatments. Although there was no significant difference in the total dry
weight per plant among the pH treatments, the total dry weight in the pH 3.6 treatment was 8.7%
smaller than that in the pH 5.7 treatment (Table 1). Similarly, the photosynthetic rate in the pH 3.6
treatment was 8.3% smaller than that in the pH 5.7 treatment. The difference in photosynthetic rates
among the three pH treatments could be attributed to differences in the stomatal conductance; thestomatal conductance in the pH 3.6 treatment was 7.5% smaller than that in the pH 5.7 treatment.
Table 1. Effect of low pH on weekly increment of plant length, leaflet area per plant and dry matter weight(Experiment 1)pH
Treatment

Weekly increment
Leaflet area
2of plant length (cm) per plant (cm )pH 5.7pH 4.5pH 3.6

2.0 a
2.0 a
2.0 a

2400.6 a2457.2 a2418.8 a

Dry matter weight per plant (g)LeafletPetiole

Root

Whole

18.2 a19.5 a17.3 a23.7 a22.0 a20.1 a8.9 a10.8 a9.0 a50.8 a52.3 a46.4 aMeans with the same letters in a given column are not significantly different at the 0.05 level by the TukeyKramer test (n=3).

Under the Al treatments, the total dry weight and total leaflet area in the 10 ppm Al
treatment were slightly larger than those in the other treatments, while those in the 200 ppm Al
treatment were significantly smaller than those in the control treatment (Table 2). Moreover, theroots of sago palm seedlings under the 200 ppm Al treatment were stunted, brownish and thick, andthe root dry weight was 58% smaller than that in the control treatment, representing a significant
difference (Table 2). Consequently, the critical value to inhibit the growth of sago palm wasconsidered to be approximately 200 ppm Al in the growth media, which is much higher than the realconcentrations in the natural soil conditions.
The concentrations of Al3+, N, P, K+, Ca2+ and Mg2+ in the leaflets, petioles, roots andwhole plants under the Al treatments are shown in Table 3. The Al3+ concentration in all the plantparts increased with the rise of the Al concentrations. Moreover, the Al3+ concentration in the leaflet
was lower than that in the petiole and tended to be significantly higher in the root than in the top
parts (leaflets and petiole) in all the Al treatments. Our current results in sago palm strongly support
the assumption that Al3+ has a high binding ability with cellular components of the root and usuallyshows slight translocation to the upper parts of the plant (Ma et al., 1997). The total N and P
concentrations in the whole plants of the 10 ppm Al-treated plant were higher than those in thecontrol and other Al-treated plants, which could lead to an increase in the growth of sago palm
under a mild Al concentration in the growth media. These results indicated that Al was unlikely tohave induced the P and N deficiency in plant tissues but the uptake of these nutrients was higherthProceedings of The 7 ACSA Conference 2011161

under a lower Al condition, as such evidence was also found in rice (Fageria, 1985) and some
native plants (Osaki et al., 1997). The K+ concentration in all the plant parts was independent of the
Al treatment. Whereas the Ca2+ and Mg2+ concentrations in whole plants decreased by the increaseof the Al concentrations in the growth media, a significant difference was clearly observed in the
200 ppm Al-treated plants. One interesting feature of these results is that Al3+ inhibited Ca2+ andMg2+ absorption more than K+ absorption in all the plant parts under the higher Al treatment. The
3+
possible mechanism to explain the different effects on cations is that the Al toxicity was+3+2+ameliorated by cations in the following order, H approximately = C > C > C+, and theamelioration was due to their binding to or screening the negative charges on the plasma
membrane (Kinraide et al.,1992).
Table 2. Effect of Al concentration on weekly increment of plant length, leaflet area per plant and dry matterweight (Experiment 2)
Alconcentration(ppm)
Control
10
20
100
200

Leaflet area
Weekly increment
per plantof plant length (cm)
2
(cm )
2.0 a
2.0 a
1.9 ab
1.9 ab1.7 b2418.8 ab
3008.6 a
2092.0 b
2151.7 b
1153.4 c

Dry matter weight per plant (g)LeafletPetiole

Root

Whole

17.3 ab23.0 a15.7 bc
15.1 bc
8.0 c

20.1 ab27.1 a16.3 b
18.0 b
11.2 c

9.0 ab
13.6 a
6.7 b
6.6 b
3.8 c

46.4 ab63.7 a38.7 bc
39.7 bc
22.9 cMeans followed by different letters within a column are significantly different at the 0.05 level by the TukeyKramer test (n=3).

According to Chenery (1948), thousands of the plant species are classified, according totheir Al concentrations in plant tissues, as Al-accumulators (≥ 1,000 mg Al kg-1 dry weight) or Al
excluders (< 1,000 mg Al kg-1 dry weight). Some plant species known as the Al accumulators may
contain more than 10 times this Al level without any Al injury. However, most plants contain no morethan 300 mg Al kg-1 dry weight. In the current study, the range of Al3+ concentrations in whole plantsof sago palm was from 9.4 to 15.6 µmol g-1 (254 to 420 mg kg-1) dry weight even under the 200 ppm
Al treatment (Table 3). Considering the result of the Al3+ concentration in whole plant tissues, sago
palm is considered to have high resistance to Al with mechanical restriction of the excess Al basedon its Al exclusion ability under acid conditions.

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Table 3. Effect of Al concentration on nutrient concentrations in the leaflets, petioles, roots and whole plants(Experiment 2)
Nutrient
concentration
3+-1
Al (µmol g )

Plant
part
LeafletPetioleRoot
Whole
LeafletPetioleRoot
Whole
LeafletPetioleRoot
Whole
LeafletPetioleRoot
Whole
LeafletPetioleRoot
Whole
LeafletPetiole
Root
Whole

-1

N (mg g )-1

P (mg g )+-1

K (µmol g )

2+

-1

2+

-1

Ca (µmol g )Mg (µmol g )Control
8.7 cB
8.9 bB
12.1 cA
9.4 b
20.9 aA
8.8 aB
10.8 aB
13.6 a
1.8 aA
2.2 aA
1.6 abA
1.9 ab93.5 aB
219.6 bA
253.4 aA
178.2 b42.4 aB
55.7 abA
28.8 abB
45.7 ab41.1 aB
56.7 abAB
63.9 aA
52.1 abAl concentration (ppm)
10
20
100
9.5 bcB
10.3 bB
10.2 bcB
10.7 abB
11.6 abB
13.1 abB
15.1 bcA
16.3 abA
17.7 abA
11.2 b
11.9 ab12.7 ab24.6 aA
23.0 aA
22.6 aA
9.1 aB
8.7 aB
7.7 aB
9.9 aB
9.8 aB
10.2 aB
14.8 a14.6 a14.1 a
1.9 aA
1.8 aAB
1.7 aAB
2.3 aA
2.2 aA
1.9 aA
1.8 aA
1.4 abB
1.1 bcB
2.0 a
1.9 ab
1.7 ab
92.6 aB
97.8 aB
93.4 aB
199.5 bA
215.4 bA
220.0 bA
209.3 bA
226.7 abA
250.7 aA
162.8 b170.7 b173.9 b45.3 aB
42.6 aB
32.8 abB
64.4 aA
65.2 aA
47.7 bA
36.1 aB
36.8 aB
30.5 abB
51.4 a51.3 a39.2 b40.3 aB
40.9 aB
34.8 abC
60.4 aA
63.0 aA
46.6 bB
65.1 aA
67.4 aA
66.8 aA
54.1 a55.1 a45.5 b200
14.3 aB
15.1 aB
19.8 aA
15.6 a21.0 aA
6.3 aB
11.3 aAB
12.6 a
1.6 aAB
1.4 bA
0.9 cB
1.4 b
98.5 aB
250.7 aA
267.0 aA
200.0 a25.5 bA
28.9 cA
21.7 bA
26.7 c29.0 bA
29.5 cA
36.2 bA
30.5 cMeans followed by different letters within a row are significantly different at the 0.05 level by the Tukey-Kramer test (n=3).
Lowercase letters indicate a comparison among the Al treatments in each part of a plant. Capital letters indicate acomparison among plant tissues under the various Al treatments.

References
Baba I and Y Takahashi. 1958. Solution culture. In Y. Togari ed., Sakumotsu Shiken Ho. Nogyo Gijutsu Kyokai,
Tokyo. 327-343.
Chenery EM. 1948. Aluminium in the plant world: I. General survey in dicotyledons. Kew Bull. 2: 173-183.
Ehara H, S Susanto, C Mizota, S Hirose and T Matsuno. 2000. Sago palm (Metroxylon sagu, Arecaceae)
production in the eastern archipelago of Indonesia: Variation in morphological characteristics and pithdry matter yield. Econ. Bot. 54: 197-206.
Ehara H. 2005. Geographical distribution and specification of Metroxylon palms. Jap. J. Trop. Agr. 49: 111-116.
Ehara H. 2009. Potency of sago palm as carbohydrate resource for strengthening food security program. J.
Agron. Indonesia 37: 209-219.
Fageria NK. 1985. Influence of aluminum in nutrient solutions on chemical composition in two rice cultivars at
different growth stages. Plant Soil 85: 423-429.
Foy CD and AL Fleming. 1978. The physiology of plant tolerance to excess available aluminum and manganesein acid soils. ASA spec. 32: 301-328.
Jong FS and M Flach. 1995. The sustainability of sago palm (Metroxylon sagu) cultivation on deep peat inSarawak. Sago palm 3: 13-20.
3++Kinraide TB, PR Ryan, and LV Kochian. 1992. Interactive effects of A1 , H , and other cations on root
elongation considered in terms of cell-surface electrical potential. Plant Physiol. 99: 1461-1468.
Ma JF, SJ Zheng, H Matsumoto and S Hiradate. 1997. Detoxifying aluminum with buckwheat. Nature 390: 569570.
Osaki M, T Watanabe and T Tadano. 1997. Beneficial effect of aluminum on growth of plants adapted to low pH
soils. Soil Sci. Plant Nutr. 43: 551-563.

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163

Osaki M, T Watanabe, T Ishizawa, C Nilnond, T Nuyim, T Shinano, M Urayama and SJ Tuah. 2003. Nutritional
characteristics of the leaves of native plants growing in adverse soils of humid tropical lowlands. Plant
Foods Hum. Nutr. 58: 93-115.
Purwanto BH, K Kakuda, H Ando, JF Shoon, Y Yamamoto, A Watanabe and T Yoshida. 2002. Nutrient
availability and response of sago palm (Metroxylon sagu Rottb.) to controlled release N fertilizer oncoastal lowland peat in the tropics. Soil Sci. Plant Nutr. 48: 529-537.
Sato T, T Yamaguchi and T Takamura. 1979. Cultivation, harvesting and processing of sago palm. Jpn. J. Trop.
Agr. 23: 130-136.

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Transplanted Sucker Stem Growth in Sago Palm (Metroxylon sagu Rottb.)
Before Trunk Formation
Satoshi Nakamuraa,*, Keita Nabeyab, Mutsumi Akamab,, Teiji Nakamurab,
Youji Nittac, Manabu Watanabed, Yusuke Gotoba

School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Miyagi, Japanb
Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japanc
College of Agriculture, Ibaraki University, Ami, Ibaraki, Japand
Field Science Center, Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan*Corresponding author: School of Food Agricultural and Environmental Sciences,2-2-1, Hatatate, Taihaku-ku, Sendai, Miyagi, 982-0215, Japan.
Tel.: +81 22 245 1278; Fax: +81 22 245 1278.
nakamurs@myu.ac.jp

Abstract
Sago palm sucker stems transplanted in fields creep along the ground surface during a rosette stage, later
forming a vertical trunk. Many suckers grow at the stem bottom before trunk formation. Thinning and pruning ofsuckers, known as sucker control, is extremely important to maintain an appropriate density of trunks and to
produce much starch in sago plantations over time. Selection of suckers with consideration of the suckers‘
creeping growth is necessary to determine the appropriate spread of trunks in a field. Nevertheless, few reports
describe the creeping growth of sucker stems. This study was undertaken to clarify the creeping growth ofsuckers transplanted in a field under sucker control before trunk formation. Suckers were transplanted in a field-1
in Mukah, Sarawak, Malaysia on September 3, 2005. The plant density was 330 plants ha . The growth of 12
suckers was monitored and the sucker stem lengths were measured every year during 2008–2011. The average
stem lengths were 74.2 ± 4.7 cm and 155.3 ± 6.3 cm, respectively, at 1,201 days and 2,120 days after-1transplantation. The length increased linearly at 31.5 cm year (Y = 0.0862X – 25.301, r = 0.993, P < 0.001,
where Y is the stem length and X is the days after transplanting). Each year, 10.5 leaves expanded. Althoughsucker growth differs among soil types, regions, and varieties, the information collected in this study is expected
to be useful for predicting the trunk position.
Keywords: creeping growth, sago palm, stem, sucker, sucker control

Introduction
Sago palm sucker stems transplanted in fields creep along the ground surface during arosette stage, later forming a vertical trunk. Many suckers grow at the stem bottom before trunk
formation. These suckers derive carbohydrates from the mother palm, the transplanted sucker, until
the photosynthetic apparatus is sufficiently developed and absorbs enough sunlight (Flack, 1977).
Emergence and growth of many suckers at the stem bottom are expected to exacerbate
competition between the suckers and their mother-palm for nutrition from soil because of theirproximity. For that reason, the mother-palm growth might be suppressed. Reportedly, the rate ofleaf emergence of non-controlled suckers is significantly lower than that of controlled suckers during
the first five months of growth (Nakamura et al., 2009). Consequently, thinning and pruning ofsuckers, known as sucker control, is extremely important to promote the growth of mother palm, tomaintain an appropriate density of trunks, and to maintain higher starch productivity in sago
plantations over time. Selection of suckers with consideration of the suckers‘ creeping growth istherefore necessary to determine the appropriate spread of trunks in a field. Nevertheless, few
reports describe the creeping growth of sucker stems. This study was undertaken to clarify thecreeping growth of suckers that had been transplanted in a field, with sucker control applied before
trunk formation.
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Materials and Methods
Suckers were transplanted in a field in Mukah (N 02°56‘31‖, E 112°18‘26‖), Sarawak,
Malaysia on 3 September 2005. The plant density was 330 plants ha-1; the plant mean area was
30.5 m-1. The growth of 12 suckers was monitored: the sucker stem lengths were measured eachyear during 2008–2011. The transplanted sucker stem length was defined as the distance from its
end to its shoot apex, which was covered with thick leaf sheaths. We were able to monitor their
growth. Therefore, the shoot apex position was estimated from anatomical features of young sago
palms sampled in the same field in 2008 (Figure1). The leaves of suckers were marked formonitoring using a marker pen. The number of expanded leaves was recorded. The number of theirgreen leaves was also recorded each year.

a
)
b
)

Figure 1. Mother palm sampled in the field in 2008 (a) and its stem, from which leaf sheaths were removed (b).
Circles show the estimated position of the shoot apex.

Results and Discussion
All transplanted suckers grew well during 2005-2011 (Figure 2). The average stem lengths
were 74.2 ± 4.7 cm and 155.3 ± 6.3 cm, respectively, at 1,201 days (17 December 2008) after
transplanting (1,201 DAT) and 2,120 DAT (24 June 2011) (Figure 3), which suggests that the length
increased linearly at 31.5 cm year-1 (Y = 0.0862X – 25.301, r = 0.993, P < 0.001, where Y is thestem length and X is the DAT). The stem length was observed to increase gradually, probably
increasing exponentially until the start of linearly incremental extension of the stem length (Goto etal., 2010). Because some suckers that start trunk formation were observed at 1,201 DAT, the rateof creeping growth of sucker is expected to decrease. The final stem length is reached soon
thereafter.

Figure 2. Mother palm, of which the stem has been creeping, at 2,120 days (in 2011) after transplantation.

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Stem length (cm)200

y = 0.0862X – 25.301
r = 0.993***

150
100
50
0
0

500

1,000

1,500

2,000

2,500

Days after transplanting (DAT)
Figure 3. Stem length of the sucker after transplanting. Vertical bars represent standard errors.

Number of green leaves

The average numbers of living leaves of the suckers were 16.0 ± 0.6 and 11.2 ± 0.5,
respectively, at 1,201 DAT and 2,120 DAT (Figure 4). The green leaf number tended to decrease,although the total and each area of leaves increased. It was observed that the size of leaves andthe number of leaflets at 2,120 DAT was greater than those at 1,201 DAT. Results of leaf position
data showed that 26.5 new leaves expanded from 1,201 DAT to 2,120 DAT, suggesting that 10.5
leaves expanded each year.
20
15
10

5
0

500

1,000

1,500

2,000

2,500

Days after transplanting (DAT)
Figure 4. Number of green leaves of the sucker after transplanting. Vertical bars represent standard errors.

Conclusions
In conclusion, the rate of creeping growth of sucker stem in a field was specified after
transplantation by monitoring the stem length. Although sucker growth is expected to differ among
soil types, regions, and varieties, the information collected in this study will be useful for predicting
the trunk position in a field.

Acknowledgements
This work was supported by KAKENHI 20405017. We are also grateful to Mr. Smith in Mukah,
Malaysia for their kind assistance.

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Proceedings of The 7 ACSA Conference 2011167

References
Flack, M. 1977. Yield potential of the sago palm and its realization. In M.A. Tankooling ed., Sago ‘76: Papers of
the First International Sago Symposium, Kuala Lumpur. 157-177.
Goto, Y., Nakamura, S., Nakamura, T., Nitta, Y. and Watanabe M. 2010. The sucker-control and the creeping
growth of sago palm (Metroxylon sagu Rottb.). Jpn. J. Crop Sci. 61 (Extra issue 1): 220-221. (inJapanese).
Nakamura, S., Nitta, Y., Watanabe, M., Nakamura, T. and Goto, Y. 2009. Effect of sucker control on the leaf
emergence of sago palm (Metroxylon sagu Rottb.) before trunk formation. Jpn. J. Crop Sci. 60 (Extraissue 1): 260-261. (in Japanese).

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Secondary Aerenchyma Formation and Root Growth Response of Soybean(Glycine max) Seedlings under Flooded Conditions
Toshihiro Mochizuki a,*, Satomi Sakazono b, Sayuri Kajihara c,
Satoshi Shimamura daFaculty of Agriculture, Kyushu University, Fukuoka, Japan
Graduate School of Life Sciences, Tohoku University, Miyagi, Japan,cSchool of Agriculture, Kyushu University,dNational Agricultural Research Center for Tohoku Region, Akita, Japan* Corresponding author: Experimental Farm, Kyushu University, Harumachi 111, Kasuya-machi, Kasuya-gun,
Fukuoka 811-2307, Japan
Tel.: +81 92 612 2865; fax: +81 92 612 2865.
mochizuki@farm.kyushu-u.ac.jpbAbstract
Most of wetland species can develop their roots into flooded soils because of the presence of
longitudinal aerenchyma channels that facilitates oxygen diffusion from shoot to root tips. Thistissue is called as primary aerenchyma because it is formed in fundamental tissues. It is also
formed in rice root, consequently rice plants can grow well in paddy field. On the other hand, it isconsidered that most of mesophytes such as field crops cannot grow under flooded and excess
moisture conditions because of their low ability to develop aerenchyma. However, we found thatsoybean plants could develop aerenchyma and grow well in flooding compared with otherleguminous crops such as Vigna and Phaseolus species. This type of aerenchyma, which isconsisted of white spongy tissue filled with gas space and is differentiated from secondary meristem(phellogen), is called assecondary aerenchyma. It plays a role in supplying oxygen from the aerial
parts to the flooded roots and nodules. In our recent study, it was observed that there was a wide
range of varietal differences on secondary aerenchyma formation and adventitious root
development in soybean seedlings under flooding.
Although the research for secondary
aerenchyma in soybean plants is on the way, it may be able to breed soybean varieties withflooding tolerance.
Keywords: anaerobiosys, flooding, secondary aerenchyma, soybean, water logging

Introduction
Flooding is a major problem in many areas of the world and most of mesophytes such as
field crops are susceptible to the stress. Therefore, an understanding of the morphologicalmechanisms of flooding tolerance is important for developing flood-tolerant genotypes. Aerenchyma
formation is one of the morphological changes that occurs in plants grown under flooded andhypoxic conditions and it is thought to enhance the internal diffusion of atmospheric andphotosynthetic oxygen from the aerial parts to the flooded roots, allowing the roots to maintainaerobic respiration (Armstrong, 1979). The two types of aerenchyma are classified. One is cortical(lysigenous or schizogenous) aerenchyma formed in the roots of rice, maize, barley, wheat andsome Rumex species. The other is white spongy tissue filled with gas spaces and is formed in thestem, hypocotyl, tap root, adventitious roots and root nodules of Glycine soja, Sesbania rostrata,
Lotus uliginosus and Viminaria juncea grown under flooded conditions. It is differentiated fromsecondary meristem (phellogen) and is called secondary aerenchyma (Jackson and Armstrong,1999). Secondary aerenchyma seems to play a role in supplying oxygen to roots and nodules. In

thProceedings of The 7 ACSA Conference 2011169

contrast to cortical aerenchyma, however, there is little information about the morphological andanatomical processes of secondary aerenchyma formation and its function.
Soybean is generally susceptible to flooding, and its growth and yield are negatively
affected by the stress. Arikado (1954) suggested that soybean had low flooding tolerance becauseof its low ability to develop aerenchyma in the shoot and roots. However, some soybean genotypes
can form lysigenous aerenchyma in the root cortex (Bacanamwo and Purcell, 1999) and secondary
aerenchyma in the hypocotyl and tap root of seedlings grow under flooded conditions (Mochizuki etal., 2000). In this paper, we introduced our recent studies of the morphological and anatomical
processes of secondary aerenchyma formation and its function as an oxygen pathway from theaerial parts to flooded roots, and we described here the relationship between aerenchyma formationand the flooding tolerance of soybean.
Secondary aerenchyma formation in flooded soybean
We investigated secondary aerenchyma formation in soybean seedlings grown underflooded conditions (Shimamura et al., 2003). As a result, within 3 weeks of flooding, large volumesof secondary aerenchyma developed in flooded hypocotyl, tap root, adventitious roots and nodules(Fig. 1). This tissue has large intercellular spaces and consists of living cells whose walls do not
become suberized, whereas phellem has no intercellular spaces and consists of dead cells whose
walls become suberized. It is differentiated from secondary meristem (phellogen) and ishomologous with cork tissue (Fig. 2). Cells were exposed to the outside through lenticels in
hypocotyl and roots where epidermis and cortex layers were collapsing. The outer part of thenodules was covered with a layer of secondary aerenchyma that was relatively thinner in the
nodules than in the hypocotyl and roots. Nodules attached to the face of roots had pink infected
tissues, which suggested the presence of leghemoglobin and the survival of nodules under flooded
conditions.

.
a, root system; b, transverse section of an adventitious root; c, transverse section of a nodule. WL, water level; SS, soil surface;
AR, adventitious roots; RN, root nodules; SA, secondary aerenchyma.

Figure 1. Root system and secondary aerenchyma development in soybean seedlings after 3 weeks of flooding.

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Figure 2. Cross sections of young hypocotyl in soybean.

Function of secondary aerenchyma in flooded soybean
Oxygen dynamics in aerenchymatous stems were investigated using Clark-type O2
microelectrodes, and O2 transport to roots was evaluated using stable-isotope 18O2 as a tracer, forplants with shoots in air and roots in flooded sand or soil (Shimamura et al., 2010).
After introducing 18O2 gas via the stem lenticels, significant 18O2 enrichment in water
extracted from roots after 3 h was confirmed, suggesting that transported O2 sustained root
respiration. In contrast, slight 18O2 enrichment was detected 3 h after treatment of stems that lacked
aerenchyma and lenticels. The results showed that hypertrophic lenticels in the lower stem ofsoybean, just above the water surface, are entry points for O2, and these connect to aerenchyma
and enable O2 transport into roots in flooded soil.

Flood Ae-Open, aerenchyma and lenticels above the water; Flood Ae-Submerged, submerged aerenchyma and lenticeles.
Values are the mean ± s.e. Means in the shoot followed by the same upper-case letter and means in the root followed by thesame lower-case letter do not differ significantly (P<0.01, Tukey-Kramer‘s test).
18

Figure 3. Volume of O2 transported from the stem to the roots with or without exposure of the aerenchyma to
18
the O2 gas in each treatment.

thProceedings of The 7 ACSA Conference 2011171

Interspecific difference in secondary aerenchyma formation of flooded leguminous crops
We investigated the secondary aerenchyma formation in hypocotyls just below the soil
surface of young seedlings in wild soybean and six summer leguminous crops grown under uplandand flooded conditions for 14 days (Mochizuki et al., 2000).
Under the upland conditions, secondary aerenchyma was scarcely observed in any
species. Under the flooded conditions, however, there was an interspecific difference in thesecondary aerenchyma formation. Secondary aerenchyma area per transverse section of
hypocotyls under the flooded conditions was largest in soybean ‗Asoaogari‘; followed by soybean‗Akisengoku‘, wild soybean ‗D5‘, cowpea ‗Sanjakusasage‘, mung bean ‗Bundomame‘, and mungbean ‗Acc. 7703‘, and those in the other crops were less than 1 mm2 (Table 1). Since a significant
positive correlation was found between dry weight ratio (the ratio of dry weight of the aerial part
under the flooded conditions to that under the upland conditions) and secondary aerenchyma area
(r=0.738*), it is suggested that the ability of secondary aerenchyma formation is related to theflooding tolerance in leguminous crops.
Table 1. Secondary aerenchyma area of hypocotyl and dry weight ratio in summer leguminous crops

Secondary aerenchyma ratio is the percentage of secondary aerenchyma area to steal area. Dry weight ratio is the percentage
of dry weight of the aerial part under flooded conditions to that under upland conditions. Values followed by the same letter ineach column are not significantly different at P<0.05 by Duncan's multiple test.

Effects of hypoxia on dry matter production and root development in soybean and wild
soybean cultivars
Using 91 soybean and wild soybean cultivars, effects of hypoxia on dry matter production
and root development were examined (Sakazono et al., 2011). Seedlings of eight days after sowing
were grown in solution culture with (control) and without (hypoxia) O 2. Seven days after treatment,
dry weight, root characters and hypocotyl diameter were measured. Root characters were
measured with Win RHZO (Regent Instruments Inc., Quebec, Canada). In this experiment, we use
the ratio of hypocotyl diameter (ratio of hypocotyl diameter in hypoxia to that of control) as anindicator of secondary aerenchyma development in hypoxia.
Effect of hypoxia was higher in root growth than shoot growth. Total root length, root
surface area and root volume were decreased by hypoxia, whereas average root diameter and

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hypocotyl diameter were increased. However, in every trait including hypocotyl diameter, there waswide variation among cultivars.
Table 2. Effects of hypoxia on dry matter production and root development in soybean and wild soybean
cultivars

Conclusions
It is clearly that the secondary aerenchyma formed in soybean plays a role in supplying
oxygen from aerial parts to roots and nodules, and there are interspecific and intraspecificdifferences in the secondary aerenchyma formation. Although the research for secondary
aerenchyma in soybean plants is on the way, it may be able to breed soybean varieties withflooding tolerance.

References
Arikado H. 1954. Different responses of soybean plants to an excess of water with special reference toanatomical observations. Proc. Crop Sci. Soc. Japan 23:28–36.
Armstrong W. 1979. Aeration in higher plants. Adv. Bot. Res. 7:225–332.
Bacanamwo M., L.C. Purcell. 1999. Soybean root morphological and anatomical traits associated withacclimation to flooding. Crop Sci. 39:143–149.
Jackson M. B., W Armstrong. 1999. Formation of aerenchyma and the processes of plant ventilation in relation
to soil flooding and submergence. Plant Biol. 1:274–287.
Mochizuki T, U Takahashi, S Shimamura and M Fukuyama. 2000. Secondary aerenchyma in hypocotyl insummer leguminous crops. Jpn. J. Crop Sci. 69:69-73. (In Japanese with English abstract).
Sakazono S, R Matsuo, S Kajihar, M Ishimoto, K Harada, R Takahashi and T Mochizuki. 2011. Flooding
tolerance variation among soybean cultivars and lines at cotyledon stage. Jpn. J. Crop Sci. 80 (special
issue 1):442-443. (In Japanese).

thProceedings of The 7 ACSA Conference 2011173

Shimamura S, T Mochizuki, Y Nada and M Fukuyama. 2003. Formation and function of secondary aerenchyma
in hypocotil, roots and nodules of soybean (Glycine max) under flooded conditions. Plant and Soil 251:351359．
Shimamura S, R. Yamamoto, T Nakamura, S Shimada and S Komatsu. 2010. Stem hypertrophic lenticels andsecondary aerenchyma enable oxygen transport to roots of soybean in flooded soil. Ann. Bot. 106:277284.

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Agronomical Performances of Soybean Cultivated under
Saturated Soil Culture on Tidal Swamps
M. Ghulamahdia, M. Melatia, S. A. Aziza, A. Junaedia , Sahuria, Y. Puspitasaria,
D. SagalabaDepartment of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University
Department of Agronomy, Faculty of Agriculture, Prof Azairin University, Bengkulu, Indonesia* Corresponding author: Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural University, Kampus IPB, Dramaga, Bogor, Indonesia 16680
Tel/fax : +62-251-8629353
mghulamahdi@yahoo.comb

Abstract
Saturated soil culture (SSC) is a cultivation technology mantaining water depth constanly to make soil layer inthe saturated condition. This paper resumes of two experiments to evaluate the effect of water depth and bed
width, and leaching time and varieties of soybean cultivated under SSC on tidal swamp. The research was
conducted at Banyu Urip, Banyuasin, South Sumatra from April to August 2010. In the first experiment, water
depth in the furrow irrigation as main-plot consisted of 10 and 20 cm under soil surface (USS) and bed widths as
sub-plot consisted of 2, 4, 6 and 8 m. The results showed that the highest grain yield was obtained on 20 cm
USS water depth and bed width 2 m (4.15 ton/ha), it was significantly different from those at bed width 4 m (2.59
ton/ha), 6 m (1.84 ton/ha) and 8 m (1.74 ton/ha). The grain yield on water depth 10 cm USS and bed width 2 mobtained was 3.43 ton/ha, it was significantly different from those at bed width 4 m (2.46 ton/ha), 6 m (1.75
ton/ha) and 8 m (1.68 ton/ha). In the second experiment, leaching time as main-plot consisted of without
leaching, every 2, 4 and 6 weeks, and soybean variety as sub-plot consisted of Tanggamus, Slamet, Willis andAnjasmoro. The results showed that interaction between leaching time and varieties did not significally affectgrain yield, varieties responded leaching time differently. The highest grain yield was obtained by Anjasmoro
variety with leaching time every 2 weeks (4.06 ton/ha) but it was not significally different from those with leachingtime every 4 weeks (3.99 ton/ha) and every 6 weeks (3.93 ton/ha). From these experiments, water depth 20 cm
USS and bed width 2 m may be recommended for SSC soybean cultivation on tidal swamp, and presumably,leacing time every six weeks will gain more effective economically and technically.
Keywords: leaching time, saturated soil, tidal swamp, water depth, bed width

Introduction
One alternative system to develop soybean cultivation in Indonesia is to optimize the useof marginal land, and tidal swamp is one of the potential ecosystem for future soybean production.
Indonesia has about 20 million ha tidal swamps, 9 millions ha out of them is appropriate for
agriculture, 2 milions is suitable for soybean (Noor and Sabur, 2007). The major constrain of
producing soybean in tidal swamp due to its high pyrite content. When pyrite is oxidized, soil pH
decreases. Djayusman et al. (2001) reported that high pyrite content suppressed the productivity ofsoybean on tidal swamps to only about 800 kg/ha. SSC is a technology in cultivation giving waterpermanently, maintains and keeps its depth constantly (about 5 cm USS). This makes soil layer insaturated condition. In SSC, watering is started from the beginning of growth to maturity stage. By
keeping the water-table constantly, soybean will be avoided from negative effect of inundation on
soybean growth, because soybean will acclimatize and improve its growth (Troedson et al., 1985).
Soil water management can be applied to reduce pyrite content where the soil is in
reductive condition and able to support soybean growth. SSC technology is one of soil water
managements studied in highland and succeed to increase soybean production (Indradewa et al.,th

Proceedings of The 7 ACSA Conference 2011175

2004; Ghulamahdi et al., 2006). This offers the chance to reduce the pyrite, hence increasesoybean production on tidal swamps. Adisarwanto (2001) suggested for soybean cultivation using
bed width less than 2 m. The addition of bed width will reduce the use of labor in the preparing land
to make the trench, but to consider the ability of water seeped from the trench into the middle ofbeds. Indradewa et al. (2002) reported that the inundation in the trench with 3-4 m wide bed was theideal plot. There was no difference in the influence of bed width on the growth and yield of soybean.
Response of soybean to saturated condition varied between varieties and the later-maturing
soybean was better than the earlier one (CSIRO, 1983; Ghulamahdi et al., 1991; Ghulamahdi,
2008; Ghulamahdi and Nirmala, 2008). Many varieties of soybean have been studied in their
response on acid soil. Alihamsyah and Ar-Riza (2006) found that Tanggamus, Wilis and Slamet
were varieties that could adapt well on inland. Leaching of land can decrease negative effect of
poisonous material (Fe, Al, Mn) to the soybean growth. The frequency of water drainage from thetrench will effect to the content of poisonous material. The objectives of the research were : 1) tostudy the effect of water depth and bed width , and 2) to investigate the effect of leaching time andvarieties to the growth and production of soybean under SSC on tidal swamps.

Materials and Methods
This research was conducted on tidal swamps land in Banyuurip Village of Tanjung Lago
Sub District, Banyuasin District, South Sumatera, Indonesia from April to August 2010. In the firstexperiment, water depth in the furrow irrigation as main-plot consisted of 10 and 20 cm under soil
surface (USS) and bed widths as sub-plot consisted of 2, 4, 6 and 8 m. This experiment used
Tanggamus variety. In the second experiment, leaching time as main-plot consisted without
leaching, every 2, 4 and 6 weeks, and soybean variety as sub-plot consisted of Tanggamus,
Slamet, Willis and Anjasmoro. Each main plot was surrounded by furrow irrigation. Water was given
at planting time and kept until the maturity stage and made plots in wet condition. Two weeks before
planting, plots were applied with 2 ton dolomite/ha, 400 kg SP18/ha, and 100 kg KCl/ha. Soybeans
were sprayed with 10 g Urea/l water at 2, 4, 6 weeks after planting to support acclimatization.
At planting date, seeds were inoculated with Rhizobium sp and treated with insecticide withactive agent Carbosulphan 25.53%. Seeds were planted in 2 x 5 m plot size, 20 x 25 cm planting
distance, 2 seeds per hole. The observed variables were : nodule, root, stalk, and leaves dry weightat 6 weeks after planting (WAP); plant height, number of branch, fill pod, and empty pod per plant,
seed productivity (ton/ha), and 100-seed dry weight at harvest time.

Results and Discussion
In the first experiment, there was the influence of water depth on the leaf, stalk, root andnodule dry weight at 6 WAP. The leaf, stalk, root and nodule dry weight were significantly higher inthe water depth 20 cm USS than that ini 10 cm USS (Table 1). This was predicted because the root
growing zone in the water depth 20 cm USS wider than 10 cm USS, so it provied adequate for
maximum root growth. According to Suwarto et al. (1994) water depth significantly influenced on theleaf, stalk, root and nodule dry weight.
Water depth affected the plant height, number of branch and fill pod per plant, but there
was no influence of water depth on the number of empty pod and 100 seed weight. Plant height,
number of branch and fill pod and productivity of soybean were significantly higher in the water
depth 20 cm USS than those in 10 cm USS (Table 2).

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Proceedings of The 7 ACSA Conference 2011

Table 1. The effect of water depth on leaf, stalk, root, and nodule dry weight at 6 WAP
Water Depth (cm)

Leave (g)Stalk (g)Root (g)Nodule (g)

10
20

3.52b
4.51a

3.64b
4,84a

0.73b
0.99a

0.33b
0.48a

Note: numbers followed by the same letter on the same column are not significantly different with Duncan multiple range test at 5%.

Table 2. The effect of water depth on plant height, number of branch and fill pod, and productivity at harvest time
Water Depth(cm)

Plant Height(cm)

Branch/Plant

Fill Pod/Plant

Productivity(ton/ha)10
2070.66b73.86a

4.22b
4.55a

67.83b71.83a

2.33b
2.58a

Note: numbers followed by the same letter on the same column are not significantly different with Duncan multiple range test at 5%.

There was the influence of bed width on the leaf, stalk, root and nodule dry weight. The leaf,
stalk, root and nodule dry weight were significantly higher in the bed width 2 m than that with theother bed widths (Table 3). This was predicted because the water seepage from the ditch into the
middle of bed highest distributed on the bed width 2 m.
Table 3. The effect of bed width on leaf, stalk, root, and nodule dry weight at 6 WAP
Variables
Leave dry weight (g)Stalk dry weight (g)Root dry weight (g)Nodule dry weight (g)Bed Width 2 m

Bed Width 4 m

Bed Width 6m

Bed Width 8m

4.57a
5.27a
1.09a
0.58a

4.29ab
4.48ab
0.83ab0.40b4.03ab
4.09ab
0.78ab
0.34cb

3.17b
3.11b
0.72b
0.29c

Note: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

At harvest time, there was the influence of bed width on the number of branch, fill pod per
plant, seed productivity, and 100 seed weight, but there was no influence on the plant height andempty pod. Number of branch, fill pod, seed productivity, and 100 seed weight were significantly
higher in bed width 2 m than those in the other bed widths (Table 4). Indradewa et al. (2002)concluded that there was no difference in the effect of bed width on soybean growth and yield.
Table 4.

The effect of bed width on number of branch and fill pod, productivity and 100 seed weight at harvest time

Variables
Branch/plant

Bed Width 2 m
4.71a
80.17aFill Pod/plant
Productivity (tones / ha)
100 seeds weight (g)Bed Width 4 m
4.41ab73.50bBed Width 6 m
4.33ab65.17cBed Width 8 m
4.05b
60.17d

3.79a

2.52b

1.79c

1.71d

11.89a11.30ab10.98b

10.96bNote: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

There was the influence of water depth and bed width interaction on the leaf, stalk, rootand nodule dry weight. The leaf, stalk, root and nodule dry weight were significantly higher in thewater depth 20 cm USS with bed width 2 m than those in the other treatments (Table 5).
At harvest time there was the influence of water depth and bed width interaction on the theplant height, number of branch, fill pod, seed productivity and 100 seed weight of soybean. There
was no interaction effect of water depth and bed width interaction on the number of empty pods.
Plant height, number of branch and fill pod, seed productivity and 100 seed weight weresignificantly higher in the water depth 20 cm USS with bed width 2 m than those in the othertreatments (Table 6).
thProceedings of The 7 ACSA Conference 2011177

Table 5.

The effect of interaction of water depth and bed width on leaf, stalk, root, and nodule dry weight at 6 WAP

Water Depth (cm) x Bed Width (m)Variables

10x2

10x4

10x6

10x8

20x2

20x4

20x6

20x8

Leaf dry weight (g)3,22bc3,68bc4,14bc

3,90b

5,92a

4,91ab3,92bc3,29bcStalk dry weight (g)Root dry weight (g)Nodule dry weight (g)3,58bc0,69b
0,34bc3,79bc0,71b0,44b

4,16bc0,84b
0,30cd

3,06c
0,65b
0,22d

6.96a
1,50a
0,82a

5,17ab0,94b0,37bc4,02bc0,72b
0,37bc3,19bc0,82b0,36bcNote: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

Table 6. The effect of interaction of water depth and bed width on plant height, number of branch and fill pod,productivity, 100 seed weight at harvest time
Water Depth (cm) x Bed Width (m)Variables
Plant Height
Branch/plant
Productivity (tonnes/ha)

10x2

10x4

10x6

10x8

20x2

20x4

20x6

20x8

73,55ab
4.30ab3.43b72,83ab
4.31ab2.46d

68,06b
4.25ab
1.75f

67,65b3.90b
1.68g

77,70a4.68a
4.15a

77,23a4.48ab2.59c

71,14a4.41ab
1.84e70,23b
4.18ab1.74f

Note: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

In the second experiment, the leaching time did not affect the variables growth, yieldcomponent, and yield of soybean dry, but that only affected the nodule dry weight. The leachingtime every 2 weeks gave the nodule dry weight per plant significantly different with that at every 4
and 6 weeks, but did not different with without leaching (Table 7).
Table 7. The Effect of leaching time on leaf, stalk, root, and nodule dry weight at 6 WAP

Nodule dry weight (g)Without
0.18abEvery 2 Weeks
0.19a

Every 4 weeks
0.11b

Every 6 weeks
0.13b

Note: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

The variety affected the root, stalk dry weight, and seed productivity, but did not affect theother variables (Table 8). Stalk and root dry weight of Slamet were highest than those of the othervarieties. The highest of seed productivity was obtained on Anjasmoro (Table 8).
Table 8. The Effect of variety on leaf, stalk, root, and nodule dry weight at 6 WAP
Variables
Stalk dry weight (g)Root dry weight (g)Productivity (ton/ha)

Tanggamus

SlametWilis

Anjasmoro

4.86ab
0.77ab
2.73b

5.60a
0.87a
2.39b

4.07b
0.69ab
2.48b

3.85b
0.63b
3.83a

Note: numbers followed by the same letter on the same line are not significantly different with Duncan multiple range test at 5%.

The leaching time and variety interaction did not affect the seed productivity (Table 9). The
highest grain yield was obtained by Anjasmoro variety with leaching time every two weeks (4.06
ton/ha), but it was not significally different from those with leaching time every four weeks (3.99
ton/ha) and every six weeks (3.93 ton/ha). This suggested that the leaching time every 6 weeks
gained more effective economically and technically. This productivity of Tanggamus in 2010 was
lower than 2009 (Ghulamahdi, 2009). It was predicted that the Tanggamus was more responsive tothe higher solar radiation than lower solar radiation. The dry climate was in 2009, and wet climate
was in 2010. Based on visualization observation in the field the size of leaf of Tanggamus in dry

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Proceedings of The 7 ACSA Conference 2011

climate was wider than that in wet climate. Irwan (2006) stated that at high temperatures and low
humidity, solar radiation stimulated the emergence of flower buds into flowers.
Table 9. The effect of interaction on leaching time and variety on the seed productivity (ton/ha)
Leaching Time
Without
Every 2 weeks
Every 4 weeks
Every 6 weeks

Tanggamus
2.31
3.08
2.80
2.73

Slamet
2.16
2.41
2.62
2.40

Willis
2.44
2.46
2.71
2.29

Anjasmoro
3.36
4.06
3.99
3.93

Note: numbers followed without letter are not significantly different with Duncan multiple range test at 5%.

References
Adisarwanto, T. 2001. Bertanam kedelai di tanah jenuh air. Bul. Palawija. 1:24-32.
Alihamsyah, T., I. Ar-Riza. 2006. Teknologi pemanfaatan lahan rawa lebak. Hal. 181-201. Dalam D.A.
Suriadikarta, U. Kurnia, H.S. Mamat, W. Hartatik, D. Setyorini (eds). Karakteristik dan Pengelolaan
Lahan Rawa. Ed Ke-. Bogor. Balai Besar Penelitian an Pengembangan Sumberdaya Lahan Pertanian,
Badan Penelitian dan Pengembangan Pertanian, Departemen Pertanian(CSIRO) Commonwealth Scientific and Industrial Research Organization. 1983. Soybean response tocontrolled waerlogging. P.4-8. In R. Lehane (ed) Rural Reseach. The Science Communication of
CSIRO‖s Beaureau of Scientific Services.
Djayusman, M., I.W. Suastika, Y. Soelaeman. 2011. Refleksi pengalaman dalam pengembangan sisatem usaha
pertanian di lahan pasang surut, Pulau Rimau . Seminar Hasil Penelitian Pengembangan Sistem Usaha
Pertanian Lahan Pasang Surut Sumatera Selatan. Badan Penelitian dan Pengmbangan Pertanian,Pusat Penelitian dan Penmbangan Tanah dan Agroklimat. Bogor, Juni 2011. Hal 1-20.
Ghulamahdi, M., F. Rumawas, J. Wiroatmodjo, J. Koswara. Pengaruh pemupukan fosfor dan varietas terhadap
pertumbuhan dan produksi kedelai pada budidaya jenuh air. Forum Pasca Sarjana. 14(1):25-34
Ghulamahdi, M., SA Aziz., M Melati, N Dewi dan SA Rais. 2006. Pengaruh genotipe dan pupuk daun terhadap
pertumbuhan dan produksi kedelai panen muda pada budidaya jenuh air. Bul. Agron. 34(1):32-38.
Ghulamahdi, M., 2008. Pengaruh genotip dan pupuk daun terhadap pertumbuhan dan produksi kedelai panen
muda pada budidaya jenuh air. J. Agripeat. 9(2): 49-54
Ghulamahdi, M., E. Nirmala. 2008. Pengaruh waktu pemetikan dan genotip tehadap pertumbuhan dan produksi
kedelai pada budidaya jenuh air. Anterior J. 8(1):6-13.
Ghulamahdi, M., M. Melati, and D. Sagala. 2009. Poduction of soybean varieties under saturated soil cultura on
tidal swamps. J. Agron. Indonesia. 37(3):226-232.
Indradewa, D., S. Notohadisuwarno, S. Sastrowinoto dan H. Prabowo. 2002. Lebar bedengan untuk genangandalam parit pada tanaman kedelai. Bul. Agron. 30(3):82-86.
Indradewa, D., S. Sastrowinoto., S. Notohadisuwarno dan H. Prabowo. 2004. Metabolisme nitrogen padatanaman kedelai yang mendapat genangan dalam parit. Bul. Agron. 11(2):68-75.
Irwan, A.W. 2006. Budidaya Tanaman Kedelai (Glycine max (I.) Merrill). Jurusan Budidaya Pertanian Faperta.
Unpad. Jatinangor. 40 hal.
Noor, A, and A. Sabur. 2007. Hasil padi varietas unggul pada dua cara penyiapan lahan di lahan pang surut
Kalimantan Selatan. Prosiding Seminar Nasional Sumberdaya Lahan dan Lingkungan Pertanian Badan
Penelitian dan Pengembangan Pertanian Departemen Pertanian 7-8 November 2007. Hal 27-35
Suwarto., W.Q. Mugnisjah, D. Sopandie dan A.K. Makarim. 1994. Pengaruh pupuk nitrogen dan tinggi muka air
tanah terhadap pertumbuhan bintil akar, pertumbuhan dan produksi kedelai. Bul. Agron. 22(2):1-5.
Troedson, R.J., R.J. Lawn, D.E. Byth, G.L. Wilson. 1985. Saturated soil culture in innovated water management
option for soybean in the tropics and subtropics. P 171-180. In S. Shanmugasundaran and E. W.
Sulzberger (Eds). Soybean in Tropical and Sustropical System. AVRDC, Shanhua, Taiwan.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011179

Agronomic Performance of F7 Large Seed Soybean Breeding Lines
in Medium Plains
Darman M. Arsyada,* AsadibaCentral Institute for Agricultural Technology Assessment and Development, Bogor, Indonesia
Research Institute for Agricultural Biotechnology and Agricultural Genetic Resources, Bogor, Indonesia* Corresponding author: Central Institute for Agricultural Technology Assessment and Development,
Bogor, Indonesia
Tel: +62-251-8351277; Fax: +62-251-8350928
dmarsyad@gmail.comb

Abstract
Hybridization between different genotypes aims to obtain descents (breeding lines) which inherits with the goodcharacters of both parents. One of the soybean breeding program is directed to obtain new varieties with higher
yield potential, wide adaptation, desirable agronomic traits and large seed. A total of 60 F7 soybean breedinglines (origin crosses of Tanggamus x Local Tegal , Sibayak x Local Tegal, and Sibayak x Argomulyo), selected
in 2007, and four large seed standard varieties (Argomulyo, Burangrang, Anjasmoro, and Panderman) were
evaluated in rice fields, Sukasono Village, Sukawening Subdistrict, Garut District, West Jawa, Indonesia (at 700
m above sea level) on the early dry season (February-May) 2008. A randomized block design with three
replicates was used. Experimental plot size was 0.9 x 4.0 m, spacing of 45 x 10 cm, one plant per hill. Intensive
techniques cultivation involves fertilizing with 75 kg urea, 200 kg SP36, and 150 kg KCl per ha, controlling of
weeds, pests and diseases were carried out. Irrigation was applied when there was no rain. The results showedthat there was variability of seed yield and agronomic traits among the breeding lines tested. Among the 60 F7
breeding lines, eight lines were significantly better yield than Anjasmoro (best standar variety), namely U-719-11, U-509-1-1, U-714-1-1, V-159-1-3, V-426-1-2, V-421-1-1, V-563-1-1, and V-570-1-2 with a yield capacity
ranged from 2.5 to 2.7 t/ha, meanwhile Anjasmoro yielded at 2.3 t/ha. Breeding lines of U-719-1-1 and U-714-11 had a moderate seed size (12.0 to 12.5 g/100 seeds); V-159-1-3 and V-570-1-2 had a slightly large seed (13.6g/100 seeds), and U-509-1-1, V-426-1-2, V-421-1-1 and V-563-1-1 had a large seed (14.6 to 15.6 g/100 seeds).
Breeding line with the U and V code were derived from Tanggamus x Lokal Tegal and Sibayak x Local Tegalcrosses, respectively. Plant height was suggested as a criterion of selection for high yield.
Keywords: soybean, high yield, large seed

Introduction
The availability of improved varieties of high yielding, early maturity or moderate, and goodseed quality are needed for increasing the domestic soybean production. Today the preferences ofusers (farmers and craftsmen tempeh and tofu) were more likely to require large varieties ofsoybean seed. Craftsmen tempeh and tofu has long accustomed to using large seed soybean
imports, so that preferences are now a lot towards the large seed. To meet user demand, thenbreeding programs to produce varieties of soybean large seed in major need of attention. A numberof varieties of soybean large seed has been available as Argomulyo, Burangrang, Anjasmoro, andGrobogan (Hermanto et al., 2009).
In an effort to produce new varieties of soybeans are superior to varieties already exist, anumber of crossing (hybridization) between the genotypes having high yield potential, adaptation isquite broad, many pods and smaller seeds with the genotypes of the large seed, few pods, narrow
adaptation and relatively low yield potential was created in 2004 (Arsyad et al. 2005). Arsyad andAsadi (2011) reported that amount of 4800 F4 lines originated from five single cross combination
were planted in ricefield, Sukawening, Garut District, West Java on early dry season 2007. By using
pedigree method of selection, 1311 F5 lines were selected and grown in similar site on late dry

180

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Proceedings of The 7 ACSA Conference 2011season 2007. Amount of 540 F6 lines were selected and grown in similar site on early rainy season
2007/2008. Sixty-two F7 lines were selected and 42 lines among them had high yield and large
seed size.
This study aims were to: (a) obtain F8 lines better than comparable variety of Anjasmoro,
having the higher yield potential, good agronomic traits, and has a large seed size or slightly larger,and (b) investigate the behavior of the relationship between traits in soybean breeding lines.

Materials and Methods
A total of 60 F7 soybean breeding lines (origin crosses of Tanggamus x Local Tegal,
Sibayak x Local Tegal, and Sibayak x Argomulyo) selected in 2007, and four large seed standard
varieties (Argomulyo, Burangrang, Anjasmoro, and Panderman) were evaluated in rice fields,
Sukasono Village, Sukawening Subdistrict, Garut District (700 m above sea level) on the early dry
season (February-May) 2008. A randomized block design with three replicates wasused. Experimental plot size was 0.9 x 4.0 m, spacing of 45 x 10 cm, one plant per hill. Intensive
techniques cultivation involves fertilizing with 75 kg of urea, 200 kg of SP36, and 150 kg of KCl perha, controlling of weeds, pests and diseases were carried out. Irrigation was applied, if there is norain. Observations were made on days to maturity, plant height, number of branches, number ofpods, seed yield, 100 seed weight. Analysis of variances was performed on the charactersobserved and followed by LSD (Gomez and Gomez 1984), and inter-character correlation and pathanalysis followed Singh dan Chaudhary (1979).

Result and Discussion
Analysis of variances showed that there were significant different among the breeding lines
tested against agronomic performances such as yield, seed size (weight of 100 seeds), plant heightand number of pods per plant (Table 1). The range of seed yield of the 60 lines tested ranged from1.53 to 2.65 t/ha, whereas the yield of four check varieties ranged from 1.47 to 2.30 t/ha. Largeseeds (based on 100 seed weight) of 60 lines tested ranged from 10.3 to 20.7 g, and four checkvarieties had 100 seeds weight from 14.7 to 19.4 g. The breeding lines selected were those with
100 seed weight over 14 g. Based on these criteria, 32 lines were selected. The best check variety(Anjasmoro) with the yield of 2.3 t/ha and 100 seed weight was 14.7 g, while three other checkvarieties gave lower yield (1.5 to 2.0 t/ha).
By using Anjasmoro as a comparison, the eight lines significantly better, namely U-719-11, U-509-1-1, U-714-1-1, V-159-1-3, V-426-1-2, V-421-1-1, V-563-1-1, and V-570-1-2. Two lines,
namely U-719-1-1 and U-714-1-1 had a moderate seed size (12.0 to 12.5 g/100 seeds), two otherlines of V-159-1-3 and V-570-1-2 had a slightly large seed size (13.6 g/100 seeds), and four otherlines of the U-509-1-1, V-426-1-2, V 421-1-1 and V-563-1-1 had a large seed size (14.6 to 15.6g/100 seeds). There were 27 other lines that have a large seed size (> 14 g/100 seeds), but theyield was lower or equal to Anjasmoro.
The results of the preliminary yield test of F7 lines showed that there were the best fivelines derived from cross of Sibayak x Local Tegal and three lines derived from cross of Tanggamusx Tegal, while lines derived from cross of Sibayak x Argomulyo nothing was selected. All F7 lines
tested in this study had a yellow seed color, and the seeds was classified as moderate, rather large
and large. Genotypes (parents) who are used to form the lines tested in this study had a medium
seed size (Sibayak), rather small (Tanggamus) and large (Local Tegal and Argomulyo).

thProceedings of The 7 ACSA Conference 2011

181

Table 1. Seed yield, 100 seed weight, number of plant harvested, plant height, number of pods per plant, anddays to maturity of F7 soybean breeding lines in Garut, early dry season 2008

No.

Breeding line

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58

U-788-1-1
U-121-1-1
U-719-1-1
U-562-2-2
U-534-3-1
U-511-1-1
U-79-2-2
U-79-2-3
U-509-1-1
U-508-3-1
U-601-1-1
U-622-1-1
U-622-1-2
U-464-1-1
U-76-2-1
U-221-1-1
U-142-1-1
U-505-1-1
U-805-1-1
U-714-1-1
U-553-1-1
U-512-1-1
U-675-1-1
U-542-1-1
U-568-1-1
U-788-1-1
V-4-1-2
V-92-1-2
V-129-1-1
V-129-1-2
V-129-1-3
V-158-2-1
V-159-1-3
V-160-1-1
V-180-1-2
V-180-1-3
V-273-2-3
V-284-2-2
V-284-2-3
V-296-1-1
V-296-1-3
V-215-1-1
V-342-1-1
V-426-1-2
V-421-1-1
V-421-1-2
V-424-1-3
V-468-1-2
V-503-1-1
V-563-1-1
V-570-1-2
V-579-1-1
V-933-2-1
V-933-2-2
V-1118-1-1
V-1118-1-3
W-104-2-1
W-38-1-1

182

Seed
yield
(t/ha)

100 seedweight (g)

1.91
1.76
2.46
1.53
2.10
1.93
1.84
2.02
2.49
2.00
1.70
2.24
2.21
2.34
2.07
2.22
2.25
2.27
2.37
2.50
2.20
2.11
2.25
2.12
1.69
2.09
2.32
2.16
1.80
2.07
2.05
2.09
2.52
2.04
2.30
1.94
1.75
2.33
2.36
1.94
1.94
2.01
2.25
2.50
2.65
2.39
1.87
2.04
1.59
2.63
2.61
2.13
1.80
1.99
1.88
1.95
1.79
2.06

12.5
12.9
12.0
12.4
15.1
12.7
13.6
14.2
15.6
15.0
13.6
13.3
13.7
12.5
12.3
12.3
13.2
14.2
14.2
12.5
12.6
14.5
13.3
14.9
13.5
10.8
14.9
16.0
14.3
16.9
14.6
13.7
13.6
13.6
15.1
15.0
15.2
14.5
14.4
15.2
15.2
13.5
13.8
14.6
15.2
14.5
15.1
14.3
14.6
15.3
13.6
13.0
14.6
20.7
16.2
16.8
12.6
14.6

Number ofplant
harvested2
per 3.6 m
42
45
50
39
38
47
42
47
48
48
47
48
46
54
52
45
53
44
46
55
47
43
51
43
40
39
48
40
38
47
46
49
51
39
56
48
39
52
48
45
38
47
48
51
46
53
43
47
38
46
49
44
41
39
42
47
44
53

Plant
height (cm)Number ofpods per plantDays tomaturity

66
59
67
52
66
74
62
55
65
69
68
73
56
64
58
58
66
67
69
63
63
54
63
58
52
61
66
70
75
77
70
68
78
68
81
69
76
81
77
67
74
73
94
82
86
71
65
66
55
72
71
71
77
69
66
72
62
83

43
53
55
56
49
51
46
46
58
45
41
49
46
59
47
42
49
52
50
56
46
44
46
38
40
63
48
44
40
30
39
45
49
58
46
39
45
37
53
45
53
40
51
52
47
44
47
48
45
40
47
51
44
36
44
39
56
46

84
84
85
84
85
85
88
89
89
90
90
87
89
88
91
92
85
90
90
86
86
92
90
83
89
93
91
92
92
91
87
86
86
86
91
91
89
89
89
93
93
89
89
89
88
93
91
91
93
89
92
91
88
86
82
94
92
93

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Proceedings of The 7 ACSA Conference 2011

59
60
61
62
63
64

W-38-1-2
W-106-1-3
Argomulyo
BurangrangAnjasmoro
Panderman
Breeding lines
LSD .05
CV (%)

2.04
2.37
1.47
2.00
2.30
1.60
**
0.12
13.1

12.7
10.3
15.9
15.7
14.7
19.4
**
1.3
6.8

51
58
51
49
44
38
**
10
13.9

74
79
52
74
76
59
**
12
10.9

42
58
22
40
35
38
**
14
19.2

95
89
80
81
85
95
-

Breeding lines of U code was from Tanggamus x Tegal cross; Breeding lines of V code was from Sibayak x Tegal cross;
Breeding lines of W code was from Sibayak x Argomulyo crossIn the plant breeding needs to investigate the relationship (correlation) between
characters. If the selection is done on a character, we need to observe how they affect othercharacters (Burton, 1983). Analysis of correlation between traits in this study showed that seedyield had the strongest positive correlation with the number of plants in both F7 populations (lines)(Table 2). The greater the number of plants the higher plants and the higher the seed yield. The
pattern of relationships among characters in Tanggamus x Tegal population seemed to differ fromthe Sibayak x Tegal population.
The study also found that seed yield did not correlate with weight (large) seed, but Susantoet al. (2001) reported that seed yield was positively correlated with large seeds. This suggested that
there were opportunities to obtain lines of high yielding and large seeds. The results also found thatthe bigger the seed the less number of pods. This indicates that it would be difficult to obtain large
seed lines by the high number of pods. To get maximum yield for the lines of large seed, let the
optimum environmental conditions, so that seed filling goes well.
Dynamic relationship between the characters can be decomposed into two components,
namely direct and indirect effects (Singh and Chaudhary 1979). The pattern of direct effect between
the two populations seem to differ (Tables 3 and 4). In the cross of Tanggamus x Tegal, a strong
direct effect was shown by the number of pods and number of plants, while in the cross of Sibayakx Tegal indicated by plant height and number of pods. Direct effect of pod number and plant numberin the cross of Tanggamus x Tegal poorly supported by the results of the other components (Table3). Direct effect of plant height in crosses Sibayak x Tegal also poorly supported by the results of
the other components, but direct effect of pod number somewhat weakened by 100 seed weight(Table 4).
Characters that can be used as selection criteria for yield is the yield component that has apositive correlation with the yield and have a large direct effect on yield, and yield components
which have a large positive direct effect (although less correlated with the outcome) (Board et al.
1997). Based on this, it appears that plant height and pod number are considered as selection
criteria in soybean. In the field work selection, the use of pod number and plant height were also
more practical compared with other characters.
Table 2. Correlation between characters of F7 soybean breeding lines in Garut, early dry season 2008
Character

Seed yield

Seed yield
100 seed weight
Number of plants
Plant height
Number of pods
Days to maturity-0,197
0,662**
0,484**
0,192-0,095100 seedweight0,090-0,272-0,119-0,526**-0,074

Number ofplants0,574**-0,1250,387**-0,0500,040

Plant
height
0,348
0,066
0,325
0,084
-0,133

Number of
pods
0,327
-0,3650,174
0,190

Days tomaturity
0,146
0,012
0,016
-0,148-0,021-0,062

Value above and below diagonal represented for Tanggamus x Tegal (N=26) and Sibayak x Tegal (N=30), respectively** significant at P. 0.01

thProceedings of The 7 ACSA Conference 2011183

Table 3. Direct effect (in diagonal) and indirect effect of yield component on yield of 26 F7 soybean breedinglines of Tanggamus x Local Tegal in Garut, early dry season 2008
Character
Number of plants
Plant height
Number of pods
100 seed weightDays to maturity

Number ofplants
0.544
0.177
0.095
-0.0680,042

Plant
height-0.133-0,408-0.078-0.027-0,299Number of
pods
0.159
0,173
0,911
-0.3330,094

100 seedweight0.008-0.0040,022-0,060-0,004Days tomaturity-0,003-0,026-0,004-0,002-0,036

Total
effect0,575-0,0880,946-0,490-0,203

Tabel 4. Direct effect (in diagonal) and indirect effect of yield component on yield of 30 F7 soybean breedinglines of Sibayak x Local Tegal in Garut, early dry season 2008
Character
Number of plants
Plant height
Number of pods
100 seed weightDays to maturity

Number ofplants-0,022-0,0090.001
0.060-0,001

Plant height
0.724
1,870
0.157
-0.223-0,249Number of
pods
0.012
0,020
0,238
-0.125-0,015100 seedweight-0.029-0.013-0,0570,108-0,008Days tomaturity
0,002-0,006-0,003-0,0030,045

Total
effect
0,687
1,862
0,336
-0,183-0,228

Acknowledgment
The authors would like to acknowledge the support of the Indonesian Ministry of Research andTechnology, for providing the funding for this research.

References
Arsyad DM and Asadi. 2011. Selection of F4, F5 and F6 soybean breeding lines for high yield and large seedthsize. Paper presented at The 7 Asian Crop Science Association Conference. Bogor, Indonesia. 27-30
September 2011.
Arsyad DM, H Kuswantoro and Purwantoro. 2005. Hybridization, and selection penggaluran
soybeans. Technical Report Research Institute for Crop Legumes and Tuber (In Indonesian).
Board JE, MS Kang and BG Harville. 1997. Path analysis identify selection criteria for yield of late planted
soybean. Crop Sci. 37 (1):879-884.
Burton JW. 1983. Quantitative genetics: Results relevant to soybean breeding, p. 211-248. In J. R. Wilcox (Ed.):
Soybean: Improvement, Production, and Uses. Second Edition, ASA, Wisconsin, No. 16 in series.
Fehr WR. 1983. Applied Plant Breeding. Dept. of Agronomy, Iowa State Univ. Ames. 174 p.
Gomez KA and AA Gomez. 1984. Statistical Procedures for Agricultural Research. 2nd Edition. Pp 680.
Heriyanto. 2006. Deployment and utilization of soybean varieties as raw materials industry and thecompetitiveness of commodities in East Java. Crop Bulletin Research Institute for Crop Legumes andTuber (In Indonesian) No.11:52-68.
Hermanto, D Sadikin and E Hikmat. 2009. Deskripsi Varietas Unggul Palawija 1918-2009. Indonesian Center for
Food Crops Research and Development. Indonesian Agency for Agriculture Research and Development.
Pp 330 (In Indonesian).
Singh ID and BD Chaudhary. 1979. Biometrical Methods in Quantitative Genetic Analysis. Kalyani Publ. New
Delhi. Pp 301.
Susanto, M. Jamaluddin, T Sanbuichi, N Sekiya, DM Arsyad and MM Adie. 2001. Large high quality seed andpromising lines selected from Mansuria as candidates of new varieties, p. 7-16. In Roesmiyanto etal. (Eds.): Forum on Soybean Seed Production in East Java. JICA-Indonesian Ministry of Agr.

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184

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Organically Production of Soybean Supported by Fertilizers Residue under
Saturated Soil Culture
Maya Melati a,*, Kalimatul Jumro aaDepartment of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, Indonesia* Corresponding author: Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural University (IPB).
Jl. Meranti, Kampus IPB Darmaga, Bogor 16680, Indonesia.
Tel.: +62 251 8629353; fax: +62 251 8629353.
maya_melati05@yahoo.com

Abstract
Organic farming system can use natural on-farm inputs that are normally available at the production site.
Saturated Soil Culture (SSC) is a technology in cultivation that gives water permanently, maintains and keeps its
depth constantly; this makes soil layer in saturated condition. SSC technology can be implemented in land withpoor drainage or in cultivating soybean on rice field in the period between two rice plantings when the soils may
still be in water saturated conditions. The experiment was conducted to study the response of two soybean
varieties to the residues of different types of fertilizer in an organic farming system. The experiment was carriedout at IPB experimental station, Bogor, Indonesia, in October 2010-February 2011. Split plot design was usedwith types of fertilizer as the main plot (chicken manure, Centrosema pubescens Benth, and Tithonia diversifolia
Hemsl.) and soybean varieties as the sub plot (Anjasmoro and Wilis). To study the effectiveness of fertilizers
residue, the current experiment applied 50% fertilizer rates (10 t chicken manure only/ha, 5 t chicken manure
mixed with 2.1 t C. pubescens/ha, and 5 t chicken manure with 2.1 t T. diversifolia/ha) of those applied in theprevious soybean planting (May-August 2010). The results showed that chicken manure yielded the highest
seed weight and filled pod number per plant. However, seed yields per hectare were not affected by fertilizer
types. The soybean plant with the application of chicken manure, C. pubescens, and T. diversifolia produced
2.37, 2.42, and 2.43 t seed/ha, respectively. Wilis had higher number of filled pod per plant than Anjasmoro, but
the yields per hectare of both varieties were not different. Production of Anjasmoro and Wilis was 2.43 and 2.38
t/ha, respectively.
Keywords: Centrosema pubescens Benth, chicken manure, Glycine max (L.) Merr., green manure, Tithonia
diversifolia Hemsl

Introduction
National demand on soybean is about 2.2 million tons per year, but only 35-40% of thisdemand can be supplied from the domestic production, and the government has to import about 1.3
t soybean/year. Low productivity, decreasing agricultural land area, and limited access of farmers totechnology and funding, are some of the factors restricting the national production of soybeans.
Organic farming is an alternative to currently used methods of farming; its use might
increase soybean production. Currently the marketing of produce from organic farms is targeted
only at consumers who are interested in it because they view it as being healthier. However,organic farming systems could be useful to farmers who have limited access to production inputs(e.g. inorganic fertilizers and pesticides) and funds. Because of the presumed lower productivity oforganic systems as opposed to conventional farming the ability of organic systems to support foodsecurity is questioned; nevertheless organic farming may be able to provide local food security.
Farmers can use on-farm inputs normally available to them on the site of production.
Organic farming system had been used to produce vegetable soybean with various typesof fertilizer, for example chicken manure, green manure of Calopogonium mucunoides, CentrosemathProceedings of The 7 ACSA Conference 2011185

pubescens, and Crotalaria juncea, rock phosphate, charcoal and ash or rice hull (Barus, 2005;
Melati and Andriyani, 2005; Sinaga, 2005; Kurniasih, 2006). Some of those organic fertilizers had
resulted significant differences in plant performances, but not in production. The experiment had
also been conducted to produce dry seed of soybean to investigate whether organic farming system
supported plant production not only at less mature seed but also at fully mature stage. Kurniansyah(2010) found that chicken manure only or its combination with green manure produced dry seed of
1.16-1.48 t/ha on upland, while Ramadhani (2011) found that soybean yield was 1.83-1.94 t/ha on
soil with saturated soil culture technology.
Saturated Soil Culture (SSC) is a technology in cultivation that gives water permanently,
maintains and keeps its depth constantly (about 5 cm under soil surface); this makes soil layer insaturated condition. In saturated soil culture, watering is started from the beginning of plant growth
to maturity stage (Hunter et al., 1980). By keeping the water-table constantly, soybean will be
avoided from negative effect of inundation on soybean growth, because soybean will acclimatize
and improve its growth (Troedson et al., 1983). Ghulamahdi (2007) found that under SSC, growthand production of soybean was improved related to the increase of ACC, ethylene, glucose content,
and neck diameter of roots, and the increase of nodules‘ weight, nitrogenase activity, and nutrient
uptakes. SSC technology can be implemented in land with poor drainage or in cultivating soybean
on rice field in the period between two rice plantings where the soils may still be in water saturated
conditions. Ghulamahdi et al. (2009) also showed that SSC can be implemented in cultivating
soybean in tidal swamp area with the production of more than double of those in upland.
The availability of nutrient provided by organic fertilizers is not as quick as from inorganic
fertilizers; this may result in available nutrient in the following cropping season. Melati et al. (2008)
showed that the residue of fertilizers supported the production of organic vegetable soybean on
upland soil. The current experiment studied the possibilities of producing dry seed of soybean withthe residue of fertilizers under SSC technology. Two types of soybean cultivar (they differ in seedsizes) were used to investigate their responses to the treatments.

Materials and Methods
A field study was conducted in October 2010-February 2011 at the Cikarawang
Experiment Station of Bogor Agricultural University (IPB), in Bogor, Indonesia. The soil is a silty clay
loam soil. The experiment was the second crop sequence. The experimental design was arandomized complete block with spilt plot arrangement and three replicates. Types of fertilizer
included in the study were chicken manure only (10 t/ha), chicken manure combined with
Centrosema pubescens (5 + 2.1 t/ha), and chicken manure combined with Tithonia diversifolia (5 +2.1 t/ha) were considered as main plots, and assigned to an area of 4 x 4 m. Subplots wereassigned within each main plot, each differing by soybean cultivar, i.e. Anjasmoro and Wilis.
Subplots dimensions were five 4-m long rows spaced 0.4 m apart and 0.1 m apart within row.
Those fertilizer rates were half of rates in the first crop sequence to evaluate theeffectiveness of fertilizers residue. In the first crop sequence, the rate of chicken manure wasapplied at the rate of 20 t/ha followed Sinaga (2005) and the rates of green manure biomass wasdetermined based on a study of Kurniasih (2006). It was expected that a yield of about 10 t
biomass/ha was obtained from 25 kg seed of C. pubescens. However, dry conditions during the
growth period of C. pubescens. (December 2009-April 2010) resulted in the production of only 3.5 t
C. pubescens biomass/ha. Although Tithonia diversifolia could be easily collected from a nearby
area, it was also applied at the rate of 3.5 t/ha to make appropriate comparisons with the use of C.
pubescens. Besides those fertilizers according to the treatments, all plots were added with 1 t ricehull charcoal and 1 t dolomite per hectare. Green manure (combined with chicken manure, rice hull
charcoal and dolomite) was applied 4 weeks before soybean planting, while chicken manure (plus

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Proceedings of The 7 ACSA Conference 2011

rice hull charcoal and dolomite) was applied 2 weeks before soybean planting. All materials wereapplied in planting rows; they were then below the position of soybean seed.
SSC technique was conducted by providing water permanently since 4 weeks after
planting (WAP), maintained and kept water depth constantly at about 5 cm under soil surface in 20cm depth furrow. Ghulamahdi (2007) found that plants normally experience chlorosis in saturated
soil, therefore, in the current experiment liquid manure (1 L liquid manure/10 L water) was added asrdththfoliar application to the plants at the 3 , 5 , and 7 day after irrigation began.
Other cultural practices were seed treatment by using Rhizobium inoculants with the rateof 6.25 g/kg seed, and the planting of Tagetes erecta and Cymbopogon nardus near soybean plants
to control plant pest and diseases. Tagetes erecta and Cymbopogon nardus had been used in
producing organic vegetable soybean (Kusheryani and Aziz, 2006).
Analysis of variance was used to analyze the data and Duncan‘ Multiple Range Test
(DMRT) was used to compare means value.

Results and Discussion
Plant characters as shown in vegetative phase and yield component in generative phasewere not different among fertilizer types. Variables were only different in number of filled pod andseed yield per plant with the highest values were in plants with the application of chicken manure
only. Since the number of harvested plant in plot with chicken manure only was lower than thosewith other fertilizers, seed yields per hectare were not different among treatments (Table 1).
Table 1. Plant characters and production with three types of fertilizer

Tithonia
diversifolia

Fertilizers
Chickenmanure only

Centrocema
pubescens

ns
ns
ns
ns

88.05
94.09
18.5
28.9

85.85
91.28
19.5
30.8

84.99
90.46
18.3
29.4

Disease intensity (%)
Dry weight of leaves (g/plant)
Moisture content of leaves (%)
Nitrogen content in leaves (%)
Phosphorus content in leaves (%)
Potassium content in leaves (%)
N uptake of leaves (mg/plant)
P uptake of leaves (mg/plant)
K uptake of leaves (mg/plant)
Generative phase
Dry weight of shoot (g/plant)

ns
ns
ns
ns
ns
ns
ns
ns
ns

8.8
5.92
64.9
3.3
0.5
2.9
19.47
3.17
17.26

10.6
6.04
64.5
3.2
0.5
2.8
19.66
3.01
16.99

8.5
7.21
67.2
3.2
0.5
2.9
23.04
3.70
21.52

ns

20.77

26.60

26.25

24.54

Numbers of filled pod/plant

*

93.0b

111.7a

99.3b

F
test

Variables
Vegetative phase
Plant height at 7 WAP (cm)Plant height at harvest/ 14 WAP (cm)
Trifoliate leaf numbers/plant
Pest intensity (%)

Means

86.30
91.94
18.8
29.7
9.3
6.39
65.5
3.2
0.5
2.9
20.72
3.29
18.59

ns

118.8

104.0

112.0

101.3
111.6

Seed weight (g/100 seeds)

ns

15.17

15.83

16.00

15.67

Seed yield (g/plant)**

25.33b32.65a28.25abSeed yield (g/4.56 m )ns1109.671082.50

1101.1728.74
1097.78

Seed yield (ton/ha)

ns

2.43

2.37

2.42

2.41

Numbers of harvested plant/4.56 m

22

Note: ns = not significant; * and ** = significant at p < 0.05 and p < 0.01, respectively. Numbers followed by letters indicating
different at α = 5% with DMRT.

thProceedings of The 7 ACSA Conference 2011187

Seed yield per plant may be related to the soil nutrient content. After the 1st crop season,
C-organic, N-total, available P and K were higher in plots with the application of chicken manure
only compared to those with other fertilizer treatments. These conditions were also detected in the
2nd crop sequence before soybeans were planted (Table 2). The amount of nutrients in the soil withchicken manure application had supported the seed yield of single plant.
Table 2. Soil characteristics

Treatments
Fertilizers

Varieties

pH
H2O

Walkly &Black

Kjeldhal

Bray lC-org

N-TotalP..(%)..

..(%)..

(ppm)

N NH4Oac pH 7.0
K

Mg

Ca

Na

KTK

..(me/100g)..

st

After 1 crop sequence
Chicken m.

Anjasmoro

6.37

2.20

0.20

25.43

0.89

2.83

6.37

0.74

16.56

Chicken m.

Wilis

6.63

2.04

0.20

17.23

0.85

2.82

7.98

0.54

15.87

Tithonia sp.

Anjasmoro

6.53

1.84

0.18

9.20

0.86

2.60

7.42

0.59

15.80

Tithonia sp.

Wilis

6.67

1.89

0.19

15.57

0.60

2.41

6.66

0.51

14.77

Centrosema

Anjasmoro

6.40

1.76

0.17

6.15

0.69

2.74

6.98

0.45

16.68

Centrosema

Wilis

6.40

1.68

0.17

5.55

0.52

2.24

6.39

0.41

15.86

n.a.

After application of fertilizers in the 2ndcrop sequence

Chicken m.

Anjasmoro

6.30

2.63

0.22

33.50

3.06

7.94

0.61

17.76

Chicken m.

Wilis

6.50

2.55

0.22

28.80

2.90

7.82

0.48

18.36

Tithonia sp.

Anjasmoro

6.40

1.60

0.15

8.50

3.57

8.72

0.47

21.45

Tithonia sp.

Wilis

6.60

1.68

0.15

12.70

3.26

8.32

0.64

18.37

Centrosema

Anjasmoro

6.60

2.00

0.19

25.00

3.78

8.46

0.80

18.16

Centrosema

Wilis

6.30

1.92

0.18

28.70

4.76

9.23

0.75

21.76

After 2nd

crop sequence

Chicken m.

Anjasmoro

7.40

2.79

0.26

31.20

1.42

3.40

8.38

1.16

19.13

Chicken m.

Wilis

7.50

2.39

0.22

20.60

1.19

3.00

8.19

1.09

17.95

Tithonia sp.

Anjasmoro

7.60

2.23

0.21

14.40

1.11

2.90

7.76

1.06

17.76

Tithonia sp.

Wilis

7.50

2.15

0.22

9.00

0.86

2.65

7.42

0.70

16.78

Centrosema
Centrosema

Anjasmoro
Wilis

7.20
7.40

2.55
2.55

0.23
0.25

5.20
16.10

0.93
1.18

2.55
2.95

7.34
8.66

1.08
1.07

18.35
18.73

Note: Soil samples were composite of 3 replicates from the same treatment, therefore they were not statistically analyzed. n.a.
= valid data were not available

There are some differences in plant characteristics and yield components of the two varieties
in this experiment. These were accordingly with the characteristics of each variety. Anjasmoro hasless number but larger leaves than those of Wilis, therefore, the nutrient uptake was higher inAnjasmoro. Anjasmoro has less number of filled pod but it has larger seed size, this resulted insimilar seed yield of both varieties (Table 3).

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Proceedings of The 7 ACSA Conference 2011

Table 3. Plant characteristics and production of two soybean varieties

Variables
Vegetative phase
Plant height at 7 WAP (cm)Plant height at harvest/ 14 WAP (cm)
Trifoliate leaf numbers/plant
Pest intensity (%)
Disease intensity (%)
Dry weight of leaves (g/plant)
Moisture content of leaves (%)
Nitrogen content in leaves (%)
Phosphorus content in leaves (%)
Potassium content in leaves (%)
N uptake of leaves (mg/plant)
P uptake of leaves (mg/plant)
K uptake of leaves (mg/plant)
Dry weight of shoot (g/plant)
Numbers of filled pod/plant2
Numbers of harvested plant/4.56 m
Seed weight (g/100 seeds)
Seed yield (g/plant)
2
Seed yield (g/4.56 m )Seed yield (ton/ha)

F test

*
ns
*
ns
ns
ns
ns
ns
ns
**
*
*
*
ns
**
**
**
ns
ns
ns

Varieties
Wilis
83.32b
91.98
20.3a
28.9
8.8
5.39
65.5
3.2
0.5
2.7b
17.47b
2.77b
14.41b
24.77
115.4a122.8a12.11b28.00
1087.11
2.38

Anjasmoro
89.29a
91.91
17.2b
30.6
9.9
7.39
65.6
3.3
0.5
3.1a
23.97a3.82a22.76a24.31
87.2b100.4b
19.22a29.49
1108.44
2.43

Means

86.31
91.90
18.80
29.70
9.30
6.39
65.50
3.20
0.50
2.90
20.72
3.30
18.59
24.54
101.30111.6015.67
28.75
1097.78
2.41

Note: ns = not significant; * and ** = significant at p < 0.05 and p < 0.01, respectively.

Conclusions
The experiment concluded that in the second crop sequence, with half rates of fertilizer, alltypes of fertilizer and both varieties could be used to produce similar seed yield per hectare oforganic soybean. In the second crop sequence, there was indication that soil characteristics had
been improved under organic farming system.

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Pertanian, Fakultas Pertanian. Institut Pertanian Bogor. Bogor.
Kurniasih, W. 2006. Pengaruh jenis, dosis benih dan umur tanaman pupuk hijau terhadap produksi kedelai(Glycine max (L.) Merr) panen muda organik. Skripsi. Agronomi dan Hortikultura, Fakultas Pertanian.
Institut Pertanian Bogor. Bogor.
Melati, M and W Andriyani. 2005. Pengaruh pupuk kandang ayam dan pupuk hijau Colopogonium mucunoides
terhadap pertumbuhan dan produksi kedelai panen muda yang dibudidayakan secara organik. Bul.
Agron. 33: 8-15.
Sinaga, YAS. 2005. Pengaruh pemberian pupuk organik terhadap pertumbuhan dan produksi kedelai (Glycinemax (L.) Merr.) panen muda yang diusahakan secara organik. Skripsi. Budidaya Pertanian. Institut
Pertanian Bogor. Bogor.
Kurniansyah, D. 2010. Produksi kedelai organik panen kering dari dua varietas kedelai dengan berbagai jenis
pupuk organik. Skripsi. Department of Agronomy and Horticulture. Bogor Agricultural University (IPB).
Bogor.
Ramadhani, E. 2011. The study of fertilizer types for organically production of two soybean varieties undersaturation soil culture technique at two crop sequences (in Indonesia: Kajian aplikasi jenis pupuk untukthProceedings of The 7 ACSA Conference 2011189

produksi dua varietas kedelai secara organik dengan sistem budidaya jenuh air pada dua musim tanam).
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Troedson, RJ, RJ Lawn, DE Byth and GL Wilson. 1983. Saturated soil culture in innovated water management
option for soybean in the tropics and sub tropics. In Soybean in Tropical and Subtropical System. Proc.
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Ghulamahdi, M. 2007. Mekanisme Fisiologi Tanaman Kedelai pada Kondisi Jenuh Air dan Kering serta
Kaitannya dengan Biosintesis Etilen. In Seminar Nasional Hasil Penelitian yang Dibiayai oleh Hibah
Kompetitif, 1-2 Agustus 2007. Institut Pertanian Bogor.
Ghulamahdi, M, M Melati and D Sagala. 2009. Production of Soybean Varieties under Saturated Soil Culture on
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Melati, M, A. Asiah, D. Rianawati. 2008. Aplikasi pupuk organik dan residunya untuk produksi kedelai panen
muda Bul. Agron. 36: 204-213.
Kusheryani, I and SA Aziz. 2006. Pengaruh jenis tanaman penolak organisme pengganggu tanaman terhadap
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Proceedings of The 7 ACSA Conference 2011

Factors Causing the Soybean Yield Gaps between Japan and USA
Keisuke Katsura a,*, Koki Homma a, Yohei Kawasaki a, Larry C. Purcell b, Randall L.
Nelson c, Taiki Yoshihira d, Tatsuhiko Shiraiwa aaGraduate School of Agriculture, Kyoto University, Japan
Department of Crop, Soil and Environmental Sciences, University of Arkansas, AR, USAc
USDA-Agricultural Research Service, University of Illinois, IL, USAdFaculty of Dairy Science, Rakuno Gakuen University, Japan* Corresponding author: Experimental Farm, Kyoto University, 12-1, Hatchonawate, Takatsuki,
Osaka, 569-0096, Japan.
Tel.: +81 72 685 0134; fax: +81 72 683 1532.
kkatsura@kais.kyoto-u.ac.jpbAbstract-1

The average soybean yield in Japan is stagnated around 1.7 t ha , which is quite low level compared to that in-1
USA (around 2.7 t ha ). The objective of this study was to reveal the factors causing this yield gap between
Japan and USA. To examine the variety effect on the yield gap, we conducted yield test at Osaka (centraloooJapan, 34 51‘N), Hokkaido (northern Japan, 43 03‘N), Feyetteville, AR (south-central USA, 36 03‘N), andoChampaign, IL (mid-western USA, 40 06‘N), in 2009 using 4–6 Japanese and 5–10 USA varieties. Averaged
yield of USA varieties was 8–59 % higher than that of Japanese varieties in each experimental site, whichmeans the yield potential of USA varieties was higher than that of Japanese varieties. To examine theenvironmental effect on the yield gap, we estimated the potential yield (Yp) at Hokkaido, Shiga (central Japan,oo
35 16‘N), Arkansas, AR (south-central USA, 34 30‘N), and Champaign, IL, by simple simulation model, where
we assumed that Yp was determined by solar radiation, air temperature and growth duration. Estimated Yp in-1-1
USA (7.34 t ha in Arkansas and 7.12 t ha in Champaign, averaged over recent 30–year) was higher than that
-1
-1
in Japan (5.84 t ha in Shiga and 4.69 t ha in Hokkaido), and this difference was mainly brought from thedifference in solar radiation intensity. Ratio of the actual yield to the potential yield (Ya/Yp) was highest inChampaign (50.7 %, averaged over recent 5–year), followed by Hokkaido (44.6 %), Arkansas (38.9 %), and the-1lowest in Shiga (24.6 %). In addition, there were increasing tendencies in Ya/Yp in USA (0.23–0.50 % year ),while no such a tendency in Japan. The increasing tendencies in Ya/Yp in USA would be brought from theimprovement of crop management in addition to breeding new varieties.
Keywords: soybean (Glycine max (L.) Merrill), Yield

Introduction
Improvement on soybean (Glycine max (L.) Merrill) production in Japan is strongly needed,because soybean self-sufficiency ratio in Japan is quite low (around 5%; MAFF, 2011a). Soybean
yield in Japan has been stagnated around 1.7 t ha-1 (MAFF, 2011b) in recent years, while that inUSA, the largest producer of soybean in the world, has increased steadily in the last few decades
and reached around 2.7 t ha-1 (USDA-NASS, 2009). It is not clear, however, how varieties,
environment and crop management have affected on this growing gap soybean yield between
Japan and USA. The objective of this study is to clarify the factors causing soybean yield gaps
between Japan and USA.
In the present study, we grew Japanese and USA commercial soybean varieties innorthern and central Japan and mid-western and south-central USA, where are the major producersof soybean in Japan and USA. The objectives of this study were (1) to examine the yield potential(Yp) of Japanese and USA varieties and study the varietal effect on the growing yield gap, (2) toestimate the changes of Yp in northern and central part of Japan and mid-western and south-central
USA using a simple simulation model, with the assumtion that Yp was determined by solar radiation,thProceedings of The 7 ACSA Conference 2011191

air temperature and growth duration, (3) to compare the Yp in Japan and USA, and (4) to study theenvironmental and crop management effects on the growing yield gap between Japan and USA
bycomparing the changes of Ya/Yp.

Materials and Methods
Eight Japanese and 14 USA commercial varieties (Suzukari, Enrei, Suzuyutaka,Tachinagaha, Sachiyutaka, Tamahomare, Toyomusume and Yuzuru for Japanese varieties andAthow, Omaha, Manokin, LD003309, 5002T, UA–4805, Osage, 5601T, Ozark, Hutcheson, Jack,
Williams82, X34 and X88 for USA varieties) were used in the present study. 4–6 Japanese and 5–
10 USA varieties were grown in Osaka (central Japan, 34o51‘N), Hokkaido (northern Japan,
43o03‘N), Feyetteville, AR (south-central USA, 36o03‘N), and Champaign, IL (mid-western USA,
40o06‘N) during the summer season in 2009. Crop managements, such as sowing date, planting
density, fertilizer application rate, and so on, were based on the common practices in eachexperimental site. Experiments were consisted of 3-4 replications. Soybean yield was determined atmaturity by harvesting more than 1m2 area from each replication.
Based on the meteorological statistics and crop progress reports, changes of Yp were
estimated by the following simple simulation model.
dY p  HI   RUE  1  exp  k  LAI   Rad n 1where, HI is harvest index, RUE is radiation use efficiency, k is light extinction coefficient, LAI is leaf
area index, and Rad is solar radiation. LAI was estimated by the logistic function of cumulative
effective temperature with the base temperature being 8 oC. RUE in Japan was setup at 1.1 g MJ-1
for pre–R1+25–day period, and 0.77 g MJ-1 for post–R1+25–day period, based on the observed
data under ideal conditions in Japan. RUE in USA was setup 10% lower than that in Japan, in
consideration that RUE decreased under intense radiation environment. HI and k were assumed tobe 0.5 and 0.6, respectively.
Potential yield (Yp) was estimated for Shiga (central Japan, 35o16‘N), Hokkaido, Arkansas,
AR (south-central USA, 34o30), and Champaign, IL. Data from 1980 to 2009 for weather, crop
calendar and statistics of soybean yield in each site were collected. Actual yield (Ya) and Ya/Yp wereanalyzed for the field with and without irrigation system separately in Arkansas, because irrigation
system has been rapidly spreading in south-central USA,

Results and Discussion
The seed yields observed in the variety tests are listed in Table 1. There was large
variation in yield, which ranged from 2.12 to 5.91 t ha-1 depending on the varieties and sites (Table
1). Averaged yield of USA varieties (4.06–5.20 t ha-1) was 8–59 % higher than that of Japanesevarieties (2.63–3.80 t ha-1) in each site, which means the yield potential of USA varieties was higher
than that of Japanese varieties.
As for the meteorological statistics in the four assessed locations, there was not so big
difference in effective growth period (98–105 days, from emergence to leaf yellowing) among thesites, while radiation intensity in USA (20.9–21.4 MJ m-2 d-1) was more than 30 % larger than that inJapan (15.5–16.1 MJ m-2 d-1) (Table 2). As a result, estimated Yp in USA (7–8 t ha-1) changed
higher than that in Japan (5–6 t ha-1), which showed the soybean potential yield in USA was higher
than that in Japan, mainly because of solar radiation intensity (Figure 1). The values of Yp on recent
30-year average in Shiga, Hokkaido, Arkansas, and Champaign were 5.84, 4.69, 7.34, and 7.12 t
ha-1, respectively.
192

thProceedings of The 7 ACSA Conference 2011

Actual yield in Japan (1.56 and 2.17 t ha-1 on recent 30-year average, in Shiga andHokkaido, respectively) was lower than that in USA (2.56, 1.72 and 3.04 t ha-1 on recent 30-year
average, in Arkansas with irrigation system, Arkansas without irrigation system, and Champaign,respectively) (Figure 1). There were increasing tendencies in actual yield in all the sites, but theincreasing rates were larger in USA (37.3, 20.2 and 35.1 kg ha-1 year-1, in Arkansas with irrigation
system, Arkansas without irrigation system, and Champaign, respectively) than those in Japan (0.9-1-1
and 12.5 kg ha year , in Shiga and Hokkaido, respectively). In addition, the increasing rates in Yp
was very small (14.5 and 35.1 kg ha-1 year-1, in USA, which means that the changes in Yp could not
explain the recent soybean yield increase in USA.
-1

Table 1. Yield (t ha ) of Japanese and USA soybean varieties grown under Osaka, Feyetteville, Champaign,andHokkaido
Osaka Hokkaido Feyetteville Champaign
in 2009
34o51'N
Japanese varieties
Suzukari
3.64
Enrei
3.93
Suzuyutaka
4.13
Tachinagaha
3.10
Sachiyutaka
4.16
Tamahomare
3.44
Toyomusume
YuzuruAverage
3.73
USA varieties
Athow
Omaha
Manokin
LD003309
5002T
UA4805
Osage
5601T
Ozark
Hutcheson
Jack
Williams82
X34 (LJ)
X88 (LJ)
Average43o03'N

5.07
5.91
5.23
5.06
4.69
5.15
5.19
5.50
5.19
5.03

5.20

36o03'N

40o06'N

3.23

2.85
2.86
2.68

4.51
3.09
3.44
3.60
3.48
3.28
4.32
3.80

3.44

4.87
4.35

2.12
2.63

3.88
4.25
4.70

3.13

4.66
5.73
4.44
4.72
5.32
4.23
4.85

4.04
4.20
3.61
4.24
4.06

4.11
3.95

4.86

4.17

Table 2. Season of soybean cropping and its climate summary
Mean peak date*
Planting

Harvesting

35 16‘
o
43 03‘

o

23 Jun23 May

4 Nov
9 Oct

Effective
growthperiod**(days)
103
102

34 30‘

o

24 May13 Octo

22 May4 Oct

Latitude
Shiga, Jpn
Hokkaido,
Jpn
Arkansas,
USA
Champaign,USA

40 06‘

Total
rainfall(mm)

Daily temperature

Daily solar
radiation-2 -1(MJ m d )

581
333

Max
o
( C)
28.4
23.9

Min
o
( C)
21.4
16.7

105

261

32.4

21.0

20.9

98

341

28.7

17.1

21.4

15.5
16.1

*, based on the crop progress report of Shiga and Hokkaido (1980-1997), East Illinois (1980-2005), and East Central Arkansas(1996-2008).
**, Emergence to leaf yellowing. The climate data were calculated for the effective growth period based on the meteorologicalstatistics from 1980 to 2009 for the districts on Japan and USA respectively.

thProceedings of The 7 ACSA Conference 2011193

(a) Shiga, Japan (35o16'N)

9

8

Ya

Yield (t ha-1)

6
5
4
3

(b) Hokkaido, Japan (43o03'N)

8

Yp

y = 0.0009x - 0.2955

5
4
3
2

1

1

Yield (t ha-1)

9

1985

1990

1995

2000

2005

y = 0.0125x - 22.739
01980 1985 1990 1995

2010(c) Arkansas, AR, USA (34o30'N)

8

7

7

y = 0.0145x - 21.546

5

4
3

Yp
Ya_irrigatedYa_non irrigatedy = 0.0373x - 71.845

6

1

2010

2010

Yp

Ya

2005

2010

3
2

2005

2005

4

1

2000

y = -0.0045x + 16.19

52

y = 0.0202x - 38.513
01980 1985 1990 1995

2000(d) Champaign, IL, USA (40o06'N)

986

y = 0.0272x - 49.445

6

2
0
1980

Ya

7

Yield (t ha-1)

Yield (t ha-1)

7

Yp

y = 0.0243x - 42.592

9

y = 0.0351x - 66.923

0
1980

1985

1990

1995

2000

Figure 1. Changes of estimated potential yield (Yp) and actual yield (Ya) in (a) Shiga, (b) Hokkaido, (c) Arkansas,
and (d) Champaign.

The Ya/Yp was the largest in Champaign (50.7 %, the recent 5 year average), followed by
Hokkaido (44.6 %), Arkansas with irrigation system (38.9 %), Arkansas without irrigation system(26.5 %), and the lowest in Shiga (24.6 %) (Figure 2). As is the case in actual yield, there wereincreasing tendencies in Ya/Yp in USA (0.23–0.50 % year-1), while no such tendency in Japan. We
assumed that the increase in Ya/Yp in USA is brought from the improvement in crop management,such as increased adoption of irrigation system, introduction of early cultivation and raised bed
planting, improvement in weed management, in addition to the developing and utilizing new highyielding varieties.
In the present study, we tried to reveal the factors causing the soybean yield gaps between
Japan and USA focusing on environmental, varietal, and technological factors. Our results showthat the greater amount of solar radiation is the major factor of higher yield in the USA and that thetechnological developments have caused the increase in yield gap between Japan and USA. Thereis still much room for improving soybean yield in Japan.

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Proceedings of The 7 ACSA Conference 2011

(a) Shiga, Japan (35o16'N)

70

70

60

60

50

y = -0.0973x + 220.78

Ya/Yp (%)

Ya/Yp (%)

50

40

40

30

30

20

20

10

10

0
1980

(b) Hokkaido, Japan (43o03'N)

1985

1990

1995

2000

2005

2010

0
1980

1985

(d) Champaign, IL, USA (40o06'N)

70(c) Arkansas, AR, USA (34o30'N)

70

60

irrigated

60

non irrigated40

y = 0.4352x - 833.06

1990

1995

2000

2005

2010

50

Ya/Yp (%)

Ya/Yp (%)

50

y = 0.0656x - 84.487

40
30

30

20

20

10

y = 0.2266x - 428.41

10

0
1980

1985

1990

1995

2000

2005

2010

0
1980

y = 0.4997x - 953.63

1985

1990

1995

2000

2005

2010

Figure 2. Changes of ratio of the actual yield to the estimated potential yield (Ya/Yp) in (a) Shiga, (b) Hokkaido,(c) Arkansas, and (d) Champaign.

References
MAFF, 2011a. Table on demand and supply of food, 2009. Association of Agriculture and Forestry Statistics,
Tokyo, Japan.
MAFF, 2011b. Statistics on crops, 2009. Association of Agriculture and Forestry Statistics, Tokyo, Japan.
USDA-NASS, 2009. Crop production reports (http://www.nass.usda.gov/).

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011195

Photosynthetic Acclimation to Elevated CO2 in Soybean
Mochamad Arief Soleha,b *, Hai Yana, Tomohiro Konnoa, Makie Kokubuna
Ryoji SameshimacaGraduate School of Agricultural Science, Tohoku University, Japanb
Faculty of Agriculture, Padjadjaran University, IndonesiacNational Agricultural Research Center for Tohoku Region, Japan
moch_amir@yahoo.com

Abstract
Previous studies on plant responses to elevated atmospheric CO2 concentration [CO2] found the occurrence ofelevated [CO2]–induced photosynthetic acclimation. Soybean cultivar Enrei was grown in pots and was-1-1
subjected to either ambient [CO2] (ca. 380 μmol mol ) or elevated [CO2] (ca. 580 μmol mol ) regime. The half ofthe plants were transferred to the other [CO2] regime at flowering. Under elevated [CO2], photosynthetic rate wasmarkedly enhanced at the early growth stage, whereas it was decreased at the later growth stage on bothtransferred and untransferred plants, indicating that the acclimation occurred at the later growth stage of theplants regardless of the transfer. Electron transport rate (ETR) curve of the plants under elevated [CO2] regime-1
became plateau at about 300 μmol mol intercellular [CO2] at the later growth stage (35 days after transfer-1(DAT)), which was less than the plateaued value under ambient [CO2] regime (320 μmol mol ), indicating thatthe acclimation of photosynthesis occurred due to decreased intercellular [CO2] which might be restricted by thedecrease in stomatal conductance, N or chlorophyll content in leaves. Moreover, the leaf N and chlorophyll
content (SPAD reading) of the plants grown under elevated [CO2] regime was lower than those of the plants
grown under ambient [CO2] regime at the later growth stage (80 days after sowing (DAS)/32 DAT). Thus theacclimation of photosynthesis of soybean appeared to be induced by decreases in stomatal conductance, N or
chlorophyll content in leaves, although other factors such as starch accumulation in leaves at the later growthstage, which can restrict Rubisco activity in leaves, might be involved in the acclimation. Further research isneeded to examine how other possible factors are involved in the acclimation of photosynthesis.
Keywords: elevated [CO2]; photosynthesis.

Introduction
Increasing [CO2] in the atmosphere will offset increased photorespiration by increasing
photosynthesis, especially in plants with C3 photosynthetic pathway. To examine environmental
effects including the effect of increasing [CO2] on soybean growth and development is very
important, because plants develop and grow properly in proper conditions environmentally andgenetically.
A number of previous studies showed that elevated [CO2] affected photosynthetic behaviorof crops such as soybean. Many previous studies showed positive responses of soybean toelevated [CO2] (Rogers et al. 1997; Kimball et al., 2002; Pritchard and Amthor, 2005; Alagarswamy
et al., 2006), but some other studies found down-regulation of photosynthesis under elevated [CO2](Sim et al. 1998; Sawada et al. 2001; Ainsworth et al. 2004; Kanemoto et al. 2009; Matsunami et al.
2009; Otera et al. 2011). The objective of the present study was to examine how the acclimation ofphotosynthesis occurs in soybean under elevated [CO2].
Phenomenon of photosynthetic acclimation in plants can be ascribed to several factors.
Several previous studies reported that acclimation of photosynthesis occurred for different reasons.
Firstly, N limitation in leaves may cause photosynthetic acclimation (Kanemoto et al. 2009; Antal etal. 2010). Secondly, starch accumulation in leaves reduced Rubisco activity and induced
photosynthetic acclimation (Sims et al. 1998; Sawada et al. 2001). Thirdly, balance in sink and

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Proceedings of The 7 ACSA Conference 2011

source affected photosynthetic acclimation (Rogers et al. 1998; Ainsworth et al. 2004). Fourthly,
decrease of chlorophyll content or Rubisco activity in leaves may affect photosynthetic acclimation(Hotta et al. 1987; Sage et al. 1989). Finally, growth temperature induced photosynthetic
acclimation in some C3 species (Yamori et al. 2005).

Materials and Methods
A soybean cultivar Enrei (normally nodulating) was used in this study. Four seeds per pot(7 L) were sown on 6 June 2010 then thinned to one plant after emergence. Every pot was applied
6 g of compound fertilizer (N-P2O5-K2O: 5-15-20) and 10 g of garden lime. The amount of eachelement per pot was N-0.3 g, P2O5-1.5 g, K2O-1.2 g and SiO2-2.8 g, Ca-0.4 g and Mg-0.4 g. The
seeds were inoculated with Bradyrhyzobium japonicum strain. The soil used was low-humic
Andosol type. The plants were grown in the temperature gradient chambers (Gradiotron) of National
Agricultural Research Center for Tohoku Region, Morioka, Japan, during June to October in 2010.
There were two levels of [CO2] inside the chamber: ambient (380 µmol mol-1) and elevated (580µmol mol-1). The half of plants were transferred into the different [CO2] regime (from ambient toelevated (A-E) or from elevated to ambient (E-A)) at flowering (48 DAS (R1)). The [CO2] andtemperature around plant canopy were monitored every half hour by sensors which were installed
above the plots.
Measurement of photosynthesis was conducted at 2 DAT and 35 DAT. Chlorophyll
fluorescence was determined by using portable photosynthesis system (LI-6400-40 with leafchamber fluorometer, LI-cor Inc., NE, USA). The air flow to the chamber was controlled at 500 µmol
s-1. The measurements were conducted at saturated PPFD (1,500 µmol m-2 s-1). The photosynthetic
measurement was conducted at varying [CO2] with a range of 100 – 1,250 µmol mol-1 (A-Ci curve).
The values used for the calculation of ETR are as follows; maximal fluorescence (Fm`), steady-state
fluorescence (Fs) and photosystem II efficiency (PhiPS2) with equation  F m` F s  . ETR was

F m` 

determined by the equation  F m` F s  .[ƒIαleaf] ,F m` 

where f is fraction of absorbed quanta that is used by PSII, and is typically assumed to be 0.5 for C 3
plants, I is incident PPFD (1,500 µmol m-2 s-1), αleaf is leaf absorptance which is assumed to be 0.85
(Genty et al. 1989). Measurements were conducted on the plants which were transferred (A-E, E-A)
and untransferred (A-A, E-E). Two to three plants for each plot were used for the measurements.

Results and Discussion
Under elevated [CO2], photosynthetic rate was markedly enhanced at the early growthstage, whereas it was decreased at the later growth stage on both transferred and untransferredplants (Fig. 1), indicating that the acclimation occurred at the later growth stage of the plants
regardless of the transfer. Electron transport rate (ETR) curve of the plants under elevated [CO2]
regime became plateau at about 300 μmol mol-1 intercellular [CO2] at the later growth stage (35days after transfer (DAT)), which was less than the plateaued value under ambient [CO 2] regime(320 μmol mol-1) (Fig. 2), indicating that acclimation of photosynthesis occurred at about 300 µmol
mol-1 intercellular [CO2] probably due to decreased RuBP regeneration (Sharkey et al., 2007) which
was affected by decrease in stomatal conductance (Fig. 3). Moreover, the leaf N and chlorophyll
content (SPAD reading) of the plants grown under elevated [CO2] regime was lower than those ofthProceedings of The 7 ACSA Conference 2011197

the plants grown under ambient [CO2] regime at the later growth stage (80 days after sowing(DAS)/32 DAT) (data not shown).

A

AE

E

EA

A

AE

E

EA

A

AE

E

EA

Measurements were conducted at saturated PPFD (1,500 µmol m-2 s-1) under respective [CO2] (A and E). Vertical bars indicate
SE of three or five plants. *: significantly different at the 5% level between A and E [CO2] regimes.

Figure 1. Photosynthetic rates of transferred (AE, EA) and untransferred (A, E) plants at the early growth stages(49, 54, and 80 DAS).

Fluorescence measurements were conducted at saturated PPFD (1,500 µmol m-2 s-1).

Stomatal conductance (µmol m-2 s-1)

Figure 2. Photosynthetic rate and electron transport rate at 2 DAT and at 35 DAT of transferred (A-E, E-A) anduntransferred (A-A, E-E) plants.

A

AE

E

EA

Measurements were made in the morning at 80 DAS/32 DAT.
Figure 3. Effects of [CO2] (A, E) on stomatal conductance of leaves.

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Proceedings of The 7 ACSA Conference 2011

References
Ainsworth EA, Rogers A, Nelson R, Long SP (2004). Testing the ―source–sink‖ hypothesis of down-regulationof ptosynthesis in elevated [CO2] in the field with single gene substitutions in Glycine max. Agriculturaland Forest Meteorology 122: 85–94
Alagarswamy G, Boote KJ, Allen LH, Jr, Jones JW (2006). Evaluating the CROPGRO–soybean model ability tosimulate photosynthesis response to carbon dioxide levels. Agronomy Journal 98: 34–42.
Antal T, Mattila H, Yatkin MH, Tyystjärvi T, Tyystjärvi E (2010). Acclimation of photosynthesis to nitrogen
deficiency in Phaseolus vulgaris. Planta 232: 887-898
Genty B, Briantais JM, Baker NR (1989). The relationship between the quantum yield of photosynthetic electrontransport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 87-92
Kanemoto K, Yamashita Y, Ozawa T, Manishi N, Nguyen NT, Suwa R, Mohapatra PK, Kanai S, Moghaieb RE,
Ito J, El-Shemy H, Fujita K (2009). Photosynthetic acclimation to elevated CO2 is dependent on N
partitioning and transpiration in soybean. Plant Science 177: 398–403
Kimball BA, Kobayashi K, Bindi M (2002). Responses of agricultural crops to free-air CO2 enrichment. Advancesin Agronomy 77: 293-368
Matsunami T, Otera M, Amemiya S, Kokubun M, Okada M (2009). Effect of CO2 concentration, temperature
and N fertilization on biomass production of soybean genotypes differing in N fixation capacity. Plant
Production Science 12: 156-167
Otera M, Kokubun M, Tabei H, Matsunami T, Maekawa T, Okada M (2011). Is yield enhancement by CO 2enrichment greater in genotypes with a higher capacity for nitrogen fixation? Agricultural and Forest
Meteorology 151: 1385-1393
Pritchard SG, Amthor JS (2005). Crops and environmental change. Food Products Press, New York. pp. 1-421
Rogers A, Fischer BU, Bryant J, Frehner M, Blum H, Raines CA, Long SP (1998). Acclimation of photosynthesis
to elevated CO2 under low-nitrogen nutrition is affected by the capacity for assimilate utilization.
Perennial ryegrass under free-air CO2 enrichment. Plant Physiology 118: 683–689
Rogers HH, Runion GB, Krupa SV, Prior SA (1997). Plant responses to atmospheric CO2 enrichment:
implications in root-soil-microbe interactions. In Allen LH, et al. eds., Advances in carbon dioxide effects
research. ASA, CSSA, SSSA, Madison. pp. 1-34
Sage RF, Sharkey TD, Seemann JR (1989). Acclimation of photosynthesis to elevated CO2 in five C3 species.
Plant Physiology 89: 590-596
Sawada S, Kuninaka M, Watanabe K, Sato A, Kawamura H, Komine K, Sakamoto T, Kasai M (2001). The
mechanism to suppress photosynthesis through end-product inhibition in single-rooted soybean leaves
during acclimation to CO2 enrichment. Plant and Cell Physiology 42: 1093-1102
Sharkey TD, Bernacchi CJ, Farquhar GD, Singsaas E (2007). Fitting photosynthetic carbon dioxide response
curves for C3 leaves. Plant, Cell and Environment 30: 1035–1040
Sims DA, Luol Y, Seemann JR (1998). Comparison of photosynthetic acclimation to elevated CO2 and limited
nitrogen supply in soybean. Plant, Cell and Environment 21:945–952
Yamori W, Noguchi K, Terashima I (2005) Temperature acclimation of photosynthesis in spinach leaves:
analyses of photosynthetic components and temperature dependencies of photosynthetic partial
reactions. Plant, Cell and Environment 28: 536–547-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011199

The Effect of Organic Materials and Decomposer on Soybean Growthand Production
Sandra Arifin Aziza*, Sri Ayu Dwi lestariaaBogor Agricultural University,
Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor, Indonesia* Corresponding author: Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor,
Jl Meranti, Kampus Darmaga, Bogor, 16680, Indonesia
Tel.: +62 0251 8629353; fax: +62 0251 8629353
sandraaziz@yahoo.com

Abstract
Field experiment was set up in Cikarawang, Darmaga Bogor, Indonesia from December 2010 to April 2011. The
objective of the study was to investigate the effect of chicken manure, rice straw, and green manure of Tithonia
diversifolia with the application of decomposer, i.e. chicken manure liquid, chicken manure + Tithonia diversifolia
liquids, and biofertilizer under organic farming system. A randomized block design was laid out with organic
materials (chicken manure, rice straw and green manure of Tithonia diversifolia) as the first factor anddecomposers (chicken manure liquid, chicken manure + Tithonia diversifolia liquid, and biofertilizer) as thesecond factor. The results showed that chicken manure gave the best effect on soybean growth and production.
Soybean productivities from organic treatment chicken manure, green manure of Tithonia diversifolia, and rice
-1
straw were 1.00, 0.85, and 0.73 ton dry seed ha , respectively. Biofertilizer gave better response on soybean
growth and production components than chicken manure + Tithonia diversifolia liquid, chicken manure liquid and-1
control, i.e. 089, 088, 0.82, and 0.98 ton dry seed ha , respectively.
Keywords: organic soybean, decomposer, chicken manure, rice straw, Tithonia diversifolia Hemsl

Introduction
Increasing the inputs of nutrients has played a major role in increasing the supply of foodto a continually growing world population. However, focusing attention on the most important
nutrients, such as nitrogen (N), has in some cases led to nutrient imbalances, some excess
applications especially of N, inefficient use and large losses to the environment with impacts on air
and water quality, biodiversity and human health. In contrast, food exports from the developing to
the developed world are depleting soils of nutrients in some countries. Better management of all
essential nutrients is required that delivers sustainable agriculture and maintains the necessary
increases in food production while minimizing waste, economic loss and environmental impacts.
More extensive production systems typified by ‗organic farming‘ may prove to be sustainable.
However, for most of the developed world, and in the developing world where an ever-growing
population demands more food, it will be essential to increase the efficiency of nutrient use inconventional systems (Gouldin et al., 2008).
The success of organic system in supporting national food security is still in doubt because
the production of organic farming system is generally less than that of conventional farming.
However, organic farming system may be able to support food security in local level because thecontinuity of plant production is possible. Farmers can use on-farm inputs that normally areavailable at production site and avoid the use of chemical fertilizer and pesticide which is relatively
expensive and negative impact to the environment.
Animal and green manure can be used as organic fertilizer for production of organic
vegetable soybean (Melati and Andriyani, 2005; Sinaga, 2005; Kurniasih, 2006). Chicken manuregave better result than sheep manure (Sinaga, 2005), while Centrosema pubescens was better
200

thProceedings of The 7 ACSA Conference 2011

than Calopogonium mucunoides and Crotalaria juncea (Sinaga, 2005; Kurniasih, 2006). Chicken
manure 10 ton ha-1 increased the vegetative growth and production in organic soybean (Melati andAndriyani, 2005).
In Indonesia, soybean is one of the crops used in the cropping pattern in the lowland. Arafah
and Sirappa (2003) stated that rice straw should be incorporated to the soil after every harvest so it
can be beneficial to the next crop in the cropping pattern. Indriani (2000) found that Tithonia
diversifolia contains high nutrients, especially Nitrogen. This nitrogen can be useful in assisting
decomposting bacteria in decomposition process.
One of the obstacles in organic agriculture is the decomposition process of the organic
matter in the soil. Local microorganisms from various organic matters (fruits, plants, fish bones,
dead animal and garbage) around us can be used as media for microorganism for decomposition
process (Indriani, 2000). This effort can be used to optimize the growth and activity of the
microorganism that decomposed the organic matter (Sutaryono dan Fauzi, 2007). Combination of
biofertilizer and various organic matters influenced the physical and biological soil properties(Mezuan et al., 2002). Bertham (2002) studied biofertilizer application that showed the increasing
total pod number, filled pod number, and root nodule number in soybean. Further Hindratno (2006)
found that decomposer application or chicken manure liquid as decomposer increased the plantheight and fresh pod of vegetable soybean weight than without decomposer application.
This research was aimed to study the influence of organic matter, i.e. chicken manure, ricestraw, and Tithonia diversifolia with decomposer, i.e. chicken manure liquid, chicken
manure+Tithonia diversifolia, and biofertilizer (Bioextrim) to the organic soybean production.

Materials and Methods
The research was carried out at organic research station Cikarawang, Bogor, West Java,
Indonesia, from November 2010 to April 2011. A randomized block design was laid out with organic
materials (i.e. chicken manure, rice straw, and green manure of Tithonia diversifolia) as the first
factor and decomposers (i.e. chicken manure liquid, chicken manure + Tithonia diversifolia liquid,and biofertilizer Bioextrim) as the second factor. Duncan‘s Multiple Range Test (DMRT) with α=5%and t-Dunnet test for comparison between treatments with control (rice straw without dekomposer)
were used in this experiment.
Liming with 2 tonnes dolomit ha-1 and 2 ton ash ricehull ha-1 were given in row for soil
conditioning 4 Weeks Before Planting (-4 WAP/Weeks After Planting). Organic matter as treatments
were given in the form of 10 tonnes chicken manure ha-1, 10 ton rice straw ha-1, and 10 tonnes ofTithonia diversifolia ha-1 and decomposer in the form of chicken manure liquid, chicken manure +Tithonia diversifolia liquid, and biofertilizer. Coconut water and red sugar, i.e. 40 l and 4 kg, wereused as the solution for each type of decomposer liquid and then fermented for 6 weeks (at -6
WAP) and given as soil drench.
Cymbopogon nardus and Tagetes erecta L. were used as the repellent plant (Kusheryani
and Aziz, 2005). Extract of Cymbopogon nardus, Cymbopogon nardus + Tithonia diversifolia, andneem (Azadirachta indica A. Juss) were used as organic pest control and sprayed every week.
Soybean of Wilis variety were planted 25 x 10 cm with the repellent plant in the middle of every plot.

Results and Discussion
The soil analysis before the experiment showed pH at 5.7 (slightly acid), C-organic at
1.20% (high), total N at 2.2 ppm (low), CEC low, base saturity of 29.26% (medium). Clay in texture(sand, loam and sand, i.e. 6.93, 23.26 and 69.81%, respectively). After the treatment the pH on
control, Tithonia diversifolia and biofertilizer were 5.7 (slightly acid). On rice straw and chickenthProceedings of The 7 ACSA Conference 2011201

manure liquid, and rice straw and biofertilizer the pH increased to 6.50, whereas the other six
treatments were neutral to base (6.60-7.30). Low total nitrogen (0.12-0.22%), high C-organic was
1.20-2.39%, P availability was 2.80-10.20 ppm, low to high K at 0.29-0.94 me/100g, high basesaturity was 64.04-96.15%.
Penta and tetra-foliate leaves found on 7 WAP, mostly on Tithonia diversifolia plots, withdark green colour, broad leaves and thicker canopy. On 5 WAP Pseudomonas syringae pv.
glycinea were found on every plant. Saleh and Hardaningsih (2007) stated that this disease wasmostly found in high altitude in Indonesia, with wet weather and high temperature.
Organic matter significantly affected seed growth percentage at 2 WAP, number ofbranches on 3, 5, 7, 9, 11, 13 WAP; number of tetra and penta-foliate on 7 WAP; shoot, root,
nodule wet and dry weight on 7 WAP, disease prevalence on 8 WAP; insect infestation on 8 and 10
WAP; number of empty pod on 13 WAP; number of plant harvested; dry seed weight per 7.5 m2;
and soybean productivity (Table 1).
Table1. Organic matter on vegetative and generative variables

Variables

Organic Matter

WAP
Chicken Manureb

Rice Straw
369.22abControlTithonia diversifolia

Growth percentage

2

336.33

Plant height (cm)

13

12.79

12.88

386.1113.53

a382.33
12.59

Number of branches

11

51.06

52.22

49.76

49.85

Number of productive branches

13

3.17

a+

2.90

b+

2.83

b+

2.47

Leaf number

3

8.73

a+

6.69

bc

7.26

b

6.37

11

10.54

a+

9.24

ab

8.43

b

8.63

Tetrafoliate

7

10.33

a+

9.12

ab

8.30

b

8.53

Pentafoliate

7

10.24

a+

9.06

ab

8.13

b

8.40

2

5-7

9.79

a

8.82

ab

7.94

b

8.27

7-9

2.77

2.78

+

2.60

NAR (g/cm /weeks)

RGR (g/weeks)

2.75

5-7

6.59

5.92

6.07

6.00

7-9

12.38

11.49

11.51

11.63

Nodule wet weight (g)7135.11

a+

77.33

c+

87.78

b+

68.00

Shoot dry weight (g)

7

37.11

a+

17.78

c+

24.56

b+

8.67

Leaf water content (%)

7

0.34

Insect infestation (%)

10

5.07

b

3.73

b

7.53

a+

4.70

Disease prevalence (%)

8

0.74

b+

0.51

c

1.03

a+

0.52

Number of filled pod

0.15

24.14

Number of empty pod

3.16

Seed dry weight (g)46.69

Number of plants harvested

217.11

0.28

19.10
a+

1.74

21.43
ab

33.28
b

241.00

0.05

2.72

19.50
a

40.36
ab

249.89a9.59

2

752.44a550.67b636.78ab738.33

1.00

a

0.73

b

0.85

ab

0.98

Productivity (ton/ha)

8.98

255.33100 seed dry weight (g)Seed dry weight /7.5 m (g)

9.15

1.07
36.58

8.65

Numbers followed by different letters in the same row significantly different at 5 and 1 % DMRT test; figures followed by (+) inthe same row significantly different at 5% Dunnet test with the control.

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Decomposer significantly affected number of branches on 3, 5 WAP; number of tetra andpenta foliate on 7 WAP; Nett Assimilation Rate (NAR) on 7-9 WAP; shoot and root wet weight on 7
WAP; shoot dry weight on 7 WAP; disease prevalence on 8 WAP; and insect infestation on 8 WAP(Table 2). Combination of organic matter and decomposer significantly affected Nett Assimilation
Rate on 7-9 WAP; shoot and root wet weight on 7 WAP; shoot and root dry weight on 7 WAP; andinsect infestation on 10 WAP.
Table2. Decomposer on vegetative and generative variables
Decomposer
Variables

WAP

Chicken Manure
LiquidChicken manure+ Tithonia
diversifolia liquid

Control
Biofertilizer

Growth percentage

2

357.67372.33361.67382.33

Plant height (cm)

13

50.83

51.29

50.93

49.85

Number of branches

3

3.04

a+

3.01

a+

2.85

5

7.8

+

7.6

+

7.3

6.4

b+

2.47

11

9.3

9.5

8.6

8.4

Number of productive
branches

13

8.9

9.2

8.5

8.3

Leaf number

3

2.8

2.8

2.7

2.6

Tetrafoliate

11

11.3

7

102.1

a+
a+

+

10.7

9.6

100.1

ab+

26.3

ab+

10.1

98

b+

25.1

b+

68

Pentafoliate

7

27.7

2

5-7

2.5x107-9

2.8x10

5-7

0.26

0.23

0.29

0.05

7-9

0.18

0.1

0.07

0.12

NAR (g/cm /weeks)

RGR (g/weeks)

-3
-3

a

1.9x10

-3

1x10

-3

-3

b

-4

7.6x108.7-4

2.4x104.5x10b

1x10-3Shoot wet weight (g)

7

4.6

b

5.16

b

6.63

a+

4.7

Root wet weight (g)

7

0.65

bc

0.7

b

0.93

a

0.52

Shoot dry weight (g)

7

1.34

b

1.36

b

1.94

a

1.41

Nodule dry weight (g)

7

0.03

0.04

+

9x10

Insect infestation (%)

8

20.89

b+

21.98

a+

29.16

b+

69.65

a+

79.46

Disease prevalence (%)

10

6.89

8

69.16

0.03
a+

19.47

ab+

68.29

7.69

7.67

-3

5.48

Seed dry weight (g)

41.49

40.3

38.54

36.58

Number of plants harvested

231.8

239.6

236.7

255.3

100 seed dry weight (g)

9.18

9.39

9.15

8.65

Seed dry weight /7.5 m (g)658

615.67666.22738.33

Productivity (ton/ha)

0.88

0.82

0.89

0.98

2

Numbers followed by different letters in the same row significantly different at 5 and 1 % DMRT test; figures followed by (+) inthe same row significantly different at 5% Dunnet test with the control

The higher result on control plots seemed to be affected by the plot placement in the fieldthat supplied more water in the beginning of the experiment than the treatment plots. Soil in thetreatment plots were hard because of the changed condition from paddy field to the rain-fed

thProceedings of The 7 ACSA Conference 2011203

condition for the soybean planting. All the organic manure was recommended for soybean planting
after paddy field. There was no need to apply decomposer after the paddy field for soybean planting
with rice straw incorporation to the soil.

References
Arafah and MP Sirappa. 2003. Kajian penggunaan jerami dan pupuk N, P, dan K pada lahan sawah irigasi. J.
Ilmu Tanah dan Lingkungan 4(1):15-24.
Bertham YH. 2002. Potensi pupuk hayati dalam peningkatan produktivitas kacang tanah dan kedelai padatanah seri Kandanglimun, Bengkulu. JIPI. 4(1):18-26.
Gouldin K, S Jarvis, A Whitmore. 2008. Optimizing nutrient management for farm systems. Phil. Trans. R. Soc.
B 363:667-680. doi: 10.1098/rstb.2007.2177.
Hindratno TP. 2006. Pengaruh Lama Dekomposisi Pupuk Hijau dan Jenis Pelapuk terhadap Budi Daya Kedelai(Glycine max (L.) Merrill) Panen Muda Secara Organik. Skripsi. Program Studi Agronomi, Fakultas
Pertanian, Institut Pertanian Bogor. 50 hal.
Indriani YH. 2000. Membuat kompos secara kilat. Penebar Swadaya. Jakarta. 62 hal.
Kusheryani I dan SA Aziz. 2005. Pengaruh Jenis Tanaman Penolak Organisme Pengganggu Tanaman
terhadap Pertumbuhan dan Produksi Tanaman Kedelai (Glycine max (L.) Merrill) yang Diusahakan Secara
Organik. Bul. Agron. 34(1):39-45.
Kurniasih W. 2006. Pengaruh Jenis, Dosis Benih dan Umur Tanaman Pupuk Hijau terhadap Produksi Kedelai(Glycine max (L.) Merrill) Panen Muda. Skripsi. Program Studi Agronomi, Fakultas Pertanian, Institut
Pertanian Bogor. Bogor. 65 hal.
Melati M dan W Andriyani. 2005. Pengaruh Pupuk Kandang Ayam dan Pupuk Hijau Calopogonium mucunoides
terhadap Pertumbuhan dan Produksi Kedelai Panen Muda yang Dibudidayakan Secara Organik. Bul.
Agron. 33(2):8-15.
Mezuan, IP Handayani, E Inoriah. 2002. Penerapan formulasi pupuk hayati untuk budidaya padi gogo : studi
rumah kaca. JIPI. 4(1):27-34.
Sinaga, YAS. 2005. Pengaruh Pemberian Pupuk Organik terhadap Pertumbuhan dan Produksi Kedelai(Glycine max (L.) Merr) Panen Muda yang Diusahakan secara Organik. Skripsi. Program Studi Agronomi,
Fakultas Pertanian, Institut Pertanian Bogor. Bogor. 35 hal.
Sutaryono, YA dan MT. Fauzi. 2007. Potensi mikroorganisme lokal dalam memacu proses pengomposan padi.
J. Agroland 14(2):134-139.

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204

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Proceedings of The 7 ACSA Conference 2011

Yield and Dry Matter Production of Japanese and US Soybean Cultivars
under Drought StressYohei Kawasakia*, Yu Tanakab, Keisuke Katsuraa, Tatsuhiko Shiraiwaa
aGraduate School of Agriculture, Kyoto University, Japan
Graduate School of Science, Kyoto University, Japan* Corresponding author: Graduate School of Agriculture, Kyoto University, Sakyo-ku, 6068502, Kyoto, Japan.
Tel.: +81 75 753 6042; Fax: +81 75 753 6065.
kwsk77@kais.kyoto-u.ac.jpbAbstract
The difference in soybean yield between Japan and US is enlarging. The authors reported that the higher yields
of US cultivars than Japanese cultivars were attributed to greater dry matter production and radiation use
efficiency and that the US cultivars tended to have higher leaf stomatal conductance to water vapor (gs). These
traits may cause different sensitivity to drought between cultivars. The objective of this study was to examine
whether the yield response to drought differed between a Japanese cv. Tachinagaha (Tc) and a US cv. UA-4805(UA) with low and high leaf gs, respectively. The two cultivars were grown on a drained paddy field in the Exp.
Farm of Kyoto Univ. (Takatsuki, Japan) and the treatment with irrigation (Control) and without irrigation(Drought) was started at 20 days after emergence. The volumetric soil water content was on average 6% lower
in the Drought than in the control plots. The leaf area development was inhibited only in UA under Drought. The
gs was higher in UA than in Tc and it decreased in both cultivars by 34~38%. UA showed a greater mean seedyield and harvest index. Drought reduced mean seed yield and total dry weight of two cultivars. The yield
reduction by drought in UA was associated with reduced radiation intercepted, while yield reduction in Tc wasassociated with reduced radiation use efficiency and harvest index. The significant G×E interaction was detectedin harvest index and mean fraction of radiation intercepted. The two cultivars did not differ in yield response to
drought, but they differed in the yield components affected by drought.
Keywords: soybean (Glycine max (L.) Merrill), yield, genotype by environment interaction, drought

Introduction
The difference in soybean yield between Japan and US is enlarging. The authors reported
that the higher yields of US cultivars than Japanese cultivars were attributed to greater dry matterproduction and radiation use efficiency (Kawasaki et al. 2010) and that the US cultivars tended tohave higher leaf stomatal conductance to water vapor (gs) (Tanaka et al. 2010). These traits may
cause different sensitivity to drought between cultivars. The objective of this study was to examine
whether the yield response to drought differed between a Japanese cv. Tachinagaha (Tc) and a US
cv. UA-4805 (UA) with low and high leaf gs, respectively.

Materials and Methods
The two cultivars were grown on a drained paddy field in the Experimental Farm of Kyoto
University (Takatsuki, Japan, Eutric Fluvisols) located at 34°51‘N and 135°37E‘. The date of sowing
was 7th of July 2010 and the date of emergence was 12nd of July. Plant spacing was 0.7 by 0.15m.
Fertilizers of N, P2O5, K2O were incorporated into the soil before sowing at 3, 10 and 10 gm-2,
respectively.
The treatment with irrigation (Control) and without irrigation (Drought) was started at 20
days after emergence. In the Control, irrigation water was evenly applied using the plastic tubethProceedings of The 7 ACSA Conference 2011

205

(Sumisansui, Sumika Agrotech Co. Ltd.) extended on the ground in every other interrow. Irrigation
was conducted when the soil matric potential declined lower than 50kPa. The volumetric soil watercontent was measured twice a week using EC-5 (Decagon Devices, Inc.).
The canopy coverage was measured by the digital image analysis using ImageJ (NIH,
USA) (Purcell 2000, Shiraiwa et al. 2011). The total above-ground plant part was harvested at 35,
47, 61, and 74 days after emergence from a 1.26 m2 land area and the dry matter weight wasdetermined after drying at 80°C for 72h. The Leaf Area Index (LAI) was estimated by measuring leaf
area of representative plants.

Results and Discussion
The volumetric soil water content was on average of 6% lower in the Drought than in theControl plots (Figure 1). The leaf area development was inhibited only in UA under Drought (Figures
2 and 3). The gs was higher in UA than in Tc and it decreased in both cultivars by 34~38% (Figures
4 and 5).

Figure 1. Change in volumetric soil water content measured by EC-5 (Decagon Devices, Inc.).

Figure 2. Change in canopy coverage measured by digital image analysis.

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Proceedings of The 7 ACSA Conference 2011

Figure 3. Change in Leaf Area Index.

Figure 4. Stomatal conductance of Tachinagaha and UA-4805 under different soil conditions.

Figure 5. Change in total above ground dry weight.

UA showed a greater mean seed yield and harvest index in both of Control and Drought.
Drought reduced mean seed yield and total dry weight of two cultivars. The yield reduction bydrought in UA was associated with reduced radiation intercepted, while yield reduction in Tc wasassociated with reduced radiation use efficiency and harvest index. The significant G×E interaction
was detected in harvest index and mean fraction of radiation intercepted (Table 1).
These results indicated that the two cultivars did not differ in yield response to drought, butthey differed in the yield components affected by drought.

thProceedings of The 7 ACSA Conference 2011207

Table 1. Yield and yield components of drought experiment at Takatsuki in 2010
Seed
yield
-1
(t ha )
Tachinagaha control
Tachinagaha drought
UA-4805 control
UA-4805 drought

3.77
2.17
4.84
3.65

Total
Dry
Weight-1(t ha )
6.22
5.34
7.12
5.74

Analisis of varience
Cultivar
Treatment
cultivar x treatment

29.75 **
35.49 **
0.77 NS

19.65 *
58.79 **
2.98 NSHI

Mean F(%)

Total radiationintercepted
(MJ)

0.521
0.341
0.585
0.531

Radiation
Use
Efficiency-2(g MJ )
0.504
0.455
0.581
0.572

72.9
69.2
71.7
58.6

1233
1170
1225
1000

27.49 **
32.50 **
8.14 *

46.75 **
4.16 *
198 NS13.87 *
28.33 **
8.72 *

8.84 *
23.13 **
7.33 NS

Total
incident
radiation
(MJ)
1690
1691
1708
1705

References
Kawasaki Y, Tanaka Y, Katsura K, Purcell LC, Homma K, Katsube-Tanaka T, Shiraiwa T. 2010. Yield and Dry
Matter Productivity of Japanese and US Soybean Cultivars－A Comparison in the Warm Regions－.
Jpn. J. Crop Sci. 79(S2):102-103.
Tanaka Y, Fujii K, Shiraiwa T. 2010. Variability of Leaf Morphology and Stomatal Conductance in Soybean[Glycine max (L.) Merr.] Cultivars. Crop Sci. 50:2525-2532.
Purcell LC. 2000. Soybean canopy coverage and light interception measurements using digital imagery. Crop
Sci. 40:834-837.
Shiraiwa T, Kawasaki Y, Homma K. 2010. Estimation of Crop Radiation Use Efficiency. Jpn. J. Crop Sci. 80:360364.

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208

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Selection of F4, F5 and F6 Soybean Breeding Lines for High Yield
and Large Seed Size
Darman M. Arsyad a*, Asadi babCentral Institute for Agricultural Technology Assessment and Development, Bogor, Indonesia
Research Institute for Agricultural Biotechnology and Agricultural Genetic Resources, Bogor, Indonesia* Corresponding author: Central Institute for Agricultural Technology Assessment and Development,
Bogor, Indonesia
Tel.: +62-251-8351277; Fax: +62-251-8350928
dmarsyad@gmail.com

Abstract
The first step in a cultivar development is to form a population with genetic variability for the characters ofinterest. This is done by hybridization of genetically different parents. Hybridization was made to combine thecharacters of high yield potential, wide adaptability, desirable agronomic characters, and large seed size byusing cv. Tanggamus, Sibayak, Argomulyo, Anjasmoro, Panderman and Local Tegal. Amount of 4800 F4 lines
originated from five single cross combination were planted in rice field, Sukawening, Garut District, West Javaon early dry season 2007. Pedigree method of selection was used. Each line was grown in one row of threemeters length and 40 cm between rows. There was a high variability on agronomic characters (plant height, pod
number, seed yield and seed size). Amount 1311 F5 lines were selected and grown in the same location on latedry season 2007. Amount of 540 F6 lines were selected and grown in the same location on early rainy season
2007/2008. Sixty-two F7 lines were selected, and 42 lines among them had high yield and large seed size.
Keywords: high yield, large seed size, soybean breeding lines

Introduction
Breeding for high yield potential is generally the major goal in any breeding program
including soybean. Breeding for high yield potential is inextricably linked with breeding for othercharacteristics. Today the preferences of users (farmers and craftsmen tempeh and tofu) weremore likely to require large varieties of soybean seed. Craftsmen tempeh and tofu have longaccustomed to using large seed soybean imports, so that preferences are now a lot towards thelarge seed.
To meet user demand, then breeding programs to produce varieties of soybean large seedin major need of attention. A number of varieties of large seed soybean have been available suchas Argomulyo, Burangrang, Anjasmoro, and Grobogan (Hermanto et al., 2009).
As an effort to produce new varieties of soybeans that are superior to the existing varieties,
a number of crossing (hybridization) between soybean genotypes of high yield potential, quite broad
adaptability, many pods and smaller seed size with the genotypes of relatively low yield potential,
narrow adaptability, few pods, and large seed size, and were carried out in 2004 (Arsyad et al.
2005).
This study aimed to: (a) obtain expected breeding lines better than the existing varieties,
which possess higher yield potential, desirable agronomic traits and has a large or slightly larger
seed size, and (b) study the behavior of the relationship among traits in soybean.

Materials and Methods
Plant Materials
Selection of F4, F5 and F6 soybean breeding lines was conducted in the rice field of
Sukasono Village, Sukawening Subdistrict, Garut District, at the growing season I (April-June 2007),season II (July-September 2007) and the season III (September-November 2007), respectively.
Selection materials that were used in the firs selection In the season I the 4800 F4 soybean
breeding lines were grown and selected. Those breeding lines were derived from a combination offive single crosses, which were Tanggamus x Tegal, Tanggamus x Anjasmoro, Sibayak x Tegal,
Sibayak x Argomulyo, and Sibayak x Panderman, made in 2005 (Arsyad et al. 2005). In season II,
thProceedings of The 7 ACSA Conference 2011209

the 1311 F5 breeding lines wich were selected from the F4 lines in season I, were grown and thenas many as 540 F6 lines were selected. Those 540 F6 lines were grown in season III.

Methods
Pedigree method based selection was used in each generation (Fehr, 1987). In eachseason, each breeding lines was grown as a single row with 1.5 meters length. Spacing between
rows (lines) were 40 cm and 10 cm within rows, and one seed/hole. Intensive techniques cultivation
involves fertilizing with 75 kg urea, 200 kg SP36, and 150 kg KCl per ha, controlling of weeds,
pests and diseases were carried out. Irrigation was applied, if there is no rain. The selection criteria
was an ideal plant type,i.e. sturdy plants (not fall), plant height > 70 cm with 4-5 branches, medium
leaf size, determinate type, more than 70 pods/plant and large pods. Selection procedure was done
as follows: First, selected the good rows of plants, and secondly, selected the 3-5 plants in selected
row that meet the selection criteria. Each selected individual plant was a new breeding line to thenext generation. Data analysis was done by creating the frequency distribution of selected breedinglines, calculate the mean and standard deviation for the observed characters of each population, as
well as t test for mean population comparation (Gomez & Gomez 1984, Sugiyono 2009).

Results and Discussion
Selection of F4 Lines
Before time to harvest, around 1600 lines was selected and then as many as 1311 lines
was selected based on yield and seed quality. Only breeding lines derived from three crossing
combinations were selected. Those were the crosses between Sibayak x Tegal (Code V),
Tanggamus x Tegal (Code U) and Sibayak x Argomulyo (Code W). The other two crosses(Tanggamus x Anjasmoro and Sibayak x Panderman) derived inferior progenies. .
Frequency distribution of selected breeding lines based on crop yield are presented in
Figures 1. The range of lines seem still very wide, ranging from less than 10 g to more than 50
g/line, with the highest frequency was in the range of 21-25 g/line. Genotype of parents produucedaverage yield of less than 20 g/genotype. This indicated the presence of transgressive segregationon the breeding materials. Variability was also found in the seed size (100 seed weight), plantheight, and number of pods (Figure 2-4). The selection would be effective when there was wide
phenotypic variability among breeding lines. This was understandable because the selected lines
were still at F4 generation and was not selected since F2. The frequency of heterozygous
individuals at a locus was 50% in F2 (Fehr 1983), and because the selection has not been done
since the F2, therefore the heterozygote frequency in the F4 was still high (50%).
Breeding lines derived from Sibayak x Tegal cross have an average higher yields, larger
seed, higher plant, but the number of pods is less than the breeding lines derived from Tanggamusx Tegal and Sibayak x Argomulyo (Table 1). No yield different between breeding lines derived from
Tanggamus x Tegal and Sibayak x Argomulyo. Breeding lines derived from Sibayak x Tegal haslarger seed size compared to the breeding lines derived from Tanggamus x Tegal and Sibayak x
Argomulyo. Breeding lines derived from Sibayak x Argomulyo had a relatively smaller seed size
compared to the other two cross combinations, but it had more pods per plant. It appears atendency that the breeding lines of a large seed pods had fewer seeds, while breeding lines ofmoderate or relatively small seed pods had more seeds.
A total of 203 breeding lines have been identified with high yield capacity, which was morethan 30 g/line (data not presented). A total of 99 breeding lines of which had large seed size(>13g/100 seeds), and the other 104 breeding lines had moderate or relatively smaller seed size.
Selection will change the frequency of genotype and phenotype of a locus (Ferhr 1983). The basisof the expected effect of the selection is the change of gene frequencies (Falconer, 1967), but we
do not know what the frequency of genes that changed, because the selection was only based on
phenotypic selection. The selection would be effective to choose recessive alleles, as consequently
in one generation all dominant alleles will be eliminated in the population. However, if the selection
is intended to eliminate the recessive alleles, it would be more difficult because it is covered bydominant alleles in the heterozygous individuals (Fehr, 1983).

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Proceedings of The 7 ACSA Conference 2011

Although the selection in this study was only based on phenotypic values, it was expected
to be quite effective to gain the best selected lines, which is expected to produce good progenies.
The progress of the selection is determined by phenotypic variability, heritability values andselection intensity (Falconer, 1967). Agronomic characters such as plant height, number ofbranches and number of pods support high capacity of selected the breeding line.

Figure 1. Frequency distribution of F4 soybean breedinglines for yield (g/plant), Garut, season I 2007.

Figure 2. Frequency distribution of F4 soybean
breeding lines for 100 seed weight (g),Garut, season I 2007.

Figure 3. Frequency distribution of F4 soybean breedinglines for plant height (cm), Garut, season I
2007.

Figure 4. Frequency distribution of F4 soybean
breeding lines for pod number per plant,
Garut, season I 2007.

Table 1. Mean and standar deviation of seed yield, 100 seed weight, plant height, and number of pods per plantof F4 lines originated of three single cross in rice field at Sukasono Village, Sukawening Subdistrict,
Garut District, early dry season of 2007
Population
V (Sibayak x Tegal)
U (Tanggamus x Tegal)
W (Sibayak x Argomulyo)

Numberof lines
490
400
401

Seed yield(g)
24a ± 7.1
22b ± 6.8
22b ± 7.3

100 seedweight (g)14.1a ± 2.1
12.6b ± 1.9
10.9b ± 2.2

Plant height(cm)
58a ± 9.2
51b ± 8.1
56a ± 7.6

Number of pods
/ plant
97b ± 25.4
100b ± 24.4
124a ± 29.5

Value in the same column followed by the same letter are not different at 0.01 probability level

Selection of F5 Lines
Before time to harvest around 700 lines was selected and, as many as 540 lines was
selected based on yield and seed quality. Frequency distribution of breeding lines on several
classes of seed yield is presented in Figure 5. The yield range of the breeding lines seemed quite
wide, ranging from less than 10 g to more than 30 g/line, with the highest frequency present in therange of 11-15 g/line. The parent produced an average yield of less than 11g. A total of 191

thProceedings of The 7 ACSA Conference 2011211

breeding lines produced higher yield than that of the best parent (Tanggamus). This indicated thepresence of the transgressive segregation, i.e. breeding line yields were higher than that of bothparents. The occurence also the same as the seed size (100 seed weight), which was as many as
88 breeding lines contained a rather large to large seed size (Figures 6). Plant height and numberof pods showed a fairly high variability (Figure 7 and 8).
Breeding lines derived from Sibayak x Argomulyo had the higher average yields, higher
plant height, and more pods per plant than those of the breeding lines derived from Tanggamus x
Tegal and Sibayak x Tegal (Table 2). In contrast, breeding lines derived from Sibayak x Tegal
produced average seed size larger than that of the breeding lines derived from Tanggamus x Tegal
and Sibayak x Argomulyo. It was a tendency that the breeding lines of larger seed correlated withfewer pods, shorter plants and lower grain yield. The selection would be effective when the highvariability occurred among breeding lines. A total of 62 breeding lines had high yield capacity (Table
3).

Figure 5. Frequency distribution of F5 soybean
breeding lines for yield (g/plant), Garut,season II 2007.

Figure 6. Frequency distribution of F5 soybean
breeding lines for 100 seed weight (g),Garut, season II 2007.

Figure 7. Frequency distribution of F5 soybean breedinglines for plant height (cm), Garut, season II
2007.

Figure 8. Frequency distribution of F5 soybean
breeding lines for pod number per plant,
Garut, season II 2007.

Selection of F6 Lines
Of the 540 F6 breeding lines that were selected in the experiment on season III 2007, 100
breeding lines were selected at the harvesting time, and the best 60 breeding lines were selected
after processing. Of the 60 F7 breeding lines, as many as 42 breeding lines had the high yield
capacity and relatively large seed (Table 4). Among the 42 breeding lines, 16 breeding lines were
derived from the cross between Sibayak x Tegal, six breeding lines were derived from the crossbetween Tanggamus x Tegal, and 20 breeding lines were derived from teh cross between Sibayakx Argomulyo cross. Those breeding lines will be tested further in the preliminary yield testing on
early dry season of 2008.

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Table 2. Mean and standar deviation of seed yield, 100 seed weight, plant height, and number of pods per plantof F5 lines originated of three single cross in ricefield at Sukasono Village, Sukawening Subdistrict,
Garut District, late dry season of 2007
Population

Numberof lines
200
200
140

V (Sibayak x Tegal)
U (Tanggamus x Tegal)
W (Sibayak x Argomulyo)

Seed yield(g)
14b ± 4.912b ± 4.1
18a ± 4.6

100 seedweight (g)11.1a ± 1.5
10.8b ± 1.5
9.9b ± 1.5

Plant height(cm)
64b ± 12.3
50c ± 7.868a ± 8.7

Number ofpods / plant
79b ± 25.579b ± 22.8
119a ± 27.2

Value in the same column followed by the same letter are not different at 0.01 probability level

Table 3. Performance of the best 62 F5 breeding lines in ricefield at Sukasono Village, Sukawening Subdistrict,
Garut District, season II 2007

Breeding line

Numberof linesV (Sibayak x Tegal)

20

U (Tanggamus x Tegal)11

W (Tanggamus x
Argomulyo)
Tanggamus
Sibayak
Tegal
Argomulyo

31
-

Plant
height(cm)
62(46-100)

Number of
branches
per plant
6.8(4-10)

Number ofpods perplant
107(77-240)

Seed yield(g)

100 seedweight (g)23.3(21.2-26.4)

11.7(9.9-14.9)

53(40-61)
69(57-102)
50
56
39
40

6.9
(4-10)
6.5
(4-8)
5
5
2
5

111
(75-145)148(109-239)
68
86
27
52

21.8
(20.6-23.4)
23.8(20.2-35.1)
10.7
10.0
4.4
8.9

10.9
(8.4-13.3)
13.0(8.8-17.40
9.2
9.8
17.2
13.3

Value in parenthese is a range

Table 4. Agronomic performance and seed yield of 42 F6 soybean breeding lines selected in ricefield at
Sukasono Village, Sukawening Subdistrict, Garut District, season III 2007
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

Breeding line
V-5-1-1-2
V-43-2-2-1
V-63-2-1-1
V-75-2-1-2
V-75-2-3-1
V-75-2-4-2
V-250-2-2-1
V-257-1-1-1
V-273-1-1-2
V-284-2-1-1
V-215-1-1-1
V-215-1-2-1
V-215-1-4-1
V-390-2-3-2
V-424-1-1-2
V-612-2-1-2
U-574-1-1-1
U-445-2-1-1
U-79-2-2-2
U-675-1-1-1
U-534-2-2-1
U-697-1-1-1
W-209-1-1-2
W-455-1-2-1
W-460-1-1-1
W-47-2-1-1
W-466-1-1-1
W-40-1-1-2
W-40-1-2-2

thPlant height
(cm)
69
95
68
69
68
65
66
62
70
66
62
62
58
59
60
66
64
60
58
56
62
54
68
98
70
68
70
67
65

Proceedings of The 7 ACSA Conference 2011

Number of
branches
5
4
7
6
4
4
7
9
7
8
9
7
8
7
10
7
8
7
5
8
4
7
8
6
5
6
7
7
8

Number of
pods
99
77
102
114
85
91
106
123
114
117
113
240
118
120
104
110
104
122
123
93
106
99
170
120
109
130
191
147
127

Seed yield
(g)
25.1
22.1
26.2
27.7
25.4
23.0
26.7
26.4
25.7
25.4
22.1
30.3
25.4
24.8
25.3
23.5
20.8
22.1
20.6
21.3
22.3
21.5
20.8
22.4
21.3
20.3
32.2
27.6
24.0

100 seed weight
(g)
14.9
14.9
13.9
14.7
14.8
13.0
13.3
14.4
13.0
13.8
14.3
13.4
14.3
13.1
13.3
13.6
13.3
14.5
12.5
12.2
12.9
13.4
13.1
15.4
13.6
13.0
13.1
14.3
13.3

213

No.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46

Breeding line
W-165-1-1-1
W-284-1-3-1
W-537-1-1-1
W-267-2-1-2
W-530-1-2-1
W-308-2-1-2
W-258-1-1-1
W-271-1-1-1
W-519-1-2-1
W-169-1-2-1
W-304-1-2-1
W-304-1-3-1
W-197-1-1-2
Tanggamus
Sibayak
Tegal
Argomulyo
V (16 lines)
U (6 lines)
W (20 lines)
Check (4 var.)

Plant height
(cm)
75
72
67
74
67
71
86
70
79
68
64
66
59
58
60
45
54
67
(58-95)
59(54-64)71(59-98)54(45-60)

Number of
branches
7
8
4
5
6
7
5
8
8
6
7
7
6
6
6
3
5
6,8
(4-10)
6,5
(4-8)
6,5
(4-8)
5
(3-6)

Number of
pods
168
239
103
100
145
180
127
158
175
160
138
176
142
68
86
36
58
114
(77-240)108(93-123)150(100-239)
62(36-86)

Seed yield
(g)
25.7
34.3
21.2
20.0
25.3
35.1
24.0
21.1
25.1
30.8
22.3
21.2
23.3
10.7
10.0
14.2
18.5
25.3
(22.1-30.3)
21.4(20.6-22.3)
24.9(20.0-35.1)
13.4(10.0-18.5)

100 seed weight
(g)
13.7
13.4
13.6
14.5
13.3
13.3
13.9
12.6
13.1
12.7
12.5
13.3
13.8
10.2
11.1
17.0
14.6
13.9
(13.0-14.9)
13.1(12.2-14.5)
13.5(12.5-15.4)
13.2(10.2-17.0)

V= Sibayak x Tegal cross
U= Tanggamus x Tegal cross
W=Sibayak x Argomulyo crossValue in parenthese is a range

Acknowledgment
The authors acknowledge the support of the Indonesian Ministry of Research and Technology, whoprovided the funding for this research.

References
Arsyad DM, H Kuswantoro and Purwantoro. 2005. Hybridization, and selection penggaluran soybeans.
Technical Report Research Institute for Crop Legumes and Tuber (In Indonesian).
Burton JW. 1983. Quantitative genetics: Results relevant to soybean breeding, p. 211-248. In JR Wilcox (Ed.):
Soybean: Improvement, Production, and Uses. Second Edition, ASA, Wisconsin, No. 16 in series.
Falconer DS. 1967. Introduction to Quantitative Genetics.Robert MacLehose and Company Limited, Glasgow.
GreatBritain. Pp 365.
Hermanto, D Sadikin and E Hikmat. 2009. Deskripsi Varietas Unggul Palawija 1918-2009. Indonesian Center for
Food Crops Research and Development. Indonesian Agency for Agriculture Research and Development.
Pp 330 (In Indonesian).
Fehr WR. 1983. Applied Plant Breeding. Dept. of Agronomy, Iowa State Univ. Ames. 174 p.
Gomez KA and AA Gomez. 1984. Statistical Procedures for Agricultural Research. 2nd Edition. Pp 680.
Sugiyono 2009. Statistika untuk Penelitian. Alfabeta, Bandung. Pp 389 (In Indonesian).

-- back to Table of Content --

214

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Proceedings of The 7 ACSA Conference 2011

Effect of Hydrogen Peroxide Spraying on Drought Stressin Soybean Plant
Yushi Ishibashia, Haruka yamaguchia, Takashi Yuasaa, Mari Iwaya-Inouea,
Susumu Arimab,c, Shao-Hui ZhengcaCrop Science Lab., Faculty of Agricluture, Kyushu University, Hakozaki, Fukuoka 812-8581, Japanb
Faculty of Agriculture, Saga University, Saga 840-8502, Japanc
Coastal Bioenvironment Center, Saga University, Karatsu, Saga 840-0021, Japan* Corresponding author: Yushi Ishibashi, Crop Science Lab., Faculty of Agriculture, Kyushu University,
Hakozaki, Fukuoka 812-8581, Japan. Tel.: +81 92 642 2824; fax: +81 92 642 2824.
yushi@agr.kyushu-u.ac.jp

Abstract
We examined whether the spraying of soybean leaves with hydrogen peroxide (H2O2) would alleviate thesymptoms of drought stress. Pre-treatment by spraying leaves with H2O2 delayed foliar wilting caused bydrought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content ofdrought-stressed leaves pre-treated with H2O2 was higher than that of leaves pre-treated with DW. Therefore,
we analyzed the effect of H2O2 spraying on photosynthetic parameters and on the biosynthesis ofoligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress,
the net photosynthetic rate and stomatal conductance of leaves pre-treated with H2O2 were higher than those ofleaves pre-treated with DW. In contrast to DW spraying, H2O2 spraying immediately caused an increase in themRNA levels of D-myo-inositol 3-phosphate synthase 2 (GmMIPS2) and galactinol synthase (GolS), which
encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In
addition, the levels of myo-inositol and galactinol were higher in H2O2-treated leaves than in DW-treated leaves.
These results indicated that H2O2 spraying enabled the soybean plant to avoid drought stress through themaintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide
biosynthesis rather than by rapid stomatal closure.
Keywords: Drought stress, Galactinol, Hydrogen peroxide, Soybean

Introduction
Plants respond and adapt to water deficits at both the cellular and molecular levels by theaccumulation of osmolytes and proteins that are specifically involved in stress tolerance. Drought
stress is the primary cause of crop loss across the globe, reducing average yields in most major
crop plants (Boyer 1982; Bray et al. 2000).
It is known that drought stress enhances the production of reactive oxygen species (ROS)
in cellular compartments such as chloroplasts, peroxisomes, and mitochondria. If drought stress is
prolonged, ROS production will overwhelm the scavenging action of the antioxidant system,
resulting in extensive cellular damage and death (Cruz de Carvalho 2008). On the other hand, ROS
are also known to function as signal molecules in plants (Foyer et al. 1997), controlling processes
such as growth, development, responses to biotic and abiotic environmental stimuli, andprogrammed cell death (Bailey-Serres and Mittler 2006).
Abscisic acid (ABA), synthesized in response to drought stress, is known to induce
stomatal closure and to reduce transpirational water loss (Schroeder et al. 2001). ABA activates thesynthesis of ROS in guard cells by a membrane-bound NADPH oxidase, and ROS mediate
stomatal closure by activating (through hyperpolarization) plasma membrane Ca2+ channels (Pei etal. 2000; Murata et al. 2001; Wang and Song 2008). In addition, it has been reported that hydrogen
peroxide (H2O2), a type of ROS, is involved in the acclimation of C. albidus to summer drought(Jubany-Marí et al. 2009) and in that of maize (Zea mays L.) to salt stress (Azevedo Neto et al.
thProceedings of The 7 ACSA Conference 2011215

2005). H2O2 also increases the soluble sugar content of melon fruits (Ozaki et al. 2009). Other
components may also be responsive to ROS as a part of a stress-activated signal transduction
pathway. We therefore focused on ROS as signal molecules by examining whether exogenous
H2O2 application (by spraying) could alleviate drought stress and by working to define the alleviation
mechanism.

Materials and Methods
Plant materials
Soybean (Glycine max L. Merrill) cv. Fukuyutaka was used as the plant material. Three
weeks after emergence, either 1 mM H2O2 or DW was sprayed only once onto the leaves of eachplant (100 mL/pot), and then irrigation was stopped. Treatments with H2O2 or DW were always
followed by drought stress. Measurements of all parameters were made after water had been
withheld for 0, 2, 4, 6, and 8 days. For an additional control treatment, we included plants with nospray treatment and with irrigation maintained throughout the experiment. The following
measurements consisted of four replicates.
Leaf relative water content
To evaluate leaf relative water content (RWC), 50 leaf discs (5 mm in diameter) from eachplant were weighed to determine fresh weight (FW), then hydrated to full turgidity by being floated in
DW for 24 h at 4 °C and weighed again to determine the turgid fresh weight (TW). Dry weight (DW)
was determined by drying for 48 h at 90 °C. RWC was then calculated as [(FW-DW)/(TWDW)]×100.
Photosynthetic measurements
Photosynthetic rate, stomatal conductance, and transpiration rate were measured insoybean leaves using an LCpro+ portable photosynthesis system (LCpro, ADC Bioscientific Ltd.,UK) at room temperature (25 °C) in the morning (8:00-11:00 am). The quantum flux density at theleaf surface, flow rate, and leaf temperature in the chamber were maintained at 1500 μmol m-2 s-1,
200 μmol s-1, and 30 ºC, respectively. The rate of CO2 assimilation in the chamber was measured at
an ambient CO2 concentration of 370 μL L-1.
RT-PCR analysis
cDNA was synthesized from total RNA (1 µg) using Rever TraACE reverse transcriptase(Toyobo, Japan) according to the manufacturer‘s protocol. cDNA (1 µL) was amplified in a reaction
containing 10 µL of Go Taq Green Master Mix (Promega, USA), 0.1 µL each of 50 µM forward andreverse primers, and 8.8 µL of water. The amplification was conducted using a Program Temp
Control System Astec PC-320 (Astec, Japan) as follows: 1 min at 94 °C; 27 cycles of 15 s at 94 °C,
30 s at 58 to 62 °C, and 30 s at 72 °C; then 5 min at 72 °C. The specific primer sequences for
GmGolS (Glyma10g28610.1, Phytozome) were 5′-GACAAGCTTAAGCAGCAGATGGGGCACGGA3′ and 5′-ATCGGATCCTGCCAGCAGCAGTGCCCCCATAAG-3′; for GmP5CS (Genbank accession
no. AY492005), the specific primer sequences were 5′-ATCAAGAGTTCCACTAAAATTCCTGTC-3‘
and 5‘-TCATATGAGAAGGTCTCTGTGAGTGTAG-3‘; for GmActin (Genbank accession no.
V00450), the specific primer sequences were 5′-GCGTGATCTCACTGATGCCCTTAT-3′ and 5′AGCCTTCGCAATCCACATCTGTTG-3′. The specific primer sequences for GmMIPSs were
determined according to Chappell et al. (2006).

216

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Proceedings of The 7 ACSA Conference 2011

Myo-inositol and galactinol contents
Myo-inositol and galactinol levels were analyzed by HPLC using a 930-RI refractive index
detector (JASCO, Japan) and a Shodex Asahipak NH2P-50 4E column (polymer-base, particle size;
5 µm, 250 mm × 4.6 mm i.d.) (Showa Denko K.K., Japan). Sugars were separated with acetonitrilewater (80:20, v/v) as an isocratic mobile phase at 0.8 mL/min using an 880PU pump (JASCO,Japan). The column was held at 40 °C.
Powdered freeze-dried leaves (50 mg) were extracted with 4 mL 80% (v/v) ethanol. The
extracts were boiled for 20 min and centrifuged for 5 min at 25 000×g to produce pellets of insoluble
material. The supernatant was removed and the pellet was extracted twice more using the same
approach. The supernatants were then combined and dried. The residue was dissolved in 1 mL of
DW and passed through a Sep-Pak C18 mini-column (Waters, USA). The extracts were filtered(0.45 µm) before HPLC injection.

Results and Discussion

Relative water content （%）

The RWCs for the three treatments are shown in Figure 1. There were no significant
differences in RWC between control, H2O2-treated, and DW-treated plants up to 2 DAT. At 4 DAT,the RWCs in control, H2O2-treated, and DW-treated plants were 80%, 60%, and 40%, respectively.
The RWC at 6 DAT was also higher in H2O2-treated plants than in DW-treated plants. By 8 DAT, the
RWCs for both the H2O2 and DW treatments were approximately 40%.
80

a

60

b

a

a
b

b

40

c
c
b

20
0
0

2

4

6

Closed squares indicate the control (irrigated, unsprayed) treatment. Open andclosed circles indicate pre-treatment with H2O2 and DW respectively, followed bydrought stress for the number of days indicated. For each day after treatment,
means followed by different letters are significantly different at the 5% level as
determined by Tukey‘s test (n = 4); error bars indicate standard deviations.

8Days after treatment

Figure 1. Relative water contents of soybean leaves during drought stress.

The net photosynthetic rate (PN), transpiration rate (E), and stomatal conductance (gs)during drought stress are shown in Figure 2. Here, although the PN, E, and gs significantly
decreased during drought stress, these parameters were significantly higher in the H2O2-treatedplants than in DW-treated plants. At 2 DAT, gs levels in control, H2O2-treated, and DW-treatedplants were 0.588, 0.508, and 0.323 mol m-2 s-1, respectively (Fig. 2C). At 4 DAT, PN levels incontrol, H2O2-treated, and DW-treated plants were 21.34, 9.36, and 5.51 µmol m-2 s-1, respectively,
and the levels of E were 7.76, 3.35, and 2.53 mmol m-2 s-1, respectively (Fig. 2A, B).
Myo-inositol plays critical and diverse biological roles in a myriad of cellular processes
including signal transduction, stress responses, cell-wall biogenesis, growth regulation, andosmotolerance (Loewus and Murthy 2000). At 2, 4, and 6 DAT, GmMIPS2 expression was markedly
higher in leaves treated with H2O2 compared with control and DW-treated leaves (Fig. 3A). At 2
DAT, GmMIPS2 expression was induced by H2O2 treatment but not by DW treatment. At 4 and 6
DAT, the expression of GmMIPS2 mRNA in H2O2-treated leaves was remarkably higher than that in
DW-treated leaves. In contrast, the expression of GmMIPS1, -3, and -4 did not vary among thecontrol, H2O2, and DW treatments. We also examined the expression of galactinol synthase (GolS),thProceedings of The 7 ACSA Conference 2011217

which acts downstream of MIPS in the synthesis of raffinose-family oligosaccharides (RFOs).
GmGolS expression was low in the control and DW-treated leaves but clearly higher in the H2O2–
treated leaves, especially at 4 and 6 DAT (Fig. 3A).

a
a

a

15

b

b

10
5

b

A

c
c

0

0

2

4

a
b

4

b

2

c

B

b

0

b

c

0

8

a

a

ab

6

b

6

a

8

2

4

C

a

0.9

a

a

0.6

6

8

a

a

0.3

b

b
c

0

Days after treatment

Days after treatment

1.2
gs (mol m-2 s-1)

a

a

20

E (mmol m-2 s-1)

PN (µmol m-2 s-1)

25

0

2

b
b

4

6

b

8

Days after treatment(A) Photosynthetic rate (PN); (B) transpiration rate (E); and (C) stomatal conductance (gs). Closed squares indicate control(irrigated, unsprayed) treatment. Open and closed circles are H2O2 and DW pre-treatments, respectively, followed by drought
stress for the number of days indicated. For each day after treatment, means followed by different letters are significantly
different at the 5% level as determined by Tukey‘s test (n = 4); error bars indicate standard deviations.

Figure 2. Photosynthetic parameters during drought stress.

A
GmMIPS1GmMIPS2
GmMIPS3
GmMIPS4

Sugar contents (mg/g DW)

Myo-Inositol

B

12

a

8

a
a

4
c

b

c

c

4

6

C

2

b
b

Galactinol

2.5

a

1.5

a

1

b

a

b
c

a

a
b
c

b
c

(A) Expression of GmMIPS genes and GmGolS.
Control (irrigated, unsprayed); H2O2, H2O2 pre-treated;
DW, DW pre-treated; d, days after treatment. GmActin
was used as a control for loading. (B) Myo-inositol
contents and (C) galactinol contents. Closed squares
indicate the control (irrigated, unsprayed) treatment.
Open and closed circles indicate pre-treatment with
H2O2 and DW, respectively, followed by drought stressfor the number of days indicated. Means followed bydifferent letters are significantly different at the 5%
level as determined by Tukey‘s test (n = 4); error bars
indicate standard deviations.

0.5

0

0
0

2

Days after treatment

8

0

2

4

6

8

Days after treatment

Figure 3. Gene expression of GmMIPSs and GmGolS and contents of myo-inositol and galactinol in leaves
during drought stress.

We also found that the myo-inositol and galactinol contents in leaves pre-treated with H2O2
were increased compared with control and DW-treated leaves (Fig. 3B, C). Myo-inositol contents
increased during drought stress, especially in H2O2-sprayed plants at 6 DAT, but decreased tostarting levels by 8 DAT (Fig. 3B). The galactinol content in leaves pre-treated with H2O2 nearly
doubled by 4 DAT, and the increased level was maintained until 8 DAT. On the other hand, thegalactinol content in leaves pre-treated with DW increased until 8 DAT, but was significantly lower
from 4 to 8 DAT than that seen in H2O2-treated plants (Fig. 3C).
218

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Stress-inducible production of galactinol synthase (GolS) plays a key role in theaccumulation of galactinol and raffinose, which function as osmoprotectants, under drought stress(Taji et al. 2002). GolS catalyzes the synthesis of galactinol from myo-inositol and UDP-galactose.
We therefore examined the transcript levels of D-myo-inositol 3-phosphate synthase1, -2, -3 and -4(GmMIPSs), which encode key enzymes in the synthesis of myo-inositol, and that of GmGolS, in
H2O2-sprayed plants. We found that among the GmMIPS genes, only the transcript level of
GmMIPS2 increased after H2O2 spraying (Fig. 3A). Chappell et al. (2006) reported that GmMIPS2
was poorly expressed in soybean leaves cultivated conventionally. The results reported heresuggest that GmMIPS2 is likely to be involved in drought stress signaling through ROS production
caused by drought stress. Treatment with methylviologen, which enhances the production of O2(another ROS), increased the transcript levels of GolS in Arabidopsis thaliana (Nishizawa et al.
2008). Overexpression of AtGolS2 in transgenic Arabidopsis thaliana caused an increase inendogenous galactinol and raffinose, and showed improved drought tolerance (Taji et al. 2002). In
soybeans, we have shown that H2O2 spraying increased the transcript level of GmGolS and thegalactinol content in leaves (Fig. 3A, C). These results suggest that H2O2 spraying enabled the leaf
to maintain a high level of RWC by regulating the osmolality in the leaf, consequently alleviating theeffects of drought stress.

References
Azevedo Neto AD, Prisco JT, Eneas-Filho J, Medeiros JV, Gomes-Filho E. Hydrogen peroxide pre-treatment
induces salt-stress acclimation in maize plants. 2005. J. Plant Physiol. 162: 1114-22.
Bailey-Serres J, Mittler R. 2006. The roles of reactive oxygen species in plant cells. Plant Physiol. 141: 311.
Boyer JS. 1982. Plant productivity and environment. Science 218: 443-8.
Bray EA, Bailey-Serres J, Weretilnyk E. 2000. Responses to abiotic stresses. In: Gruissem W, Buchannan B,
Jones R, editors. Biochemistry and Molecular Biology of Plants. Rockville, MD. American Society of Plant
Physiologists Pp1158-1249.
Chappell AS, Scaboo AM, Wu X, Nguyen H, Pantalone VR, Bilyeu KD. 2006. Characterization of the MIPS genefamily in Glycine max. Plant Breed.125: 493-500.
Cruz de Carvalho MH. 2008. Drought stress and reactive oxygen species: production, scavenging and signaling.
Plant Signal Behav. 3: 156-65.
Foyer CH, Lelandais M, Kunert KJ. 1994. Photooxidative stress in plants. Physiol. Plant. 92: 696-17.
Jubany-Marí T, Munné-Bosch S, López-Carbonell M, Alegre L. 2009. Hydrogen peroxide is involved in theacclimation of the Mediterranean shrub, Cistus albidus L., to summer drought. J. Exp. Bot. 60: 107-20.
Murata Y, Pei Z-M, Mori IC, Schroeder JI. 2001. Abscisic acid activation of plasma membrane Ca2+ channels in
guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive
oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 13:2513-23.
Nishizawa A, Yabuta Y, Shigeoka S. 2008. Galactinol and raffinose constitute a novel function to protect plantsfrom oxidative damage. Plant Physiol.147: 1251-63.
Ozaki K, Uchida A., Takabe T, Shinagawa F, Tanaka Y, Takabe T, Hayashi T, Hattori T, Rai AK, Takabe T.
2009. Enrichment of sugar content in melon fruits by hydrogen peroxide treatment. J Plant Physiol .166:
569-78.
Pei Z-M, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI. 2000. Calcium channels
activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406: 731-4.
Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D. 2001. Guard cell signal transduction. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 52: 627-58.
Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K. 2002.
Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance inArabidopsis thaliana. Plant J. 29: 417-26.
Wang P, Song C. 2008. Guard-cell signalling for hydrogen peroxide and abscisic acid. New Phytol.178: 703-18.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011219

Productivity of Several Lines of Soybean in Majalengka,West Java, Indonesia
Alia Astutia, Ence Darmo Jaya Supenaa,b, Suharsonoa,b,*a

Department of Biology, Bogor Agricultural University, Indonesia
Research Center for Bioresources and Biotechnology, Bogor Agricultural University, Indonesia*Corresponding author: PPSHB IPB, Gd PAU, Jl. Kamper, Kampus IPB Darmaga Bogor 16680, Indonesia
Tel: +62 251 8621257; fax: +62 251 8621724.
E-mail: sony-sh@ipb.ac.id; sony.suharsono@yahoo.comb

Abstract
Soybean (Glycine max L. Merr) is a very important crop in Indonesia. The creation of new elite varieties is one
approach to increase the national production of soybean. We have developed several potential lines of soybean
to be released as a new varieties. Before releasing as new varieties, the productivity of these lines has to beevaluated in several locations. Therefore the objective of this research was to evaluate the productivity of sixlines of soybean resulted from the cross between Slamet and Nokonsawon varieties, i.e. KH8, KH9, KH31,
KH38, KH55, and KH71 in Majalengka, West Java, Indonesia. Four national elite varieties i.e. Anjasmoro,
Slamet, Tanggamus and Wilis were used as standard. The experiment was conducted in two seasons by using
randomized block design, with three blocks as replication. The result showed that based on the seed production
per plant in two seasons, all six lines have higher productivity than Anjasmoro variety. The seeds of these sixlines are bigger than that of standard varieties. Analysis of production stability in two seasons showed that KH71
is the most stable genotype in Majalengka. By comparing to Anjasmoro variety in two seasons, all six lines havea potential to be released as new varieties with high productivity and big seed.
Keywords: lines, productivity, seed size, soybean

Introduction
Soybean (Glycine max L. Merr) is a very important crop in Indonesia. Every year, Indonesia
imports more than 1.2 million tons of grains of soybean for food. The creation of new elite varieties
is one approach to increase the production of soybean in Indonesia. The creation of tolerantvarieties to acid soil containing high concentration of aluminum is very important to extend thecultivation onto the marginal land with with this condition. Sunarto (1995) had created a variety of
Slamet which is tolerant to acid soil. Eventhough variety of Slamet has a high productivity and is atolerant to acid soil, it has a relatively small seeds and its hilum is black which are undesirable traits
for tofu and tempeh industry. To improve these traits, we had crossed this variety with
Nokhonsawon variety which has a big seeds, then followed by selection based on high productivity
and seed size traits (Suharsono et al., 2006, 2007; Jambormias et al., 2011). From this cross, we
obtained 18 potential lines to be released as a new variety.
Before releasing a line of soybean as new variety, the productivity of this line has to beevaluated in several locations. In this experiment, six of 18 potential lines of soybean were
cultivated in the irrigated rice field in Majalengka, West Java, Indonesia. Majalengka is a center ofsoybean production in West Java. So, the objective of this research was to evaluate the productivity
and seed size of six lines, i. e. KH 8, KH 9, KH 31, KH 38, KH 55, and KH 71, with four elite nationalvarieties, i. e. Anjasmoro, Slamet, Tanggamus, and Wilis as standard in two seasons in Majalengka,West Java, Indonesia.

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Materials and Methods
Six lines of soybean i.e. KH8, KH9, KH31, KH38, KH55, and KH71 were used in thisexperiment. Four national cultivars i.e. Slamet, Wilis, Anjasmoro and Tanggamus were used as
standard. The experiment was carried out in Randomized Block Desain with three replications, so
this experiment had 30 experimental units or plots. The size of plot is 4 m x 5 m, planting distance is
40 cm x 20 cm with two plants per hole, so the productivity per hectar is productivity per plant x
250,000. The plants were fertilized by 10 tons of manure, 100 kg urea, 150 kg SP3, and 100 kg KCl
per ha in the beginning of cultivation. The evaluation of plant productivity was carried out by
sampling. For sampling, 10 plants were ramdomly chosen per plot. Seed size was mesured byweighing 100 dry seeds which were ramdomly chosen.
The cultivation was carried out in two seasons, i. e. wet and dry seasons. The cultivation inwet season (season I) was caried out in December 2009 - March 2010, and in dry season was donein May - August 2010. The data of seed productivity per plant and seed size were analyzed by
Duncan Multiple Range Test. The clustering of genotype based on seed productivity per plant andseed size was carried out by Important Performance Analysis (IPA). The analysis of stability of seedproductivity in two season was carried out by Additive Main Effect Multiplicative Interaction (AMMI).

Results and Discussion
Seed productivity and seed size
The seed productivity of all genotype (KH lines and standard varieties) in the wet season
was very high, between 22.9 and 35.6 g per plant, equivalent to 5.7-8.9 tons per ha. This
productivity is higher than that in dry season that is between 15.35 and 20.45 g per plant equivalent
to 3.8- 5.1 tons per ha (Table 1). Based on the description of Ministry of Agriculture (Deptan, 2011),
the productivity of Anjasmoro, Slamet, Tanggamus, and Wilis is 2.25-2.3, 2.26, 1.22, and 1.6 tons
per ha respectively. This productivity is lower than that of the same variety in Majalengka that ismore than 5 tons per ha (Table1). This result indicates that the environment of cultivation in
Majalengka was very favorable for soybean. The soil of this experiment has a pH 5.9 and contains
65,3 ppm of P which are very favorable for soybean growth.
Table 1. The productivity of several genotype in wet and dry seasons
Productivity in wet season

Genotype
KH8
KH9
KH31
KH38
KH55
KH71
Anjasmoro
Wilis
Tanggamus
SlametProductivity in dry season

Average of
Productivity

g/plant*

kg/hag/plant*

kg/hag/plantkg/ha

24.74 ab30.58 bc
22.89 a29.36 b35.62 c30.27 bc
23.43 a29.64 b30.56 bc
24.74 ab

6,185
7,645
5,723
7,340
8,905
7,568
5,858
7,410
7,640
6,185

17.7 ab20.45 c19.02 bc
19.43 bc
17.71 ab17.06 ab17.30 ab16.19 a15.35 a15.42 a

4,425
5,113
4,755
4,858
4,428
4,265
4,325
4,048
3,838
3,855

21.22
25.52
20.96
24.4
26.67
23.67
20.37
22.92
22.96
20.08

5,305
6,380
5,240
6,100
6,668
5,918
5,093
5,730
5,740
5,020

*the number followed by the different letter in the same column is significantly different.

Comparing to Anjasmoro which is elite national variety, all lines have equal or higher
productivity in wet and dry seasons. This result indicates that all lines have a potential as elitethProceedings of The 7 ACSA Conference 2011

221

national variety with a potential yield more than 5 tons per ha. In wet season, KH55 line has ahighest productivity, and in dry season KH9 has a highest productivity.
If we compare between wet and dry seasons, all genotypes have a higher yield in wet
season than in dry one. In this experiment, the precipitation during wet season in Majalengka was
about 411,53 mm/month, and during dry season was about 202 mm/month. Based on Calvino &Sadras (1999), the precipitation more than 300 mm/month is better for production of soybean.
In average, all lines and Anjasmoro variety have a big seed, and bigger than the seeds ofWilis, Tanggamus and Slamet varieties (Table 2). The big size is very important in the production of
tofu because the rendement is depend on the endosperm, and the endosperm of bigger seeds isbigger than small ones. So, based on the size of seeds, all lines have a potential to be released asa big seed varieties. As the yield, the size of seeds was also bigger in the wet season than that in
dry ones. The size of seeds may be affected by the enironment of growth. KH31 and KH38 havebigger seeds compared to other genotypes.
Table 2. The size of seed of several genotype in two seasons
Size of seed (g/100)
Genotype

Wet season

KH8
KH9
KH31
KH38
KH55
KH71
Anjasmoro
Wilis
Tanggamus
SlametDry season

21.76 e22.88 e21.71 e22.82 e19.00 bc
20.74 de18.50 c14.42 ab12.60 a15.42 bAverage

16.80 bcd
15.50 b19.50 e18.46 cde
16.03 b18.70 de16.60 bc
10.83 a10.76 a12.30 a

19.28
19.19
20.61
20.64
17.52
19.72
17.55
12.63
11.68
13.86

Clustering of genotype
In wet season, except KH31 line, other lines have higher seed productivity and seed size
compared to Anjasmoro variety. In dry season, KH8, Kh31 and KH38 lines have higher productivity
and seed size compared to Anjasmoro variety (Figure 1). This result indicated that KH8 and KH38
lines are consistently better than Anjasmoro variety.
Dry season (M2)

Seed productivity per plant (g)Seed productivity per plant (g)Wet season (M1)

Seed size (g/100 seeds)

Seed size (g/100 seeds)

Figure 1. Clustering genotype of soybean based on seed productivity and seed size in wet and dry seasons.

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Analysis of stability of seed productivity in two seasons showed that KH71 is the most stablegenotype in Majalengka. KH9, KH38 and KH55 lines were more stable than Anjasmoro variety in
wet and dry seasons than Anjasmoro Variety (Figure 2). If we consider to the seed productivity, thesize of seeds and the stability of seed productivity, KH38 and KH71 are a potential lines to be
developed to become elite national varieties.
BIPLOT AMMI I
M1

KU1 of Seed productivity
Principle component

3
SLAMET

2
TANGGAMU
WILIS

1

KH55
KH71

0

KH9
KH38

KH8
ANJASMOR

-1

KH31

-2
M2

-3
16

18

20

22

24

26

28

30

32

rata-rata (g)Average of seed productivity

Figure 2. Stability of seed productivity in wet and dry season of soybean in Majalengka. M1= wet season, M2=
dry season.

Acknowledgement
This research was financially supported by I-MHERE B2c IPB entitled: ―Test of adaptability of several
elite lines of soybean in several locations in the frame of the creation of new elite cultivars‖, contract No:
13/13.24.4/SPP/I-MHERE/2010 on behalf of Suharsono.

References
Calvino PA, Sadras VO. 1999. Interannual variation in soybean yield: interaction among rainfall, soil depth andcrop management. Field Crops Research 63: 237-246.
[Deptan] Departemen Pertanian. 2011. Daftar varietas unggul kedelai, http://pangan.litbang.deptan.go.id/index.
php?bawaan=varietas/gerbang&proses=daftarstok&komoditas=05025&varietas=1 [05 Juli 2011].
Jambormias E, Sutjahjo SH, Jusuf M, Suharsono. 2011. Using information from relatives and path analysis toselect for yield and seed size in soybean (Glycine max L. Merrill). Sabrao J. Breeding Genet 43 (1): 4458
Suharsono, Jusuf M, Paserang AP. 2006. Analisis ragam, heritabilitas, dan pendugaan kemajuan seleksi
populasi F2 dari persilangan kedelai kultivar Slamet x Nokonsawon. J Tanaman Tropika 9:86-94.
Suharsono, Jusuf M, Dasumiati. 2007. Analisis ragam, dan seleksi populasi F3 dari persilangan kedelai kultivar
Slamet x Nokonsawon. J Tanaman Tropika 10: 21-28.
Sunarto. 1995. Pemuliaan kedelai untuk toleransi terhadap tanah masam dan keracunan Aluminium. Jurnal
Tanaman Industri dan Pangan 12(2): 98-99.

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Paclobutrazol Application Effectiveness on Growth of Two Peanut(Arachis hypogaea L.) Varieties
Iskandar Lubisa,*, Aries Kusumawatib, Munif Ghulamahdic, Heni Purnamawatia, Yudiwanti
WE Kusumod, Ahmad Ghozi Mansyurie, Sri Astuti RaisfaLaboratory of Crop Production, Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural Universityb
Depatment of Agronomy, Faculty of Agriculture, Andalas UniversitycLaboratory of Ecophysiology, Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural UniversitydLaboratory of Plant Breeding, Department of Agronomy and Horticulture, Faculty of Agriculture,
Bogor Agricultural UniversityeLegume and Tuber Crop Research Center, Indonesian Ministry of Agriculturef
Gen Resource and Biotechnology Research and Development Center, Indonesian Ministry of Agriculture*Corresponding author: Lab of Crop Production, Department of Agronomy and Horticulture,
Faculty of Agriculture, Bogor Agricultural University.
Jalan Meranti 1, Kampus IPB Dramaga, Bogor 16680. Indonesia
Tel/Fax: +62-251-8629353.
iskandarlbs@yahoo.com

Abstract
Peanuts productivities in Indonesia during 1986–2003 ranged 0.7 to 1.2 ton/ha dry seeds, although some newvarieties having potential yield from 2.0 to 2.5 ton/ha or more, farmers‘ productivity reached only 50–60% of theyield. This research was conducted to investigate the effectiveness of paclobutrazol on the growth andproduction of peanuts. Experiment was conducted at Bogor Agricultural University farms at Cikabayan. Planting
was conducted in April to July 2009, using 2 peanut Varieties, namely Sima and Kelinci. Design of experiment
was Split-split plot. Paclobutrazol was applied at 6 and 8 Weeks after Planting, WAP as main plot, Varieties(Sima and Kelinci) was used as sub plot and concentrations of Paclobutrazol (0, 100 and 200 ppm) was used as2
sub-sub plot with three replicates. Size of experimental unit was (3 x 5) m , with planting density of 125 000
plants/ha. Urea, SP36 and KCl were applied at planting date at 33.75 kg N, 36 kg P 2O5 and 45 kg K2O per ha,
respectively. Observation covered growth and production, such as dry matter production, leaf area index, pod
number, pod weight, non-structural carbohydrate and total nitrogen in the stem. The results showed thatpaclobutrazol concentration at 200 ppm increased pod weight and productivity of peanuts, however, did notaffect the total soluble carbohydrate and nitrogen content in the stem at 10 WAP. Sima had vigorous growththan that of Kelinci variety showing bigger leaf area and higher chlorophyll.
Keywords : Peanut, paclobutrazol, growth

Introduction
Peanuts productivities in Indonesia during 1986–2003 ranged from 0.7 to 1.2 ton/ha of dry
seeds (Kasno, 2004), although some new varieties having potential yield from 2.0 to 2.5 ton/ha or
more have been released, farmers‘ productivity reached only 50–60% of the potential yield.
Productivity is related with empty and or imperfect pod filling. Bell and Right (1998) found that
peanut population in Indonesia is relatively high, however, many of pod produced empty or partly
filled. Lukitas (2005) and Khasanah and Purnamawati (2007) reported that only 26-68% (in average46%) of gynophores became filled pod. Physiological modification of plant growth by controlling thevegetative growth is one of method overcomes the problem. Growth regulator is used in order tomaintain vegetative and reproductive growth balance. It can be suppressed the competition
vegetative and generative growth which may decrease assimilate distributed to the sink (CruzAguado et al., 1999; Arzani and Roosta, 2004).

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225

Peanut is a semi determinate crop has vegetative growth continuously even if reach theflowering stage. In order to maximize assimilate for filling the pod, it is necessary to investigate
source size modification and sink-source balance in peanut. Therefore, retardant as a growth
regulator can be used for regulating plant growth balance. Banon et al. (2002) reported that one of
growth retardant widely used is paclobutrazol. Moreover, Seeno and Isoda (2003) reported thatfoliar application of paclobutrazol at 100 and 200 ppm in the stage of initial pod formation and initialand medium pod filling increased seed production of peanuts.
This research aims were to investigate the effect of time application of paclobutrazol on thegrowth of peanuts; to optimize the paclobutrazol concentration to growth and production of peanuts;
and to investigate the growth pattern and production of peanuts.

Materials and Methods
Experiment was conducted at Bogor Agricultural University farm at Cikabayan. Planting
was conducted for four months from April to July, 2009. Analysis was done at laboratory of RGCI
(Research Group of Crop Improvement), Department of Agronomy and Horticulture, Faculty ofAgriculture, Bogor Agricultural University. Materials for experiment were peanut seed of Kelinci andSima varieties, Urea, SP36 and KCl as fertilizers, KCl was applied at planting date at 33.75 kg; N at
36 kg; P2O5 of 45 kg K2O per ha, respectively. Paclobutrazol was used as retardant.
Design of experiment was Split-split plot design where Time of Paclobutrazol applications(at 6 and 8 Weeks after Planting, WAP) was used as main plot, Varieties (Sima dan Kelinci) wasused as sub plot and Dosage of Paclobutrazols (0; 100 and 200 ppm) was as sub-sub plot withthree replicates, Size of experimental unit was (3 x 5) m2, with planting density of 125 000 plants/ha.
Data were analyzed by F test at 5% level, and advanced test used Duncan Multiple Range Test
(DMRT) in 5% level.
Observation included physiological character, growth and production. Growth component
covered total dry matter, stem and leaf weight and leaf area index (LAI). Production component
covered total number of pod, pod weight and productivity, while physiological character covered
chlorophyll, total non-structural carbohydrate (TNC) and stem nitrogen content.

Results and Discussion
The results showed that the single treatment affected the growth of peanut. There was nointeraction among time of application, concentration of paclobutrazol and variety. Time of
application had significant effect on 8 WAP of peanut. Effect of variety was significant for plantheight, total dry matter, culm and leaf dry weight, number of gynophores and pod, 100 seed weightand harvesting index, respectively. Paclobutrazol application had significant effect on plant height,
stem dry weight, leaf number, pod number per plant, pod weight per plant, seed weight per plant,
seed number per plant and plant productivity, respectively.

Leaf Area Index (LAI)
Statistical analysis showed that at 8, 10 and 12 WAP, LAI was significantly affected byvariety. At 12 WAP, Sima had LAI of 4.48 which was significantly higher than that of Kelinci (3.55)(Figure 1). Slow initial growth in peanuts was performed by slow LAI development. Then LAI
reached value of 5.5 and 7.0, respectively. Although LAI was higher, assimilate supply still restrictedseed yield, this could be to its used for maintaining higher LAI and compensating dead leaf. Then
growth of leaf may compete with the development of the sink organ (Gardner et al., 1991).

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Figure 1. Leaf Area Index of Peanut varieties. Number followed by similar letter is not significant in 5% level.

Pod Number per Plant
Statistical analysis showed that time application of paclobutrazol and varieties had no
significant effect on pod number per plant. There was only the concentration of paclobutrazol having
significant effect on the pod number per plant. The highest number of pod represented by 200 ppm
paclobutrazol reaching 14 pods per plant, while 0 and 100 ppm produced similar number (12) ofpods per plant, respectively, as shown in Figure 2.

Figure 2. Total number of pod per plant after paclobutrazol treatment.

Productivity
Peanut productivity in this experiment represented by pod dry weight per ha. Statisticalanalysis showed that paclobutrazol concentration significantly affected peanut productivity. The
highest productivity performed by 200 ppm paclobutrazol concentration which was 1.18 ton per ha,
while concentration 0 and 100 ppm had no significant differences in productivity (Figure 3).

Figure. 3 Productivity after paclobutrazol treatment.
thProceedings of The 7 ACSA Conference 2011227

Chlorophyll content
Time of application and paclobutrazol concentrations did not have significant effect on thechlorophyll content. While variety significantly affected the chlorophyll content, and Sima has higher
chlorophyll content than Kelinci (Figure 4). Yudiwanti (2007) stated that peanut which has darkgreen color has higher chlorophyll content and has high yield potential and tolerant to bacterial leaf
blight. It is also supported by Taiz and Zeiger (2002) stated that highest content of chlorophyll
visually performed by greenness of leaf, and dark green leaf will more efficient to catch solar
radiation for photosynthesis.

Figure 4. Chlorophyl content of peanut varietyof Sima and Kelinci.

Total Soluble Carbohydrate and Nitrogen Contents
There was no significant effect of paclobutrazol concentration on the content of totalsoluble carbohydrate (TNC) and Nitrogen content in the stem at 10 WAP, however, the content of
TNC in 200ppm was absolutely higher than that of 0 or 100 ppm of paclobutrazol, and in the
opposite nitrogen content in 200 ppm was absolutely lower than that at 0 and 100 ppm
paclobutrazol, respectively (Figures 5a and 5b).
The effort for increasing potential yield of peanuts is important role in order tobreakthrough barrier from physiological factors. The first step is to assess growth of yield organ, is it
limited by availability of source or by sink capacity. Recently, genotype differences in allocation andmobilization pattern of assimilate in the stem of wheat has been sown (Cruz-Aguado et.al. 1999).

(a)

(b)

Figure 5. Soluble carbohydrate (a) and Nitrogen (b).content in the stem at 10 WAP as affected by paclobutrazol
concentration.

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Proceedings of The 7 ACSA Conference 2011

Conclusion
Paclobutrazol concentration at 200 ppm increased pod weight and productivity of peanuts,
however, it had no significant effect on total soluble carbohydrate and nitrogen content in the stemat 10 WAP. Sima had vigorous growth than that of Kelinci variety showed by bigger leaf area andhigher chlorophyll content in the leaf.

References
Arzani, K., H.R. Roosta. 2004. Effect of Paclobutrazol on Vegetatif and Reproductive Growth and Leaf Mineral
Content of Mature Apricot (Prunus armeiaca L.) Trees. J.Agri.Sci.Technol. 6:43-55.
Banon, S., A. Gonzales, E.A. Cano, J.A. Franco, J.A Fernandes. 2002. Growth, Developmentand and Colour
Response of Potted (Dianthus caryophyllus cv. Mondriaan to Paclobutrazol Treatment. Scientia
Horticulturae 94: 371 – 377.
Bell, M.J., G.C. Wright. 1998. Groundnut Growth and development in Contrasting Environment. 1. Growth andPlant Density Responses. Ekperimental Agriculture 34 : 99 – 112.
Cruz-Aguado, J.A., F. Reyes, R. Rodes, I. Perez, M. Dorado. 1999. Effect of Source-to-Sink Ratio on
14
Partitioning of Dry Matter and C-photoassimilates in Wheat during Grain Filling. Annals of Botany 83:
655 – 665.
Gardner, F.P., R.B. Pearce, R.L. Mitchell. 1991. Fisiologi Tanaman Budidaya. UI Press. Jakarta. 428 hal.
Khasanah, U., H. Purnamawati. 2007. Pertumbuhan dan Produktifitas 5 Varietas Kacang Tanah. [Makalah
Seminar Skripsi]. Departemen Agronomi dan Hortikultura Fakultas Pertanian, Institut Pertanian Bogor. 7
hal.
Kasno, A. 2005. Profil dan Perkembangan Teknik Produksi Kacang Tanah di Indonesia. Seminar Rutin
Puslitbang Tanaman Pangan. Bogor. 15 hal.
Lukitas, W. 2005. Uji Daya Hasil Lima Kultivar Kacang Tanah. Skripsi. Departemen Budidaya Pertanian
Fakultas Pertanian IPB. 39 hal.
Seeno, S., A. Isoda. 2003. Effect Paclobutrazol on Dry Matter Distirbution and Yield Peanut. Plant Production
Science Vol. 6. P90-94.
rdTaiz, Zeiger. 2002. Plant Physiology (3 edition). Sinauer Associates, Inc. Massachutes, USA.
Yudiwanti. 2007. Galur Kacang Tanah Berdaun Hijau Tua: Keunggulan dan Pengendalian Genetiknya.
Prosiding Seminar Nasional Hasil Penelitian yang dibiayai oleh Hibah Kompetitif; Bogor, 1 – 2 Agustus
2007. Hlm 143 – 146.

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Effect of Source or Sink Restriction on Flowering, Podding, and Yield
Performance in Field-Grown Adzuki Bean
Shinya Kasajimaa,*, Hirotake Itoha, Yasuo Nakamarua, Hozumi Yoshidaa, Hitoshi Satoba

Faculty of Bioindustry, Tokyo University of Agriculture, Hokkaido, Japan
Hokkaido Research Organization Tokachi Agricultural Experiment Station, Hokkaido, Japan* Corresponding author: Faculty of Bioindustry, Tokyo University of Agriculture, Yasaka 196, Abashiri,
Hokkaido 099-2493, Japan.
Tel.: +81 152 48 3823
s3kasaji@bioindustry.nodai.ac.jpbAbstract
The effect of source or sink restriction on flowering, podding, and yield performance in field-grown adzuki bean
cv. ‗Erimo-shozu‘ was investigated. Defoliation and defloration treatments were performed as source and sink
restriction during the flowering period. Defoliation treatment reduced the flower and pod numbers and shortened
the period of flowering and podding. Defloration increased the flower and pod numbers. Defoliation treatment
was shown to reduce the seed yield and yield components. The 100-grain weight in defoliation treatment wassignificantly the lowest among all treatments. These results suggest that the seed yield of adzuki bean depends
on dry matter production during the flowering period. Furthermore, a compensatory effect in sink restriction
indicated by an increase in flower number was also noted.
Keywords: adzuki bean, defloration, defoliation, flowering, podding, yield component

Introduction
Adzuki bean (Vigna angularis (Willd.) Ohwi et Ohashi) is the second most important
leguminous crop in Japan after soybean (Glycine max (L.) Merr.). It is widely used for the
production of a sweet bean paste called Ann, which is a major component in traditional Japanese
sweets (Murata, 1999). It has been commercially produced in Hokkaido, Northern Japan, wherecrop breeding program is mainly carried out in that area (Shimada, 2006). In recent years,
improvement of yield stability is an important issue to be considered since the production efficiencyof adzuki bean grown in Hokkaido varies drastically each year because of higher rates of damage
due to cold weather (Shimada, 2006). However, up-to-date information with regard to the yielddetermining process of adzuki bean has been limited because of its minor role worldwide.
In adzuki bean, as in many other leguminous crops, knowing the habit of flowering andpodding is considered to be important for analyzing the yield-determining process. Compared to thesoybean, the adzuki bean has a long flowering period and a low podding efficiency (Takahashi,1958). In general, source-sink relationships play an important role in the yield-determining process.
For example, defoliation or defloration treatments of soybean plants accelerated the development offlower buds and podding (Saitoh et al., 2001). However, there is limited information about the effectof source or sink restriction on flowering, podding, and yield performance of adzuki bean. In order toevaluate the relative importance of source or sink on yield-determining process, we examined thechanges in flower and pod numbers and yield components in field-grown adzuki bean plants asaffected by defoliation or defloration treatments.

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Materials and Methods
The field experiment was conducted in the Tokyo University of Agriculture farm at Abashiri,
Hokkaido, Japan (43°53′44″N, 144°21′45″E). The soil type was andosol, and the preceding crop
was barley (Hordeum vulgare L.). Adzuki bean cv. ‗Erimo-shozu‘, which is a most famous leading
variety of adzuki bean in Hokkaido, was used in this study. Four seeds per hill were seeded on June2, 2010, at 16.6-cm spacing in approximately 3.5-m-long rows with 72-cm row spacing. The
seedlings were thinned to two seedlings per hill three weeks after seeding. Compound fertilizer(N:P2O5:K2O = 5:25:14%) at 80 g m−2 was applied as the basal application. Although the summer
air temperature in 2010 was higher than that in the average year, this experiment was performed
under normal climatic conditions.
Source or sink restriction treatment was initiated on July 26, after flowering began. Source
restriction treatment consisted of removing two lateral leaflets of every trifoliate leaf when flowering
began. Sink restriction treatment consisted of removing every flower on each branch. After initiation,
defoliation and defloration were continued until flowering ceased. Six experimental plots including acontrol were set up according to a randomized block design with two replications.
The change in flower and pod numbers was investigated for 5 plant samples in each plot.
The flowering date was recorded on a small label (9 mm in width and 22 mm in length) for eachplant, and then the label was wrapped around the pedicel of the flower. The labels were attached on
all flowers that had formed pods by the time of maturity. The podding rate was calculated from theflower and pod numbers during the entire flowering period based on the label data. At maturity, 20
standard plants from 10 hills in each plot were sampled except for the plant with attached labels.
After air-drying, the seed yield and yield components—namely, pod number, seed number per pod,and 100-seed weight—were examined.

Results and Discussion
The average temperatures during the vegetative and reproductive stages were 17.5°C and19.6°C, respectively. The growth and yield of adzuki bean are known to be susceptible to cold
weather damage (Shimada, 2006), e.g., the low temperature during vegetative growth induced
abortion of main stem elongation, and low temperature before flowering induced podding injury in aprevious study (Aoyama et al., 2009). In the present experiment, air temperature during every
growth phase tended to be higher than that in the average year. The main stem length and top dry
weight at maturity in control treatment condition were 83.4 cm and 640 g m−2, respectively. It was
confirmed that in the normal plant type, flowering and podding were not affected by low
temperatures.
The change in flower and pod numbers per plant differed among treatments (Fig. 1). In
control treatment, the cumulative flower and pod numbers per plant were 104.0 and 56.4,
respectively (Fig. 1A). In source restriction treatment, however, the flower and pod numbers werereduced and the period of flowering and podding was shortened compared to control (Fig. 1B). Data
indicated that dry matter production after flowering affects the flower and pod numbers.
Furthermore, this study clarified that the reduction in dry matter production also affects the floweringand podding periods. Conversely, the flower and pod numbers in sink restriction treatment werehigher than those in the control but the podding rate was lowest among all treatments (Fig. 1C).
These results suggested that sink restriction treatment was compensated by a drastic increase in
the flower number. In this study, sink restriction treatment consisted of removing all flowers from allbranches. The effects of source and sink manipulations on the flower numbers of soybean variedwith flower position between the main stem and branches (Saitoh et al., 2001). Thus, further studies
are required to clarify the source-sink relationship between the main stem and branches of adzuki
bean.
th

Proceedings of The 7 ACSA Conference 2011231

Table 1 showed the effect of source or sink restriction on the seed yield and yieldcomponents of adzuki bean. The seed yield tended to be lower in source restriction treatment thanin control and sink restriction treatments, although no significant difference was observed. Similartendencies were observed in yield components. The 100-seed weight in source restriction treatment
was significantly lower than the weights in other treatments. The decline in 100-seed weight bydefoliation suggested that the seed yield of adzuki bean might be limited by source size, which wasconsistent with previous reports on soybeans (Egli & Leggett, 1976; Saitoh et al., 2001).
Table 1. Effect of source or sink restriction on the seed yield and yield components of adzuki bean
-2-2

Treatment

Seed Yield (g m )Pod no. (no. m )Seed no. per Pod

Control
Source restriction
Sink restriction

358 a
305 a
338 a

487 a
438 a
463 a

5.52 a5.42 a5.48 a100-seed weight(g)
12.9 a12.1 b
13.0 aValues with different letters in a single column are significantly different at the 5% significance level by Tukey–Kramer‘s test.

Flower number

Pod number

5
Flower no. per plant : 104.0
Pod no. per plant : 56.4
Podding rate (% ) : 54.2

(A)
4

3
2

Number of flowers and pods per plant

1
0
7/25

7/30

8/4

8/9

8/14

8/19

8/24

8/29

5

(B)

Flower no. per plant : 73.2
Pod no. per plant : 41.8
Podding rate (% ) : 57.1

4
3

2
1
0
7/25

7/30

8/4

8/9

8/14

5

8/19

8/24

8/29

Flower no. per plant : 113.2
Pod no. per plant : 58.8
Podding rate (% ) : 51.9

(C)
4
3

2
1

29-Aug24-Aug

19-Aug

14-Aug

9-Aug

4-Aug

30-Jul

25-Jul0

Data shows 5-days moving average with 10 plants of 2 replicates. The flower number, pod number, and podding rate in eachfigure show the values of entire examination periods.

Figure 1.

232

The change in flower and pod numbers per plant in control (A), source restriction (B), and sink
restriction (C).

thProceedings of The 7 ACSA Conference 2011

In conclusion, this study suggested that the seed yield of adzuki bean depends on dry
matter production during the flowering period. A compensatory effect in sink restriction treatment
indicated by an increase in flower number was also observed. Moreover, defoliation treatment
shortened the flowering and podding periods. This result may be related to early maturity. In the
future, we plan to conduct additional experiments using early maturing varieties.

References
Aoyama S, H Shimada, N Hasegawa, K Murata, S Fujita and I Matsukawa. 2009. A new early maturing adzuki
bean variety ―Kita-roman‖ with cool-weather tolerance and soil-borne disease resistance. Bulletin of
Hokkaido Prefectural Agricultural Experiment Station 94: 1-16**.
Egli DB and JE Leggett. 1976. Rate of dry matter accumulation in soybean seeds with varying source-sink
relations. Agronomy J. 68: 371-374.
Murata K. 1999. Breeding of adzuki bean cultivar ―Erimo-shozu‖ and cultivars with large seeds or resistance tosoil-borne diseases in Hokkaido. Breed. Res. 1: 173-179*.
Saitoh K, S Isobe, Y Seguchi and T Kuroda. 2001. Effects of source and/or sink restriction on the number offlowers, yield and dry-matter production in field-grown soybean. Jpn. J. Crop Sci. 70: 365-372**.
Shimada H. 2006. Circumstances of adzuki bean production in Hokkaido and future direction of breeding adzuki
bean. J. Agr. Sci. 61: 11-15*.
Takahashi N. 1958. Ecological studies of fruiting behaviours of adzuki beans (Phaseolus angularis W. F. Wight).
Proc. Crop Sci. Soc. Jpn. 26: 43-44***.
*In Japanese.
** In Japanese with English summary.
*** In English with English abstract.

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233

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Analysis of Genes Expressed during the Early Maturation of Sesame Seeds
Motonobu Yoshida, Akihiro Kishikawa, Takayuki Nakano, Eiji Tanesaka
Lab of Plant Breeding and Agrobiotechnology, Department of Agricultural Science
Kinki University
Nakamachi 3327-204, Nara 631-8505, Japan
Tel: 81-742 43 5245, Fax: 81-742 43 1155
yoshida_m@nara.kindai.ac.jp

Abstract
Sesame seeds of cultivated species, Sesamum indicum, contain abundant oil, in particular, large amounts of
unsaturated fatty acid oleic and linoleic acids. Total amounts of oleic and linoleic acids in sesame seeds keep
constant among varieties of S. indicum. However, the regulation of the mechanism is unknown. Therefore, thisstudy attempted to clarify these points through transformation of Arabidopsis thaliana with novel genes insesame seeds. Oil contents increase rapidly in sesame seeds within 4 weeks after flowering. A full-length cDNA
library prepared from sesame seed of 1 to 3 weeks oldwas substracted with cDNAs from plantlets of 4 weeks
after germination. The results showed that the cDNA library was expressed specifically during the earlymaturation.The 1,545 cDNA clones were sequenced. Of these, 30.3% of the clones were responsible for protein
destination, 16.7% of the clones for the metabolism, and 6.9% of the clones for the cell growth and division.
Among them, 13 genes for a transcription factor were identified, four were identified as a transcription factor
involved in ethylene signaling. In addition, nine genes: osmotin-like protein, expansin-like protein, aquaporin-likeprotein, MADS-box protein 4, novel putative uncharacterized protein, ethylene response factor 2, ethylene
responsive element binding protein 3, AP2 containing transcription factor and lipid transfer protein wereanalyzed by overexpression of A. thaliana. The A. thaliana overexpression strain for novel putative
uncharacterized protein and aquaporin-like protein, respectively showed the increase of unsaturated fatty acids.
The amount of oleic acid per seed grain in a strain for novel protein was 1.7 fold, and that of linoleic acid 1.5
fold, respectively.
Keywords: Sesamum indicum, fatty acids, full-length cDNA, Arabidopsis transformation

Introduction
Sesame seeds of cultivated species, Sesamum indicum, contain abundant oil, in particular,large amounts of unsaturated fatty acid oleic and linoleic acids. These unsaturated fatty acids lower
the cholesterol level in the body (Satchithanandam et al., 1993). Because of their effects andantioxidant substances, attention has been paid to sesame seeds as health food (Budowski andMarkley, 1951). Total amounts of oleic and linoleic acids in sesame seeds are constant among
varieties of S. indicum. However, the regulation of the mechanism is unknown. It is necessary to
clarify these points to breed novel varieties with high content of unsaturated fatty acids. Oil contentsof sesame seeds increase rapidly within 4 weeks day after flowering (DAF). Therefore, this study
attempted to analyze the function of novel genes expressed specifically during the early maturationof sesame seeds through transformation of Arabidopsis thaliana with novel genes of sesame seeds.

Materials and Methods
Plant materials and growth conditions
Sesamum indicum seeds were obtained from Toyama University. Arabidopsis thaliana
plants were grown under continuous light at 22-23oC and used for transformation.

thProceedings of The 7 ACSA Conference 2011235

Preparation of a full-lengh cDNA library and cDNA materials
Total RNAs were prepared from S. indicum seeds of 1 to 3 weeks old. A full-length cDNA
library was prepared using pCMVFL3 vector as described previously (Uenishi et al., 2004). Total
RNAs from young plants were extracted four weeks after germination. After purification of poly(A)+RNA, cDNA materials were synthesized with cDNA synthesis kit M-MLV version (TaKaRa). A
full-lengh cDNA library expressed specifically during the early maturation was prepared bysubtraction of the following methods. A full-lengh cDNA library from S. indicum seeds of 1 to 3
weeks old was amplified with SP6 primer, T7 primer and the PCR programme which was 16 cyclesof 98oC for 10 sec, 56oC for 5 sec and 72oC for 3 min, followed by 72oC for 3 min. They were used
as a tester cDNA in subtraction. The subtraction was carried out with DsDD cDNA subtraction kit(Wako) using amplified cDNAs from a full-length cDNA library as a tester cDNA and cDNA materials
from young plants of four weeks after germination as a driver cDNA. Subtracted cDNA clones were
amplified with the PCR programme which was 35 cycles of 98oC for 10 sec, 56oC for 5 sec and72oC for 3 min, followed by 72oC for 7 min. After removing cDNA clones of the lower molecularweight with Chroma spinTM-200 (Clontech), they were selected in size with Chroma spinTM-400, andpooled into four groups (fraction No. 4, fractions No. 5-7, fractions No. 8-14, fractions No. 15-30).
Each pooled cDNA clone was amplified with EX-taq polymerase (TaKaRa), SP6 primer, T7 primer
and the PCR programme which was 25 cycles of 98oC for 20 sec, 50oC for 30 sec and 72oC for 3
min, followed by 72oC for 5 min. They were ligated into pCR8/GW/TOPO vector (Invitrogen).
Sequence of cDNA clone
The cDNA clones from three groups (fraction No. 1-4, fraction No. 5-7, fraction No. 8-14)
were sequenced with ABI Prism® 3100-Avant Genetic Analyzer (Applied Biosystems) using BigDye®
terminator v3.1 cycle sequencing kit (Applied Biosystems). Homology analysis was carried out with
BLASTX (DDBJ; http://www.ddbj.nig.ac.jp/welcome-j. html ).
Transformation of Arabidopsis thaliana
The cDNA clones ligated into pCR8/GW/TOPO vector were transferred into pBI-OX-GW(Inplanta Innovations Inc.) by using Gateway System (Invitrogen). Each cDNA clone transferred into
pBI-OX-GW was used for the transformation of Arabidopsis thaliana which was done as describedin a previous paper (Clough and Bent, 1998), with minor modification.

Results and Discussion
To obtain cDNA clones expressed specifically during the early maturation of S. indicum
seeds, the subtraction was carried out using amplified cDNAs from a full-length cDNA library from
S. indicum seeds of 1 to 3 weeks old as a tester cDNA and cDNA materials from young plants offour weeks after germination as a driver cDNA. The subtraction efficiency was confirmed with the
PCR programme which was 22 cycles of 98oC for 20 sec, 50oC for 30 sec and 72oC for 1 min,followed by 72oC for 3 min. The primers were GAPC-F primer (5‘-CCAACGCTAGCTGCACCAC-3‘)
and GAPC-R primer (5‘-AGGTCAACAACTGAGACATC-3‘) of the gene for cytosolic glyceraldehyde3- phosphate dehydrogenase (gapC) which is one of house keeping genes (Figure 1). Signals wereobserved in both a full-length cDNA library from S. indicum seeds of 1 to 3 weeks old and cDNA
materials from young plants of four weeks after germination, while no signals were observed in thesubtracted cDNA library, suggesting that subtraction was efficiently performed.

236

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Proceedings of The 7 ACSA Conference 2011

bp

M

1

2

3
M.

λ / Eco T14 digest

Lane 1. cDNA library from immature seedsof 1-3 weeks after flowering
Lane 2. cDNAs from young plantsof 4 weeks after germination

421
GAPC

Lane 3. Subtracted cDNA

GAPC : Cytosolic glyceraldehyde-3- phosphate dehydrogenase

Figure 1. Confirmation of subtraction efficiency.

The subtracted cDNA library was amplified and separated in size into four groups (fraction
No. 4, fractions No. 5-7, fractions No. 8-14, fractions No. 15-30). Fraction No. 15-30 mainly contains
cDNA clones of less than 400 bp. Therefore, 1,545 cDNA clones were sequenced from fractions
No. 4-14. Properties of cDNA clones in fraction No. 4, fractions No. 5-7 and fractions No. 8-14 weresummarized as the following; the average size 1,043.7 bp in fraction 4; 996.7 bp in fractions 5-7;
706.4 bp in fractions 8-14. The 1,545 cDNA clones were categorized and summarized withclassification of each function (Table 1). The cDNA clones responsible for protein destination
formed the largest percentage of 30.52%. Next, the cDNA clones responsible for metabolism
formed the second largest percentage of 16.36%, the cDNA clones for cell growth, division were
7.01%, the cDNA clones for transcription and RNA processing were 4.48%. Unknown and novel
cDNA clones were 30.39%. Comparison of our results with results by Suh et al. (2003) showed that
subtraction was effectively performed from the fact that the occupation percentage of house
keeping genes was low, and that of unknown novel genes was high at the present experiment. Of
them, functional analysis of nine cDNA clones: osmotin-like protein, expansin-like protein,aquaporin-like protein, MADS-box protein 4, novel putative uncharacterized protein, ethylene
response factor 2, ethylene responsive element binding protein 3, AP2 containing transcription
factor and lipid transfer protein was carried out by overexpression of A. thaliana. Southern blot
analysis in transformed A. thaliana using each DIG-labeled sesame gene as a probe showed thateach sesame gene was indeed introduced into transformed A. thaliana (data not shown). The A.
thaliana overexpression strain for novel putative uncharacterized protein and aquaporin-like protein
showed the increase of unsaturated fatty acids in A. thaliana seeds. The amount of oleic acid perseed grain in the overexpression strain for novel putative uncharacterized protein was 1.7 fold, andthat of linoleic acid 1.5 fold. In a strain for aquaporin-like protein, the amount of oleic acid was 1.4
fold, and that of linoleic acid 1.5 fold.

thProceedings of The 7 ACSA Conference 2011237

Tabel 1. Functional category of identified genes
Function%Protein destination
Metabolism
Cell growth, division
Transcription and RNA processing
Cell resue, defense, senescence and deathTransport
Signal transduction
Energy generation
Protein synthesis
Intracellular trafficking
Cellular organization/biogenesis
unknown

30.52
16.36
7.01
4.48
4.16
2.27
2.14
1.10
1.04
0.32
0.19
30.39

References
Budowski P. and K.S. Markley. 1951. The chemical and physiological properties of sesame oil. Chem. Rev. 48:
125-151.
Clough S.J. and A.F. Bent. 1998. Floral dip: a simplified method for Agrobacterium- mediated transformation of
Arabidopsis thaliana. Plant J. 16: 735-743.
Suh M.C., M.J. Kim, C-G Hur, J.M. Bae, Y.I. Park, C-H. Chung, C-W. Kang and J.B.
Ohlrogge. 2003. Comparative analysis of expressed sequence tags from
Sesamum indicum and Arabidopsis thaliana developing seeds. Plant Mol. Biol. 52: 1107-1123.
Satchithanandam S., M. Reicks, R.J. Calvert, M.M. Cassidy and D. Kritchevsky. 1993. Coconut oil and sesame
oil affect lymphatic absorption of cholesterol and fatty acids in rats. J. Nutr. 123: 1852-1858.
Uenishi H., T. Eguchi, K. Suzuki, T. Sawazaki, D. Toki, H. Sinkai, N. Okumura, N. Hamashima and T. Awata.
2004. PEDE (Pig EST Data Explorer): construction of a database for ESTs derived from porcine fulllength cDNA libraries. Nucleic Acids Res. 32: 484-488.

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Proceedings of The 7 ACSA Conference 2011

Effects of Different Seeding Dates on Pattern of Internode Length inSorghum Variety „Kazetachi‟
Akihiro Fujiia*, Satoshi Nakamurab, Mitsuo Saitob ,Yusuke Gotoaab

Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Miyagi, Japan* Corresponding author: Graduate School of Agricultural Science, Tohoku University,
1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, Miyagi, 981-8555, Japan
Tel.: +81-22-717-8639
b0am1131@s.tohoku.ac.jp

Abstract
Sorghum (Sorghum bicolor Moench) stems, which have many internodes, are the main organs harvested forfeed and biofuel materials. Sorghum reportedly has three types, characterized by individual internode length
based on the internode position from base to top: 1) ever-increasing type, with internode length increasing fromthe base to the top internode; 2) unimodal type, with internode length in approximately the middle internode
position that is longer than in lower or upper positions, except at the neck internode of the panicle; 3) bimodal
type, for which the internode lengths alternately increases and decreases twice before reaching the peduncle.
Sorghum variety ‗Kazetachi‘, an ultralate-maturity variety in Japan with strong lodging tolerance, showedpatterns with many peaks. It is a multimodal pattern in internode length. Furthermore, this variety shows large
variation in the number of peaks per plant and the internode position with a peak among plants. This report
describes internode length pattern analysis of ‗Kazetachi‘ and the assessment of seeding date effects on thepattern. Seeds were sown on 20 May, and 3 and 18 June in 2009 in the northern part of Japan. Internode
lengths were measured at harvest in early November. Peaks in the internode length pattern of each plant were
4–7, including both distinct and vague peaks. Pattern analysis using the internode position with a distinct peak
contributed to the distinction among patterns on each seeding date. Although internode positions showing a
distinct peak differed among patterns of different seeding dates, the period during which the internode with
distinct peaks elongated rapidly appeared to be almost identical among them, suggesting that the internode
length in ‗Kazetachi‘ is characterized more by environmental factors than by genetic factors.
Keywords: internode length, multimodal pattern, pattern of internode length, Sorghum bicolor Moench

Introduction
Sorghum (Sorghum bicolor Moench) stems, consisting of many internodes, are the mainorgan harvested for feed and making syrup. Sweet sorghum has attracted attention recently as abiomass energy crop because its stems accumulate large amounts of sugars, which are convertible
into ethanol: bioethanol. However, little information about development of cultivation techniques toincrease stem yield in sweet sorghum exists because most research efforts have emphasized
increasing of grain yields for grain sorghum. Stem size of sweet sorghum is the important factor that
determines the stem yield including sugar yield (Tsuchihashi and Goto 2004; Sato et al., 2008;
Nakamura et al., 2009).Increasing sweet sorghum stem yields necessitates research into internodeformation in stems. Three types of sorghum can be characterized by individual internode length
based on the internode position from the base to top (Ayyangar et al., 1938). 1) For the everincreasing type, the internode length increases from the base to the top internode. 2) The unimodal
type has internode length in approximately the middle internode position that is longer than in lower
or upper positions, except at the neck internode of the panicle. 3) In the bimodal type, the internode
lengths increase gradually, then decrease and increase slightly, and decrease again, with one moreincrease before reaching the peduncle. However, ‗Kazetachi‘, ultralate-maturity variety, with strong
tolerance of lodging in Japan, shows new patterns with many peaks: a multimodal pattern ofthProceedings of The 7 ACSA Conference 2011239

Internode lengthinternode length. This variety also shows large variation in the number of peaks per plant and theinternode position, with the peak among plants (Figs. 1, 2) (Nakamura et al., 2011). This study
assesses a method to analyze the internode length pattern in ‗Kazetachi‘ and examines effects ofthe seeding date on the pattern.

ever-increasing type
unimodal typebimodal typemultimodal typeInternode position
Figure 1. Schematic depiction of three internode length patterns––the ever-increasing, unimodal, and bimodal
type––in sorghum, with the multimodal type shown for ‗Kazetachi‘.

Figure 2. Short internodes among upper internodes of a stem of sorghum variety‘ Kazetachi‘.

Materials and Methods
This study was conducted at the experimental field of the School of Food, Agricultural andEnvironmental Sciences, Miyagi University, Japan. Seeds were sown on 20 May (S1), 3 June (S2),and 18 June (S3) in 2009 with three replications. Seedlings were thinned to one per hill at 0.85 m
distance between the lines and to 15.0 cm between the plants. A basal fertilizer of 16–14–16 g NPK
per m-1 was applied. The internode length was measured for each of the 18 plants at harvest in the
beginning of November.
The (n)-th internode (IN n) was defined that between the (n+1)-th node and the (n)-th node
where the (n)-th leaf is attached. The internode position of the peak in the internode length pattern
was defined as the internode position that was longer than just upper and lower internodes.

Results and Discussion
The average of the total leaf number on the main stem was 30.7 ± 0.1 (S1), 29.8 ± 0.2(S2), 29.1 ± 0.2 (S3). Figure 3 shows the internode length of each internode position through IN8–
IN28 at each seeding date. The peaks per plant in the internode length patterns shown in S1, S2,
and S3 were 3–5, from 3–6, and from 3–5, respectively. The average numbers of peaks were 3.6 ±
0.2 (S1), 4.4 ± 0.2 (S2), and 3.8 ± 0.2 (S3) (Fig. 4). Distinct and vague peaks existin the internode

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Proceedings of The 7 ACSA Conference 2011

length patterns of all plants, but the average patterns had very similar shapes. Three distinct peaks
were observed in the average pattern, irrespective of the seeding date (Fig. 3). According to thisresult, these peaks were designated acropetally as peak I, peak II, and peak III. The respective
internode positions with peak I in S1, S2, and S3 were IN12–IN14 (13.1 avg.), IN14–IN17 (14.7
avg.), and IN11–IN13 (12.0 avg.) (Fig. 5 and Table 1). The respective internode positions with peak
II in S1, S2, and S3 were IN20–IN21 (20.4 avg.), IN18–IN21 (19.4 avg.) and IN16–IN20 (17.7 avg.).
The respective internode positions with peak III in S1, S2, and S3 were IN25–IN27 (26.1 avg.),
IN23–IN26 (24.8 avg.), and IN21–IN26 (23.4 avg.). These results show that the average internode
positions of peak II and peak III shifted lower with a later seeding date, excepting peak I.

15

a)

S1

II

I

III

10

5

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Internode length (cm)

15

b)

II

S2
I

III

10

5

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
15

II

c) S3

III

I

10

5

0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Internode position
Figure 3. Internode lengths through IN8–IN28 in S1 (a), S2 (b) and S3 (c).

thProceedings of The 7 ACSA Conference 2011241

Number of stem

12
10
8

S1
S2
S3

6
4
2
0
3

4
5
Number of peaks per stem

6

Figure 4. Number of internode length pattern peaks on each seeding date.
a)

S1

15
Ⅰ
Ⅱ
Ⅲ

10
5
0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30b)

Number of stem

15

S2

Ⅰ
Ⅱ
Ⅲ

10
5
0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
c)

S3

15
Ⅰ
Ⅱ
Ⅲ

10
5
0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Internode position
Figure 5. Internode positions with peak I – peak III in S1 (a), S2 (b), and S3 (c).

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Proceedings of The 7 ACSA Conference 2011

Table 1. Internode position and average internode position for peak I – peak III for S1, S2, and S3Range of internode position
plot

Average of internode position

I

II

III

I

II

III

S1

12 – 1420 – 2125 - 27

13.1+ 0.1

20.4+ 0.3

26.1+ 0.2

S2

14 – 17

18 – 2123 – 26

14.7+ 0.1

19.4+ 0.2

24.8+ 0.3

S3

11 - 1316 - 20

21 – 26

12.0+ 0.1

17.7+ 0.2

23.4+ 0.4

According to an investigation of the internodes as they increased rapidly in each plot on 5
September 2009, the positions of the internode in S1, S2 and S3 were estimated respectively as
IN20, IN19, and IN18. These internode positions correspond to internodes with peak II in each plot,
indicating that the internodes with peak II in S1, S2 and S3 were elongating at almost the same
period. These results suggest that the internode length in sorghum variety ‗Kazetachi‘ ischaracterized more strongly by environmental factors than by genetic factors.

References
Ayyangar, G.N.R., V.P. Pao and T.V. Reddy. 1938. Studies in sorghum internodes and leaf sheaths. Proc.
Indian Acad. Sci. 7: 161-176.
Nakamura, S., N. Nakajima, A. Sato, M. Saito, Y. Nitta and Y. Goto. 2009. Accumulation of dry matter and sugarin the stem of sweet sorghum (Sorghum bicolor Moench) after heading. Tohoku J. Crop Sci. 52: 43-44.
(in Japanese)
Nakamura, S., A. Fujii, K. Aoki, T. Tagaya, K. Hasegawa and Y. Goto. 2011. A method of analysis for themultimodal pattern in internode length of sorghum variety ‗Kazetachi‘. Jpn. J. Crop Sci. 80 (Extra issue1):
300-301. (in Japanese)
Sato, A., N. Nakajima, S. Nakamura, M. Saito, Y. Nitta and Y. Goto. 2008. Effect of fertilizer application on
characteristics of sweet sorghum (Sorghum bicolor Moench) internodes. Jpn. J. Crop Sci. 77 (Extraissue 2): 106-107. (in Japanese)
Tsuchihashi, N. and Y. Goto. 2004. Cultivation of sweet sorghum (Sorghum bicolor (L.) Moench) anddetermination of its harvest time to make use as the raw material for fermentation, practiced during rainyseason in dry land of Indonesia. Plant Prod. Sci. 7: 442-448.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011243

Pranajiwa as Raw Material for Bio-Oil
Endang Yuniastuti, Susanti Indriya Wati
Agricultural Faculty, Sebelas Maret University Surakarta, Indonesia
e.yuniastuti_ratu@yahoo.co.id

Abstract
The high increase in the number of human population every year causes the increase of human need forenergy. However, it unfortunately still does not meet the production of energy sources.. This situation forces us
to seek new sources of energies which are certainly eco-friendly, such as utilization of natural resources forbiofuels. One of altenative solution is the use of Pranajiwa plants as a source for production of bio-oil. Pranajiwa
or Pranajiwa (Sterculia foetida L.) is one species of plants in Indonesia originally from East Africa, Tropical Asia
and Australia. This kind of tree crops with a kind of tall trees between 30-40 m, the tree is often found in forest.
Pranajiwa seeds contain vegetable oils that can be used as an ingredient for industrial products such as
cosmetics, soaps, shampoos, fabric softeners, paints, plastics and as an adaptive agent biodiesel. Pranajiwa
seeds contain vegetable oils comprising a fatty acid that is sterculat acid. This plant has several advantages
over other alternative crops as sources for oil production. Pranajiwa plants is not used for consumption that isdfferent from oil palm, sugarcane, coconuts, yams or cassava that has a primary function for consumption.
Pranajiwa plants had fairly high oil yield that was greater than 40%. Event from a research conducted with thepurification using ether, the yield of oil could reach more than 70%. Whereas using direct processing, theaverage oil yield rearched 45%. For biofuels, Pranajiwa seeds can then be extracted and used as raw materialof bio-oil.
Keywords: Pranajiwa, bio-oil, alternative energy

Introduction
Increase in population leds to the increase of human need for energy. The energy currently
comes from fossil and no longer it will run out. This situation encourages the Indonesian nation to
immediately provide and build a populist-based alternative energy that is environmentally friendly byutilizing natural resources and existing plants as a source of energy that can be harnessed anddeveloped. One form of energy is Biofuel (BBN).
Some plants such as jatropha, sweet potatoes, cassava, sugarcane, oil palm, coconut,
sunflower, kapok, and pranajiwa or commonly known as a plant "Pranajiwa" can be developed as a
biofuel or biofuel farming and to support energy programs or green energy. , The crop plant"Pranajiwa" (Sterculia foetida Linn.) is considered more appropriate to be used as Biofuel (BBN) or
biofuels, especially as the bio-oil (lubricating oil) and bio-diesel (diesel substitute). This is supportedby the fatty acid content which can be also used as an adaptive biodiesel.
However, the existence of plants "Pranajiwa" is now rarely found, even the presence statusof plants "Pranajiwa" included a rare (Yuniastuti, 2008). This is partly because these plants havegrowing requirements at altitude less than 500 m above sea level, and exacerbated by logging andillegal use of timber for furniture materials or raw materials made of paper without rejuvenation. The
remaining plants are only in sacred places because people are afraid to cut them down. Cultivationof crops "Pranajiwa" is very little or not at all and eventually the plant is just a legacy from ancestors
that will be extinct. In this condition, it is necessary to give serious attention for "Pranajiwa" that has
a potential as a producer of alternative energy (bio-oil). Cultivation "Pranajiwa" can be either
generative or vegetative.
Pranajiwa (Sterculia foetida) is a kind of tree crops with high stature plants between 30-40
m, the tree is often found in the woods. Nomenclature is taken from the Roman myth, the name of
god or Sterquilinus Sterculius fertilizer. Together with the species name, foetida (meaning, hardsmelling, foul) scientific name refers to the unpleasant odor released by these trees, especiallyflowers. In some areas in Indonesia, this plant is also used as a drug. Plant leaves can be used asan herbal remedy laxative, diuretic and mosquito deterrent; bark as a cure of some diseases; tree
wood can be used as building construction; other than edible seeds can also be used as a coughmedicine (Yuniastuti, 2010). Pranajiwa oil benefits directly related to the fatty acid content sterculat
which is unsaturated fats in seeds (Anggraeni, 2005)

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Pranajiwa as an alternative energy-producing plant has several advantages over otheralternative energy crops, such as oil palm, sugarcane, coconuts, yams, and cassava. Pranajiwa is
not a crop for consumptions; while oil palm, sugarcane, coconuts, yams or cassava has the primaryfunction for consumption. Also compared to other alternative energy crops, Pranajiwa has high oilyield that is greater than 40%, even from research conducted by refining petroleum ether the yield
can reach more than 70%. While using direct processing (compound) average oil yield reached45%. As Fuel Plant (BBN), plant seeds and billowing retrieved and extracted can then be used as
raw material for lubricating oils (bio-oil) (Yuniastuti, 2010).

Materials and Method
Locations of research
Research locations were in 10 districts in Central Java, namely:
1. Karanganyar District: Karangmojo, Karangwuni, Derman, Ngaliyan1, Bibis, Kodokan,
Teak, Ngaliyan 2, Ngetal, Jetak
2. Sukoharjo District: Shower, Cangkol, Macanan, Temulus, Pondok 1, Pondok 2, Minggiran,Nderso, Palur, Triyagan.
3. Boyolali District: Tegalmuncar, Klabang, Kendal big, Tlatar, Jagoan, Tagung, Badan,
Gatak Malangan, Pojok, Bulu
4. Purworejo District: Kroyo, Trukan, Doplang, Sindurjan, Tegal Kuning, Pogung Juru
Tengah, Borowetan, Bedug, Paduroso, Bayan
5. Wonogiri District: Krisak, Bulak, Godean, Donorejo, Kaloran, Nambangan, Gerdu, Brajan,
Gebang Wetan, Mblimbing
6. Klaten District: Kurung, Kalikebo, Jetis, Talang, Jerukan 1, Jerukan 2, Kebon, Sabrang,
Pandanrejo, Planggu.
7. Sragen District: Plalar, Toyogo, Bero, Pengkol, Sambung Macan, Mojopahit, Pucang,
Drojo, Tunjung Semi, Demakan.
8. Grobogan District: Gendingan, Tambirejo, Nambuhan, Bakalan, Candisari, Kepayu,
Penawangan, Getasrejo, Kronggen, Rejosari.
9. Blora District: Brengus, Dlongos, Keboan, Gagakan, Pudak, Loworejo, Jambangan, Putat,
Sukorame, Sambiroto
10. Semarang District: Ngener, Mbatan Kidul, Tawang, Reksosari, Muncar Jaten, Mrakas,
Durenan, Sambiroto, Dadapayam
Analysis of Morphology, Cytology and Molecular conducted at the Central Laboratory of theSebelas Maret University, Laboratory of Plant Breeding, Physiology and Biotechnology Faculty ofAgriculture, Sebelas Maret University, Surakarta.
Plant materials
Plant materials used in this study were Pranajiwa or billowing plants (Sterculia foetida
Linn.) existing in the region of Central Java which covers 10 districts of Sragen, Boyolali, Klaten,
Wonogiri, Sukoharjo, Karanganyar, Semarang, Purworejo, Grobogan and Blora.
Activities of the experiment included:
1. Identification of Morphology
2. Cytology
3. Analysis of Chemical Components
4. Seeding and In Vitro Plant Propagation Pranajiwa (Sterculia foetida Linn)

Results and Discussion
In the habitus of the plants, grendruwo is a tree with height reaching 45 meters. Plant
leaves of "Pranajiwa" grow at the end of branches, is compound leaves, and palmatus shaped
leaves with 7-8 children (foliolum). unfoliolatus-shaped leaves, long strands of leaves (laminae)
between 10-17 cm with a smooth leaf surface. Flower crop "Pranajiwa" grow at the end of branches
and branching to form clumps, and yellow to purplish red. Flower is unisexual with diametersthProceedings of The 7 ACSA Conference 2011245

ranged between 2 – 2.5 cm. "Pranajiwa" Fruits are large enough with a length of about 10 cm, ovalshaped, smooth and woody rind. Seeds of black fruit have a length of between 1.5 to 2.0 cm.
"Pranajiwa" fruits are green when still young, then become red and brown when old and will fall toground. "Pranajiwa" fruits are also unique and strange that are comprised of five lumps (locus),
quite large, weighing 1-3 kg that further strengthens the community,s assumption that the fruit is afruit of "Pranajiwa" (devil fruit). The seeds of "Pranajiwa" fruits will fall and not be utilized optimallybecause many people are afraid to use it because of the above reason.
Ecologically, the "Pranajiwa" plants serve as a micro-habitat of animals and certain
animals. It is reported in Komodo Island the population of yellow-crested parrot (Cacatua subphurea
parvula) are protected and use ―Pranajiwa‖ trees as a nest. "Pranajiwa" plants have crowns androots that are fairly large and it can function as a regulator of the hydrological cycle because theroots can hold soil water in a large enough capacity. Internally the "Pranajiwa" plants are difficult tobreed through generative ways because its seeds have thick-though skin and dormancy (Sutopo,2002). However, in plant nurseries soaking in warm water (temperature + 45 °C) for 50-10 hours
could help to break seed dormancy (Yuniastuti, 2008). "Pranajiwa" seeds soften the harshexperience, and can germinate 3 days after planting. In addition to the generative seed,multiplication can also made through in vitro approaches. This is done as a form of plant germplasm
conservation of ―Pranajiwa‖. To overcome Pranajiwa from extinction, conservation efforts should bedone through both in-situ and ex situ approaches.
Morphological identification of ‖Pranajiwa‖ plants that were found in Central Java could beclassified into two types, namely large fruit and fruit respectively. Grouping of seeds was based on
the average weight of 10 seed parents between 1.5 to 2.0 grams (large seeds) and 1.0 to 1.5 grams(small seeds). From the observation, the flowers appeared on the last branch or subsidiary to the
4th to the 6th. Similarly, the leaves, which also appeared in young leaves of the branch to the 4th to
the 6th. It was found that the percentage of fruit-set was quite low where in a flower stalk that
contains 20-30 flowers is only 1-2 pieces standing. In some areas the observations that had beenpreviously done for such research in Navan this plant was not found, it relates to altitude of thelocation that is less than in accordance with the terms of Pranajiwa crops to grow. Pranajiwa growbest at altitudes of less than 500 m above sea level. Meanwhile, in some locations of Semarang
regency Pranajiwa also have not been found anymore because it was felled and no effort for the
rejuvenation of this plant has been done.
Observations in some locations in Blora and Grobogan District Pranajiwa plants have been
started to be used for reboitation by the District Government and Blora through the Grobogan
Department of Public Works (MPW) by planting plants on the curb and the edge of the irrigation
channels.
In May to July pranajiwa plants begin to form flowers and young fruits become old fruit,
which is ready for harvesting. In general, pranajiwa plants flowering and fruiting are throughout theyear. This situation allows pranajiwa plants to be intensively cultivated and used as biofuel
feedstock (bio-oil).
Observation of pranajiwa chromosome number indicated that the number of chromosomes
is 2n = pranajiwa 2X = 32. This number gives the assurance that the number of pra
najiwachromosomes is 32 not 40. The number of chromosomes is reinforced by images of cells ofdifferent pranajiwa (Setyawan, 2009) The previous opinion that pranajiwa chromosome number was
40 may be due to that the number of chromosomes of the genus Sterculia is 40 (six of nine speciesof the genus Sterculia species that have been identified and has a number of chromosome 40 is S.
acerifolia A. Cunn., S. A. St Chicha .- Hil., S. rubiginosa Vent., S. striata A. St .- Hil., S. urens Roxb.,
and S. villosa Roxb.). another reason is that the method used by previous researchers was lessprecise that might cause a low quality of the picture of chromosome (eg cell rupture that resulted in
the chromosomes of neighboring cells join counted that made the chromosome number increased).
Karyotype was based on chromosome length, and shape of chromosomes. Based on thesimilarity of the size and shape of chromosomes could be seen that pranajiwa chromosomes are
diploid. Based on the size and curve of the centromere is known that each pair of chromosomes
pranajiwa is two. This is supported by the similarity of the size and shape of a sorted chromosomesthat are homologues of each chromosome.
Standard chemical analysis consisting of analysis of terpenoid compounds, flavonoids, andsteroids showed a very low content even not exist at all. Further analysis for tannins, saponins andalkaloids also showed a very low content. This could facilitate the purification pranajiwa seed oil as

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a lubricating oil feedstock (bio-oil). Common proximate analysis consisted of water, fat content,
protein, carbohydrate, ash, and others.
To determine the amount of oil content (yield results) seeds was carried by two methods
that are compress (compound) and of purification with ether methods. On purification with ether, oilcontent (yield results) was obtained nearly 70%. While the direct compression method yield theresults was obtained about 40%. High oil yield from pranajiwa seeds indicate a huge potential forfurther development as a lubricating oil (bio-oil).

References
Anggraini, L. 2005. Karakteristik Produk Hidrogenasi Ester-Ester (Monoalkil) Asam-Asam Lemak Minyak
Pranajiwa ( Sterculia foetida Linn.) sebagai Adaptif http://abstraksi-ta.itb.ac.id./abstraksi-1&detail-1&id760& tahun-2005 Pada hari Senin, 20 Nopember 2006.
Setyawan, B. 2009. Analisis Sitogenetika Tanaman Pranajiwa ( Sterculia foetida L). Skipsi S1. Fakultas
Pertanian Universitas Sebelas Maret.Surakarta.
Sutopo, L. 2002. Teknologi Benih . Rajawali. Jakarta.
Yuniastuti. 2008. Identifikasi dan Seleksi Keragaman Tanaman Pranajiwa ( Sterculia foetida Linn.) Serta
Teknologi Perbanyakan Tanaman Secara In Vitro Untuk Penyediaan Bahan Baku Biofuel. Laporan
Penelitian Riset Insentif Dasar. Kementrian Ristek. Universitas Sebelas Maret. Surakarta.
-----------. 2010. Identifikasi dan Seleksi Keragaman Tanaman Pranajiwa (Sterculia foetida Linn).
http://lppm.uns.ac.id. Diakses pada Rabu, 15 September 2010. 2010.

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Selection and Evaluation Characteristics of Six Candidate Varities of
Cucumber (Cucumis sativus) in the Dry Season Planting
Rahayu, S.Ta,*, U.Sumpena a, Ali Asgar aaIndonesian Vegetables Research Institute (IVEGRI)* Corresponding author: Indonesian Vegetables Research Institute (IVEGRI)
Jl.Tangkuban Perahu no 517 Lembang, Bandung
Tel.: 0222786245; fax: 0222786416.
ayyuks@gmail.com

Abstract
Cucumber is one of vegetables that are rich in vitamin C. The purpose of this study was to select and toevaluate physical and chemical characteristics of six varieties of cucumber that will be released as new varieties.
Planting sited was chosen in plain medium during the dry season in Bandung. The design used was randomized
block design with three replicates. Observation was done by determining physical characteristics including
length, diameter, weight, texture, and colour of fruit. Chemical characteristics observed were moisture andcontent of vitamin C. The results showed that two varieties were not significantly different from thecharacteristics of the control, so both can be recommended to be released as new varieties.
Keywords: characteristics, cucumber, selection, varieties.

Introduction
Cucumber (Cucumis sativus) is one type of vegetable widely grown in Indonesia.
Cucumber has a fairly broad market both modern and traditional markets. Cucumber production inIndonesia in 2009 reached 583,139 tons per hectare with an area of 56,099 hectares planted.
Production mostly in West Java, Central Java and North Sumatra provinces (Anonymous, 2010).
Production is still potential for further improved.
Cucumber planted in low, medium, or high plains with optimal rainfall ranges between 200400 mm/month. Too high rainfall is not suitable for growing vegetables, as it will abort flowers(Sumpena, 2007). Cucumber grow well at the acidity of the soil ranged from 5.5 to 6.5, on alluvial,
latosol, and andosol soil type. Cucumber planted in the lowlands can be harvested faster thancucumbers grown in the highlands. Cucumber harvest age ranged from 30 to 50 days after planting(Arief, 2009). Cucumber is one of horticultural commodity easy to handle because it is grown withsimple maintenance, inexpensive, and short-lived cycle when it is compared to other vegetables
such as tomatoes or peppers. Fluctuations in market prices are low compared to other vegetables,
so it would be more profitable for farmers (Sumpena, 2007).
Cucumbers can be consumed directly in fresh as a salad and for pickles. The slicing
cucumber are peeled, sliced and served with vinegar or dressing or as an ingredient of salads. In
South-East Asia, the young shoots are eaten as raw or steamed (Siemonsma and Piluek, 1994).
Cucumber is very important in South East Asia as it is in temperate regions. Breeding work should
be aimed to produce improved cultivars resistence to diseases and pests attack in tropical lowland
condition (Siemonsma and Piluek, 1994).
The nutrient content per 100 grams of cucumber is water at 96 g, protein at 0.6 g, fat at 0.1
g, carbohydrates at 2.2 g, Calcium at 12 mg, Iron at 0.3 mg, vitamin C at 12 mg. Cucumber fruit
consists of 24 mg of P, 45 IU of Vitamin A, 0.03 mg of Vitamin B1, 0.02 mg of Vitamin B2, 0.3 mg of
Niacin, and 15 mg at magnesium, respectively (Siemonsma and Piluek, 1994). In addition,thProceedings of The 7 ACSA Conference 2011249

cucumber are also rich in fiber. The purpose of this study was to select and to evaluate physical andchemical characteristics of six varieties of cucumber that will be released as new varieties.

Materials and Methods
The research was conducted in plain medium during the dry season at Ciwastra, Bandung
400-700 m above sea level. Chemical analysis was performed at the laboratory of plant physiology
at IVEGRI in Lembang, Bandung. Materials used in this experiment were three candidates of newvarieties and three varieties of cucumber production of private firms as control.
Each treatment was planted in mulched beds at 9,6 m2 using a plant distance of 40 x 60
cm. The fertilizer applied was NPK at 125 kg/ha and goat manure at 20 ton/Ha. The remaining dose
given as a supplementary fertilizer twice during the plant growth. To control pests of plants (OPT)
pesticides were used intensively twice a week, starting at two weeks after planting. Pesticides were
adjusted to the type of pests that attacked.
Research was carried out by Randomized Design Group consisted of three replicates.
Observation physical parameters included length, diameter, weight, colour, and texture of the fruit.
Chemical parameters measured were moisture and vitamin C. Texture observation was done usinga QTS 25 Texture Analysis, and colour observations used a chroma meter. Statistical tests were
performed with the PKBTSTAT analysis followed by Tukey test at 5% level.

Results and Discussion
There are two types of cucumbers were observed in this study, i.e. green and white
cucumbers. Green cucumber fruit is longer than the local cucumbers. Table 1 shows that thevarieties of fruit length of variety H1 was not significantly different with the comparison varieties H5.
Varieties H2, H3, and H4 were not significant different.
Tabel 1. Analysis variance for length, diameter, weight,colour, texture of cucumber fruit, moisture, and vitamin Cof six virieties
Variety

Length(cm)

Diameter(cm)

Weight
(g)

H1

20.89

a

H2

16.53

H3
H4

3.37

b

121.47

a

0.89

b

0.12

ab

95.30

b

19.16

b

b

3.87

a

146.70

a

0.93

b

0.12

ab

95.68

a

17.87

c

14.43

bc

4.22

a

150.44

a

1.05

a

0.14

a

95.14

b

19.06

b

16.44

b

3.93

a

144.98

a

0.94

b

0.14

a

95.38

ab

17.28

c

H5

21.27

a

3.27

b

140.83

a

0.89

b

0.10

b

94.49

c

20.06

a

H6

12.66

c

3.83

a

145.63

a

1.05

a

0.12

ab

95.66

a

17.86

c

HSD 5%

2.19

0.40

71.08

Colour
(N/mm)

0.10

Texture(mm/10sec/50g)

Moisture
(%)

0.03

0.31

Vitamin C(mg/100g)

0.79

Table 1 also shows that the diameter of varieties H1 was not significantly different with H5,meanwhile among varieties H2, H3, and H4 were not significantly different. Diameter would affectthe weight of fruit. Type of fertilizer affect the production of cucumber (Sutater and Supriyadi, 1989).
Cucumber weight of six varieties were not significantly different at level 5% at Turkey test.
Cucumber weight affected the total production of cucumber. Factors planting and type of fertilizer
used affected plant growth and produced cucumber (Hilman and Rosliani, 2004). Irigation andcomposition of the N on the fertilizer also affected the weight of cucumber (Zhan et al., 2011).

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The colour of varieties H1, H2, H4, and H5 were not significantly different from each other,
they were more dominant to the colour of green. Variety H3 was not significantly different comparedwith variety H6, having the dominant colour of white. Cucumber colour ranges from dark green,green, light green, whitish green to white depending on the type varieties (Sumpena, 2007).
Texture of varieties H1, H2, and H6 were not significantly different among them. Similar
results were also found for varieties H3, H4, and H5. Higher texture value indicated softer texture,and conversely lower texture values indicated tougher texture. Texture value difference was also
influenced by varieties and fertilization. K elements made harder texture, while the elements of N
make the texture softer (Wiranatakusumah et al, 1992). Texture of consumer software was notpreferred. Storage temperature affected the texture of the cucumber. Storage at low temperature
will maintain the texture of the cucumber (Kohyam et al, 2009).
The highest moisture content was found in the variety H2, while the lowest was found atvariety H5. Variety H1 was not significantly different compared with variety H3. Moisture will affectthe shelf life of these products. The presence of water in food is a good medium for the growth of
microorganisms. The high moisture is held within the product by osmotic force within cells, mostly
as free water, althought a small proportion is chemically bound, therefore, this is more tightly held
and more stable (Will, et al., 1989). Variety H5 had the highest of Vitamin C content , while variety
H4 was the lowest. The vitamin C in vegetables is affected by the type of varieties and types ofseasons. In the rainy season Vitamin C content is higher level compared with in the dry season(Hanson et al, 2011).

Conclusions
Caracteristic cucumber was effected by the kind of varieties. Varieties H1 and H3 hadgood caracteristics to be released in public, because they had low moisture and high in the Vitamin
C content.

References
Anonymous, 2010. Statistik Indonesia. Biro Pusat Statistik. Jakarta.Pp.220-225
Appel LJ, Moore TJ, Obarzanek E .1997. A Clinical Trial of the Effects of Dietary Patterns on Blood Pressure.
DASH Collaborative Research Group. J Med.16:1117-1124.
Arief P.S., 2009. Agribisnis Mentimun. Penerbit CV.Pustaka Grafika, Bandung.Pp. 1-34
Hanson, P., et al, 2011. Carotenoids, ascorbic acid, minerals, and total glucosinolates in choysum (Brassica
rapa cvg. parachinensis) and kailaan (B. oleraceae Alboglabra group) as affected by variety and wet anddry season production. Journal of Food Composition and Analysis. Article in Press.
Zhan H-X, D-C Chi, Q Wang, J Fang, and X-Yu Fang.2011. Yield and Quality Response of Cucumber to
Irrigation and Nitrogen Fertilization Under Subsurface Drip Irrigation in Solar Greenhouse. Agricultural
Sciences in China. 10 (6) :921-930.
Hilmam, Y and Rosliani, R. 2004. Pemanfaatan cacing tanah untuk meningkatkan kualitas hara limbah organik
dan hasil tanaman mentimun. Jurnal Hortikultura 12(3): 148-157.
Kohyam ,K., A Nagata, Y Tamaki, and N Sakurai. 2009. Postharvest Biology and Technology. 53(2): 243-246
Siemonsma, J.S., and Piluek, K.1994. Plant Resources of South-East Asia 8 Vegetables. Prosea Foundation .
Bogor, Indonesia. 157-160
Sumpena, U 2007. Budidaya Mentimun Intensif dengan Mulsa, Secara Tumpang Gilir. Penebar Swadaya.
Jakarta.Pp 16-17
Sutater, T and Supriyadi, A., 1989. Pengaruh pemberian kapur pertanian, pupuk kandang dan P terhadap
produksi mentimun di tanah latosol masam. Bul.Penel.Hortikultura XVIII(3): 27-32.
Will, R.B.H., McGlasson, W.B., Graham, D., Lee,T.H., 1989. Postharvest. An Introduction to Physiology andHandling of Fruit and Vegetables. An Avi Book. Van Nonstrand Reinhold.New York. Pp 53-56.
Wiranatakusumah, M.A., Abdullah, K, Syarief, AM. 1992. Sifat Fisik Pangan. PAU IPB. Bogor. Pp 225 -232.

-- back to Table of Content -thProceedings of The 7 ACSA Conference 2011251

Blue Light Induced the Stem Growth in Vegetable Water Spinach
Futoshi Sasakia,*, Mikio Sekinumaa, Futoshi Katob and Naoto InouebaInterdisciplinary Graduate School of Science and Technology, Shinshu University, Minamiminowa, Japanb
Faculty of Agriculture, Shinshu University, Minamiminowa ,Japan.
Tel.: +81 265 77 1314.
10st504f@shinshu-u.ac.jp

Abstract
A local variety of water spinach in Taiwan was grown under various artificial light conditions to clarify the suitable
growth condition in a plant factory. In this experiment, water spinach shoots were grown under various artificial
light spectra controlling plant growth. Eight plots with conditions of 8 different light qualities were used tocompare the growth of the water spinach plant. Different light qualities were established by combining 3 types offluorescent light selected from 4 types of light emitted by white, blue, purplish-red, and far-red fluorescent lamps,
respectively, and irradiation treatments were performed for 10 days in each plot. The results showed that theplant length and stem diameter increased in the plot in which 1 white and 2 blue fluorescent lamps were set.
Increase in plant length and stem diameter were positively correlated with the energy of blue light (400-480 nm),but were negatively correlated with energy of green, red and far-red light (520-780 nm). The size of the stem
cavity negatively correlated to the amount of energy of red light (600-660nm) and positively correlated to theamount of energy of blue light (480 nm). The results suggest that blue light promotes and red light inhibits stem
growth of the water spinach.
Keywords: artificial light, light quality, plant length, stem diameter, water spinach

Introduction
Water spinach (Ipomoea aquatica Forsk.) is a popular vegetable in Japan because of its
good texture and high nutrient value. This vegetative has a high content of protein (2.2 g/100 g),
calcium (74 mg/100 g) and fiber (3.1 g/100 g) (JATCC, 2009). Moreover, many researchers haveevaluated the chemical characteristics of this functional food (Miean and Mohamed, 2001; Prasad
et al., 2005; Dasgupta and De, 2006). However, the duration of cultivation of this plant is limited tosummer because the frost resistance of this plant is low. For obtaining a stable yield throughout theyear, water spinach must be cultivated in an artificial environment that provides suitable conditions
for its growth. The species can grow easily in hydroponic cultures, and it shows a high nutrient
absorption. Therefore, the best growth conditions (light, temperature, nutrition) required for thisspecies must be investigated in detail to maximize the production and sales of this vegetable. In thisexperiment, the light spectra that controls stem growth were investigated to establish a year-roundculture under low energy input condition.

Materials and Methods
Plant materials
A local variety of water spinach from Taiwan was used in this indoor experiment. The
seeds were scarified and soaked in water for 3 days, and the germinated seeds were sown on
culture soil in 12 cm poly pot. After sowing, the seedlings were grown under the light of fluorescentlamps (FL40SBR-A, NEC Co. Ltd., Japan) for 7 days. After 7 days, we removed these fluorescentlamps and set other fluorescent lamps producing different light colors, therefore, these seedlings
were grown under different light quality conditions by using artificial light.

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Proceedings of The 7 ACSA Conference 2011

Energy density（μW cm-2 nm-1）

Treatments and analytical method
Four different fluorescent lamps (white, blue, purplish red and far red) were used indifferent combinations to provide different lightings in 8 plots (L1-L8). Fig.1 shows the light spectrumof the 4 fluorescent lamps. The spectral distribution of the light in each plot was measured with aspectroradiometer (HSU-100S, Asahi Spectra Co. Ltd., Japan). Table 1 shows the energy
distribution in each spectral band (UVA, B, G, R and FR). The air temperature was maintained at
27°C in the room, and all fluorescent lamps were continuously irradiated. The photosynthetic photon
flux density (PPFD) was adjusted to 200 µmol m-2 s-1 at the top of the seedlings in each plot. Tendays after the treatments, the main stem length, stem diameter and cavity size were measured(Figure 2), and the average of all these parameters were calculated from the data of 15 stems.
The correlations between the morphological traits of the stems and the light quality in eachplot were obtained. The analytical method of statistics referred to Kasajima et al. (2007). The ratioof energy (RE) was defined as the light energy in a specified wavelength width (50 nm) to the total
light energy in the whole spectral range (250-1000 nm).
1.6
B

1.4

W

1.2

PR

1

FR

0.8
0.6

0.4
0.2

0
300

400

500

600

700

800

Wavelength（nm）

B = Blue (Caribbean Blue, 15W, Sudo Co. Ltd., Japan), W = White (Mellow White, 15W, Toshiba Co. Ltd., Japan), PR = Purplish
red (Exotic Rose, 15W, Sudo Co. Ltd., Japan),
FR = Far-red (FL20SFR74, 20W, Toshiba Co. Ltd., Japan)

Figure 1. The emission spectra of four kinds of fluorescent lamps used in the experiment.

Table 1. The light quality in experimental plots (L1-L8)

Plot

Combination of lamps1)

L1
L2
L3
L4
L5
L6
L7
L8

W+W+W
W + PR + PR
W+B+B
W + PR + B
W + W + FR
W + PR + FR
W + B + FR
PR + B + FR

UVA
0.018
0.007
0.007
0.007
0.010
0.005
0.010
0.004

RE in each spectral region2)
B
G
R
0.314
0.457
0.182
0.410
0.351
0.220
0.697
0.224
0.051
0.523
0.317
0.132
0.263
0.345
0.146
0.302
0.307
0.155
0.390
0.265
0.090
0.491
0.183
0.139

FR
0.020
0.010
0.004
0.011
0.188
0.210
0.204
0.170

1) Refer to Figure 1.
2) Ratio of energy in UVA (320-400nm), B(400-500 nm), G(500-600 nm), R(600-700 nm) and FR (700-800nm) range tothat in whole spectral range (250-1000 nm).

thProceedings of The 7 ACSA Conference 2011253

a, Stem diameter, b, Cavity size

Figure 2. Stem-cross section in water spinach.

Results and Discussion
The plant length and stem diameter of water spinach increased in the plot in which 1 whiteand 2 blue fluorescent lamps were set (L3 in Table 3). Figure 3 shows the correlation coefficient
between REs in various spectral ranges and the morphological traits of stems. Plant length andstem diameter were positively correlated with the energy of blue light (400-480 nm), however, they
had negative correlation with the energies of green, red and far-red lights (520-780 nm). In
particular, stem diameter was significantly correlated with the energies of light at wavelengths of
480 nm and 660 nm. The size of the stem cavity was negatively correlated to the amount of energyof red light (600-660nm) and positively correlated to the amount of energy of blue light (480 nm).
These results showed that the stem diameter, stem length and cavity size growth were
promoted by blue light and inhibited by red light. Blue light had a positive effect on the stem growthof water spinach cultivated under artificial light conditions. However, some studies showed different
results. Minanizawa and Kitta (1977) reported that greenish blue light of 450-550 nm suppressed
the growth of mulberry (Morus alba L.). In morning glory (Ipomoea nil L), the elongation of plant
length decreased under the blue light compared with that under green or red lights (Yamazaki et al.,2003). Kasajima et al., (2008) reported that red and green light play an important role in regulating
hypocotyl elongation in common buckwheat (Fagopyrum esculentum Moench) and Tartary
buckwheat (Fagopyrum tataricum Gaertn.). In petunia (Petunia × hybrid Vilm.), blue light had noeffect on the plant height (Fukuda et al., 2002). These reports suggested that the light response
systems of some plants differed from those of water spinach.
On the other hand, stem elongation of eggplant (Solanum melongena L.) and sunflower(Helianthus annuus L.) were promoted under blue light (Hirai et al., 2006). Goto (2003) summarized
the findings of previous studies in order to evaluate the significant effects of the quality of artificial
light on plant growth and reported that blue light had various effects on plant growth. Therefore,
these reports and our results suggest that the effects of light quality on stem growth differed among
plant types and species. In future, these factors should be studied in detail for controlling the plant
growth under artificial light.

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Proceedings of The 7 ACSA Conference 2011

1
0.8

Stem diameter

0.6
0.4
0.2
0

-0.2
-0.4
-0.6
-0.8

-1

Main stem
length

0.6
0.4
0.2
0

-0.2
-0.4
-0.6
-0.8

500

600

700

Cavity size

0.6
0.4
0.2
0
-0.2

-0.4
-0.6
-0.8
-1

-1
400

Correlation coefficient (r)1
0.8

Correlation coefficient （r）

Correlation coefficient （r）

1
0.8

400

Wavelength （nm）

500

600

700

Wavelength （nm）

400

500

600

700

Wavelength (nm)

Significant level: |r| > 0.83 (p < 0.01)

Figure 3. Correlation coefficient between RE in various spectral ranges and morphological traits of stem.

References
Nabasree D and B De 2007. Antioxidant activity of some leafy vegetables of India: A comparative study. Food
Chem. 101: 471-474.
Naoya F, M Kobayashi-Yoshinaka, M Ubukawa, K Takayanagi and S Sase 2002. Effects light quality, intensityand duration from different artificial light sources on the growth of petunia (Petunia × hybrida Vilm.). J.
Japan. Soc. Hort. Sci. 71:509-516
Eiji G, 2003. Effects of light quality on growth of plants under artificial lighting. Environ. Control in Biol. 41: 121132.
Tadayoshi H, W Amaki and H Watanabe 2006. Effects of monochromatic light irradiation by LED on theinternodal stem elongation of seedlings in eggplant, leaf lettuce and sunflower. J. SHITA. 18: 160-166.
(In Japanese, with English abstract)
JATCC 2009. Standard Tables of Food Composition in Japan. Pp305.
Shin-ya K, N Inoue, M Shirai, R Mahmud and K Fujita 2008. Effect of various light qualities on hypocotyl
elongation in common and tartary buckwheat. Fagopyrum. 25: 37-43.
Shin-ya K, N Inoue, R Mahmud, K Fujita and M Kato 2007. Effect of light quality on developmental rate of wheat
under continuous light at a constant temperature. Plant Prod. Sci. 10: 286-291.
Koo MH and S Mohamed 2001. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content ofedible tropical plants. J. Agric Food Chem. 49: 3106-3112.
Kichisaburo M and F Kitta 1978. Effect of light quality on the growth of mulberry. J. Sericult. Sci. Japan. 47: 9-14.
(In Japanese, with English abstract)
Nagendra PK, S Divakar, GR Shivamurthy and SM Aradhya 2005. Isolation of a free radical-scavenging
antioxidant from water spinach (Ipomoea aquatica Forsk). J. Sci. Food Agric. 85: 1461-1468.
Keisuke Y, Y Isii, S Matsui and I Tanaka 2003. Effects of light quality, daylength, and growing temperature on
flowering in morning glory (Pharbitis nil Choisy). Environ. Control in Biol. 41: 211-219.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011255

Effects of Genotypes and Storage Time on Quality Parameters ofChinese Flowering Cabbage (Caisim) Planted in Subang
Rahayu,S.T.a* R.KiranaaaIndonesian Vegetables Research Institute (IVEGRI)
Jl.Tangkuban Perahu no 517 Lembang Bandung 40391* Corresponding author: Jl.Tangkuban Perahu no 517 Lembang, Bandung, Indonesia
Tel.: 0222786245; fax: 0222786416.
swarnit@yahoo.com

Abstract
Chinese flowering cabbage (Brassica rapa parachinensis) is one type of leaf vegetables which are rich in fiber,
minerals, and vitamins. Postharvest handling techniques and storage will affect the quality of chinese floweringcabbages. This experiment aimed to determine the effect of variety and storage time on quality parameters ofchinese flowering cabbages. Chinese flowering cabbage was planted in Subang. The experiment was designed
using Randomized design group (RAK) with three replications and two factors. The first factor was variety
consisting of 10 varieties and the second factor was storage time were 5 and 10 days. Observations were madeoon 0, 5, and 10 days. Chinese flowering cabbage was placed in a cold storage at a temperature of 10 C withhumidity at 98%. Observing parameters included texture, colour, moisture, ascorbic acid, fiber, and chlorophyll
content. The results showed that three varieties on storage for 5 days had good quality parameters.
Keywords: chinese flowering cabbages, quality, storage time, variety

Introduction
Chinese flowering cabbages that is one type of leaf vegetables grows well in Indonesia.
Chinese flowering cabbage production and other types of mustard in 2009 were 562.838 tons with aplanting area of 56,414 hectares. The plants are most widely grown in West Java, Central Java andNorth Sumatra provinces (Anonymous, 2010).
There were 22 varieties of chinese flowering cabbages or caisim that have been released
by private sectors through introduction. Eventhough IVEGRI has not released any variety of chinese
flowering cabbages, the seeds of chinese flowering cabbages line (LV-145) have been distributed tousers. LV-145 already has a uniform appearance, but its quality has not been documented well.
Chinese flowering cabbages contain many vitamins and minerals that essential are for our
body needs. The nutrient content per 100 gram edible fresh portion moisture 95 g, protein 1.2 g, fat
0.2 g, carbohydrates 1.2 g, vitamin A 5800 IU, vitamin B1 0.04 mg, Vitamin B2 0.07 mg, Niacin 0, 5
mg, ascorbic acid 53 mg, 102 mg Ca, Fe 2 mg, mg 27 mg, P 37 mg, K 180 mg, Na 100 mg. The
brassica family includes broccoli, cabbage, kale, cauliflower and brussels sprouts, to prevent many
common diseases such as cancer, heart disease, diabetes and hypertension (Siemonsma andPiluek, 1994).
The purpose of storage to maintain the price stability, inhibit the development of biologicalagents and preserve the quality of the produce in minimal moisture of loss and reduce therespiration (Dris et al.,2003).
The purpose of this experiment was to determine the quality of LV-145 and other chinese
flowering cabbages line as a preliminary testing.

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Proceedings of The 7 ACSA Conference 2011

Materials and Methods
The preliminary testing of chinese flowering cabbage quality was conducted in IVEGRI
Research Station in Subang, 100 m above sea level. The treatments were 10 genotypes (fiveIVEGRI line and five released varieties as control). The experiment was designed using
Randomized Design Group (RAK) with three replications. Differentiation among treatments todetermine (genotypes) using the F test at 5% level of test. If there was a significant the analysis wascontinued using LSD test. Plants were grown in mulched beds measuring 3m2 using a plantdistance of 20 cm x 20 cm with a population of 60 plants for each replication. The first application offertilizers given were goat manure (10t/ha) and half dose of NPK 16:16:16 (1.5 t/ha). The remaining
dose of NPK was given twice as a supplementary fertilizer during the plant growth. In addition, lime,
dolomite (1 ton/ha), was applied a week before planting. Pesticides were applied twice a week tocontrol pests, starting two weeks after planting. Pesticides were adjusted to the type of pests that
attacked.
Chinese flowering cabbages were kept in cold storage at a temperature of 100 C with RH
98%. Observations were conducted on 0, 5 and 10 days. Physical and chemical analyses wereconducted in IVEGRI Laboratory. Physical analysis covered texture and colour, while chemical
analysis included moisture, ascorbic acid, chlorophyll content, and fiber.

Results and Discussion
Table 1. Analysis of variance of texture, colour, moisture, ascorbic acid,chloropyll, and fiber for genotype

Genotype

Texture(mm/sc/gr)

Colour
(N/mm)

Moisture(%)

Ascorbic acid(mg/100gr)

Chlorophyll(µg/mg)

1

2.07

a

1.18

a

92.37

c

93.94

d

262.42

2

2.08

a

1.21

a

92.36

c

94.12

d

3

2.02

a

1.26

a

92.97

a

81.74

g

4

2.05

a

1.20

a

93.13

a

87.04

f

245.84

5

2.03

a

1.23

a

92.60

b

92.72

d

6

2.02

a

1.13

a

92.27

c

90.25

e

7

2.16

a

1.23

a

92.74

b

86.27

8

2.00

a

1.22

a

92.66

b

96.19

9

2.04

a

1.20

a

92.18

c

10

2.09

a

1.21

a

91.39

d

HSD 5%

0.30

0.16

0.21

cd

0.99

216.63d

1.10

232.05

d

1.18

cd

1.14

340.11b

1.38

376.50

ef
d
c

cd
a

b

1.00

cd

0.96

c

1.04

e

347.62b

1.32

b538.55a1.36

f245.74c282.82106.62b112.24

a

1.84

Fiber
(%)

50.17

ef
f

ab

0.05

Table 2. Analysis of variance of texture, colour, moisture, ascorbic acid, chlorophyll, and fiber for storage time

Storage Time

Texture(mm/sc/gr)

0 hari

1.81

b

0.89

c

92.10

b

77.43

c

5 hari

1.91

b

1.25

b

93.41

a

85.80

b

10 hari

2.45

a

1.49

a

91.89

c

HSD 5%

0.12

thColour
(N/mm)

0.06

Proceedings of The 7 ACSA Conference 2011

Moisture(%)

0.08

Ascorbic acid(mg/100gr)

119.110.73

Chlorophyll(µg/mg)

Fiber
(%)

c

0.98

c

53.99

a

498.70a1.00

b373.79

b

1.46

a

19.88

0.02

257

Figure 1. Effect of genotypes on chinese flowering cabbage texture.

Figure 1, the hardest texture was shown by genotype number 6, while the value of the soft
texture was shown by genotype number 7. However, from the results of LSD test at 5% level showsthat there is no significantly different for all varieties (Table 1). Duration storage affected the textureof chinese flowering cabbage. This was indicated by the value of texture where 5 days storage wassignificantly different from 10 days storage (Table 2). The longer storage time, the more evaporationfrom the material occurs that is due to the process of respiration.

Figure 2. Effect of genotypes on chinese flowering cabbages colour.

Figure 2 shows that genotype number 6 had the lowest value of the colour (dark green),
whereas genotype number 3 had the highest colour value (light green).However, the test of LSD atthe level of 5% all types of varieties were not significantly different in colour values. The colour
influenced by the content of chlorophyll is one of the parameters that affect the appearance of the
product. Genotype number 3 had lower chlorophyll content than genotype number 6. Furthermore,colour values were significantly different for 0, 5 and 10 days-storage. During storage, chinese
flowering cabbage colour changes because the process of respiration continues. Table 3 shows the
genotypes no 3,4, and 5 were not significantly different from control genotypes number 9 and 10 for
the colour values in 5 days-storage.

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Proceedings of The 7 ACSA Conference 2011

Table 3. Texture, colour, moisture, ascorbic acid, chlorophyll, and fiber for storage time * genotype
Texture(mm/sc/gr)

ST*VarColour
(N/mm)

Moisture(%)

Ascorbic acid(mg/100gr)

Chlorophyll(µg/mg)Fiber(%)

ST 0 D VAR1

1.79

ghij

0.89

e

92.68

cd

76.34

kl

45.91

k

0.92

jklm

ST 0 D VAR2

1.73

hij

0.90

de

92.06

fghij

78.44

jk

54.67

k

0.97

jkl

ST 0 D VAR3

1.72

ij

0.91

cde

92.55

cde

73.19

lmno

43.27

k

0.85

mno

ST 0 D VAR4

1.74

hij

0.89

e

92.81

c

72.36

mno

41.44

k

0.90

jklm

ST 0 D VAR5

1.81

fghij

0.89

e

91.78

ijk

88.36

h

50.89

k

1.01

ij

ST 0 D VAR6

1.91

bcdefghij

0.87

e

91.72

ijk

67.34

p

58.06

k

0.95

jklm

ST 0 D VAR7

1.91

bcdefghij

0.88

e

91.68

ijk

76.34

kl

55.91

k

0.96

jklm

ST 0 D VAR8

1.81

fghij

0.89

e

91.90

ghijk

75.24

klmn

57.46

k

0.98

ijk

ST 0 D VAR9

1.89

cdefghij

0.87

e

92.23

efgh

81.44

ij

59.18

k

0.88

klm

ST 0 D VAR10

1.75

hij

0.88

e

91.62

kl

85.22

hi

73.14

k

1.35

de

ST 5 D VAR1

1.88

defghij

1.24

abcd

91.82

hijk

97.25

g

551.44

de

1.08

hi

ST 5 D VAR2

2.17

abcdefghij

1.24

abcd

93.88

b

71.63

no

339.03

ghi

0.77

no

ST 5 D VAR3

1.84

efghij

1.29

ab

94.05

ab

72.68

lmno

393.23

fg

1.21

fg

ST 5 D VAR4

1.97

abcdefghij

1.27

ab

94.32

a

75.82

klm

375.21

fgh

0.94

jklm

ST 5 D VAR5

1.84

efghij

1.27

ab

94.02

ab

84.71

hi

577.60

cd

1.33

de

ST 5 D VAR6

1.64

j

1.12

bcde

93.88

b

71.11

op

410.63

fg

0.75

o

ST 5 D VAR7

2.04

abcdefghij

1.24

abcd

93.72

b

75.81

klm

409.41

fg

0.59

p

ST 5 D VAR8

1.81

fghij

1.25

abc

93.72

b

83.14

i

457.04

ef

0.87

lmn

ST 5 D VAR9

1.92

bcdefghij

1.26

ab

92.65

cde

100.91g

629.84

bcd

1.19

gh

ST 5 D VAR10

2.02

abcdefghij

1.29

ab

92.08

fghi

124.96d843.56

a

1.30

def

ST 10 D VAR1

2.55

a

1.40

ab

92.63

cde

108.24f

189.89

j

0.96

jklm

ST 10 D VAR2

2.35

abcdefgh

1.48

a

91.15

m

132.28b

256.19

ij

1.56

b

ST 10 D VAR3

2.50

abcd

1.56

a

92.31

defg

99.35

259.65

ij

1.47

bc

ST 10 D VAR4

2.45

abcde

1.44

ab

92.27

defg

112.94e

320.86

ghi

1.58

b

ST 10 D VAR5

2.43

abcdef

1.54

a

92.00

fghijk105.10f

391.84

fg

1.79

a

ST 10 D VAR6

2.49

abcd

1.42

ab

91.20

lm

132.29b660.82

bc

1.31

def

ST 10 D VAR7

2.52

ab

1.55

a

92.81

c

106.67f

271.89

hij

1.33

de

ST 10 D VAR8

2.40

abcdefg

1.51

a

92.37

def

130.20bc

333.96

ghi

1.26

efg

ST 10 D VAR9

2.32

abcdefghi

1.48

a

91.65

jk

137.52a353.84

fghi

1.88

a

ST 10 D VAR10

2.50

abc

1.47

a

90.47

n

126.54cd

698.94

b

1.41

cd

HSD 5%

0.62

th

0.34

Proceedings of The 7 ACSA Conference 2011

0.43

3.87

g

105.600.11

259

Figure 2. Effect of genotypes on chinese flowering cabbages colour.

Figure 2 shows that genotype number 6 had the lowest value of the colour (dark green),
whereas genotype number 3 had the highest colour value (light green).However, the test of LSD atthe level of 5% all types of varieties were not significantly different in colour values. The colour
influenced by the content of chlorophyll is one of the parameters that affect the appearance of the
product. Genotype number 3 had lower chlorophyll content than genotype number 6. Furthermore,colour values were significantly different for 0, 5 and 10 days-storage. During storage, chinese
flowering cabbage colour changes because the process of respiration continues. Table 3 shows the
genotypes no 3,4, and 5 were not significantly different from control genotypes number 9 and 10 for
the colour values in 5 days-storage.

Figure 3. Effect of genotypes on chinese flowering cabbages moisture.

Chinese flowering cabbage moisture was significantly affected by genotypes. Figure 3
shows genotype number 4 was the highest water content, whereas the lowest levels of watercontained was genotype number 10. Moisture was inversely related to chlorophyll and fiber content
of which is a component of dissolved solids in chinese flowering cabbages. The moisture of
genotype number 5 was not significantly different from control genotype number 7 and 8.
Table 2 also shows that moisture values were significantly different between 5 and 10 days-storage.

Figure 4. Effect of genotypes on chinese flowering cabbages ascorbic acid

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Figure 4 shows that the ascorbic acid content in genotype number 5 was the highest value.
Ascorbic acid is affected by the type of varieties and seasons (Hanson et al.,2011). Genotypes
number 1,2 and 5 were not significantly different in ascorbic acid contents. Ascorbic acid is one of
the components with high nutritional value in chinese flowering cabbages as other cabbage typevegetables such as broccoli (Jagdish Singh et al.,2007). During storage ascorbic acid becomes
unstable (Hounsome et al., 2009) as also indicated in Table 2. However, genotypes number 3.5, 6,
and 7 showed the values of ascorbic acid contents were relatively stable for 5 days-storage (Table
3).

Figure 5. Effect of genotypes on chinese flowering cabbages chlorophyll content

Genotype number 10 had the highest chlorophyll content (Figure 5). The chlorophyll
content affected the colour of the leaves that made it become greener. The chlorophyll content ofgenotype number 5 was not significantly different from control genotype number 6 and 9. While
genotypes number 1 and 4 were not significantly different from control genotype number 7.
The content of chlorophyll rapidly declines during storage. Storage in the dark room will decrease
the speed of leaf colour change from green to yellow (Zhang et al., 2011, In Press).

Figure 6. Effect of genotypes on chinese flowering cabbages fiber

Fiber content of genotype number 10 had the highest value. Genotype number 5 was not
significantly different from genotype number 10, as well as the interaction 5 days-storage andgenotype varieties were not significantly different for number 5 and 10.

Conclusion
1.
2.
3.

Genotypes of chinese flowering cabbage gave effects on moisture, ascorbic acid,chloropyll, and fiber of chinese flowering cabbages
Storage time gave effects on texture, colour, moisture, ascorbic acid, chlorophyll, and fiberof chinese flowering cabbage.
Three genotypes still had good qualits up to 5 days on cold storage.
th

Proceedings of The 7 ACSA Conference 2011261

References
Anonymous, 2010. Statistik Indonesia. Biro Pusat Statistik. Jakarta.220-225.
Dris, R., Niscanen, R., and Jain, S. M. 2003. Crop Management and Postharvest Handling of Horticultural
Product Volume II Fruits and Vegetables. Science Publishers, Inc, Enfield NH. USA. Pp 1-22 and 89106
Hanson P, Yang, R-Y., Chang, L-C., Ledesma, L. ,and Ledesma, D. 2011. Carotenoids, ascorbic acid, minerals,
and total glucosinolates in Choysum (Brassica rapa cvg. parachinensis) and Kailaan (B. oleraceae
Alboglabra group) as affected by variety and wet and dry season production. Journal of Food
Composition and Analysis. 24(&): 950-962.
Hounsome, N., Hounsome, B., Tomos, D. and Edwards-Jones, G.2009. Changes in antioxidant compounds inwhite cabbage during winter storage. Postharvest Biology and Technology 52(20):173-179.
Jagdish Singh, A.K. et al. 2007. Variability of carotenes, vitamin C, E and phenolics in brassica vegetables.
Journal of Food Composition and Analysis. 20 (2): 106-112
Siemonsma,J.S. and Piluek,K(Editors).1994. Plant Resources of South- East Asia 8 Vegetables. Prosea
Foundation . Bogor, Indonesia. Pp.123-126
Zhang, X., Zhang, Z., Li, J., Wu, L., Guo, J., Ouyang, L., Xia, Y., Huang, X., and Pang, X. 2011. Correlation ofleaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested chinese
flowering cabbage (Brassica rapa var. parachinensis). Journal of Plant Physiology. Article in Press.

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Influence of Organic Fertilizer on Growth and Vitamin E Content of
Traditional Vegetable, Codonopsis lanceolata
Futoshi Kato a,*, Naoto Inoue a, Seishi Yoshida babFaculty of Agriculture, Shinshu University, Nagano, Japan
Nagano Prefecture Vegetable and Ornamental Crops Experiment Station, Nagano, Japan* Corresponding author: Faculty of Agriculture, Shinshu University. 8304 Minamiminowa,
Nagano 399-4598, Japan.
Tel.: +81 265 77 1409 ; fax: +81 265 77 1409.
fkato@shinshu-u.ac.jp

Abstract
Codonopsis lanceolata (Sieb. et Zucc.) Trautv. is wild vegetable found in East Asian countries and consideredvaluable, especially among the Korean people. The effects of organic fertilizer application on the yield andquality of rhizome were investigated under Andosol condition. Commercial barnyard manure fermented withcrushed bark, beef cattle dung, and leaf mold of Japanese oak were applied at the rates of 0, 5, 10, 15, and 20-2
g·m . Barnyard manure enhanced the fresh weight of rhizome, but decreased the vitamin E content. The C/Nratio in the soil surface up to the depth of 30 cm, which was controlled by the application ratio of barnyard
manure and leaf mold, drastically influenced the rhizome yield. Total N, soluble P, exchangeable K, Ca, and Mg,
however, did not significantly affect the rhizome yield. There was significantly negative correlation between the
fresh weight of rhizome and the α-tocopherol (vitamin E) content at harvest time. The highest vitamin E content-2-1
was observed with the leaf mold application rate of 10 g·m , and the contents achieved at 9–10 mg·DM . The
nitrogen supply from the soil to plants influenced primarily the yield and quality as vegetable or drug. The vitamin
E content under higher soil C/N ratio was one of the highest among commercial vegetables in the common
Japanese food market.
Keywords: α-tocopherol, East Asia, fertilizer application, wild vegetable

Introduction
Food must always be safe and nutritious. Traditional vegetables grown on organic farms
are becoming popular in markets in East Asian countries. The organic cultivation of vegetables has
increased because organic farming benefits human health and is also effective for the conservationof agricultural environments.
Codonopsis lanceolata (Sieb. et Zucc.) Trautv. is a perennial climbing herb that is a
popular fancy vegetable in high demand among organic foods in Korea. On the other hand, it isrecognized as a rare and quaint wild plant mainly inhabiting mountainous areas in Japan (Figure 1).
C. lanceolata is chemotaxonomically similar to Codonopsis pilosula (Wang et al., 1995), which is amajor source of the traditional Chinese drug ―dangshen‖ (Iwai et al. 1992; Namba et al., 1992a,
1992b). The natural population of C. lanceolata is distributed throughout shaded and humid sites in
Korea, China, and Japan. Many farmers domesticate, produce, and sell C. lanceolata for its
rhizome vegetable, which is used in a distilled spirit, traditional medicine, or as a foodstuff in Korea.
The Korean common name for this plant is ―deo-deog.‖ Korean and Chinese people have been
carefully protecting and fostering C. lanceolata as a useful vegetable and drug since ancient times(Park and Lee 1991; Pemberton and Lee 1996; Sakamoto 1998).
C. lanceolata is also used as a minor vegetable and in a distilled spirit in Central Japan(Inoue 1998a; 1998b). Furthermore, the native Ainu people use it as a familiar vegetable, drug, andmedicinal plant (Shiraoi Ainu Museum 1989; Annetai et al. 1996; Fukuoka and Sato 1995; Hayashi
1968). The common Japanese name for C. lanceolata is ―tsuru-ninjin;‖ ―tsuru‖ means vine andthProceedings of The 7 ACSA Conference 2011263

―ninjin‖ means rhizome. The Ainu names are ―chir-muk‖ and ―tope-muk‖. Local names in Central
Japan include ―tou-do,‖ ―to-dog,‖ and ―jii-sob‖.
The cultivation area of C. lanceolata has increased to over 1000 ha in South Korea
because the edible rhizome has a good aromatic flavor (Lee et al. 1996), good texture, and highantioxidant activity (Maeng and Park 1991). However, the chemical composition of the rhizome
fluctuates according to the cultivation conditions; at present, the optimal soil conditions for
producing high-quality rhizomes are unknown. For providing a high-quality vegetable for healthyfunctional foods, productivity testing is important to clarify the effects of organic fertilizer application
rate on the yield and vitamin E content, which is responsible for the antioxidant activity of C.
lanceolata rhizomes.
In this study, field experiments were carried out to investigate the effects of commercial
barnyard manure fermented with crushed bark, beef cattle dung, or leaf mold of Japanese oak on
the rhizome yield and α-tocopherol (vitamin E) content in C. lanceolata rhizomes at the end of 1
growing season.

A: inflorescence and vine; B: capsule and single seed; C: rhizome
The plant sample was collected atu Yong Weoul in Kangwon province of Korea

Figure 1. Codonopsis lanceolata (Sieb. et Zucc.) Trautv. (family Campanulaceae).

Materials and Methods
The experimental field was located at Shinshu University, Minamiminowa Village, Nagano
Prefecture, Japan (alt. 740 m, 35°N and 138°E). The soil taxonomy and texture are Andosol andclay loam according to the FAO/UNESCO and international systems, respectively. Commercial
barnyard manure that was fermented with crushed bark (80%), cattle dung (20%), and leaf mold ofJapanese oak was prepared before seeding. Table 1 shows the chemical compositions of the soil
and organic fertilizer. Two organic fertilizers were applied at rates of 0, 5, 10, 15, or 20 g·m-2 andplowed to a depth of 35 cm below the soil surface just before transplanting the seedlings. The
experimental design was a random block design with 4 replicates. For all treatments, the plot size
was 100 × 100 cm with 15 cm spacing between individuals. A nylon net was placed to allow thevines to climb.
Samples from the local population in Young Weoul, Kang Weon Province, South Korea wereprepared, and seedlings with 4 leaves were transplanted into each plot in early May. Rhizomes
were harvested in early November.
Soil samples were collected to a depth of 30 cm before and after fertilization and air-dried atroom temperature. The total carbon and nitrogen contents of the soil were determined using a C-N
corder (Yanaco, MT700). Ammonium-N and NO3-N in the soil were extracted with 2 N KCl solution
and measured by Bremner‘s method. Soluble P in the soil was determined by Truog‘s method.
Exchangeable K, Ca, and Mg were extracted with 1 N acetic ammonium solution and measured by

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atomic absorption spectrochemical analysis. Total α-tocopherol (as vitamin E) content in therhizome was measured by high-performance liquid chromatography (HPLC).
Table 1. Chemical properties of soil fertilizer
Constituent

Soil

pH (H2O)EC (ms)CEC (me)
Total N
Total CC/N ratio
Inorganic N (NO3)
Inorganic N (NH4)Soluble P
Exchangeable K
Exchangeable Ca
Exchangeable Mg

6.3
0.06
22.5
0.5
9.3
17.3
2.4
0.7
17.7
41.6
398.4
50

Organic fertilizer
Barnyard manure
Leaf mold
7.1
6.8
2.2
0.5
36.7
44.6
2.2
0.9
23.8
23.8
10.9
25.9
11.6
0.2
5.8
1.8
787.8
58.9
56.6
25.1
302.3
239.1
29.2
18.7

Table 2. Correlation between chemical component in soil and rhizome yield
Constituent
Total N
Total C
Soulble P
Exchangeable K
Exchangeable Ca
Exchangeable Mg
C/N ratio

Correlation coefficient
0.58
0.22
0.47
0.23
0.05
0.04
-0.90

significant
P < 0.100

P < 0.001

Results
During the growth period, the precipitation on the site was 600 mm, mean air temperature
ranged from 10–24°C, and mean solar radiation was approximately 15000 J·cm-2·day-1.
The application ratio of barnyard manure and leaf mold strongly influenced the rhizome
yield at harvest time. The barnyard manure increased the rhizome yield, whereas fermented leaf
mold decreased the yield. The correlation between the soil chemical conditions after fertilization andrhizome weight at harvest time was analyzed using all of the data (Table 2). There was a significant
negative correlation between C/N ratio and rhizome weight (Figure 2). On the other hand, total N,
total C, soluble P, and exchangeable K, Ca, and Mg in the soil after fertilization did not significantly
affect the rhizome yield. The C/N ratio in the soil from the surface to a depth of 30 cm, which wascontrolled by the application ratio of barnyard manure or leaf mold, strongly influenced the rhizome
yield.
Barnyard manure increased the fresh weight of rhizomes but decreased the vitamin E
content (Figure 3). There was a significant negative correlation between the fresh weight andvitamin E content of rhizomes (p < 0.001). The highest vitamin E content occurred with theapplication of 10 g·m-2 leaf mold (9–10 mg·DM-1). The nitrogen supply from the soil to plants
primarily influenced the yield and quality of the rhizome as a vegetable or drug. The vitamin E
contents produced under higher soil C/N ratios are one of the highest among commercial
vegetables available in the Japanese food market.

thProceedings of The 7 ACSA Conference 2011265

Barnyard manurey = -2.42x + 56.4

20
15
10

Leaf mold5

y = -8.70x + 167.6

0
14

15

16

17

18

19

C/N ratio in soil (0-30 cm)
Figure 2. Relationship between C/N rations soil andrhizome yield.

α-tocopherol (mg / 100g fresh matter-1)

Fresh weight of rhizome (g / plant-1)

25

3.5

3

2.5
y = -0.063x + 3.718(p < 0.001)

2
0

5

10

15

20

25

Fresh weight of rhizome (g / plant-1)

Figure 3. Relationship between rhizome yield and αtocopherol.

Discussion
The natural habitat of C. lanceolata is cool shaded forest floor in mountainous areas in
Korea and Japan. In this experiment, moderate amounts of rhizomes were harvested in only 1
growing season and despite a lack of shade, suggesting that the climate of the experimental field isfavorable for both vegetative growth and rhizome production. Lee et al. (1996) reported that shading
decreases the growth but improves the aromatic constituents. Our results and the previous reportsuggest that shading and the fermented organic fertilizers favor production of high-quality
vegetables.
Our survey in South Korea (Sakamoto et al., 1998) revealed that organic farmers in
Kangwon Province have developed an organic fertilizer consisting of saw dust, rice bran, and ricechaff; it is mixed and fermented over 1 year and applied at a rate of 6 g·m-2 before seeding. The key
technique of the organic manure production is considered to maintain the soil C/N ratio around 17%
in the case of Andosol soils according to our results.
Wang et al. (1995, 1996) report that C. lanceolata is chemotaxonomically similar to C.
pilosula and contains many polysaccharides that exhibit immunomodulatory effects. Maeng and Lee(1991) also report that the ethanol extract from C. lanceolata rhizome possesses effective
antioxidant activity that is stronger than that of extracts from Panax ginseng C. A. Meyer. In
addition, they point out that C. pilosula rhizome exhibits protective action on experimentally induced
gastric ulcers in rats (Wang et al., 1997). These reports and our present results suggest that therhizome of C. lanceolata is a healthy food. Despite the trade-off between rhizome yield and quality,
organic farming with an organic fertilizer that supplies nitrogen slowly is considered valuable incommercial food production.
C. lanceolata is also a traditional wild vegetable eaten by the Ainu people in Northern
Japan. The screening research of the chemical composition in wild plants used by the Ainu
revealed that the species has the highest vitamin E content among 67 tested species (Annetai etal., 1996). It is also reported that most edible wild plants have vitamin E contents less than 0.5
mg·100 g-1 fresh weight; on the other hand, vitamin E content of the rhizome of C. lanceolata is 2.38
mg·100 g-1 fresh weight. Our results show that greater amounts of vitamin E are present in smaller
rhizomes produced under higher C/N ratios due to the fermented leaf mold. Since the vitamin E
contents of C. lanceolata are one of the highest among commercial vegetables in the Japanesefood market, the vegetable is expected to be gradually accepted as a worthwhile healthy food.

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Many wild species of Campanulaceae are believed to be useful ingredients in drugs thatmaintain the human health while being functional foods among Asian peoples. For example, many
species of Platycodon, Campanula, and Adenophora are popular as traditional crude drugs andfoods. Many Korean people and researchers of Korean herbal medicines believe that C. lanceolata
is a functional food that has anti-cancer activity. Hata et al. (1998) point out that many edible wild
plants in Akita prefecture, Japan induce cell differentiation, including the activity of the humanleukemia cell line (HL60), which is a useful model system for drug screening. However, Inoue(2003) reports that the ethanol-soluble fraction from the rhizome of C. lanceolata does not exhibit
the nitroblue tetrazolium reduction activity for the human promyelocytic leukemia cell line HL60 or
neutrophil activity. In addition, the rhizomes do not inhibit melanine synthesis in mouse melanoma
cell line B16. More investigation is required from other perspectives regarding the anti-cancer
activity of C. lanceolata rhizomes to establish its status as a healthy food.

Acknowledges
We thank emeritus Professor Dr. Sadao Sakamoto of Kyoto University and Professor Dr. Choel-Ho
Park, Kang Weon National University in Korea.

References
Annetai, M. Ogawa, H., Hayashi, T., Aoyagi, M., Chida, M., Muraki, M., Yasuda, C., Yabunaka, T., Akino, S. andYano, S. 1996. Studies on wild plants traditionally used by the Ainu people. Part I. Contents of vitamin A,
C and E in edible plants. Report of Hokkaido Eisei Kenkyushyo 46: 34-39.
Fukuoka, I. and Sato, S. 1995. Botanical Memories of Ainu. Sofukan, Tokyo. p.177-180.
Hata, K., Ishikawa K. and Hori, K. 1998. Differentiation - inducing activities of human leukemia cell line (HL60)
by extracts of edible wild plants in Akita. Natural Medicines 52(3): 269-272.
Hayashi, Y. 1968. Foods and life of Ainu people. In Education Board of Hokkaido ed., Ethnological researchnote of Ainu. 92-113.
Inoue, N. 1998a. Traditional use of wild plants in Okumibu, Shinshu. Agricultural Technology and Culture 21:
159-191.
Inoue, N. 1998b. Turu-ninjin (Codonopsis lanceolata). In Fujimaki, H. ed., Dictionary of biological resources inJapan. Noubunkyo, Tokyo. p.215-216.
Inoue, N. 2003. Traditional use and quality as food of edible wild plant Codonopsis lanceolata Trautv. in themountainous area of Japan and Korea. The Annals of Environmental Science, Shinshu University 25:
29-44.
Iwai, M., Komatsu, K. and Namba, T. 1992. Pharmacognostical Studies on the Codonopsis plants. (3) On the
botanical origin of Chinese crude drug ―Danshen‖ derived from sect. Electae plant (I). Shoyakugaky
Zassi 46(3): 217-223.
Lee, S. P., Kim, S. K., Nam, M. S., Choi, B. S. and Lee, S. C. 1996. Effects of shading and organic matter
applications on growth and aromatic constituents of Codonopsis lanceolata. Korean J. Crop Science
41(4):496-504.
Maeng, Y. S. and Park, H. K. 1991. Antioxidant activity of ethanol from Dodok (Codonopsis lanceolata). Korean
J. Food Sci. Technol. 23(3): 311-316.
Namba, T., Komatsu, K., Iwai, M. and Xu, G. F. 1992a. Pharmacognostical Studies on the Codonopsis plants.
(1) On the anatomical characteristics of the root of sect. botanical origin of Chinese crude drug
―Danshen‖ derived from sect. Codonopsis plants. Shoyakugaky Zassi 46(2): 156-164.
Namba, T., Komatsu, K. and Iwai, M. 1992b. Pharmacognostical Studies on the Codonopsis plants. (2) On the
botanical origin of Chinese crude drug ―Danshen‖ and its related crude drugs derived from sect.
Codonopsis. Shoyakugaky Zassi 46(2): 165-173.
Park, C. H. and Lee, K. C. 1991. Production of edible wild plants in Korea. Sun Jin Mum Hwa Sa, Seoul.
Pemberton, R. W. and Lee, N. S. 1996. Wild food plants in South Korea; market presence, new crops, andexports to the United States. Economic Botany 50(1): 57-70.

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Sakamoto, S. 1998. Comparative ethnobiological studies in the area surrounding the Japan Sea, especially in
Korea and Japan. Bulletin of the interdicipinary Research Institute of Environmental Sciences 17: 79-95.
Shiraoi Ainu Museum .1989. Chiru-Muck. In Shiraoi Ainu Museum ed., Ainu people and plants. Shiraoi Ainu
Museum, Hokkaido 21.
Wang, Z. T., Ma, G. Y., Tu, P. F., Xu, G. J. and Ng, T. B. 1995. Chemotaxonomic study of Codonopsis (family
Campanulaceae) and its related genera. Biological Systematics and Ecology 23(7/8): 809-812.
Wang, Z. T., Ng, T. B., Yeung, H. W. and Xu, G. J. 1996. Immunomodulatory effect of a polysaccharide-enrichedpreparation of Codonopsis pilosula root. Gen. Pharmac. 27(8): 1347-1350.
Wang, Z. T., Du, Q., Xu, G. J., Wang, R. J., Fu, D. Z. and Ng, T. B. 1997. Investigation on the protective actionof Codonopsis pilosula (Dangshen) extract on experimentally-induced gastric ulcer in rats. root. Gen.
Pharmac. 28(3): 469-473.

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Review on the Effect of Postharvest Treatment on Potato Quality
Ali Asgar
Indonesian Vegetables Research Institute
Jl. Tangkuban Perahu No. 517 Lembang 40791
Tel.(022)2786245, PO BOX 1586 Bandung, Indonesia
asgar1957@yahoo.com

Abstract
Research on tuber quality, storage, and processing was carried-out. Research emphasized on the evaluation ofclones for processing quality, on-farm evaluation of promising storage technologies, and application oftechniques to control postharvest losses due to pest and diseases, transportation, storage and marketing to aid
in the management control at pest and disease and in the process upgrade of participating of laboratories,
stores and farmers practices. Research on improving of processing method is needed to increase the quality of
product including flavor. Research on packaging is mainly concentrated on the development of suitable
packaging system for the product.
Keywords: post-harvest treatment, potato, quality, income.

Introduction
Among major vegetables, potato is potential to increase small farmer income, since potato
is economically competitive vegetable. Potatoes are highly demanded by food industries such as
french fries restaurants and potato chips industries. As estimated by World Bank, vegetable andfruit consumption in Indonesia increased approximately 3.9% during the period of 1995-2010(Pasandaran & Hadi 1994). Demand projection to overall vegetable increases by 4.1% every year
(8.2 to 12.3 million tons every year, Van Lishout, 1992).
The major challenge for Indonesian agriculture is how to produce high quality of potato ataffordable prices that fit into the demands as rapidly growing population. It was clear that AARD
plays an important role on vegetables quality. Postharvest technology increases product quality andreduces losses. Loss assessment of poor postharvest technology is estimated about 20 – 40%(Winarno & Aman, 1981).
Basic research program has to emphasize on strengthening the vegetable quality by
improving efficiency of farming techniques, utilizing of the valuable domestic resources wisely toendure food safety, efficiency on management, food security and agribusiness development.
Determination of some pre and postharvest treatment were evaluated for potato quality. Pre-harvest
treatment commonly concerned with variety and harvest time, whereas postharvest concerned withfresh handling and processing.
In Indonesia, there are a few research on postharvest aspect of potato have been carriedout. Postharvest experiments so far concentrated on the storage and handling of seed and potato
tubers. In this paper we summarized the postharvest treatment affected the quality of potato.

Material and Methods
Several reports provided by IVEGRI were available fragmentally. Reserach started frompotato varieties up to postharvest have been done, however, they need more improvement.
Sequence of postharvest information were hardly found, however, some crop varieties, somepostharvest experiments on various crops and some agronomic aspect methods were describedthProceedings of The 7 ACSA Conference 2011269

briefly. Field testing design commonly used were randomized block design, whereas in thelaboratory most research were carried-out by complete randomized design with sufficient replication
related to number of treatments. Postharvest treatment methodology related to the some vegetablevarieties testing were mainly based on physical characterization, chemical analysis method andorganoleptic test.
For potato varieties testing, 2-5 g sample was placed in 250 ml flask added with 50 ml ofalcohol 80% and stirred for 1 h, then they were filtered and cleaned. The filtrate of 250 ml containedcarbohydrate. Starch in filtrate as a residue were put on filter paper, and washed 5 times in 10 ml ofether, then let these samples to evaporate and they were rewashed by 150 ml of alcohol 10% toobtain free dissolved carbohydrate. Residue was removed and placed in an erlenmeyer flask
through washing by 200 ml of aquadest then added with 20 ml of HCl 25%, covered with cooler lid,
and boiled for 30 min. After that they were cooled down by NaOH 45% and diluted to 500 ml.
Filtered and determined the sugar content as glucose from the filtrate. Determination of starchcontent was calculated by glucose weight times factor 0.9. Crop hardness was measured bypenetrometer, and organoleptic test was carried-out by hedonic method.

Results and Discussion
Storage of seed potatoes
An experiment was conducted to compare the effect of diffuse light and dark storage oftuber seed on yield of patato by Sihombing (1986). This experiment was carried out at hight land
Ciwidey at West-Java, (1080 m above sea level) on a farmer field in both dry and wet season. A
randomized block design of non-factorial was applied with two storage method having 12 replicateson each season. The results indicated that the seed tubers stored in diffuse light storage showed
better plant vigour and gave yielded increases from 16.9 to 26.9%, compared with tubers stored in
the dark.
The effect of storage method and delay in planting of tuber seed on yield and quality ofpotato tubers was carried-out by Nainggolan (1993). A randomized block design with three
replicates was used in this experiment. Two methods of storage used tuber seed (in dark anddiffuse light) and planting periods (5 months of storage as control). The results showed that storageof tubers seed in diffuse light storage increased the yield up to 15.02% and the quality of tuber was
better compared to dark storage. The delay in planting of tuber seed decreased the yield to 27.50
g/seed/plant and showed lower quality tuber.
Potato seed storage in mid-elevation area with diffuse light storage was done by Asgar
and Asandhi (1994). The planting time of mid-elevation potato was in May to June, while the tuber
seed used was harvested in December, long storage of potato seed affected poor quality of seed.
One way to overcome the problem was by using diffuse light storage. Six varieties of potato useddiffuse light storage. Six varieties of potato (Granola, Red Pontiac, Cosima, Monza, Cipanas, andBerolina) were stored in diffuse light storage and dark storage by using randomized completely
block design with six replicates. The results showed that after four months storage the percentageof rotten tubers of Granola was 69%, although diffuse light storage was better than that in darkstorage in terms of higher number of sprout, shorter, bigger and more healthy.
Storage of ware potatoes
Study on storage of ware potato and the loss due to storage method was conducted by
Asgar and Asandhi (1990). Thirty farmers was sampled for interview. The results showed that some
farmers stored their ware potato for several days until two months when the market price as low(Asgar, 1990). Two method of storage was used in the field (either covered delaying the harvest or
pilled beside the field and covered with canvas) or in the storage which was usually seed storage.

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The average loss was 7.1% for farmers having less than 1 ha land and 9.3% for farmers having 1
ha or more land. The highest loss was recorded at 25%.
Improvement of ware potatoes store method was carried-out by Asgar and Asandhi(1991). A two-layer of box was made in the storage. The distance between the two layers was 14
cm and filled up with rice husk. The box was divided into 12 parts to place the experimental unit
consisted of four treatments and three replicates. The size of the experimental unit was 104 cm
length x 77 cm width x 44 cm depth. The design used was randomized completely block design.
The treatment was pilling potato tubers with: 1) vertical without ventilation, 2) horizontal without
ventilation, 3) vertical with ventilation and 4) horizontal with ventilation. The ventilation made fromproliferated tube was put vertically into the pile. The size of the tube was 74.5 cm of length, 8.5 cm
of diameter, and the number of holes was 20. From this study could be concluded that ware potato
storage using double layers of wall and filled up with rice straw could be introduced to farmers in thehighland, since the temperature in the tubers piles was not significantly different with the room
temperature. However, pilling the tubers horizontally was better than vertically as done by thefarmers.
Experiments on the harvest time and storage period was done by Asgar and Marpaung(1997). Seed tubers with the size of 25 to 45 g/tuber collected from Pangalengan were planted at
the experimental area at the Indonesian Vegetables Research Institute. The seed tubers were
72/plot. Fertilizing and maintanance at the field was conducted at the Indonesian Vegetables
Research Institute recommendation. Manure was given side of planting with dosage of 30 tons/ha,
ZA at 100 kg/ha, TSP at 250 kg/ha and KCl at 300 kg/ha. Seed tubers were planted with the sproutof 1 to 2 cm length and planting distance 80 x 30 cm on matches. A split plot design was used andplants were harvested on day 70, 80, 90, 100, and 110 days. Sub plot was storage period consistedof 1, 2, 3, 4, and 5 day. The result showed that harvest time of 100 after planting yielded goodquality as evaluated by panelist. The best characteristic was found for tuber harvested on 100 days
after planting and stored for two days.
Processing
The best variety for chips was Atlantic as investigated by Asgar and Kusdibyo (1997) using
method as follows: seed tubers of 25–45 g/tuber collected from the farmers in Pangalengan and
Wonosobo were planted at the experimental area of IVEGRI. The seed tubers were 72 per plot.
Fertilizing and maintenance at the field used IVEGRI recommendation (1994). Manure was givenside of planting with dosage of 30 tons/ha. Artificial fertilizer used at planting time were Urea at 200
kg/ha, ZA at 100 kg/ha, TSP at 250 kg/ha, and KCl at 300 kg/ha. Seed tubers were planted with the
sprout of 1–2 cm in the length and planting distance was 80 x 30 cm on matches. A split plot design
was used in this research and every treatment combination was repeated three times. A main plot
was varieties consisted of Atlantic, Latif, and Granola. A sub-plot was harvest time consisted of 90,
100 and 110 days. The results showed that the quality of Atlantic variety and harvest time of 100
days were the best than the others in terms of the colour, brittleness, taste and appearance.
The chipping quality of 45 advanced potato clones was determined by Asgar and Chujoy(1999). The results showed that best clones with chipping scores ranged from 1.0 to 3.4 (lightyellow to dark yellow) which were: I-853, ASN-691, Muziranzara, CFJ-691, CEW-691, Atlantic, ABZ69-1, TS-2, VC24.16, Cruza-148, LT-5, Monsama, P-4, TS-13, 379706.34, 283232.11, CCN-69-1,
MF-II, CFM-69-1, 3806010.14, 378501.3, Chiqita, Graso-28, I-1150, AGB-69-1, Desiree, I-11035,
Cruza-27, and Precodepa.
Research on the evaluation of 12 potato processing clones was conducted by Basuki et al.
(2003). Experimental area used was at 4 locations i.e: Pangalengan, Garut, Batur (Middle Java),
and Tosari (East Java). Industry for potato chips modern scale, two home industries, and 42
farmers were interviewed to evaluate the production character, tuber quality, specific gravity,
appearance and process product taste from 12 potato processing clones. The results showed that
380584.3 and FBA clones were preferred by farmers, home industry, and consumer. FBA-4 cloneth

Proceedings of The 7 ACSA Conference 2011271

coled be developed for modern industry with good handling. TS-2 and MF-II were received bymodern industry processor as raw materials. These clones could be released as new variety forprocessing. The product of TS-2 was equal to Atlantic and Panda and product of MF-II was higher
than that of the Atlantic and Panda.
Basuki (2005) showed that according to resistance of the leaf rotting diseases and swollen
root nematodes, 380584.3, TS-2, FBA-4, I-1085 and MF-II clones were resistance. This figure wasachieved from ten experimental areas which were 380584.3 resulted in 33.5 t/ha, TS-2 was 22.4
t/ha, FBA-4 was 28.1 t/ha, I-1085 was 25.3 t/ha and MF-II was 30.1 t/ha, respectively. Furthermore,
he stated that the use of FBA-4, TS-2 and MF-II clones were suitable for raw materials of potatoes
chips for modern industry, while 38058.4.3 and I-1085 were suitable for home industry and middle
class.
Research on distribution of taste on potato slices was conducted by Kastaman et al.
(2000). A completely randomized block design was used in this research. First factor was potato
cultivar consisted of: Atlantic and Granola. Second factor was Sodium Metabisulfite concentration at
500, 750, and 1000 ppm. Third factor was ingridients consisted of: 1) salt, 2) salt and broth, 3) salt
and chicken curry, 4) salt and meat simmered in spices and coconut milk, and 5) salt and pepper.
The results showed that for Atlantic cultivar, dipping with sodium metabisulfite treatment was not
significantly different on the colour, flavor, hardness and taste of potato chips. On Granola, there
was a significantly different on organoleptic characters (colour, taste, appearance). Increasing inIndonesian spices such as kaldu ayam (chiken broth), kari ayam (chiken curry), rendang (meatsimmersed in spices and coconut milk), and pepper gave significantly different to increase in
consumer preference on organoleptic test. There was an interaction between cultivar and sodium
metabisulfite concentration on the quality. Economic analysis for potato chips on the estimation of
cost price production of Atlantic cultivar was Rp. 15.711/kg. The price of chips was Rp. 16.750/kg,
whereas the price of raw materials was Rp. 4000/kg. For estimation of cost price of Granola cultivar
was Rp. 13.211/kg and a sell price of chips was Rp. 14.240/kg with buying price of raw material at
farmer level Rp. 1500/kg.
Quality assessment for several clones has already done by Asgar et al. (2010). The
objective of this research was to test for clones/cultivars selected from the yield and quality. Quality
test of 10 selective clones was determined. This research was conducted from July to September
2010. The research was arranged in randomized block design. The results showed that chips whichhaving value between 2.00 and 2.36 (yellow uniform) for potato chips was clone 7 (391011.17 x
385524.9). Reduction sugar content from this clones were assumed to lower than reduction sugar
content of the other potato clones having dark colour.

Conclusions
Considering the increasing demand for processed potato products, a systematic evaluationof cultivars and clones for processing quality would decrease high research priority. On-farmevaluation of promising storage technologies for seed and ware potatoes that have already beentested elsewhere should also be emphasized. Application of technologies to control postharvest
losses due to pest and disease, transit, storage, and marketing were to improve the management
control at pest and disease and in the process upgrade of participating of laboratories, stores andfarmers practices. Research on improving of processing product is needed to increase in the quality
including taste pass through giving spices (flavour). Research on packaging is mainly concentrated
on the development of suitable packaging system for the product.

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References
Asgar, A. and A.A. Asandhi, 1990. Cara Penyimpanan dan Kehilangan Hasil Kentang Konsumsi diPangalengan. Bul. Penel. Hort. XX(1):1-7
Asgar. A. and A.A. Asandhi, 1991. Improvement of Ware Potato Store Method. Bul. Penel. Hort. XX(4):138-142
Asgar, A. and Kusdibyo, 1997. Pengaruh Varietas dan Umur Panen terhadap Kualitas Umbi Kentang (Solanum
tuberosum L.) sebagai Bahan Baku Pembuatan Keripik Kentang. Prosiding Seminar Nasional Teknologi
Pangan. Editor : Slamet Budijanto, Fransisca Zakaria, Ratih Dewanti Haryadi dan Budiatman
Satiawiharja. Perhimpunan Ahli Teknologi Pangan Indonesia. Kantor Menteri Negara Urusan Pangan RI.
Hal:251-262
Asgar, A. and A.A.Asandhi, 1994. Penyimpanan Umbi Bibit Kentang di Dataran Medium dengan Tipe Gudang
Terang. Bul. Penel. Hort. XXVI(2):151-159
Asgar, A. and L. Marpaung, 1997. Pengaruh Umur Panen dan Lama Penyimpanan terhadap Kualitas Umbi
Kentang. J.Hort. 8(3):1208-1216
Asgar, A., S.T. Rahayu, M. Kusmana, and E. Sofiari, 2011. Uji Kualitas dari Beberapa Klon Kentang (Solanum
tuberosum L.) Dataran Tinggi Lembang. In Press.
Basuki, R.S., Kusmana, A. Dimyati, Nur Hartuti, E. Sofiari and U. Jayasinghe, 2003. Evaluation of Processing
Potato Clones in Indonesia. Progress in Potato and Sweet-potato Research in Indonesia. Edited by Keith
O. Fuglie. International Potato Center (CIP) and Indonesian Agency for Agricultural Research andDevelopment (IAARD).
Basuki. R.S., Kusmana and A. dimyati, 2005. Analisis Daya Hasil, Mutu dan Respons Pengguna terhadap Klon
380584.3, TS-2, FBA-4, I-1085 dan MF-II sebagai Bahan Baku Keripik Kentang. J.hort. 15(3):160-170
Kastaman, R., D.N. Wahyu, Sudaryanto, B.D. Sofiah, C.S. Achyar, A. Asgar and Kusdibyo, 2000. Studi
Pemberian Beberapa Rasa pada Irisan Kentang yang Dikeringkan dengan Metode Penggorengan
Vakum untuk Mendapatkan Mutu Keripik Kentang yang Bernilai Jual Tinggi. Lembaga Penelitian UNPAD
Bekerja Sama dengan Agricultural Research Management Project II. Badan Penelitian dan
Pengembangan Pertanian.
Kusdibyo and L. Marpaung, 1998. Pengaruh Jenis dan Konsentrasi Lilin terhadap ketahan Simpan Kentang
Segar. Laporan Balai Penelitian Tanaman Sayuran.
Nainggolan, P., 1993. Pengaruh Methode Penyimpoanan dan Penundaan Tanam Umbi Bibit terhadap Hasil dan
Mutu Umbi Kentang. Bul. Penel. Hort. XXIV(4):87-92
Pasandaran, E. and P.U. Hadi, 1994. Prospek Komoditi Hortikultura di Indonesia dalam Kerangka
Pembangunan Ekonomi. Penyusunan Prioritas dan Design Penelitian Hortikultura. Solok 17-19
November 1994.
Sihombing, P., 1986. Penyimpanan Umbi Bibit di Ruang Terang terhadap Hasil Kentang. Bul. Penel. Hort.
XIV(2):27-29
Van Lishout, 1992. Consumption of Fresh Vegetables in Indonesia. Internal Comm. No.48. ATA-395 Project.
LEHRI-The Netherlands.
Winarno, F.G. and M. Aman, 1981. Fisiologi Lepas Panen. Sastra Hudaya, Jakarta.

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Perception, Attitude and Factors Influencing Household‟s Acceptance toTransgenic Late Blight Resistant Potatoes
Asma Sembiring*, Eri SofiariIndonesian Vegetable Research Institute, Lembang, Indonesia*Corresponding author: Indonesian Vegetable Research Institute (BALITSA)
Jl.Tangkuban Perahu No.517 Lembang, Bandung-40395, Indonesia
Tel : +62 22786245; Fax : 62 22786416
asma_h_1999@yahoo.com

Abstract
The objective of this study was to identify the perception, attitude and factors influencing the household‘s
acceptance to the transgenic late blight resistant potatoes. The research used data taken from 101 respondents
in Bandung West Java, Indonesia from September to November 2009 using stratified random sampling. The
surveys showed that most of the respondents (99%) never heard about transgenic potatoes. About 36%
respondents said that transgenic potatoes were safe to be consumed. At least, 55.4% respondents were willingto consume transgenic potato as long as there was no negative effect by consuming it. In addition, 44.6%
respondents said that they were not willing to consume the potato because they did not know it. Result of Chisquare test showed that the most factors influencing household acceptance to transgenic potato wereconsumption frequency and consumers‘ potato criteria. Statistical analysis using logit binomial test showed that
education and consumers‘ potato criteria were the most important factors for the household to accept thetransgenic late blight resistant potatoes.
Keywords: consumers’ acceptance, consumers’ attitude, potato transgenic

Introduction
Potato is the fourth source of carbohydrate in the world and has a big chance as analternative food diversification because it contains high nutrient. Potato production centers inIndonesia commonly locate in high land, with high rainfall and humidity. The condition is very suitable
for late blight (Phytopthora infestans-Mont de Barry disease). Yield loss caused by diseases reached
60 to 80% (Wattimena, 1994). In severe attacked, the loss reached 100%.
The development of resistant potato varieties are one of the solution to reduce yield loss
caused by late blight disease. Several sources of potato resistant to late blight have been found insome species of wild potatoes such as S. demissum, S. bulbocastanum, S. stoloniferum, and S.
microdontum (Hawkes, 1994). Resistant genes to the late blight from S. bulbocastanum have beencloned and used to produce potato resistant late blight. The crossing between Katahdin Rb with
Granola and Atlantik resulted in resistant filial to late blight (Song et al., 2003). In 2004, trial to
Katahdin transgenic potatoes was conducted. The results showed that Katahdin could control lateblight at the early-season. Field trail to examine potatoes resistant characteristic was held in 2007
and 2008 and several filials (F1) resistant to late blight infection.
Kotler and Susanto (2001) reported that several factors influence consumer behavior,including psychology. Psychology aspects involve perception, knowledge, and attitudes become
important aspects in consumer acceptance to the product. Study on consumer behavior to thetransgenic products has been conducted in some countries. The consumer acceptance to thetransgenic products depends on their perception to the benefit and the risk of the transgenic (Byrne,2006). Supporting of transgenic products come from United State of America and Japan consumers
in agriculture and healthy sectors. The more information consumers get, the more positive response
give to transgenic crops (Hoban, 1999). Other studies showed that respondents‘ knowledge related

274

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Proceedings of The 7 ACSA Conference 2011

to transgenic crops and labeling has a negative impact to consumer acceptance (Jill et al., 2001). In
Korea, the consumers tend to reject transgenic food, especially women, people who get higheducation and persons who have enough information about transgenic (Onyango et al., 2004).
In Indonesia, the issue about transgenic crops is still in debates. Pros and cons emerge as
the development of transgenic information and the willingness to plant the products with several
reasons. Studies to observe factors influencing household acceptances‘ to transgenic crops has
been done in three big cities in Indonesia, i.e. DKI Jakarta, East Java and North Sumatra concluded
that the less information about transgenic crops consumers have, the easier for consumers toaccept transgenic food. Meanwhile, in economic term, consumers from middle and higher economic
strata are more willing to accept transgenic crops than the consumer from lower economic. Public
acceptance to transgenic crops are also influenced by direct and indirect risk perception fromtransgenic food, benefit, risk and the credibility of policy institution in evaluating safety crops andthe environment (Riska, 2009).
Issues on pros and cons about transgenic crops in Indonesia developing bigger than the
product itself. The opponent was greater seen from media coverage rise out the negative effect oftransgenic crops and it will affect consumer acceptances‘ to the transgenic potatoes. In fact, theproduct has not been released and sole in Indonesia. The commercialization of transgenic potatoes
shall go through several trial tests in advance such as limited testing of varieties, limited testinglaboratories (bio safety containment) and limited field test (bio safety confainment) based on Bio
safety Commission regulation.
So far, there have been several studies conducted to determine perception, knowledge,
attitude and consumer acceptances‘ to the transgenic cops/foods, yet specifically for Phythopthora
resistant potato varieties. So, the study is expected contribute to increase knowledge related toconsumer acceptances‘ to transgenic crops.

Materials and Methods
The survey was conducted on September to November in 2009. Household respondents
were chosen as stratification of 101 persons in Bandung municipal, Indonesia. The stratification was
based on income strata. Lower level with income was from IDR 1,000,000 to 2,000,000; middle
level was from IDR 2,000,001 to 3,000,000 and high level was more that IDR 3,000,000.
The variables were characteristic of respondents, perception, attitude and respondentacceptances‘ to the potatoes transgenic. Data for perception and attitude were counted byqualitative statements on the ordinal scale (1 to 5) using Likert scale. Whereas, acceptance‘s data
variable were measured by nominal scale (yes or no). Furthermore, the data were analyzed with
Descriptive statistic method, Chi-square test and Logit binomial.

Results and Discussion
Demographic and Socioeconomic of the Respondents
The sums of respondents were 101, dominated by ages between 31 and 60 years, 78
respondents (78%), with average level of education were senior high school (34%). Most of the
respondents were house wife (67%). Level of income per month were 1-2 millions rupiah (68%).
The potatoes household average consumption renged from 1 to 2 times per week (64%) withnumber of consumption was 1,05 kg.
Consumer Perceptions‟ to the Potatoes
Descriptive statistic showed that more than 93% respondents stated that the potatoes was
nutritious vegetable, made healthy, delicious and affordable by consumers, available in the market
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and easy to be processed for any types of cuisines. As many as 88,1% respondents also said thatthe potatoes could be used as staple food (Table 1).
Table 1. Likert scale from household perception to the potatoes
Statements

Percentage (%)
2
3
4
91,1
2,0
0,0
87,1
0,0
1,0
89,1
3,0
1,0
88,1
3,0
2,0
78,2
1,0
10,9
68,3
0,0
0,0
86,1
1,0
1,0

1
6,9
11,9
6,9
6,9
9,9
31,7
11,9

The potatoes is nutritional food
The potatoes is a healthy food
The potatoes is delicious food
The potatoes price is affordable
The potatoes can be used as staple food
The potatoes is available in the market
The potatoes is easy to be processed

5
0,0
0,0
0,0
0,0
0,0
0,0
0,0

(1=strongly agree; 5=strongly not agree, N = 101)

The Consumers‟ Potatoes Criteria
Taste is the most important criteria for the consumers. The results were consistent withprevious study concluded that the consumers noticed more to potatoes tastes (Ameriana et al.,1998). The color, taste and nutrition were also more important criteria‘s for the potatoes comparedto the price, aroma and the safety to be consumed with value of 72,3; 24 and 50, 5%, respectively(Table 2).
Table 2. Likert scale from the consumer important criteria‘s‘ to the potatoes
Statements

Percentage (%)
1

Taste
Nutritional
Appearance
Price
Color
Aroma
Safety to be consumed

4,0
9,9
13,9
5,0
12,9
2,0
22,8

2
89,1
83,2
83,2
67,3
83,2
21,8
26,7

3
2,0
5,0
1,0
4,0
1,0
4,0
4,0

4
5,0
2,0
2,0
23,8
3,0
72,3
46,5

5
0,0
0,0
0,0
0,0
0,0
0,0
0,0

(1=strongly agree; 5=strongly not agree, N = 101)

Consumers‟ Perception and Attitude toward Transgenic Potatoes
Table 3 described that 70,3% of respondents always seeked the information of potatoes
consumption. The information included the origin of the potatoes (respondents more likely the
potatoes from Dieng-West Java), safety while be consumed, taste, size and the color.
Table 3. Likert scale on consumers‘ perception and attitude toward the potatoes. Statements
Always seek information of potatoes consumption.
Knowing transgenic foods on the market
Knowing transgenic potatoes (raw/processed) on the market.
Transgenic potatoes are safe to be consumed
Transgenic labeling should be attachedCirculation of food made from transgenic potatoes need to be
informed on community
Pros and cons related to transgenic including to potatoes are
occurred because of lack of information on consumer
socialization.
Transgenic potatoes need to be introduced on the consumers

1
3,0
2,0
0,0
2,0
7,9
8,9

Percentage (%)
2
3
4
67,3
2,0
27,7
38,6
12,9
46,5
0,0
7,9
91,1
33,7
20,8
42,6
80,2
3,0
7,9
78,2
6,9
5,9

5
0,0
0,0
1,0
1,0
1,0
0,0

0,0

84,2

10,9

5,0

0,0

6,9

90,1

1,0

2,0

0,0

(1=strongly agree; 5=strongly not agree, N = 101)

More or less 40,6% of respondents ever heard the circulating transgenic food on themarket. Specifically for the potatoes, most of the consumers (99%) were not informed. Therefore,276

thProceedings of The 7 ACSA Conference 2011

definition of potatoes transgenic should have been explained to the respondents at the beginning.
After that, questions related to transgenic were asked. After the explanation, 40 respondents (40%)
said that they would be looking for more information associated with the transgenic potatoes, while
60% of respondents were not decided yet to seek transgenic potatoes information because they
were unaware with the product, beside that, they have already familiar with the potatoes sole on themarket. All consumers were not aware toward transgenic potatoes circulation in the market whether
they were in raw or processed product.
The survey also indicated that fewer consumers (36%) stated that the transgenic potatoes
were safe and edible. Transgenic potatoes were derived by placing a gene from naturally blightresistant wild potato, so the origin was still from the potatoes, less pesticide and guaranteed fromthe government (Curtis et al, 2004) become the reasons of these arguments. Reducing pesticide
becomes essential factor for consumer acceptances‘ to transgenic food (Wen and Rickertsen.,
2002; Hoban, 1999). The positive response from consumers are might because the respondent did
not have enough information about transgenic products (Onyango et al, 2004; Arianti., 2009). Study
conducted by Jill et al (2001) indicated that they had less knowledge about biotechnology, they
though more positive about the use of biotechnology in food production. The rest repondents had
no opinion regarding to the transgenic potatoes since they had no knowledge what transgenic was.
Respondent opinions to the importance of transgenic labeling placed 88%. Consumers
also argued that transgenic potatoes information should be disseminated to the communities.
Furthermore, regarding the potatoes transgenic pros and cons, most of respondents‘ response theissues emerged because the community awareness toward the issues were still low due to the lackof information. Therefore, almost all consumers agreed that information about the transgenic
potatoes should be shared to the communities.
Consumer Acceptances‟ toward the Potatoes Transgenic
The consumer acceptances‘ to transgenic potatoes based on the consumer beliefs that thetransgenic potatoes were safe to be eaten, consumer willingness to consume transgenic potatoes
and the influence of debating transgenic potatoes to consumer decision resulted that 55.4% ofconsumers were willing to accept transgenic potatoes as long as no negative side effect. Moreover,the debate about transgenic crops does not affect consumer decisions to eat the product. About
44.6% of respondents were avoid to accept transgenic potatoes because they had no information,never heard and seen the potatoes, so that they were still reluctant to consume it.
Chi-square to test the connection between two variables among age, education,
occupation, income, consumption frequency, perception, an important potato criteria‘s according toconsumer and attitude showed that only consumption frequency and consumers‘ potato criteria
significant was related to consumer acceptances‘ to transgenic potatoes with value 0,032 and 0,021(table 4).
Table 4. Chi-Square test (significant in 5%, N=101)
No
1
2
3
4
5
6
7
8

Statements
Age
Education
Occupation
IncomeConsumption frequency
Perception
Criteria‘s
AttitudeChi-square value
0.781
0.148
0.687
0.549
0.032*
0.226
0.021*
0.303

Significant in 5%.

Meanwhile, logit binomial test indicated that the relationship between variables age,education, occupation, income, consumption frequency, perception, an important potato criteria‘saccording to consumer and attitude overall to consumer acceptance‘s to transgenic potatoes
resulted that age, occupation, income, consumption frequency and perception did not affectconsumer acceptance‘s to transgenic potatoes. The variables affected consumer acceptance‘s
were education and an important potato criteria‘s with value of 0,028 and 0,013, significant in 5%,
respectively (Table 5).

thProceedings of The 7 ACSA Conference 2011277

Table 5. Logit binomial test
No
1
2
3
4
5
6
7
8

Statements
Age
Occupation
IncomeConsumption frequency
Perception
Attitude
Education
Criteria‘s

Logit value
0.515
0.367
0.679
0.102
0.216
0.315
0.028*
0.013*

Significant in 5%.

Conclusions
About 55,4% of consumers were willing to eat transgenic potatoes as long as no negativeside effect, while 46% of them were not willing to consume it because they did not have any
information about the products. The most factors influencing household acceptance to transgenic
potato were consumption frequency and consumers‘ potato criteria. Moreover, logit binomial test
showed that education and consumers‘ potato criteria were the most important factors for thehousehold to accept the transgenic late blight resistant potatoes.

References
Ameriana M, W Adiyoga, L Sulistyowati and D Ma‘mun. 1998. Perilaku konsumen rumah tangga dalam menilai
kualitas kentang. J.Hort.7(4):944-951.
Arianti GD. 2009. Penerimaan petani terhadap produk transgenik. Tesis, Sekolah Pasca sarjana Institut
Pertanian Bogor.
Byrne PF. 2006. Safety and public acceptance of transgenic products. Crop Sci.46 :113-117.
Curtis KR, Jill JMC and TI Wahl. 2004. Consumer acceptance of genetically modified food products in thedeveloping world. AgBioForum, Vol.7 No 1 & 2, article 13.
Hawkes JG. 1994. Origin of Cultivated Potatoes and Species Relationships in Bradshaw JE, Mackay GR, editor.
Potato Genetics. CAB International. Pp 3-42.
Hoban TJ. 1999. Consumer acceptance of biotechnology in the US and Japan. Food Technology 53(5) 50-53.
Institute of Food Technologies.
Kotler P dan AB Susanto.2001.Manajemen pemasaran Indonesia, Buku 2, Jakarta. Salemba Empat.
Onyango B, R Govindasamy, W Hallman, Ho-Min and VS Puduri. 2004. Consumer acceptance of geneticallymodified foods in Korea : factor and cluster analysis. Northeast Agricultural and Resource of Economics
Association and Canadian Agricultural Economics Society Annual Meeting, Canada. June 20-23, 2004.
Riska, 2009. Penerimaan ibu rumah tangga terhadap produk transgenik. Tesis, Sekolah Pasca Sarjana Institut
Pertanian Bogor.
Song J, JM Bradeen, SK Naess, JA Raasch, et al. 2003. Gene RB cloned from Solanum bulbocastanum
Confers Broad Spectrum Resistance to Potato Late Blight. Proc. Natl. Acad. Sci. USA, 100: 9128-9133.
Wen SC and KRickertsen. 2002. Consumer acceptance of GMO: Survey results from Japan, Norway, Taiwan,
and the United States. The Ohio State University Working Paper: AEDE-WP-0026-02.

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Relations Between the Amyloplast Sedimentation in Tubers andthe Morphogenesis of Tubers in Yams
Michio Kawasaki a*, Seito Hosokawa a, Hayuru Yoshida a,
Kyoko Takahashi a, Hiroki Suto aaFaculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan*Corresponding author: Faculty of Agriculture and Life Science, Hirosaki University,
3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan
Tel.: +81 172 39 3815; fax: +81 172 39 3815.
kawasaki@cc.hirosaki-u.ac.jp

Abstract
Tubers which have different forms in Japanese yam (Jinenjo) and Chinese yams were used in this study. In
developing tubers of all materials, amyloplasts were locally formed at the part beneath the stele in tuber apexes.
However, the numbers of total amyloplasts in each cell and settling amyloplasts in each cell at the parts ofelongating tubers of Nagaimo and Jinenjo were greater than their numbers in the parts of thickening tubers of
Genkotsujirou and Iseimo. When tubers of all materials matured, few amyloplasts were observed at the part. In
this study, we discussed about the relations between the amyloplast sedimentation in tubers and themorphogenesis of tubers in yams.
Keywords: amyloplast, gravitropism, morphogenesis, starch granule, yam

Introduction
The tuber of Dioscorea is generally regarded as a tuber but it has some characteristics
different from conventional tubers. Therefore the tubers of Dioscorea are also regarded as
rhizophores. The tubers have various shapes. Even in a cultivar or a line, the form of tubers may bevariable. The inconstant nature causes the quality of tubers and the work efficiency to decrease.
Previously we reported that many amyloplasts were locally formed and settled down by gravity at
the apexes of elongating tubers in Chinese yam (Kawasaki et al. 2008). In this study, relations
between the amyloplast sedimentation in tubers and the tuber morphogenesis were investigated byusing some Chinese yam (Dioscorea opposite Thunb.) and Japanese yam (Dioscorea japonica
Thunb.).

Materials and Methods
Japanese yam (Jinenjo) and Chinese yam, cv. Nagaimo which elongate until their harvest
times to form long tubers, Chinese yam, cv. Iseimo which spherically thickens until harvest time,
Chinese yam, cv. Genkotsujirou which elongates in the initial stage and then spherically thickens,were used. Developing and mature tubers were sampled and observed with a light microscope.
Morphological characterization related with amyloplasts in tuber apexes was mainly investigated inthis experiment.

thProceedings of The 7 ACSA Conference 2011279

Results and Discussion
In developing tubers of all materials, amyloplasts were locally formed at the part beneaththe stele in tuber apexes. However, at the part beneath the stele in tuber apexes, the numbers ofamyloplasts in each cell and amyloplasts which settled down in each cell were not the same among
materials. The numbers of amyloplasts and settling amyloplasts at the parts in elongating tubers ofNagaimo and Jinenjo were greater than their numbers at the parts in thickening tubers of
Genkotsujirou and Iseimo (Fig.1). In Genkotsujirou, the number of amyloplasts which settled downat the part of tubers in thickening stage more decreased than it in elongating stage. In tubers ofIseimo, both the amyloplasts in each cell and the amyloplasts which settled down in each cell at thepart were lower level throughout growing period. When tubers of all materials matured, few
amyloplasts were observed at the part. While amyloplasts had been accumulated in parenchyma ofstele in tubers with growing, their directional bias was not observed in each cell in all materials.
Crystal cells and tannin cells dispersed in the part beneath the stele in tuber apexes as in cortex
surrounding the stele in all materials. The crystals and the tannin bodies were not also ubiquitous ineach cell at the part in each material (Fig.1).
It was shown that the amyloplast sedimentation in tuber apexes was common structuralcharacteristic in some Dioscorea materials. In root caps of plants, amyloplasts have been reported
to act as gravisusceptors (Blancaflor et al., 1998; Perbal, 1999) and it is known that they are related
to morphogenesis of the roots. The results from this study indicated that the amyloplast
sedimentation would be concerned in gravity perception and the characteristic of the sedimentation
possibly was concerned in the morphogenesis of tuber in each material. We are also carrying out
further research with a lot more cultivars and lines in Dioscorea by using electron microscopes.

(a) Diagram of longitudinal section of tubers. (b) Longitudinal sections of the part beneath the stele in tips of developing tubers.
The sections were stained with toluidine blue O. Arrowheads indicate amyloplasts. Cc, crystal cell; G, the direction of gravity; Tc,
tannin cell. Genkotsujirou was investigated at thickening stage.

Figure 1. Amyloplast distribution in tips of developing tubers.

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References
Blancaflor EB, JM Fasano and S Gilroy. 1998. Mapping the functional roles of cap cells in the response of
Arabidopsis primary roots to gravity. Plant Physiol. 116: 213-222.
Kawasaki M, M Taniguchi and H Miyake. 2008. Dynamics of amyloplast sedimentation in growing yam tubers
and its possible role in graviperception. Plant Prod. Sci. 11: 393-397.
Perbal G. 1999. Gravisensing in roots. Adv. Space Res. 24: 723-729.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011281

Optimalising Potato Productivity in Sembalun Highlands,
Nusa Tenggara Barat – Indonesia
Baiq Nurul Hidayaha*, Sudjudia, Lia Hadiawatia, Dwi Praptomo Sudjatmikoa,
Peter Dawsonb, Terry Hillb, Julie M. WarrenbaInstitute for Assessment of Agricultural Technology, Nusa Tenggara Barat Province, Indonesiab
Department of Agriculture and Food, Western Australia*
Corresponding author: Balai Pengkajian Teknologi Pertanian (BPTP) Nusa Tenggara Barat,
Jalan Raya Peninjauan Narmada, Lombok Barat – NTB, Indonesia 83010
nurul.hidayah@yahoo.com

Abstract
Potato cultivation in Nusa Tenggara Barat (NTB) Province of Indonesia locates in the Rinjani Valley highlands at
1,050 – 1,250 m above sea level. Farmers grow the Atlantic variety for a crisp processing company. The yield of
Atlantic is low which is about 18.2 ton/ha, however, this is a profitable crop for farmers. Elsewhere in Indonesia
the frequent cropping of potatoes has led to soil quality decline. To reduce the decline, potato farmers in NTB
used compost and superphosphate to increase potato productivity. They also applied a new management
technique. This is important because potatoes are a new crop in NTB and specific management suited to local
conditions which has not yet been developed. The research was conducted at six farmers‘ fields and involving
six Farmer Field School (FFS) groups. The compost treatments applied were local compost at 3,000 and 5,000
kg/ha. The super phosphate treatments applied were 300 and 600 kg/ha. Each treatment had six replicates by
having six different farmer groups which had the same experimental design in their fields. The results showedthat there was no significant difference in yields of super phosphate at 300 and 600 kg/ha which were 33.0 and
33.1 t/ha, respectively. Similarly there was not a significant difference in yield between the compost treatmentswith 3,000 kg/ha producing 33.0 t/ha, meanwhile with 5,000 kg/ha of compost produced 32.7 t/ha.
Keywords: productivity, potato, compost, farmer initiated learning, Nusa Tenggara Barat

Introduction
Potato (Solanum tuberosum) is an important vegetable commodity in Indonesia
having a good market, both as a vegetable and raw material for food processing industries.
Because of the high demands, potatoes are expected in the future for diversification ofcarbohydrate sources that may increase sustainability and overcome poverity in Indonesia. In
Indonesia, potatoes are produced in 21 provinces, the biggest are in Sumatra and Java with the
cropping area are around 64,148 ha. Demand for table and processing potatoes increases every
year. The value of imported processing potatoes to Indonesia in 2007 was US$ 40 million whch was
about 43,477 tonnes. In 2008 (from January to September) processing potato imports reached29,187 tonnes with value of US$ 28 million. In 2009 Indonesia imported 48,000 tonnes with value of
US$ 33 million. Indonesian domestic potato production has increased steadily at over 3% perannum since 1997 and reached 1,176,304 tonnes in 2009 (Badan Pusat Statistik, 2011).
Potato production is generally conventional and traditional or continuing from previous
generations, so that yields are still low which is from 14.9 to16.4 tonnes/ha (average yield inIndonesia). Many problems are found to increase potato production in Indonesia including: (1) low
quality and quantity of seed potatoes, which forms the main concern in the effort to increase potato
production in Indonesia (Fuglie et al. 2006), (2) current conventional cultivation techniques(Kuntjoro, 2000), (3) topographic factors, where high areas with suitable temperatures for growing

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potatoes are very limited (Kuntjoro, 2000), (4) the tropical conditions are optimum for thedevelopment of pests and disease of potato crops (Kuntjoro, 2000).
In NTB, potatoes are mainly cultivated in the Sembalun sub-district on the slopes of Mount
Rinjani, from about 1,050 to 1,250 m asl. Potatoes are now the most important horticulturalcommodity for the Sembalun community. The potato variety grown by Sembalun farmers is Atlantic
with a yield of 18.2 tonnes/ha (BPTP NTB, 2009) which is still relatively low because in several
potato studies in Indonesia the yield has been 35 tonnes/ha (BPTP NTB, 2009). Sembalun farmers
can grow potatoes in the dry season as well as the wet season. In the wet season potatoes areplanted in dryland that has a potential area of more than 1,500 ha and in the dry season potatoes
are planted in paddy fields after the rice harvest in the months of June and July with a potential areaof 1,105 ha. In 2010, the percentage of the paddy area used to produced potatoes was just 15 %.
The main constraint to development of Atlantic potatoes in Sembalun is sub-optimal
application of integrated crop management (ICM). Farmers still predominantly use chemical fertiliser
and do not use organic fertiliser although there is the potential to produce and develop the localorganic fertiliser. Farmers are also accustomed to control pests and diseases with chemical
pesticides without observing threshold levels indicating whether control is required or not, also apply
mixtures of various pesticides together without paying attention to the active ingredients causing an
impact on the important natural predators. The dominant use of chemical fertiliser and pesticides is
the reason why potato enterprises have high costs. In fact the constant use of chemical fertiliser will
damage the soil structure and make the soil hard (Nurmayulis and Maryati, 2008). Furthermore, theexcessive use of chemical pesticides will destroy the insect biodiversity and lead to the death of
insects and other microorganisms antagonistic to pests and pathogens (Nurmayulis and Maryati,2008).
The constant practising of conventional potato production systems will reduce theprofitability of farmers (BPTP NTB, 2009), and in the long term will cause environmental damage
and the loss of biodiversity in this region (BPTP NTB, 2009). Because of this, a sustainable andenvironmentally friendly plan to support agricultural development through the study of optimalising
potato yield in the Sembalun highlands is needed. Sembalun is a small, isolated potato production
area without specialist potato support services and so research into optimising potato production
inputs will have to be carried out by the farmers themselves with local extension workers.
To overcome the contraint of sub-optimal crop management a technique was required thatenabled farmers to become their own researchers. The technique used was a modification to the
FFS methodology. The aim was to instigate demonstration plots that allowed the impact of single
management changes to be measured by farmers. Previously the potato FFSs had compared an
ICM plot versus a conventional plot. This resulted in a range of management changes between theplots which made it difficult to identify the cause of improvements in profits between the treatments.
We call this improved methodology Farmer Initiated Learning (FIL).
This research aimed to help farmers develop and put into use a less costly potato
production system that will significantly increase the ability of small farmers to take up potato
production in the Sembalun highland.

Materials and Methods
Experimental set-up
Simple FIL experiments to test one variable were introduced to Sembalun Farmer Field
Schools in 2009. The research was conducted at six farmers‘ fields involving six Farmer InitiatedLearning groups. At Sembalun six farmer groups compared the use of superphosphate withcompost. The plots were around 1000 m2 and all had a 50 m2 yield assessment sub-plot pegged in
the center. This meant that the yield measured by each farmer group came from the same size plot.
The results from each farmer group were used as replicates. Results were presented on a t/hathProceedings of The 7 ACSA Conference 2011283

basis. The experiment was located at the Sembalun highland of NTB. The FIL groups were allmembers of the overarching farmer group Kelompok Horsela. The experiment was conducted at
one growing season from June to November 2009.
Cultural practices and conditions
The compost treatments applied were local compost at 3,000 and 5,000 kg/ha. The
superphosphate treatments applied were 300 kg/ha and 600 kg/ha. Other fertilisers applied were
the same and included; NPK 600 kg/ha and sulphate of ammonia 300 kg/ha. The plot design isshown in Figure 1.

Farmer‘s conventional
practicee.g. 600 kg/ha super
phosphate

b
o
r
d
e
R

FIL treatment, 300
kg/ha super phosphate

0.5 m
To the left the farmers‘ usual rate of superphosphate was applied. To the right a lower experimental rate of superphosphate wasapplied. Plots varied in size according to individual. Yield was determined by harvesting a 50 m 2 sub-plot in the centre of eachtreatment.

Figure 1. Example of experimental plot design.

Statistical analysis
The simple experiments were executed to plan by the six farmer groups. Yields were
measured and recorded from the yield sub-plots. The results from each of the six farmer groups
were used as replicates in an ANOVA. All the data were statistically analysed using analysis ofvariance (ANOVA). The significance of treatment effect was determined using F-test, and to
determine the significance of the difference between the means of the two treatments, leastsignificant differences (LSD) were estimated at the 5% probability level.

Results and Discussion
There was no significant difference in yields from superphosphate applied at 300 and 600
kg/ha. The respective yields were 33.0 and 33.1 t/ha (Table 1). Similarly there wasn‘t a significant
difference in yield between the compost treeatments with 3,000 kg/ha producing 33.0 t/ha while
5,000 kg/ha produced 32.7 t/ha (Table 1). This indicated that farmers can improve their efficiency ofphosphate and compost, therefore, it also meant to improve income through the reduction in the
input costs.
Super phosphate cost was 2000 Rp/kg (BPTP NTB 2009) and the average farmer uses
was 433 kg/ha. The finding that 300 kg of super phosphate is sufficient for potato production in the
paddy areas of Sembalun means that they can save 133 kg of super phosphate or 266,000 Rp/ha
which will improve farmers‘ income through reduced input costs.
Compost at 5,000 or 3,000 kg/ha did not significantly affect the yield of potatoes. Manure
costs 497 Rp/kg (BPTP NTB 2009) and the average farmer uses 3,192 kg/ha. The finding that
3,000 kg of compost is sufficient for potato production in the paddy areas of Sembalun means that
there can be a saving of 192 kg of compost or 95,425 Rp/ha for the average farmer which will also
improve farmer income because of reduced input costs. Farmers who use above average organic284

thProceedings of The 7 ACSA Conference 2011

manure, the savings will be greater. For example, farmer who previously used 5,000 kg/ha ofcompost then he reduced to 3,000 kg/ha, the savings would be 994,000 Rp/ha.
Table 1. Results of Farmer Initiated learning-by-doing plots investigating the effect of lower super phosphateand compost rates – NTB 2009

Treatment
Super phosphate
300 kg/ha
600 kg/ha
SignificanceLSDnCompost3,000 kg/ha
5,000 kg/haSignificanceLSDnDescription

Yield(t/ha)

experimental rate
farmers‘ usual application rate

33.0
33.1
ns
1.4
6.0

experimental rate
farmer‘s usual application rate

33.0
32.7
ns
2.8
6.0

Conclusions
There was no significant difference in yields resulted from superphosphate at 300 and 600
kg/ha which produced 33.0 and 33.1 t/ha respectively. Similarly there wasn‘t a significant differencein yield between the compost treatments at 3,000 kg/ha producing 33.0 t/ha while at 5,000 kg/ha
produced 32.7 t/ha. Therefore farmers can improve their efficiency of phosphate and compost
inputs to improve income through reduced input costs. The six FIL groups within Kelompok Horsela
demonstrated that these farmers and their agricultural extension workers now have the capacity toplan and coordinate a series of simple though specialized potato experiments. Sub-group results
could be analyzed as replicates in an ANOVA of the combined results. This means that this isolatedgroup of farmers can now undertake their own objective testing of new management techniques.

Acknowledgments
We would like to thank the Australian Centre for International Agricultural Research (ACIAR), Balai
Pengkajian Teknologi Pertanian (BPTP) Nusa Tenggara Barat Province and the Department of Agriculture andFood, Western Australia (DAFWA) for funding this project. The assistance of collaborator farmers from
Kelompok Horsela and extension workers from Dinas Pertanian, Kecamatan Sembalun, is gratefully
acknowledged too.

References
Badan Pusat Statistik, 2011. Produksi Sayuran di Indonesia. Accessed 9 March 2011.
http://www.bps.go.id/tab_sub/view.php?tabel=1&daftar=1&id_subyek=55&notab=15.
BPTP NTB, 2009. Sosial Ekonomi Kentang Sembalun. (Balai Pengkajian Teknologi Pertanian NTB, Narmada)English translation attached to ACIAR Final Report AGB/2005/167 Appendix 2 Economic Baseline Surveyof Potatoes. http://aciar.gov.au/publication/FR2011-24
Fuglie, KO, W Adiyoga, R Asmunati, S Mahalaya and R Suherman. 2006. Farm demand for quality potato seedin Indonesia. Agricultural Economics 35: 257-266
Kuntjoro, A.S., 2000. Produksi Umbi Mini Kentang G0 Bebas Virus melalui Perbanyakan Planlet secara Kultur
Jaringan di PT. Intidaya Agrolestari (Inagro) Bogor – Jawa Barat. Skripsi. Jurusan Budidaya Pertanian
Fakultas Pertanian, Institut Pertanian Bogor. Pp 62.

thProceedings of The 7 ACSA Conference 2011285

Nurmayulis and Maryati, 2008. Content of Nitrogen and Yield of Potato Supplied with Fermented Organic Matter,
Azospirillum sp., and Nitrogen Fertilizer in Cisarua, Lembang, West Java. Jurnal Tanah Trop., Vol 13, No.
3, Pp 217-224-- back to Table of Content --

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Transformation and Transient Ekspresion Analysis of L-HBsAg DNA in
Fruits of Musa acuminata Colla cultivar „Ambon Lumut” and „Mas‟ using
Agrobacterium tumefaciensSony Suhandono*, Lailatul B, Ernawati Giri R, Putri DU, Dina RR, Ima MZ
School of Life and Sciences, Bandung Institute of Technology, Bandung, Indonesia* Corresponding author: Bandung Institute of Technology. Jl. Ganesa No 10, Bandung 40132,
West Java, Indonesia.
Tel./fax.:+62-22-2534107, +62-22-2511575.
sony@sith.itb.ac.id

Abstract
Conventional HBsAg is still expensive for most Indonesia citizens. Recently, scientists developed plant-based
vaccine which will give easy delivery, flexible production scale up, and produce cheaper vaccine. Agrobacteriumtransformation has been widely used in genetic engineering. This research was conducted to transform andanalyze the L-HBsAg DNA expression in banana fruit using Agrobacterium tumefaciens and vacuum infiltration
method. Unripe Musa acuminata Colla cultivars ‗Ambon Lumut‘ (AAA) and ‗Mas‘ (AA) were used in thistransformation. The slices of banana were co-cultivated with A. tumefaciens strain AGL1 for Ambon lumut and
GV3101 for Mas banana in three days at 25˚C±1˚C in the dark. Both strains contain a plasmid pCAMBIA 1390
with MeEF-1α promotor and L-HBsAg gene. Expression of L-HBsAg was confirmed by using RT-PCR with genespecific primers of L-HBsAg. A 1,323 bp band of L-HBsAg was observed in gel electrophoresis as a result of RTPCR. The sequence of recombinant plasmid was read with specific primers for L-HBsAg. These steps were
performed using MACROGEN services. These results indicated that the cDNA sequences of samples andprotein-coding DNA sequences of L-HBsAg were identical. Therefore, it can be concluded that the L-HBsAg
DNA was inserted and expressed in unripe fruits of Ambon lumut and Mas banana.
Keywords : transformation, Agrobacterium tumefaciens, L-HBsAg gene, Musa acuminata, ambon lumut banana,mas banana

Introduction
Hepatitis B is infectious disease caused by Hepatitis B virus (HBV). It was estimated that
more than 400 million people worldwide suffer from this disease (Piramanayagam et al., 2008).
Effort to tackle this problem like vaccination has been available since 1980 (European Centre for
Disease Prevention and Control, 2008). However, the vaccination program does not run well indeveloping countries like Indonesia because of the high price of the vaccine and syringe andinadequate facility for the storage and distribution of vaccines (Streatfield and Howard, 2003). Oneof the solutions developed to overcome these problems is the production of vaccine in a plant.
Research on the production of Hepatitis B vaccine in a plant is constantly being developed.
Large hepatitis B surface antigen or L-HBsAg is the latest variant of HBV vaccine. It consists of preS2, pre-S1, and S antigen (Lou et al., 2007). It is expected that vaccination using the L-HBsAg maytrigger immune responses effectively in preventing HBV infection.
Agrobacterium-mediated transformation is a method to be used to transform plant withforeign DNA fragments encoding L-HBsAg into the plant genome. A segment of Agrobacterium
tumefacines Ti-plasmid called T-DNA is moved from a bacterium cell into the host plant
chromosomal genome. The T-DNA region is bordered by two 25 bp direct repeat sequences, called
left border and right border. After entering the plant host cells, the T-DNA sequence will express
genes that incorporated into it. For this reason, the targeted recombinant gene usually is inserted in
the T-DNA region (Taiz and Zeiger, 2002).
thProceedings of The 7 ACSA Conference 2011

287

One of the plants that can be used to express the hepatitis B vaccine is banana. Banana
trees can be grown in tropical regions and many people from various backgrounds and ages
consumed the fruit. Bananas can be directly consumed so that the degradation process of thevaccine (protein) due to heating can be avoided (Radji, 2004). In this study expression analysis of
the gene encoding L-HBsAg proteins in Ambon lumut and Mas banana fruit was made transiently
because it requires less time and the result in transgene expression levels could be higher thanstable expression in transgenic plant (Sheludko, 2008).

Materials and Methods
Plant material
Unripe banana used in this experiment was Ambon lumut and Mas fruits. The banana was
specially ordered from a farmer. Age of the fruit was about three months from flower formation. The
fruit skin color is green and gummy when peeled. While the fruit flesh is white and hard. The fruit
was cut with a thickness of about 2-3 cm. Then the skin of the fruit was peeled off. The pieces were
immersed in 1% sodium hypochloride for 10 min. After that, it was rinsed twice using sterile distilledwater. The soaked pieces were cut again with a thickness of 2 mm. Each of the banana slices as
thick as 2 mm was further divided into 8 sections.
Agrobacterium tumefaciens
Strain A. tumefaciens that used in transformation were AGL1 for Ambon lumut and
GV3101 for Mas banana. Both strains contain a plasmid pCAMBIA 1390 with MeEF-1α promotor
and L-HBsAg gene. The Agrobacterium strain with no binary plasmid was used as a control for theexperiment.
Bacteria were activated on solid YEP medium with antibiotic for three days at 25˚C±1˚C,
250 rpm, in dark condition. Antibiotics for strain GV3101 were rifampicin at 50 ppm and kanamycin
at 50 ppm. Antibiotics for AGL1 were carbenicilin at 100 ppm, rifampicin at 50 ppm and kanamycin
at 50 ppm. While the bacteria lacking-plasmids were inoculated into the same medium, but without
kanamycin.
Transferred of L-HBsAg gene into banana fruit
The vacuum infiltration method used in this experiment was modified from Matsumoto et al(2009). Single colony of bacteria was inoculated into 10 ml of liquid YEP medium containing
antibiotics until the cell density has reached an OD600 of 0.5. An aliquot of 10% of theAgrobacterium cell suspension was again subcultured in 20 ml of same medium at same condition
until the OD600 of 0.8. After that, the cells were collected by centrifugation at 2000 g for 5 min andsuspended in infiltration medium on ½ MS (Murashige and Skoog) containing 2% of sucrose, 200µM of acetosyringone, and 0.01% of Silwet L-408.
The banana slices cut and divided into eigth pieces, were put into 1.5 ml microtube
containing 1 ml of the Agrobacterium cell suspension (OD600 = 0.8) in the infiltration medium. Then
those were subjected to vacuum for 10 min, followed by co-cultivation for three days at 25˚C±1˚C indark condition.
Expression analysis of L-HBsAg gene in banana fruit
To analyze the gene expression of L-HBsAg, the RNA isolation was performed using themethod from Kansas et al (2008). The presence of RNA was confirmed by the protocol on agarose
gel electrophoresis of RNA concentration of 2% (w/v) containing EtBr in 1x TAE buffer. DNase wasadded to the RNA preparation to eliminate genomic DNA isolation. Then the reverse transcriptasePCR (RT-PCR) was made to convert total RNA into spesific cDNA of L-HBsAg gene in the banana
fruit.
288thProceedings of The 7 ACSA Conference 2011

The iScript cDNA Synthesis Kit from Bio Rad Laboratories, Inc. were used to reverse
transcript RNA into cDNA. PCR using specific primers of MeEF-1α promotor in 25 cycles was done
to ensure the absence of genomic DNA from the total RNA. PCR was performed with Applied
Biosystem 2720 Thermal Cycler. Primer sequences used were Me-EF1α forward (5‘
AAGCTTCCAGTGAATGGTCA 3‘) and Me-EF1α reverse (5‘TGTGAACCTTCTCTCTAGACATTGTTAGT 3‘).
The PCR cycles consisted of 5 min at 94oC, 30 sec at 94oC, 30 sec at 50oC, 1 min at 72oC, and 7omin at 72 C.
An order to amplify the expression of L-HBsAg from cDNA , the primers LHB-forward(5‘GGATCCTGATGAAAATGAAGGTCCTTGTTTTCTTCGTTGCTACAATTTTGGTAGCATGGCAATGCCATGCGATGGGAGGTTGGTCCTCAAAACC-3‘) and LHB-reverse (5‘GGTCACCTTAAATGTATACCCAAAGAC-3‘)

were used. The series of cycles used were five min at 94oC, 30 sec at 94oC, 45 sec at 55oC, 1 min
and 30 sec at 72oC, and 7 min at 72oC.The PCR reaction composition were dNTP mix at 0,2 mM,
Taq buffer 1x, MgCl2 1,5 mM, of primer forward at 1 µM, primer reverse at 1 µM, sample cDNA at 1
µg, DNA polymerase at 1 unit, and deion.
cDNA from the RT-PCR was cloned using the vector pGEM ®-T Easy vector system from
Promega. Restriction analysis with EcoRI was made to confirm the clone in the plasmid. Restriction
results were visualized on agarose gel electrophoresis. A nitrogen base sequence of cDNA wasread as the last stage using specific forward and reverse primer L-HBsAg from MACROGEN.

Results and Discussion
Transformation of L-HBsAg gene into banana fruit
RNA isolation results indicated the presence of two RNA bands that dominant in the midagarose gel, which was ribosomal RNA (rRNA) 28S and 18S (Figure 1a). Quantification of total
RNA with a spectrophotometer showed that the ratio λ A260/λ A280 in samples isolated RNA was
1.36 for Ambon lumut and 1.35 for Mas banana. The ratio obtained indicated that the purity of theisolated RNA was low because there may contain the impurities, such as genomic DNA andproteins. Electrophoresis results were also seen on the tape at the top indicating the presence ofgenomic DNA. Therefore, the isolation of total RNA were added DNase to eliminate genomic DNA
so it was not interfere the next process.

a.

b.

Figure 1. (a) Electrophoresis result of mRNA and (b) RT-PCR product from Ambon lumut (a) and Mas (b)bananaConfirmation the presence of L-HBsAg gene was made using PCR spesific primer of LHBsAg. Electrophoresis results of RT-PCR products is shown in Figure 1b obtained after threeconsecutive PCR. This suggests that the gene encoding L-HBsAg was successfully inserted andexpressed in unripe Ambon lumut and Mas banana.
thProceedings of The 7 ACSA Conference 2011289

Confirmation the presence of L-HBsAg gene was also made by restriction analysis andreading the sequence of nucleotide bases from cloned RT-PCR result. The results of restriction(Figure 2b) indicate that the presence of two bands of DNA size at 1300 and 3000 bp in Ambon
lumut and Mas banana.
Final stages was reading the sequence of recombinant plasmid with specific primers for LHBsAg. These steps were performed using MACROGEN services. The results from MACROGEN
shows that the cDNA sequences of samples and protein-coding DNA sequences of L-HBsAg are
identical. Therefore, it can be concluded that the cDNA sample was a cDNA of L-HBsAg. Thismeans the L-HBsAg transgene successfully expressed at the mRNA level (transcription) in unripe
ambon lumut and mas banana in transient system.

ab
Figure 2. (a) restriction map of clone vector, (b) electrophoresis result from white colony that restricted using
EcoRI. Sample 1 was ambon lumut while sample 2 was mas banana

Acknowledgements
Our sincere thanks to PT Mata Pelangi and Mommentive for The Silwet L-408 and Silwet L-77
donation.

References
European Centre for Disease Prevention and Control. 2008. Ann. Epidemiological Report on Communicable
Diseases in Europe. Pages 3–7, 76–85, 146–150.
Lou X, Yao Q, Zhang Z, Peng R, Xiong A and Wang H. 2007. Expression of the Human Hepatitis B Virus Large
Surface Antigen Gene in Transgenic Tomato Plants. Clinic. and Vacc. Immun. 14 (4): 464 – 469.
Matsumoto K, Cabral GB, Teixeira JB and Monte DC. 2009. Agrobacterium-mediated Transient Expression
System in Banana Immature Fruits. African J. of Biotech. 8 (17), pp. 4039-4042.
Piramanayagam P, Choudhuri G and Singh SP. 2008. Hepatitis B: News from the research world. Hep. B.
Annual. 5:146-62.
Radji M. 2004. Pemberian Vaksin Melalui Tanaman Transgenik. Majalah Ilmu Kefarmasian 1 (1).
Sheludko YV. 2008. Agrobacterium-Mediated Transient Expression as an Approach to Production of
Recombinant Proteins in Plants. Recent Patents on Biotech. 2: 198-208.
Streatfield S J and Howard J A. 2003. Plant-Based Vaccines. Intern. J. for Parasit. 33:479-493.
Taiz L and Zeiger E. 2002. Plant Physiology, 3nd Edition. Massachusets : Sinauer Association, Inc.

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The Study and Early Evaluation of Resistance of Banana Accessions forWilt Disease Caused by Fusarium oxyporum f.sp.
cubense VCG 01213/16 (TR4)
Agus Sutantoa,b,*, Sudarsonoa, Dewi Sukmaa, Catur HermantobaDepartment of Agronomy and Horticulture, Bogor Agricultural Institute, Bogor, IndonesiabIndonesian Tropical Fruit Research Institute, Solok, Indonesia*Corresponding author: Indonesian Tropical Fruit Research Institute,
Jl. Raya Solok-Aripan Km. 8, PO. Box 5 Solok, West Sumatera Indonesia,
Tel: +62 755 20137, Fax: +62 755 20592
bagusutanto_02@yahoo.com

Abstract
Fusarium wilt is one of main diseases of banana in Indonesia. This disease has destroyed banana plantation inalmost all parts of Indonesia and it is difficult to be managed by agronomic and chemical controls. However,some species/cultivars show tolerance or resistance to Fusarium wilt. It indicates that those species/cultivars
have resistance genes in their genomic DNA. The evaluation of banana plants for Fusarium wilt resistance canbe carried out artificially using young plants from tissue culture. The objectives of this research were to evaluate
young acclimatized tissue culture plants for Fusarium wilt resistance and to study the resistance mechanism ofplant to Fusarium wilt disease. The experiment used five banana accessions; there were Calcuta-4 (AAw),
Ketan (AAB), Klutuk (BB), Kepok (ABB) and Ambon Hijau (AAA), and double compartment for planting system.
6
Before planting on the plastic cup containing sterile sand medium, roots of the plantlets were dipped in 10
conidia suspension of Fusarium oxysporum f.sp. cubense for five minutes. All of the cups containing inoculated
plants were put on the plastic trays. The data were collected five weeks after planting. Base on the value of DSI(disease severity index) of RDI (rhizome discoloration index) and LSI (leaf symptom index), accessions will be
categorized into highly susceptible, susceptible, tolerant and resistant. Result showed that Klutuk, Calcuta-4,
Ketan and Kepok were categorized as tolerant, while Ambon Hijau was susceptible. All of tolerant accessions
had symptom on leaves (LSI) and/or rhizome (RDI) at low level, and they still grew well.
Keywords: banana, resistance, fusarium wilt, early evaluation

Introduction
Banana is the most important fruit in Indonesia. Based on Ministry of Agriculture database(http://aplikasi.deptan.go.id/bdsp/hasil_kom.asp) banana contributed 31.10% of national fruit
production in 2009, with the production reached 6.37 million ton. This potecy can support threemain programmes of agricultural development; food security, development of agribussiness andprosperity. However, the successfull of those programmes are constrained by pests and diseases
development that affect banana plantation with vary disease intensity rank from 0.08-100%(Hermanto et al., 2011). One of destructive banana diseases in Indonesia is fusarium wilt diseasecaused by Fusarium oxysporum f.sp. cubense (Foc). Fusarium wilt control using practical cultures such as
chemical, soil treatments, crop rotation, organic amendments may reduce the severity of the disease but
relatively difficult to be adopted commercially (Pegg et al.,1993), therefore, the use of resistance cultivars isthe best alternative for controlling this disease.
Field evaluation is the most reliable menthod for disease resistance selection, however, it requires
high cost, manpower, space and facing the risk of environmental stress. Afforts to simplyfy the evaluation
procedures have been carried out such as the use of young plant or in vitro calus as selected materials(Chand et al., 2008), specific pathogen race or toxin as selective agent (Hadrami et al., 2005), and screenhouse or in vitro condition as selection method (ŠVábová and Lebeda, 2005). The objectives of thisthProceedings of The 7 ACSA Conference 2011291

research were to evaluate young acclimatized tissue culture plants for fusarium wilt resistance andto study the resistance mechanism of plant to fusarium wilt disease

Materials and Methods
Planting Material, Foc Inoculation and Planting
The experiment used 10-15 cm in size of acclimatized in vitro plantlets of five banana
accessions; Calcuta 4 (AAw), Ambon Hijau (AAA), Ketan (AAB), Kepok (ABB) and Klutuk Wulung(BB). Plantlets were gently uprooted and only those with healthy roots will be used for experiments
by dipped in the Foc suspension (106 conidia/mL) for 5 min before replanted into cups containing
sterile sand and placed in the trays for maintenance and observation. Plantlets were watered
everyday and fertilized using nutrient solution (Hyponex®) every week.
Evaluation
Disease symptoms on leaves were recorded after the first two weeks, four weeks. Finalevaluation was observed at fifth week based on the leaf symptom index (LSI) and rhizome
discolouration index (RDI) (Mohamed et al., 2001).
Scales for leaf symptom index (LSI) were:
1. No streaking or yellowing of leaves. Plant appeared healthy
2. Slight streaking and/or yellowing of lower leaves
3. Streaking and/or yellowing of most of the lower leaves.
4. Extensive streaking and/or yellowing on most or all of the leaves.
5. Dead plant.
Scales for rhizome discolouration index (RDI) were:
1. No discoloration of tissue of stellar region of rhizome or surrounding tissue.
2. No discoloration of stellar region of rhizome; discoloration at junction of root and rhizome.
3. Trace to 5% of stellar region discolored.
4. 6-20% of stellar region discolored.
5. 21-50% of stellar region discolored.
6. More than 50% of stellar region discolored.
7. Discoloration of the entire rhizome stele.
8. Dead plant.
After collecting data of LSI and RDI, the overall Disease Severity Index (DSI) for leaf
symptoms and rhizome discoloration for each accession was calculated as follows:

Furthermore DSI of LSI and RDI were translated into four categorize; resistant, tolerant, susceptible
and highly susceptible (Table 1).
Table 1. Translation of DSI scales
DSI Scales for LSI

292

DSI Scales for RDI

Translation

1

1

Resistant

Between 1.1 and 2

Between 1.1 and 3

Tolerant

Between 2.1 and 3

Between 3.1 and 5

Susceptible

Between 3.1 and 4

Between 5.1 and 8

Highly susceptiblethProceedings of The 7 ACSA Conference 2011

Results and Discussion
Early Evaluation of Banana Accessions toward Fusarium Wilt VCG 01213/16 (TR4)
Susceptible cultivar ‗Ambon Hijau‘ produced disease symptom within two weeks after
inoculation, with leaves chlorosis were started from older leaves to the younger leaves. Tolerant
cultivar ‗Klutuk Wulung‘ produced no symptom on leaves until five weeks after inoculation. The DSIof both LSI and RDI of five accessions and their susceptibility or tolerance status are shown in
Table 2.
Table 2. The status of susceptibility or tolerance of five banana accessions
Calcuta-4
LSI
RDI
1
1
1
1
1
1
1
1
1
1
1
1
3
3
1
1
2
3
1
1
1
1
1
1
1.25
1.33
Tolerant

Samples
1
2
3
4
5
6
7
8
9
10
11
12
DSI
Status
Notes:

LSI
RDI
DSI

Ketan
LSI
RDI
1
1
1
1
1
1
3
3
2
3
2
2
2
3
1
1
2
3
1
1
1
1
1
1
1.50
1.75
Tolerant

Klutuk Wulung
LSI
RDI
1
1
1
1
1
1
1
4
1
1
1
1
1
1
1
1
1
3
1
1
1
1
1
1
1. 00
1.42
Tolerant

Ambon Hijau
LSI
RDI
2
4
2
2
1
1
4
5
2
4
2
5
3
5
2
4
2
5
2
4
2
3
2
3
2.17
3.75
Susceptible

Kepok
LSI
RDI
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1.17 1.17
Tolerant= Leaf Symptom Index= Rhizome Discoloration Index= Disease Severity Index

Base on DSI of both LSI and RDI (Table 2), all accessions except Ambon Hijau werecategorized as tolerant. Only one inoculated plant of Ambon Hijau had no symptom, while others
showed symptom on leaves (LSI from 2 to 4) and rhizome (RDI from 2 to 5) (Figure 1B). Ambon
Hijau is the member of Cavendish subgroup, which is naturally susceptible to Foc TR4. This finding
was consistent with Hermanto et al. (2011), reported that 81% Foc-infected Ambon Hijau inIndonesia was caused by TR4 (VCG 01213/16). External symptom of infected leaves and internal
symptom of Ambon Hijau were shown at Figure 1B. Meanwhile, only two out of twelve plants ofCalcuta-4 showed Foc symptoms (DSI of LSI=1.25 and RDI=1.33, respectively) and the status of
this accession was tolerant (Figure 1C). Calcuta-4 is wild-seeded species (Musa acuminata sp.
burmanica) and they are often used for Fusarium wilt resistant breeding program (Tomekpe et al.,2004).
Five out of twelve plants of Ketan showed symptom on leaf (LSI from 2 to 3) and rhizome(RDI from 2 to 3), however, DSI values of LSI and RDI were translated as tolerant (Figure 1E).
Ketan also showed tolerant to Foc in some areas in Lampung, West Sumatera and West Java.
Ketan had some synonyms in some places; Janten (Lampung), Jantan (West Sumatera), Uli (WestJava), and Ketip (Nusa Tenggara Barat). This cultivar is popular as cooking banana. An interesting
occurrence was shown by Klutuk Wulung. Two out of twelve plants produced symptom on rhizome(RDI=3), nevertheless, all plants showed no symptom on leaves (Figure 1A), therefore, DSI of LSI
and RDI of this accession was translated as tolerant. Klutuk Wulung is seeded Musa balbisiana andscattered in Java, which usually people use leaves of it for wrapper and male bud flower forvegetable.

thProceedings of The 7 ACSA Conference 2011293

Another tolerant accession was Kepok, which two out of twelve plants showed symptom
on leaves and rhizome (Figure 1D), and translation of DSI was tolerant. Kepok is very popular
cooking banana and grows in whole part of Indonesia. Cases of infected plant of Kepok were foundin West Java, Yogjakarta, Central Kalimantan and South Kalimantan. Kepok was not only infected
by VCG 01213/16 (TR4), but also VCG 0120 and 01218 (Hermanto et al., 2011).

A

B

C

D

E

Figure 1. External (leaves) and internal (rhizome) symptoms of Fusarium oxysporum f.sp. cubense VCG
01213/16 on Klutuk Wulung (A), Ambon Hijau (B), Calcuta-4 (C), Kepok (D) and Ketan (E).

Resistance Mechanisms of Plant to Fusarium Wilt Disease
Resistance mechanism of plant against pathogen was started before the infection ofpathogen into plant tissues. Fusarium oxysporum f.sp. cubense infects banana plant through rootsystem. Since conidia attach hairy roots, they will germinate and penetrate into the epidermal cellsof roots. Roots of resistant cultivar produce exudates that inhibit germination and growth of conidia;
otherwise, exudates from roots of susceptible cultivar induce germination and growth of conidia (Liet al., 2011).
Fungal pathogen is capable to penetrate plant roots through invading root epidermal cells
directly, epidermal cell of root caps and elongation zone, natural wound in the lateral root base.
During invasion, fungal hyphae produce cell wall degradation enzymes and penetrate to intercellularspace, grow and develop branches and penetrate to other cells. Besides enzymes, fungal pathogen
also produces micotoxins such as fusaric acid and beauvericin that affect trans-membrane electric
potential, electrolyte leakage and respiration root cells (Pavlovkin 2006). Cell membrane damage
causes the production of reactive oxygen species (ROS) and elicit the production of antioxidants
such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) (Kuzniak 2001), andtransduction signal molecules that will trigger the production of pathogenesis related proteins such

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as chitinase and β-1,3-glucanase. These enzymes will degrade fungal cell wall and inhibit growthand development of pathogen in the plant cells (Wu et al. 2008).
The case in Klutuk Wulung, which infection symptom appeared in the rhizome but nosymptom on the leaves (Figure 1A) indicated that resistance mechanism of plant against pathogen
occurred. The development of fungi was localized only in part of rhizome and blocked for further
expansion.
This evaluation technique was adequate for screening of Fusarium wilt resistant cultivars
and the expression of the disease can be obtained within 6-8 weeks. Using small plants forevaluation can reduce space requirement when compared to field evaluation.

References
Chand R, D Sen, KD Prasad, AK Singh, BM Bashyal, LC Prasad and K Joshi. 2008. Screening for disease
resistance in barley cultivars against Bipolar sorokiniana using callus culture method. Indian J Exp Biol.
46:249-253.
Hadrami AE, AE Idrissi-Tourane, ME Hassni, F Daayf, IE Hadrami. 2005. Toxin-based in-vitro selection and its
potential application to date palm for resistance to the bayoud Fusarium wilt.C. R. Biologies 328:732–
744.
Hermanto C, A Sutanto, Jumjunidang, HS Edison, JW Daniells, WTO Neill, VGO Sinohin, AB Molina and P
Taylor. 2011. Incidence and distribution of fusarium wilt disease of banana in Indonesia. Proc Int ISHSProMusa Symp on Global Perspective on Asian Challenges. Acta Hort. 897:313-322.
Kuzniak E. 2001. Effects of fusaric acid on reactive oxygen species and antioxidants in tomato cell cultures. J
Phytopathol 149(10):575-582.
Li C, S Chen, C Zuo, Q Sun, Q Ye, G Yi and BZ Huang. 2011. The use of GFP-transformed isolates to study
infection of banana with Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol. 131:327-340.
Mohamed AA, C Mak, KW Liew, and YW Ho. 2001. Early evaluation of banana plant at nursery stage for
Fusarium wilt tolerant. In: AB Molina, NH Masdek, KW Liew, Eds. Banana Fusarium Wilt Management:
Toward sustainable cultivation, Los Banos, INIBAP-ASPNET. Pp:174-185.
Pavlovkin J, I Mistríková, M Luxová, I Mistrík. 2006. Effects of beauvericin on root cell transmembrane electric
potential, electrolyte leakage and respiration of maize roots with different susceptibility to Fusarium. Plant
Soil Environ. 52(11):492–498.
Pegg KG, Moore NY, Sorensen S. 1993. Fusarium wilt in the Asian Pacific region. In: Valmayor RV, Hwang SC,
Ploetz RC, Lee SW, Roa NV, eds. Procceeding of International Symposium Recent Developments in
Banana Cultivation Technology. Los Baños: International Network of Banana and Plantain/ASNET.
Pp:225-269.
ŠVábová L and A Lebeda. 2005. In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens.
J Phytopathol. 153(1):52-64
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Wu HS, XM Yin, DY Liu, N Ling, W Bao, RR Ying, YY Zhu, SW Guo, QR Shen. 2008. Effect of fungal fusaricacid on the root and leaf physiology of watermelon (Citrullus lanatus) seedlings. Plant and Soil 308: 255266.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011295

Response of Plant Roots to Drought Stress
Atsushi Kato, Saki Hoshiyasu, Akiho Yokota and Kinya Akashi*
Graduate School of Biologiccal Sciences, Nara Institute of Science and Technology*Corresponding author: Graduate School of Biologiccal Sciences, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara, 630-0192 Japan
Tel.: +81 743 72 5561; fax: +81 743 72 5569.
akashi@bs.naist.jp

Abstract
Around the world, terrestrial plants are strongly affected by abiotic stresses such as drought, high or low
temperatures, high light and salt stress. Among them, drought is one of the major factors limiting distribution andproductivity of plants. Understanding the mechanisms for drought resistance in plants is important for breedingnew crop varieties which can grow under water deficit conditions. In particular, plant root plays pivotal roles inwater uptake and maintenance of water status of plants, thus is an important research target for plant
physiologists and molecular biologists. Responses of plant roots to drought have been extensively investigatedin model plants such as Arabidopsis, which have offered basic knowledge on the molecular mechanisms
underlying the multilateral physiological responses in the roots. Moreover, recent physiological and molecularstudies on drought-resistant plants have shed lights on how these ‗xerophytes‘ cope with severe water deficit
conditions by developing unique survival strategies.
Keywords: root development, drought stress, abscisic acid, xerophyte, wild watermelon

Morphological changes of roots in response to drought stressDrought stress severely inhibits plant growth (Larcher, 1995). However, development ofroot is usually less affected than that of shoot under drought. In maize, it is reported that root growthis maintained at lower water potentials, while shoot growth is completely inhibited (Sharp et al.,2004). This report also showed that elongation activity is highly retained in root tip regions underwater deficits. On the other hand, in the case of Brassicaceae species including Arabidopsis,
inhibition of primary root elongation under drought is accompanied with morphogenetic
differentiation process in the lateral roots. In this process, lateral roots remain short, hairless andoften take a tuberized shape with concomitant accumulation of starch and proline (Vartanian et al.,
1994; Xiong et al., 2006). This so-called ‗short-roots‘ then switch to a dormant mode under
prolonged drought periods, until the resume of growth upon rehydration conditions (Wasilewska etal., 2008). These observations therefore offer an illustrative example on the flexibility of root systemarchitecture in plants under fluctuating water availability in the soils.
Abscisic acid (ABA) signaling and root morphogenesis
ABA is one of the major plant hormones which mediate adaptation of plants toenvironmental stresses. Many plants synthesize a large quantity of ABA in every tissue under stress(Shinozaki et al., 2003). In the leaves, ABA is well known to stimulate stomatal closure and thus
reduce transpirational loss of water under drought. In the roots, on the other hand, ABA inhibits
elongation of primary roots (Leung et al., 1997; Bai et al., 2009) and decreases a number of lateralroots (Xiong et al., 2006). Moreover, ABA is shown to be implicated in the development of the‗short-roots‘ (Schnall and Quatrano, 1994). Furthermore, analysis of Arabidopsis mutants revealed
that ABA-deficiency inhibits morphological changes of roots in response to the stress, which
impaired stress resistance in these mutants. It is suggested that ABA and ABA signaling elements

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are important factors for regulating morphological changes of the roots under drought stress(Wasilewska et al., 2008).
Compatible solutes in the roots
In many plant species, responses to drought stress involve changes in the fluxes of
specific metabolic pathways, which lead to the accumulation of compatible solutes such as sugar
derivatives and amino acids. Previous studies suggested that proline accumulation in the roots
contributes to the osmotic adjustment and fortification of water uptake (Sharp et al., 1990; Voetberg
and Sharp, 1991). In a recent study using Arabidopsis mutants impaired in proline metabolism, it isdemonstrated that proline biosynthesis, transport and degradation are regulated in tissue- andstage-specific manners during drought (Sharma et al., 2011). Under drought condition, proline ismainly synthesized in photosynthetic tissue, and a part of the proline is transported to root
meristems, which is further catabolized to various metabolites to support continued growth at low
water potential. Arabidopsis mutants deficient in ABA biosynthesis show impaired proline
metabolism and root growth elongation under water deficits, which is reversed by exogenous ABA(Sharma et al., 2011). These observations suggested that proline metabolism is partly regulated by
ABA signaling pathway under drought, and these molecular networks coordinately contribute to theadaptation response of plants under stress conditions.
Root tropism under drought
In response to environmental signals, plants exhibit tropism to control the direction of their
growth. Root system development is largely directed by the interaction of gravitropism andhydrotropism, which are the responses to gravity and moisture gradient, respectively (Gilroy and
Masson, 2008). Gravitropism is dominantly orientated on primary roots, which is mediated by
sedimentation of starch anchors in the amyloplasts of columella cells. The lateral gradient of auxin
in the root cap is also known to be implicated in the root gravitropism. In water-stressed roots,hydrotropism is accompanied by the rapid degradation of amyloplasts in columella cells, which
reduces responsiveness to gravity (Takahashi et al., 2003; Eapen et al., 2005). Recent genetic
analysis in Arabidopsis revealed that hydrotropism is mediated by novel factors such as MIZ1 and
GNOM, the latter encoding guanine-nucleotide exchange factor for ADP-ribosylation factor-type G
protein (Kobayashi et al., 2007; Miyazawa et al., 2009), suggesting that GNOM-mediated vesiculartrafficking plays a pivotal role in hydrotropism.
Root development in xerophytes
Plants which survive in water-deficit environments are called xerophytes, which develop
unique drought resistance mechanisms (Wickens, 1998; Graham and Nobel, 1999; Akashi et al.,2008). Most xerophytes are characterized by their deep root system architecture. A global-scale
investigation of 253 woody and herbaceous plant species reported that plants in the arid and semiarid regions have more developed roots, with an average of 9.5±2.4 m for desert, and 15.0±5.4 m
for tropical grassland/savanna, which are deeper than those for more humid regions such astemperate deciduous forest (2.9±0.2 m), temperate grassland (2.6±0.2 m) and tundra (0.5±0.1 m)(Canadell et al., 1996). Notably, maximum rooting depth was found to be 68 m for Boscia
albitruncas in the central Kalahari Desert. These results illustrated that development of deep rootsystem is advantageous for reaching to the deep water table in the soils, thereby enabling thesurvival in the water-limiting environments.
Interestingly, some xerophytes are characterized by their very shallow root systemarchitecture (Graham and Nobel, 1999). For example, African baobab (Adansonia digitata) reaches
to a height of 25 m with a trunk of up to 10 m diameter; nevertheless, roots of mature trees arethProceedings of The 7 ACSA Conference 2011297

significantly shallow and rarely elongate beyond 2 m (Sidibe and Williams, 2002). Similarly, cactus(Opuntia ficus-indica) and agaves (Agave deserti) develop shallow and broad root system in which
lateral roots are extensively produced. It has been discussed that these shallow roots areadvantageous for capturing water derived from light rains, which are notorious for their infrequency
in arid regions. These observations therefore suggest that contrasting strategies are employed byvarious xerophytes, to survive in the competitive environments for water resources.
Wild watermelon as a xerophyte model to study root response under drought
Deep root system observed in xerophytes is a favorable trait for future molecular breeding
program towards water-saving agriculture. However, molecular mechanisms underlying this trait
have not been fully elucidated. Wild watermelon is a xerophyte inhabiting the Kalahari Desert in
Botswana, despite carrying out C3-type photosynthesis (Kawasaki et al., 2000; Miyake and Yokota,
2000), therefore offers an interesting model for how C3 plants can cope with severe drought
conditions. Previous physiological and molecular analyses demonstrated that root development ofwild watermelon is significantly enhanced at the early stage of drought stress, which isaccompanied with a dynamic change in the root proteome in this plant (Yoshimura et al., 2008).
Drought-induced proteins include those for root morphogenesis such as actin and α-tubulin. Various
enzymes for carbon/nitrogen metabolisms such as triosephosphate isomerase, malate
dehydrogenase and methionine synthase are also induced under the stress, suggesting a globalchange in the cellular metabolism in the roots to support root growth at the early stage of drought
stress. Interestingly, further changes in root proteome are observed at the later stage of drought
stress, where factors for stress tolerance such as lignin synthesis-related proteins and molecularchaperones are preferentially induced at this stage. These results, therefore, suggested thepresence of complex molecular networks for regulating abundance of respective proteins in thisplant, in a temporally-programmed manner.
Towards understanding the molecular mechanisms for developing deep root system,
experimental systems for exploring genetic resources in a given xerophyte should offer great
opportunities to characterize genes involved in their root growth. A system for genetic engineeringhas been recently established in wild watermelon, where hairy root transformation techniqueenables to integrate a given foreign gene construct to both wild and domesticated varieties ofwatermelon (Kajikawa et al., 2010). Moreover, generation of a large-scale EST database, as well astranscriptome analysis are ongoing for wild watermelon, which should offer valuable platforms forscreening and mining useful genes in this xerophyte. It is anticipated that these approaches would
unravel the molecular mechanisms of root vigor in this xerophyte under drought.

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-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011299

Growth and Development Characteristics of Hoya multiflora Blume
Sri Rahayu
Bogor Botanical Gardens, Indonesian Institute of Sciences
Jl. Ir. H. Juanda 13 Bogor, Indonesia
srirahayukrb@yahoo.com

Abstract
Hoya multiflora is one of the valuable germplasm in Indonesia utilized as ornamental and medicinal plants. Thisepiphytic plant faces problems in decreasing habitat, while the culture technique of this species has notestablished yet in Indonesia. It is important that all useful plant can be maintained to prevent the exploitationfrom the wild. Biological data on growth and development will provide a guideline to establish good culture
techniques. The research aim was to investigate the growth and development of the seedlings of Hoya multiflorain two different conditions and growing media i.e. (1) in the natural habitat at the Bodogol Research Station of
Gunung Gede Pangrango National Park, and (2) in a shade house of the Bogor Botanical Gardens as artificial
habitat. Three different growing media was used in observation, i.e. (1) its phorophytes observed at natural
habitat, (2) organic matter conducted at the shade house (3) tree fern log conducted at the shade house. The
results showed that general, the growth and development of Hoya multiflora started by seed germination
characterized by opened cotyledon, seedling establishment characterized by formation of alternate leaves
arrangement, followed by growth of young plant characterized by formation of opposite leaves arrangement,
growth and development of adult plant, flowering and fruiting. The critical point was at the seedlingestablishment phase, which best reached at the cocopeat medium. When the seedling established anddeveloped to adult, there were no differences at the different growing media used. The best recruitment, growthand development of Hoya multiflora was at the cocopeat media located in the shade house.
Keywords: Gede Pangrango National Park, Hoya multiflora, growth and development, Bogor Botanical Gardens

Introduction
Hoyas (Apocynaceae: Asclepiadoideae) are becoming increasingly popular as ornamental
plant, particularly in Europe, USA and Australia (Goyder, 2008). The international trade of thisgenus is increasing, but their existence in the nature obtains less attention, eventhough theconservation status was not state yet. In contrary, their habitat as epiphyte rapidly decrease mainlyby the deforestation i.e. logging, burning, and forest conversion into plantation. The rapid increaseon palm oil plantation especially prompted by the potential use as biofuel will multiply habitat lost for
Hoyas as epiphyte.
The genus naturally originated from India, distributed to Australia and the Pacific Islands,
with the greatest diversity is now found in South East Asia, particularly in Indonesia (Kleijn &Donkelaar, 2001; Wanntorp, 2006). There are about 150-200 Hoya species in the world (Hoffman
et all, 2002), 50-60 of which are in Indonesia (Rahayu, 2001). H. multiflora widely distribute from
India to New Guinea (Goyder, 2008) from 50-1500 m above sea level (Rahayu et al., 2010). This
species is characterized by its short (non vein) plant, leathery (non succulent) oblong leaves andarrow head form white corona with yellow tip of the corolla. Flowers arranged in umbel appear atinterpetiolar node. This plant produces white latex from all of its part.
Some indigenous people in the country have been utilizing some species of Hoyas as
traditional medicine. The uses are for treat burns, cuts, convulsions, coughs, asthma, pneumonia,
elephantiasis, encephalistis, fractures, gonorrhea, hemorrhoids, inflammation, insect
bites/poisonous fish stings, orchitis, phthisis (tuberculosis), Pyroderma (skin disease caused by
microorganisms), rashes, rheumatism/arthritis, swellings, traumatic injures/wounds, stomach and

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Proceedings of The 7 ACSA Conference 2011

intestinal ailments, and childbirth tonic (Zachos, 1998). A recent study shows their high potential as
insecticide to the ―Dengue‖ mosquito. Hoya multiflora Blume is one of the economic important as
ornamental plant, and has been used traditionally as medicine, particularly to treat arthritisrheumatism (Burkill, 2002) and stomach/intestinal ailments (Ambasta, 1986). The active compoundof this plant unrevealed yet, but it could be Indomethacine like compound. Indomethacine, acommon non-steroidal anti inflammatory drug (NSAID), has been used for more than 30 years totreat symptomatic pain of rheumatoid arthritis. Recently, this compound has been tested as a newclass of anti HIV drug (Bourinbaiar & Lee-Huang, 1994) and seems to be specific since no toxicity
at clinical doses.
Despite of their high economic importance, little is known about their cultivation especiallyin Indonesia. As this plant is loved as ornamental plant and has a medicinal property, theknowledge on its growth and development is needed. The knowledge will be useful for thecultivation and living collection management for ex- situ conservation purposes. This research
aimed to observe the growth and development characteristics of Hoya multiflora Blume.

Materials and Methods
The observation on the growth and development characteristics of H. multiflora wasconducted both at their own natural habitat at the Bodogol Research Station, Gunung Gede
Pangrango National Park, West Java and in a shade house at the Bogor Botanical Gardens. There
were 10 mature, 5 young and 3 clumps of seedling observed at Bodogol RS. Observation at the
Bogor Botanical Gardens was focused on the flowering and fruiting time using 20 accession
collected from the Bodogol RS (2004-2006); and seedling growth and development from the fruit
produced (2005). The observation on growth and development conducted at the following
parameters:
Germination
The experiment was conducted at the Bogor Botanical Gardens shade house. The shadehouse condition was under the paranet having 75% canopy. The seeds were shown on the 2different kinds of growing media, namely (1) cocopeat at the plastic plate and (2) tree fern log. The
seeds used were from the harvested mature fresh pod in the green house. Each of 20 seeds wereshown on the media two times. The seedlings were watered daily. Parameters observed wereseedling percentage, time of opened seed coat, time of hypocotyle, time of first leaf appearance,
and time of opposite leaf formation.
Seedling establishment
Observation was conducted using the growing seedling taken from the seed growth
germination experiment. The parameter observed was started at the established seedling growth tothe young plant, recorded by the formation of opposite leaf arrangement.
GrowthThe young plant stated as the seedling formed the first opposite leaves. The observation
was done from the growth of shoot and leaf (number of leaf) to the plant formed the firstinflorescence.
Flowering and fruiting
Observation was conducted to record the flower formation, time of flowering, and lower
product. The process of flowering and fruiting were recorded. Observation also made on the fruit
ratio to the number of flower.

thProceedings of The 7 ACSA Conference 2011301

Results and Discussion
Generally, the growth and development of Hoya multiflora started from (1) seedgermination characterized by opened cotyledon, (2) seedling establishment characterized byformation of alternate leaves arrangement, (3) growth of young plant characterized by formation of
opposite leaves arrangement, (4) growth and development of adult plant, (5) to flowering andfruiting. The process of growth and development at the different condition are as follows:
Germination (in shade house condition)
The germination process of H. multiflora started from seed coat opening, followed bycotyledone opening until the first leaf forming. H. multiflora evolve the epigeal germination, which
cotyledone above soil surface. According to Copeland & McDonald (2001), there are two
germination types based on location of storage reserves. The research showed that the seed of H.
multiflora grew 2-3 days after showing and had the high viability up to 100% at the cocopeat media.
The seed of H. multiflora was similar to H. parasitica without dormancy (Rahayu & Sutrisno, 2007).
Showing seed at the tree fern media resulted in the lower viability (80%) and took longer time (7-30
days) to germatinate (Table 1). Overall, the germination process was faster at the cocopeat media
than that at the fern tree fern log. There was microclimate different condition between cocopeat
media and tree fern log, especially on the media humidity and water capacity. Cocopeat media wasreported to be able to absorb more water and keep wet for a longer time than the log. The time ofgermination is faster at the cocopeat media, as more water stimulate the seed imbibitions. Besides
that, the cocopeat media also provided ready to use nutrient. Media humidity and water content are
the main factors for seed germination. Germination of seed involves the imbibitions of water, a rapid
increase in respiratory, mobilization of nutrient reserves and initiation of growth in the embryo(Fenner & Thompson, 2006). Water is a basic requirement for germination as it is essential for
enzyme activation, breakdown, translocation and use of reserve storage material (Copeland &McDonald, 2001).
Table 1. Seed germination of H. multiflora at the two different kinds of media

Media

Seed coat
opening (days)

Cotyledone opening(days)First Leaf
Formation (days)

Tree fern log

7-30

14-45

41-60

80

Cocopeat

2-3

5-7

13-20

100

Seed viability

Seedling establishment
The seedling is characterized by alternate leaf formation. Seedling establishment wasreached 12 month after seed germination. Seed on the cocopeat media had sorter time to grow(Table 2) and the highest establishment (80%). In contrary, at the tree fern log resulted the lowest
growth and establishment (30%). Natural phorophyte was better to support seedling establishmentof H. multiflora compared with the tree fern log media. Tree fern log had the lowest water holding
capacity and nutrient availability compared with the cocopeat media and natural phorophyte. At thenatural phorophyte, the seedling of H. multiflora grew on the ant nest (Rahayu et al, 2010), which
provided nutrient and water holding capacity for the requirement of the H. multiflora to grow. During
the seedling establishment, nutrient requirement increase from the former phase (germination). The
tree fern log had the most limiting factor for seedling establishment of H. multiflora. According toFenner & Thompson (2006), factor limiting establishment are competition between seedlings andabiotic hazards i.e. occurance of physical damage and lack of moisture. The limiting factor at tree
fern log was dominantly by lack of moisture which caused seedling desiccation. Seedlingdesiccation is a particularly acute hazard in the branches of trees (epiphytes) and the main cause of

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Proceedings of The 7 ACSA Conference 2011

seedling mortality (Fenner & Thompson, 2006). While the limiting factor at the natural phorophyte
was dominantly by competition because many seeds were germinated at one hole (ant nest).
Table 2. Time of seedling growth of H. multiflora at different media
Media

Tree fern log

Number ofalternateleaves12-13

Time of first opposite
leaf formation(months)
14 ± 2,0

Plant height at the timeof first opposite leaf
formation (cm)18,4 ± 1,2

Successful
seedling
percentage
30

Cocopeat12-13

12 ± 1,0

25,6 ± 0,780

Natural phorophyte

13-17

13 ± 1,5

19,8 ± 1,550Young to adult
At this phase, plant growth was determined after seedling establishment, characterized by
opposite leaf formation. At this phase, growth and development of plant were dominated by theincrease in the node number followed by the increase in leaf number. There are two leaves(opposite formation) at each node. The new node emerged at about every one month. When 12
nodes completed (after one year), new branch developed at the shoot base and followed by the
emergence of peduncle. The time at this phase was quite similar to the occurance at different
growing media (Table 3). This condition indicated the stability of the plant after seedlingestablishment phase.
Table 3. Growth and development of H. multiflora at different growing media

12±1Time of firstflowering(Months after
germination)
20-28

Plant heightat the firstflowering
(cm)
30±5

Number of
branch at
the first
flowering2

28 ± 7

12±1

18-30

30±2,50-2

29 ± 8

12 ±1

18-30

40±10

0-2

Media

Time of node
growth(days)

Number of opposite
leaves‘ node at thefirst flowering

Tree fern log

30 ± 9Cocopeat
Natural phorophyte

Flowering and fruiting
All plant at the different growing media and location developed and produced flowers andfruit. The time of flower and fruit development was quite similar (Table 4). This condition indicatedthe time for seedling establishment and development to adult plant, when the reproduction process
occured, i.e. plant produced flower and fruit. It means all growing media provided nutrient and basic
requirement for the plant development. While fruiting process occurred at both different
environments i.e. at the shade house condition and at the natural habitat. The pollination process in
H. multiflora needs the presence of pollinator (Rahayu et all, 2010) and Chasanah (2010) has been
identified that Vespidae was the pollinator of H. multiflora at the Bodogol forest.
Table 4. Flower and fruit development of H. multiflora at different condition

Tree fern log

Peduncledevelopment(days)
30 ± 1028 ± 514 ± 10.01

Fruitdevelopment(days)
35 ± 4

Cocopeat

29 ± 927 ± 414 ± 10.01

34 ± 5Natural phorophyte

30 ± 1129 ± 514 ± 10.01

36 ± 5Mediath

Bud development(days)

Anthesis(days)

Percentage offruit set

Proceedings of The 7 ACSA Conference 2011303

References
Ambasta S.P. 1986. The usefull plants of India. Publ. & Inform. Dir. Council of Scientific and Industrial Research.
New Delhi, India
Bourinbaiar AS; Lee-Huang, S. 1995. The non steroidal anti inflammatory drug, Indomethacin, as an inhibitor of
HIV replication. FEBS Letters 360 (1):85-88
Burkill IH. 2002. A dictionary of economic product of Malay Peninsula. Vol. 2. Ministry of Agriculture Malaysia,
Kuala Lumpur.
Chasanah LR. 2010. Diversity and frequency of pollinator insect visit and its influence to the fruit set in Hoya
multiflora. [S2 thesis]. Bogor Agric. Univ. Bogorth
Copeland, LO and McDonald, MB. 2001. Principles of seed science and technology. 4 Ed. Kluwer Acad. Publ.
LondonFenner, M and Thompson, K. 2006. The ecology of seeds. Cambridge Univ. Press, Cambridge
Goyder D. 2008. Hoya multiflora Blume. Curtis Bot. Magazine 7(1): 3-6.
Hoffman C, van Donkelaar R, Albers F. 2002. Hoya R.Br. In Albes F & Meve U (Eds.) : Illustrated Handbook of
Succulent Plants: Asclepiadaceae. Berlin: Springer-Verlag. pp: 146 - 158
Kleijn D. and R van Donkelaar. 2001. Notes on The Taxonomy and ecology of the genus Hoya (Asclepiadaceae)
in Central Sulawesi. Blumea 46: 457 -483
Rahayu S. 2001. Genetic diversity of Hoya in Sumatra. [S2 thesis]. Bogor Agric. Univ. Bogor
Rahayu S, Sutrisno (2007) Potency of Hoya seed in propagation and conservation: case study on Hoya
parasitica Wall. Buletin Kebun Raya10 (2) : 33-39 [Indonesian]Rahayu S, Kusmana C, Abdulhadi R, Jusuf M, Suharsono (2010) Distribution of Hoya multiflora Blume at
Gunung Gede Pangrango National Park, Indonesia. Journal of Forestry Research 7 (1): 42-52
Wanntorp L, Koycan A, Renner S (2006) Wax plants disentangled: A phylogeny of Hoya (Marsdenieae,
Apocynaceae) inferred from nuclear and chloroplast DNA sequences. Molecular Phylogenetics andEvolution 39: 722-733
Zachos E. 1998. Practical uses of various Hoya species. The Hoyan 19 (4)/20 (1): part II: 6-10/3-8-- back to Table of Content --

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Proceedings of The 7 ACSA Conference 2011

Effects of High Water Table and Waterlogging on Sunflower Growth, Yield
and Seed Quality
Satoko Yasumotoa,*, Yukari Terakadob, Morio MatsuzakiaaBiomass production and processing team, National Agricultural Research Center, Tsukuba, Japan,b
Ibaraki Prefectural Agricultural Experimental station, Daitoku, Ryugasaki, 301-0816, Japan* Corresponding author: Institute of National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Japan,
Tel./fax: +81 298 838 7038
ysatoko@affrc.go.jp

Abstract
Understanding the response of sunflower (Helianthus annuus L.) to high water table and waterlogging isimportant for successful cultivation in monsoon Asia. We examined the physiological responses of sunflower
cultivars to these conditions. In the rotational paddy field, where the water table depth was shallow, growth of theplant was suppressed, seed yield was decreased and harvest quality was deteriorated. In the artificially inclined
field, the ditch surrounding the sloped plot was constantly filled with water, the growth of plant and oilconcentration were decreased significantly when the water table was shallower than about 30 cm. In the pot
experiment, where water was logged at different growth stages, the relationship between seed yield and theunderground dry weight was significant (r = 0.87, p < 0.01). In cultivars with vigorous growth of undergroundpart, many adventitious roots were appeared. Screening for the appearance of these roots may be an effective
way to select sunflower cultivars that are capable of tolerating high water table and tolerating waterlogged
conditions and avoiding decreases in seed and oil yield.
Keywords: sunflower, water table, waterlogging, seed quality, adventitious roots

Introduction
Regarding the effects of short-term waterlogging on sunflower, there were some reports
about that to the growth, yield and quality. Orchard and Jessop (1984) reported on sunflower andsorghum that growth stages were greater importance than the duration of waterlogging. And
Orchard and So (1985) reported that waterlogging at the vegetative and floral initiation stages
reduced the root growth and it reduced the water use and nutrient uptake (Orchard et al. 1986).
Grassini et al. (2007) reported waterlogging during grain filling determines direct physiologicalresponses that decrease grain yield. Regarding the fatty acid, there were some reports on thefactors changing their compositions. The temperature condition was widely reported as one of thefactors. Nagao et al. (1984) and Sobrino et al. (2003) reported that the oleic/linoleic acid ratio wasincreased with higher temperature during grain filling. But there are few reports about the effects ofshallow water table to sunflower growth and quality.
We are studying the cultivation of sunflower on rotational paddy field for the purpose ofhuman consumption, and for the use of bio-diesel fuel. In the field, the shortening of maturing
periods and reductions of stem length, leaf number and disk diameter were observed in many
cultivars. And the decrease of seed yield and oil content, oleic acid composition and the increase oflinoleic acid composition were also observed. The objective of this paper is to elucidate the effectsof shallow water table on the growth, yield and quality and to elucidate the effects of waterlogging indifferent stages and in different cultivars of sunflower.

thProceedings of The 7 ACSA Conference 2011305

Materials and Methods
Field and pot experiments
Experiments I was conducted in 2007 and 2008 on an artificially sloped plot at Ibaraki
Agriculture Institute (Ryuugasaki, N: 35°54‘, E:140°12‘)The slope had 8.3 m in length and 0.86 m inheight at one end. It could set of 10 rows with different water table. Their water table depths werefrom 0 cm to 86 cm in 2007, and from 3.7 cm to 78 cm in 2008. The row spacing was 0.95 m in2007 and 0.90 m in 2008 and the distance between plants was 0.2 m in both years. The ditch
surrounding the sloped plot was constantly filled with water after establishment of seedling until thefinish of the experiment. Soil moisture content was measured by a soil moisture probe (Profile
Probe PR2, Daiki Rika Kogyo Co. Ltd., Tokyo). Two hybrid varieties were used. One was traditional
type; the name was Hybrid sunflower (Hy.) (Kaneko Seeds Ltd. Gunma), and the other was midoleic type, 63M80 (Pioneer Hi-Bred International, Inc., USA). They were sown on 7 June in 2007
and on 5 June in 2008 as early sowing and on 26 June in 2007 and in 2008 as late sowing. Before
planting, a chemical fertilizer was applied at the rate of N-P2O5-K2O = 8.4-8.4-8.4 g m-2, in 2007 and2008. Root growth of the two cultivars at flowering (22 Aug.) was examined by excavating the wholeroot systems of three plants in late sowing block in 2008 at two water depths (3.7 cm and 20.2 cm).
Stem length, leaf nitrogen, number of seeds per a flower disk, flowering date, and maturing datewere measured. Soil moisture content was measured by a soil moisture probe (Profile Probe PR2,
Daiki Rika Kogyo Co. Ltd., Tokyo). Yield of each plot was determined using the same method asreported by Izquierdo et al. (2002). Thousand kernel weight, oil content and fatty acid composition
were measured by the same method as reported by Yasumoto et al. (2011). All treatments werereplicated two or three times depending on the varieties.
Experiments II was conducted in 2010. It was examined effects of waterlogging on
sunflower at different developmental stages by a pot experiment. The pots were 50 cm long, 65 cm
wide and 45 cm deep. Seven hybrid varieties were used. In the cultivars, 3 were traditional type, 4were mid-oleic type. Before planting, a chemical fertilizer was applied at the same rate as inExperiment I. Waterlogging treatments were imposed at different developmental stages,
establishment and flower bud visible. The control was irrigated according to the necessary. Because
the flowering time was delayed by waterlogging at the stage of establishment, the seeds of theplants for this treatment were sown on 25 May, while the others were sown on 4 June in 2010. The
duration of the waterlogging was the same as in the experiments reported by Wample and Davis(1975), namely 4 days in each treatment. The parameters as in Exp. I were measured. And root dry
weight and total dry weight were also measured. Yield and seed quality were measured as in Exp. I.
The sampled roots were carefully washed using a colander. The roots and other samples weredried at 60 °C until constant weight (for about 48 hr.) in a forced-air circulation oven, to determine
the dry weight.
Sample and data analysis
Sampled seeds were air-dried. Two g seeds from each sample were crushed and the oil
was extracted with n-butyl alcohol. The measurement of oil concentration and fatty acids
composition in total fatty acid were determined by the method of Caviezel (Pendl et al., 1998) usinga gas chromatograph ( B-820,NihonBüchCo.Ltd.,Tokyo ). The concentrations of fatty acids were
calculated from their peak areas. And the percentage of the total fatty acid content was calculated
from their peak areas.
Statistical analysis
The results were analyzed by ANOVA. All statistical analyses were performed with SPSS
11.0 for Windows (SPSS, 2001). All values are expressed as mean values. Significant differenceswere established by the Tukey‘s test at P<0.05. A correlation was calculated and the significancelevels (P<0.01) are based on the Pearson coefficients.
306

thProceedings of The 7 ACSA Conference 2011

Results
Exp. I. Effects of water table depth on growth, yield and quality
In both years, stem length, disk diameter and seed and oil yield were significantly reducedwith shallower water table. Even in the condition that the depth to water table was 3.7 cm, Hy. grew
more vigorous roots near the ground surface than 63M80 (Fig. 1a-b). Fig. 2 showed the decrease ofleaf nitrogen, number of seeds per a disk, thousand kernel weight, seed yield. That was clearer
upper than about 30 cm of the depth to water table. Oil concentration was also decreased upper
than about 30 cm of the depth to water table. And oil concentration was some higher in early sowing
block. The decrease in oleic acid and the increase in linoleic acid with rising water table were also
somewhat clearer in the plants sown on early sowing block. Their results were shown about sametendency in 2007 and in 2008.
ab

Figure 1. Root growth of sunflower grown at water table depths of 3.7 cm (left) and 20.2 cm (right). (a) Hy. (b)63M80.

Leaf nitrogen (% )Thousand kernel weight (g)Number of seeds per a disk

1600

8

1200

6
4

2
0
0

30
60
Water table depth (cm)

90

800
400
0
0

30
60
Water table depth (cm)

60

30

0
0

30
60
Water table depth (cm)90

60

Oil concentration (% )Seed yield (g plant -1)

120

90

90

90
60

30
0

0

30
60
Water table depth (cm)

90

40

20

0
0

30
60
Water table depth (cm)90◆: Hy., ▲: 63M80 in early sowing, ◇:Hy., △: 63M80 in late sowing.

Fig. 2. Growth, Yield and seed quality in the inclined plot in 2007.

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Proceedings of The 7 ACSA Conference 2011307

Exp. II Effects of waterlogging on sunflower at different developmental stages
There were significant differences between the stages of waterlogging (Table1). The
sunflower was affected by waterlogging (Table1). In many cultivars, seed yield was decreased bywaterlogging at the flower bud initiation stage. The response of waterlogging to root dry weight wasdifferent between cultivars. In many cultivars, their root weight was decreased by waterlogging at
the flower bud initiation stage. In some cultivars, adventitious roots were formed after waterlogging
treatment. The high significant relationship between seed yield and plant characters as the disk
diameter and the root dry weight were observed through the different stages of water logging
treatments (Fig.3).
Table1. Effects of waterlogging treatment on the growth, yield and harvest quality in 2010
Growth stage atTypewaterlogging
Establishment

ThousandOilkernelconcentration
weight
（％）
(g)
24.6
38.7

Shoot
Root dry
Oleic Linoleic
dryweightacid acidweight(g）（％） （％）(g）36.1 50.7 39.5
6.3

Sep. 2

36

1143

126

13

13.0

North Queen

July 28

Aug.29

33

984

121

8

10.6

27.2

25.5

42.1

42.9

28.2

2.4

IS3011

July 28

Aug. 30

34

1009

104

14

17.0

36.7

35.2

40.2

47.7

47.4

6.0

63M80

July 28

Sep. 2

37

1170

67

9

7.1

19.5

12.2

68.5

19.0

13.9

2.3

Hysun521

July 26

Aug.26

32

935

89

10

9.4

27.6

30.3

58.0

27.9

29.4

3.9

Hysun 511

July 26

Aug.26

32

945

93

10

13.7

32.9

31.5

67.5

18.9

29.3

3.8

Hysun530

July 25

Aug.26

33

992

115

12

14.9

26.0

34.6

69.3

17.3

34.7

4.6

Trad. Hybrid sunflower July 31

Sep.1

33

1039

102

13

9.8

25.0

33.4

35.1

49.7

41.6

4.2

North Queen

July 28

Aug.26

30

871

132

11

9.0

24.6

30.8

43.6

42.1

50.6

3.7

IS3011

July 30

Aug.28

30

864

79

9

4.4

18.1

28.8

37.9

48.3

33.4

2.9

63M80

July 30

Sep.2

35

1124

119

14

8.9

36.6

40.5

72.6

15.1

47.2

5.8

Hysun521

July 29

Aug.28

31

982

89

8

5.9

21.1

29.3

65.0

21.8

26.0

2.1

Hysun 511

July 25

Aug.26

33

962

78

9

11.0

19.5

30.5

67.8

18.3

33.0

3.2

Hysun530

July 30

Aug.25

27

908

72

6

2.1

17.8

29.1

70.5

15.7

8.6

0.8

Trad. Hybrid sunflower July 31

Sep.1

34

1080

138

14

19.9

34.2

34.7

38.7

47.1

39.7

5.3

North Queen

July 30

Aug.26

28

826

144

11

12.7

27.4

29.6

43.6

41.3

57.0

3.7

IS3011

July 30

Sep.1

34

1048

121

15

24.2

31.6

37.9

35.2

52.9

54.9

7.9

63M80

July 29

Sep.2

36

1141

139

15

23.9

35.8

43.0

72.3

17.6

62.8

10.6

Nusun

Control

Plant
Disk
Yield
height diameter(g plant-1）
(cm)
(cm)

Trad. Hybrid sunflower July 28

Nusun

Flower bud
initiation

Ripening Sum.
Flowering Maturing
period Temp.
date
date
(days) (℃）

Cultivar

Nusun

Hysun521

July 29

Aug.31

34

967

114

14

19.3

28.8

37.1

63.2

24.1

62.1

7.1

Hysun 511

July 26

Aug.26

32

761

107

14

20.8

24.6

32.6

61.0

25.9

62.7

6.3

Hysun530

Aug. 1

Aug.31

32

876

113

12

19.4

26.5

30.2

74.2

11.0

61.3

6.4

Stage

**

**

**

**

**

**

*

ns

ns

**

**

Cultivar

**

**

**

ns

*

**

**

**

**

ns

ns

Stage×Cultivar

**

**

**

ns

ns

ns

ns

ns

ns

ns

*

ns, not significant. **, * indicated statistically significant at P<0.01 and 0.05.
ns, not significant. ** indicated statistically significant at P<0.01.
3025

r=0.60**

20
15
10
5

Establishment
Flower bud initiation
Control2530

r=0.82**
Seed yield (g・ plant－１）

Establishment
Flower bud initiation
Control

Seed yield (g・ plant－１）

Seed yield (g・ plant－１）

30

20

15
10
5

0

0
0

50

100

150

200

Plant ｌength (cm)

0

5

10

15

Disk diameter (cm)20

Establishment
Flower bud initiation
Control25

r=0.87**

20

15
10
5

0
0

5

10

15

Root dry weight (g D.W.・ plant-1)** indicated statistically significant at P<0.01.

Figure 3. Relationship seed yield and plant characters after each water logging treatment.
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thProceedings of The 7 ACSA Conference 2011

Discussion
In Exp. I, the plant growth and seed yield were reduced with shallower water table while
the oil concentration was highest in the plot with a water table depth of 30 cm. It was quite shallow.
And from this result, it was thought that the growth, yield, and seed quality of sunflower wereaffected by soil moisture conditions. In Exp. II, there were differences in the response towaterlogging between cultivars. In some cultivars, adventitious roots were formed after
waterlogging. Kramer (1951) reported the adventitious roots contributed to plant survival during
flooding. Wample and Davis (1975) reported that adventitious roots formation was one of the
responses to flooding. And Jackson (1955) reported that when the original root system wasflooded, adventitious root developed to prevent injury to the shoot and to promote shoot recovery
from flooding. And in Exp. I, the differences of root growth between cultivars was also observed.
Even in the condition that the depth to water table was 3.7 cm, Hy. grew more vigorous roots near
the ground surface than 63M80. So this difference of appearance of the root was thought to berelated to differences in the ability of these cultivars to tolerate flooding and high water table.
These results of this study suggested that the water management was important for
improving the growth and quality of sunflower. Further research is needed to identify thephysiological mechanisms responsible of sunflower to excess soil moisture.

References
Grassini P., Indaco, G.V., Pereira, M. L., Hall, A. J. and Trapani, N. 2007. Response to short-term waterlogging
during grain filling in sunflower. Field Crops Res. 101:352-363.
Izquierdo, N., Aguirrezabal, L., Andrade, F. and Pereyra, V. 2002. Night temperature affects fatty acidcomposition in sunflower oil depending on the hybrid and the phenological stage. Field Crops Res. 77:
115-126.
Jackson, W. T. 1955. The role of adventitious roots in recovery of shoots following flooding of the original rootsystems. Am. J. Bot. 42:816-819.
Kramer, P.J. 1951. Causes of injury to plants resulting from flooding of the soil. Plant Physiol. 26:722-736.
Nagao, A. and Yamazaki, M. 1984. Effect of temperature during maturation on fatty acid composition ofsunflower seed. Agric. Biol. Chem.48:553-555.
Orchard, P. W., and Jessop, R. S. 1984. The response of sorghum and sunflower to short-term waterlogging. I.
Effects of stage of development and duration of waterlogging on growth and yield. Plant and soil 81:119132.
Orchard, P. W., and So, H. B.1985. The response of sorghum and sunflower to short-term waterlogging. II.
Changes in the soil environment under waterlogged conditions. Plant and soil 88:407-419.
Orchard, P. W., Jessop, R. S. and So, H. B. 1986. The response of sorghum and sunflower to short-termwaterlogging. IV. Water and nutrient uptake effects. Plant and soil 91:87-100.
Pendl, R., Bauer, M., Caviezel, R. and Schulthess, P. 1998. Determination of total fat in foods and feeds by theCaviezel method, based on a gas chromatographic technique. J. of AOAC International 81 (4): 907-917.
Sobrino, E., Tarquis, A. and Dias, M. C. 2003. Modeling the oleic acid content in sunflower oil. Agron. J. 95:329334.
Yasumoto, S., Terakado, Y., Matsuzaki M. and Okada, K. 2011. Effects of High Water Table and Short-Term
Flooding on Growth, Yield, and Seed Quality of Sunflower. Plant Prod. Sci. 14(3): 233-248.
Wample, R. L. and Davis, R. W. 1975. Effect of aeration on the flood-induced formation of adventitious roots andother changes in sunflower (Helianthus annuus L.). Planta 127: 263-270.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011309

Population Genetics of Hoya multiflora at Sukamantri of Gunung Salak,West Java, Indonesia Based on Isozyme Analysis
Sri Rahayua* and Rochadi AbdulhadibaBogor Botanical Gardens, Indonesian Institute of Sciences (LIPI), Jl Ir. H. Juanda 13 Bogor.IndonesiabResearch Centre for Biology, LIPI, Cibinong Science Centre. Jl Raya Cibinong Bogor, Indonesia*Corresponding author: Bogor Botanical Gardens, Indonesian Institute of Sciences (LIPI),
Jl Ir. H. Juanda 13 Bogor. Indonesia. Telp/fax 0251-8322187.
srirahayukrb@yahoo.com

Abstract
Hoya multiflora Blume is tropical epiphytic plant reported having some medicinal properties in the ruralcommunities in Asiatic counties i.e India and Malaysia. In Europe and USA, this plant has been internationallytraded as exotic ornamental plant. The traded plant was directly extracted from the wild habitat, which imply on
the population equilibrium. Population genetic study on epiphytic Hoya multiflora plant has been conductedusing the plant population at Sukamantri at gunung Salak, West Java Indonesia. The study was based on the
isozyme analyses by using PER, EST, MDH, ADH, AAT. The results showed that analyses using POPGENE
software indicated that the genetic differentiation was low, but the estimation on gene flow was high.
Keywords: Hoya multiflora, isozyme, population genetic

Introduction
Hoya multiflora Blume (Apocynanceae:Asclepiadoideae) is one of the economic important
species as ornamental plant, and has been used traditionally as medicine, particularly to treat
arthritis-rheumatism (Burkill, 2002) and stomach/intestinal ailments (Ambasta, 1986). The active
compound of this plant is unrevealed yet, but it is predicted to be Indomethacine like compound.
Indomethacine, a common non-steroidal anti-inflammatory drug (NSAID), has been used for morethan 30 years to treat symptomatic pain of rheumatoid arthritis. Recently, this compound has beentested as a new class of anti HIV drug (Bourinbaiar & Lee-Huang, 1994) and it seems to be specificsince no toxicity at clinical doses.
The distribution of H. multiflora ranges from India to New Guinea (Goyder, 2008) from 50
to1500 m above sea level (Rahayu et al., 2010). This species is characterized by its short (nonvein) plant, leathery (non succulent) oblong leaves and arrow head form white corona with yellow tipof the corolla. Flowers is arranged in umbel appear at interpetiolar node. This plant produces white
latex from all of its part (Rahayu 2006). In Europe and USA, this plant has been internationaly
traded as exotic ornamental plant. The traded plant was directly extracted from the wild habitat,
which imply on the population equilibrium.
All of Hoya plant species including H. multiflora are epiphyte. In the natural habitat, thepresence of this plant depends on the presence of host plant, which defined as phorophyte(Benzing 2008). So this species faces problems in decreasing habitat due to the increase of
deforestation. The decrease on the number of forest tree will imply on the population decrease ofthis species, which in turn will imply on the genetic erosion toward extinction. Little is known about
population genetic of this species. The study on population genetic will provide data to support
conservation strategies and development of this species as economic plant. Genetic markers
generally have contributed to the study of plant biology by providing methods for detecting genetic
differences among individuals. There are some important ecological topics which often use
allozymes as powerful markers, especially to assess the genetic differentiation among populations

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(Zeidler, 2000). This research was aimed to assess the genetic differentiation of Hoya multiflora
populations at Sukamantri of gunung Salak, West Java Indonesia based on the isozyme analysis.

Materials and Methods
Sample source
A total 50 samples were obtained from three populations of H. multiflora from Sukamantri.
All populations were located at 700 – 800 m above sea level. Population 1 was from Buper
Sukamantri with the total sample of 16, population 2 was from Tapos with the total sample of 16 andpopulation of 3 were from Bobojong with the total sample of 18. All of sample were collected asstem cutting and planted at the greenhouse at Bogor Botanical Gardens as pot plants. After 2
months of acclimation, the plant produced new shoot and young leaves. Young leaves with the size
less than 3 cm long were used as enzyme source.
Isozyme extraction
The method of enzyme extraction followed Soltis and Soltis (1989). Leaves were
processed at the same day they were collected from the plants. Approximately 400 mg of samples
were placed in a mortar, extract buffer were added than it was grinded well. The liquid of extract
was adsorbed by a 0.5 x 0.5 cm filter paper and ready to placed in to the prepared gel.
Electrophoreses
Starch was prepared as follows: equal amounts of the two starch types (16.5 + 16.5 g)
were mixed well and fully suspended in 100 mL the appropriate gel buffer in a flask. The remaining
200 mL of this buffer were brought to a boil before being added to the suspension. The buffer
systems used depended on the enzyme activities to be assayed, and included the following: AAT(Aminoacid transferase), ADH (Alcohol dehidrogenase), EST (Esterase), PER (Peroksidase), MDH(Malate dehidrogenase), ACP (Acid phosphatase). The gel were mold to become firm. The wells(0.5 x 0.5 cm) were prepared and fill with the samples and indicator (bromphenol blue). The
electrophoresis was run in a refrigerator at 4oC for 3 h at 100 V at the beginning and increase to
200V.
Staining and visualization
After electrophoresis the gels were sliced producing three replicas that were placed indifferent enzyme staining solutions. Gels were placed on a white light transilluminator to improve
the identification of bands colocalizing with the specific enzyme activities.
Data Analysis
The polymorphic bands were transformed into numeric data as co-dominant data, and run
by using POPGENE software (Yeh et all, 1999). The genetic variation intra and inter population
were measure with assumption population at Hardy–Weinberg‘s equilibrium, p2+2pq+q2 = 1.
Genetic variation (Ht) in a population was measure by Nei‘s (1978), while genetic variation interpopulation (genetic diferentiation=Gst) was measure as fixation index (Fst) of (Wright 1978); Gst =Fst = (Ht-Hs)/Ht.

Results and Discussion
There were four alleles found with the various distributions. The common two allele was A
and B which express at the all isozymes. Four alleles were found at the EST, three alleles at theADH, ACP, MDH, and two alleles at PER and AAT (Table 1). Among the four alleles, A was thethProceedings of The 7 ACSA Conference 2011311

most frequent in EST, ADH, and ACP, and express more than 50% (dominant). The allele C wasonly found at EST with the lowest frequency (0.06) and categorized as a rare allele. Allele O
express in EST, ADH, ACP and MDH in low frequency (below 0,3).
Table 1. Allele frequency from total populations
Allele \ Locus

EST

PER

ADH

ACP

MDH

AAT

Allele A
Allele B
Allele C
Allele O

0.56000.14000.06000.24000.44000.5600-0.84000.16000.28000.56000.16000.04000.30000.66000.24000.38000.3800-Allele heterozygocity as shown at Table 2, was range from 0.2303 to 0.5631 with the mean
was 0.4197 (under 50%). The highest was at EST (0.5631), and the lowest was at ADH (0.2303).
The result express the low heterozygocity wich mean its low genetic diversity.
Table 2. Allele heterozygocity
Locus
EST
PER
ADH
ACP
MDH
AAT
Mean
St. Dev

Sample
size
50
50
50
50
50
50
50

Obs-HomObs-hetExp-hom*

Exp Het*Nei**

Ave Het0.60000.76000.68000.84000.48000.72000.68000.12650.40000.24000.32000.16000.52000.28000.32000.12650.38200.49710.72570.40570.51760.33310.47690.14050.61800.50290.27430.59430.4824
0.66690.52310.14050.60560.49280.26880.58240.47280.65360.51270.13770.56310.33070.23030.42200.46410.50830.41970.1218* Homozygocity and heterozygocity estimation (Levene ,1949)**Number of polymorphic loci = 6; Proportion of polymorphic loci = 100.00 %The genetic differentiation (Gst = Fst) as shown at Table 3, was very large, which
performed by the Fst = 1.1485. A very large genetic differentiation was performed by Fst more than
0.25.
Table 3. F statistic and gene flow estimation
Locus

Sample size

Fis

Fit

Fst

Nm*

EST
PER
ADH
ACP
MDH
AAT
Mean

50
50
50
50
50
50
50

0.28470.2581-0.4273
0.6161-0.1472
0.44440.22680.33390.5015-0.1967
0.7165-0.1293
0.56630.36500.06880.32800.1616
0.26170.0156
0.21940.17883.38630.51211.29700.705315.78170.8896
1.1485*Nm = Gene flow estimated from Fst = 0.25(1 - Fst)/Fst.
According to Wright (1978), genetic differentiation classified as follows:
Fst = 0 – 0,05 indicated low genetic diffrentiation
Fst = 0,05 – 0,15 indicated moderate genetic differentiation
Fst = 0,15 – 0,25 indicated large genetic differentiation
Fst > 0,25 indicated very large genetic differentiation
Gene flow (Nm) was estimated from genetic differentiation (Gst) as follow:
Nm = 0.5(1 - Gst)/Gst

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A very large genetic differentiation, mean there were very large differentiation between(inter) populations, and indicated low genetic diversity at intra populations. This is also indicated thevery low of gene flow. At this condition, conservation strategy was need to conserve of all
population, and sampling was need from representative of all population.

References
Ambasta S.P. 1986. The usefull plants of India. Publ. & Inform. Dir. Council of Scientific and Industrial Research.
New Delhi, India
Bourinbaiar AS; Lee-Huang, S. 1995. The non steroidal anti inflammatory drug, Indomethacin, as an inhibitor of
HIV replication. FEBS Letters 360 (1):85-88
Burkill IH. 2002. A dictionary of economic product of Malay Peninsula. Vol. 2. Ministry of Agriculture Malaysia,
Kuala Lumpur.
Goyder D. 2008. Hoya multiflora Blume. Curtis Bot. Magazine 7(1): 3-6.
Hoffman C, van Donkelaar R, Albers F. 2002. Hoya R.Br. In Albes F & Meve U (Eds.): Illustrated Handbook of
Succulent Plants: Asclepiadaceae. Berlin: Springer-Verlag. pp: 146 - 158
Kleijn D. and R van Donkelaar. 2001. Notes on The Taxonomy and ecology of the genus Hoya (Asclepiadaceae)
in Central Sulawesi. Blumea 46: 457 -483
Rahayu S. 2001. Genetic diversity of Hoya in Sumatra. [S2 thesis]. Bogor Agric. Univ. Bogor
Rahayu S, Sutrisno. 2007. Potency of Hoya seed in propagation and conservation: case study on Hoya
parasitica Wall. Buletin Kebun Raya10 (2) : 33-39 [Indonesian]Rahayu S, Kusmana C, Abdulhadi R, Jusuf M, Suharsono. 2010. Distribution of Hoya multiflora Blume at
Gunung Gede Pangrango National Park, Indonesia. Journal of Forestry Research 7 (1): 42-52
Wanntorp L, Koycan A, Renner S. 2006. Wax plants disentangled: A phylogeny of Hoya (Marsdenieae,
Apocynaceae) inferred from nuclear and chloroplast DNA sequences. Molecular Phylogenetics andEvolution 39: 722-733
Zachos E. 1998. Practical uses of various Hoya species. The Hoyan 19 (4)/20 (1): part II: 6-10/3-8
Soltis DE, Soltis PS. 1989. Isozymes in plant biology. Dioscorides Press, Portland, Oregon .
Zeidler M. 2000. Electrophoretic analysis of plant isozymes. Acta Univ. Palacki. Olomuc. BIOLOGICA 38 : 7–16
Yeh FC, Yang Rc , and Boyle T. 1999. POPGENE VERSION 1.31 Quick User guide. Univ of Alberta and
CIFOR. Canada-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011

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Endophytic Bacteria as an Alternative Agent for the Biological Controlof Plant Parasitic Nematodes on Black Pepper
Abdul Munif a,*, Rita Harni, SiswantobaDepartment of Plant Protection, Bogor Agricultural University ,Bogor, IndonesiabResearch and Development Agency, Ministry of Agriculture, Indonesia*Corresponding author: Department of Plant Protection, Bogor Agricultural University,
Jl. Kamper Kampus IPB Darmaga, Bogor, Indonesia 16680.
abdulmunif@ipb.ac.id

Abstract
Plant parasitic nematodes cause damage and losses significantly to black pepper in Indonesia. Control of plantparasitic nematodes with pesticides is often restricted due to their high toxicity and negative impact on theenvironment. The need for environmentally safe control strategies has increased interest in developing biologicalcontrol measures. Endophytic bacteria are ubiquitous in most plant species and reside within healthy planttissue without producing symptoms of damage. The internal plant habitat provides several advantages forendophytic bacteria as biological control agents: 1) colonization of an ecological niche also used by plant
pathogens, 2) less competition with other microorganisms, 3) sufficient supply wirth nutrients, 4) less exposure
to environmental stress factors, and 5) better translocation of bacterial metabolites throughout the host plant.
The objective of this work was to evaluate the effect of endophytic bacteria isolated from black pepper to control
Meloidogyne incognita on black pepper. The results showed that some endophytic bacteria were able to reduce
the root galls caused by Meloidogyne incognita and juveniles of nematodes in the soil up to 70% as well as topromote the growth of pepper seedlings.
Keywords: endophytic bacteria, Meloidogyne incognita, black pepper, root gall

Introduction
Black pepper (piper nigrum L) is one of the important export commodities in Indonesia.
However, the production of black pepper is now threatened by pests and diseases. One of the maindisease on black peper is yellow diseasae caused by plant parasitic nematodes i.e. Meloidogyne
sp. and Radopholus similis. Several control methods have been developed to combat thenematodes, but they are still a serious problem of black pepper especially in Bangka Island,
Indonesia. The use of chemical pesticides with persistent pesticides can result in negative impacts
to the environment, pathogens become more resistant, disruptive presence of beneficial microbes inthe soil, and human health. Use of pesticides also provides residual effects on pepper which iscurrently an one obstacle for consumers, mainly for export purposes that are very concerned about
the health and environmental aspects. In connection with the need to develop a system ofagricultural production, including plant disease control systems are environmentally sound one ofthem by optimizing the use of biological agents.
Endopyitic bacteria are bacteria living inside plant tissues without doing symptoms onthese plants. As the internal plant habitat, endophytic bacteria provide several advantages as
biological control agents: 1) colonization of an ecological niche also used by plant pathogens, 2)less competition with other microorganisms, 3) sufficient supply with nutrients, 4) less exposure toenvironmental stress factors, and 5) better translocation of bacterial metabolites throughout the hostplant. Several studies have shown that endophytic bacteria isolated from various plant tissues areable to suppress plant parasitic nematodes Meloidogyne incognita on cotton and tomato plants(Hallmann 2001) as well as to control plant nematode Pratylenchus sp on patchouli (Harni 2010). In
addition, some bacterial isolates endophytes have been reported to enhance plant growth becausethProceedings of The 7 ACSA Conference 2011315

it can increase the availability of nutrients to induce plant resistance. The objective of this research
was to determine the potential of biological agents bacterial endophytes isolated from pepper forcontrolling Meloidogyne incognita on pepper and their effects on the plant growth.

Materials and Methods
Isolation of Endophytic Bacteria
A total of 10 samples of healthy pepper plants were taken at random each of pepper
plants and were cultivated in Bangka, Bogor and Sukabumi. The pepper plant roots weretransported to the laboratory for immediate processing. The roots were washed with running tap
water to remove adherent soil particles then blotted dry on tissue paper. The root material was
weighed and surface sterilized with alcohol at 70% for 30 sec and in 2% of sodium hypochlorite(NaOCl) containing 0.01% of Tween 20 for 3 min, followed by four rinses with sterile 0.01 M of
potassium phosphate buffer (PB) at pH of 7.0 (80 g NaCl, 2 g KCl, 11.5 g Na2HPO4, 2 g KH2PO4).
To confirm complete surface sterilization (sterility check), the surface disinfected roots were
imprinted on Tryptic Soy Agar (TSA). If bacterial growth occurred within 48 h, samples werediscarded. The pepper roots were then macerated with a sterile mortar and pistil in three times PB(w/v). The macerate was decanted into sterile conical flasks and shaken for 30 sec. A dilution series
was made and 100 µl of each dilution was plated onto 1/10 strength of TSA on petri disk. Petriplates were incubated at 24°C for 2-3 days and colony forming units (cfu) were determined. Threereplicates were made for each dilution. On each petri plate containing approximately 10 bacterial
strains was marked and all bacterial strains from this zone were transferred and purified on fullstrength of TSA. The bacterial strains were stored in Tryptic Soy Broth (TSB) plus 20% of glycerol
at -20°C. Two isolates collection of endophytic bacteria were used in this experiment.
Inoculum of Parasitic Nematodes
Inoculum of nematode Meloidogyne incognita was isolated from the infected roots pepperof nematodes in Central Bangka, Province Bangka-Belitung Indonesia. Subsequently, thenematodes were cultured and propagated on susceptible tomato plants (cv. Ratna) for 2 months.
After that plant was uprooted and the nematodes was extracted and used as a source of inoculum.
Effect of Endophytic Bacteria on Meloidogyne incognita
The effectiveness of biological agents against M. incognita on pepper seedlings was donein a greenhouse. Two months pepper cuttings with one segment were treated with isolates ofendophytic bacteria. Eight isolates of selected bacterial endophytes isolated from root pepper andpatchouli isolate MER7, AA2, HEN1, HEN3, MER9, ANIC, TT2 and EH11 were used in this study.
The bacterial isolates were grown on TSA medium 100% for 24-48 h at room temperature. A single
colony of the bacterial isolate was transferred into 100 ml of liquid TSB medium and shaken for 2days with a speed of 150 rpm at a room temperature. Furthermore, the bacterial suspension wascentrifuged at 11.000 rpm for 15 min at -4oC to separate the supernatant/culture filtrate with abacterial cell culture. Suspension of bacteria was made by diluted the bacterial cell with sterilewater. Roots of 3 months old of pepper seedlings were soaken for 1 h in bacterial suspension with apopulation density of 109-1010 CFU. The treated pepper was planted subsequently in pots. One
week after the bacterial treatment, the plants were inoculated with 1000 larvae of the nematode M.
incognita per plant. Each treatment was repeated for 5 times and arranged in a completely
randomized design, with positive control was the plant only nematode inoculation, while negative
control was plants no inoculation with nematodes. Three months after inoculation the plants were
harvested and the number of galls and the population of nematodes in the roots and the soil wereobserved as well as the plant height and weight, root weight, number of branches and number ofleaves.
316

thProceedings of The 7 ACSA Conference 2011

Results and Discussion
Effect of Endophytic Bacteria against M. incognita on Black Pepper
In this study, eight isolates of endophytic bacteria isolated from root pepper and patchouli
were tested on seedling of black pepper against M. incognita in the greenhouse. All of isolates ofbacterial endophyte were able to reduce gall and juveniles of M. incognita. Four out of eight
isolates, i.e EH11, HEN1, HEN3 and TT2 significantly reduced gall nematodes compared to thecontrol. Isolate EH11 showed the highest in reducing gall nematodes compared to other isolates(Table1). The isolates of bacteria were also able to suppress juveniles of nematode in the soil
compared to control. Six isolates of EH11, AA2, HEN1, MER9, ANIC and TT2 significantly reduced
number of juveniles compared to control. The highest influence in suppressing number of juvenilesof M.incognita was showed by isolate HEN1 and followed by EH11. Five Isolates of HEN1, EH11,
TT2, ANIC, and AA2 were able to reduce the population of M. incognita in the soil at more than75%.
Table 1. Effect of isolates of biological agents bacterial endophyte against the number of galls and the
population of nematodes larvae
Treatments
Isolate MER 7
Isolate EH11
Isolate ANIC
Isolate MER 9
Isolate AA2
Isolate HEN1
Isolate HEN3
Isolate TT2Control + (with nematode)

Number ofgalls
70,4 ab
9,0 c
66,0 ab57,4 ab
48,8 bc
42,2 bc
38,4 bc
32,6 bc
101,0 aPopulation of thejuveniles
1212 b23 ef
312 cde504 c
352 cd
13 f
1176 b50 ef1432 abPopulation of
reduction (%)
15,36
96.97
78,21
64,80
75,41
99,00
17,87
96,50
-

Values followed by same small letters on the same column are not significantly different at 5% by DMRT.

Biological agents can suppress the development of plant diseases through a mechanismof competition, predation and the resulting antibiotics (Kloepper et al. 1991). Some research
indicates that the use of biological agents endophytic bacteria through seed treatment reduce 3050% of the amount of gall of M. incognita on cotton plants (Hallmann et al. 1997). Some bacterial
endophytes isolated from the roots patchouli suppress populations of Pratylenchus brachyurus at
73.9% on patchouli plants in the greenhouse (Harni et al. 2007). Biological agents, including
endophytic microbial may protect plants against plant-parasitic nematodes through various ways
such as by producing toxic compounds that are nematicidals (Sikora et al. 2007; Yang et al. 2011).
In addition, certain bacteria can also suppress the development of plant diseases due to the abilityof bacterial endophytes in binding Fe (III) and to produce HCN (Keel et al. 1992). A treatment of
culture filtrate of endophytic bacterial isolate isolated from patchouli was also able to reduce
Pratylenchus barchyurus larvae up to 100%, 24 h after the treatment (Harni 2007).
Application of several bacterial endophytes can increase plant growth of black pepper.
Isolate MER7 increased plant hight of pepper, number of branches and number of leaves (Table 2).
Isolates EH11, MER9, TT2 increased plant height compared to control, while isolate HEN1
increased the number of brances compared to control. Vetrivelkalai et al. (2010) reported thatbacterial endophytes suppressed the gall number of nematodes on bhendi, but not all of bacterial
isolates increased the plant growth.

thProceedings of The 7 ACSA Conference 2011317

Table 2. The influence of endophytic bacteria on the growth of pepper plants inoculated with M. incognita 3
months after treatment

Treatments
Isolat MER7
Isolat EH11
Isolat MER9
Isolat TT2
Isolat AA2
Isolat HEN1
Isolat HEN3
Isolat ANIC
Control + (with nematode)

Plant height addition(cm)
24,20 a21,60 ab
20,40 ab
20,40 ab
18,80 ab
17,60 ab
16,40 ab
14,76 b
16,00 b

Numbers ofbranches increment
4,6 a
2,0 bcd
2,0 bcd
1,6 bcd
2,0 bcd
2,8 abc
2,2 bcd
1,2 cd0,4 d

Numbers ofleaves increment
12,6 ab
9,4 abc
10,0 abc
7,8 bc7,8 bc9,2 abc
9,2 abc
5,2 c
6,4 c

Values followed by same small letters on the same column are not significantly different at 5% Duncan test.

Inoculation with nematodes caused damage from the stabbing stylet and secretion of
enzymes released nematodes when the nematodes feed. Hallmann et al. (1997) reported thatnematodes are taking root cells can reduce the ability of plants to absorb water and nutrients fromthe soil and cause symptoms such as lack of water and nutrients. Besides, the reduced
concentration of plant growth regulators such as auxins, cytokinins and gibberellins can occur
because the nematode secretes enzymes cellulose and pectinase that may degrade the cell up to
the root tip injuries and broken, this leads to auxin is not active then growth of plant will be stunted.

Acknowledgements
The authors would like to thank the leader of the program at the Secretariat of Research andDevelopment. The research was made possible with the support of funding from the State Budget of Researchand Development Agency, Ministry of Agriculture.

References
Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW. 1997. Bacterial endophytes in agricultural crops.
Canadian Journal of Microbiology 43:895-914
Hallmann J, Quadt-Hallmann A, Miller WG, Sikora RA, Lindow SE. 2001. Endophytic colonization of plant by
biocontrol agent Rhizobium etli G12 in relation to Meloidogyne incognita infection. Pyhtopathology
91:415-422
Harni R, Munif A, Supramana, Mustika I. 2007. Pemanfaatan bakteri endofit untuk mengendalikan nematodepeluka akar (Pratylenchus brachyurus) pada tanaman nilam. Jurnal Hayati 14 (1): 7-12.
Keel C, Schneider U, Maurhofer M, Voisard C, Laville J, Burger U, Withner P, Hass D, Defago G. 1992.
Suppression of root disease by Pseudomonas fluorescens CHAO: Importance of the bacterial secondary
metabolite 2,4 diacetylphloroglucinol. Mol. Plant-Microbe Interaction 5:4-13.
Kloepper JW, Zablotowiz RM, Tipping EM, Lifshiftz R. 1991. Plant growth promoting mediated by bacterial
rhizosphere colonizers. In: Keister DL and Cregan PB.(Eds.). The Rhizosphere and Plant Growth.
Kluwer Academic Publisher. Dordrecht, The Netherland. P:315-326.
Sikora RA, Schafer K, and Dababat AA. 2007. Modes of action associated with microbially induced in planta
suppression of plant parasitic nematodes. Australasian Plant Pathol. 36:124-134.
Vetrivelkalai P, Sivakumar M, Jonathan FI. 2010. Biocontrol potential of endophytic bacteria on Meloidogyne
incognita and its effect on plant growth in bhendi. Journal of Biopesticides 3 (2): 452-457
Yang CJ, Zhang XG, Shi GY, Zhao HY, Chen L, Tao K, Hou TP. 2011. Isolation and Identification of endophytic
bacterium W4 against tomato Botrytis cinerea and antagonistic activity stability. African Journal of
Microbiol. Res. 5 (2): 131-136.

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The Effect of Organic and Inorganic Fertilizers on Growth and Yield
of Red Ginger (Zingiber officinale Rosc.)
Rudiyanto* and Tri Muji Ermayanti
Research Center For Biotechnology LIPI
Jl. Raya Bogor Km. 46 Cibinong Bogor 16911, Indonesia
Phone: +62-21-8754587, Fax: +62-21-8754588*Corresponding author: rudidinasty@yahoo.com

Abstract
Ginger (Zingiber officinale Rosc.) has been used for medicinal purposes in Southeast Asia since ancient times.
Yield of ginger is affected by soil where the crop grows, climatic conditions and its cultivation management.
Similar to other tuber crops, ginger needs high nutrients on soil. The objective of this experiment was toinvestigate the growth and yield of red ginger plants grown in media suplemented with organic and inorganic
fertilizers. The research was conducted in a glasshouse in the Research Center for Biotechnology LIPI. The
treatments tested were factorial between the medium and fertilizers used, with the total of treatments was 16
with 5 replicates of each treatment. Treatment M1 was soil : manure : grit (1:1:1); M2 was soil : manure : chaff(1:1:1); M3 was soil : manure : grit (1:2:1); M4 was soil : manure : chaff (1: 2:1); P0 was no addition of fertilizer
control treatment); P1 was NPK granule (18:9:10) at 5 g/plant; P2 was NPK (20:20:20) at 1 g/l given once a
week; P3 was NPK (32:10:10) at 1 g/lt giving once a week. Growth and yield parameters measured were height,
number of leaves, number of shoots, stem diameter (from 0 to 24 weeks), leaves fresh and dry weight, stem
fresh and dry weight, root fresh and dry weight, and leaf area recorded at month 4 and 6. After 9 months, redginger was harvested to determine its yield. The results indicated that treatment with M2P2 was the besttreatment for height, number of leaves and stem diameter, of red ginger, meanwhile M4P3 resulted in thehighest number of shoots among the others (11.25). The highest production of rhizome of red ginger plant after
9 month growth was achieved by M1P2 treatment (174.058 gr fresh weight/ polybag and 19.75 dry weight/
polybag). Physiological parameters showed that the highest Leaf Area Index (LAI) value was from M2P2
treatment (3.83 after 4 months of growth), and from M4P3 (6.415448, after 6 month of growth); M3P2 treatment
produced the highest net assimilation rate (NAR) at 0.01, M3P1 had the crop growth rate (CGR) value at
0.031548. The best value of Harvest index (HI) was reached by M3P0 treatment at 2.038.
Keywords :Zingiber officinale Rosc., red ginger, organic and inorganic fertilizers, growth, yield

Introduction
Ginger (Zingiber officinale Rosc.), a monocotyledon belonging to family Zingiberaceae andin the natural order Scitamineae, is herbaceous perennial, usually grown as annually for its pungent
rhizome. It is native of Southeast Asia and one of the earliest oriental spices known to Europe(Parthasarathy et al., 2003; Ravindran and Babu, 2005; Kandiannan et al., 2009). Ginger normally
propagates by its rhizome. Ginger has been used for medicine purposes in Asia since the ancient
times. For example, it is used as folk medicine, as a carminative, stimulant of the gastro-intestinal
tract, and counter-irritant. The rhizome is believed to have diaphoretic and diuretic effects, and antiinflammatory. Extracted ginger rhizome contains gingerol inhibits the growth of Helicobacter pylori
CagA+ strains in vitro, and may contribute to chemopreventative effects (Mahady et al., 2003).
Ginger is also widely used as a spice in forms of fresh ginger, dried whole or powdered ginger, andpreserved ginger.
Crop yields is a function of three major factors; namely the soil where the crop is grown,
climatic conditions and management practices. Like other root and tuber crops, ginger has a highthProceedings of The 7 ACSA Conference 2011319

nutrient demand on soils. It does well in farmlands newly opened from long fallows, making use ofnutrients reserves accumulated during the fallow period (Attoe et al., 2009)Management practices used in agriculture will influence soil properties, nutrient use
efficiency and crop production. Conventional crop management systems that rely on inorganic
fertilizers and agrochemicals have, in recent years, increased agricultural productivity, albeit at ahigh environmental cost (Pimentel, 2005). Organic farming systems which depends on organic
sources of nutrients (i.e., animal manure, crop residues, green manure crops and catch crops) may
sustain productivity at reduced environmental cost by enhancing microbial nutrient turnover(Watson et al., 2002).
The objective of our study was to investigate the growth and yield of red ginger plants
grown in media suplemented with organic compared with inorganic fertilizers.

Materials and Methods
The research was conducted in a glasshouse in the Research Center for Biotechnology
LIPI. The treatments tested were factorial between the medium and fertilizers used, with the total oftreatments was 16 with 5 replicates of each treatment. Table 1 showed the combination treatment
of inorganic and organic fertilizers used in this experiment.
Table 1. Treatment of Combination between organic and inorganic fertilizer
Code

P0

P1

P2

P3

M1
M2
M3
M4

M1P0
M2P0
M3P0
M4P0

M1P1
M2P1
M3P1
M4P1

M1P2
M2P2
M3P2
M4P2

M1P3
M2P3
M3P3
M4P3

Note: M1: soil : manure : grit (1:1:1)
M3: soil : manure : grit (1:2:1)
P0: no addition of fertilizer (control treatment)
P2: NPK (Hyponex 20:20:20) at 1 g/l given once a week

M2: soil : manure : chaff (1:1:1)
M4: soil : manure : chaff (1: 2:1)
P1: NPK granule (Decastar 18:9:10) at 5 g/plant
P3: NPK (Growmore 32:10:10) at 1 g/l giving once a week

Seedlings of 1 month old were used in this experiments. Seedlings were grown in
polybags of 25 x 30 cm. Each polybag contained growth medium with different trreatment of
fertilizers as shown at Table 1. Growth and yield parameters measured were height, number ofleaves, number of shoots, stem diameter recorded from 0 to 24 weeks. However, since the data
were plenty, only data at week 24 was presented. After 9 months, red ginger was harvested todetermine its yield as fresh and dry weight of the rhizomes.

Results and Discussion
Growth of red ginger after 24 weeks planting on different combinations of fertilizer types
and concentratitons was shown at Table 2. The results indicated that treatment with M2P2 was thebest treatment for height, number of leaves and stem diameter, of red ginger (Table 2), meanwhile
M4P3 resulted the highest number of shoots among other treatments. This indicated that the best
medium for growing red ginger until 24 weeks was a combination between M2; soil: manure: chaff(1:1:1) and NPK fertilizer containing NPK (20:20:20) at 1 g/l that was given once a week.
Unlike the case with other treatments, Slow Release fertilizer (P1: NPK granule (18:9:10)
at 5 g/plant) actually had no significant effect on shoot growth performance, which were indicatedby the low values of plant height, leaves number, shoots number and the plant diameter. Ginger isan herbaceous plant that needs nutrient appropriately. When it was fertilized with the low supply ofnutrients from the granular fertilizer, the optimum vegetative growth could not be achieved.

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Nutrient management is always an important consideration for ginger since large quantities
of nutrients is required for optimal growth, especially for N, P and K nutrients. However, farmers
generally overuse N, P and K and ignore manure fertilization. They are not used to apply
appropriate fertilization in many crops, therefore they need knowledge about balanced fertilization
between inorganic and organic fertilization (Li et al., 2010). Organic and conventional farming
practices differ in the use of several agriculture management strategies, including the use of covercrops, green manure, and fertilization, which may influence soil properties, greenhouse gas
emissions and productivity of agroecosystems.
Table 2. Growth of red ginger after 24 weeks of planting treated with different combinations of fertilizer type andconcentration
Treatment code*

Height of plant (cm)

M1P0
M1P1
M1P2
M1P3
M2P0
M2P1
M2P2
M2P3
M3P0
M3P1
M3P2
M3P3
M4P0
M4P1
M4P2
M4P3

70.33
54.07
82.38
74.83
82.33
99.13
100.6380.025
79.23
34.00
75.63
63.38
81.83
82.13
94.50
93.25

Number of leaves
36.00
42.00
94.75
63.75
50.33
111.00104.25
99.00
66.67
18.00
53.00
42.75
84.00
98.25
116.25
123.50

Number of shoots

Stem diameter (cm)

5.67
4.33
9.00
6.00
5.33
10.00
9.00
9.50
7.33
4.00
6.75
7.50
8.00
9.50
9.00
11.25

0.50
0.37
0.58
0.48
0.50
0.55
0.63
0.55
0.57
0.30
0.48
0.48
0.57
0.63
0.63
0.53

Note : *description of each code of treatment is shown at Table 1.

7

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

Leaf Area Indexs

Leaf Area Indexs

6
5
4
3
2
1
M1P0
M1P1
M1P2
M1P3
M2P0
M2P1
M2P2
M2P3
M3P0
M3P1
M3P2
M3P3
M4P0
M4P1
M4P2
M4P3

M1P0
M1P1
M1P2
M1P3
M2P0
M2P1
M2P2
M2P3
M3P0
M3P1
M3P2
M3P3
M4P0
M4P1
M4P2
M4P3

0

A

Treatment

B

Treatment

Note : each code of treatment is presented on Table 1.

Figure 1.Leaf Area Index (LAI) of red gingeron 4 (A) and 6 month (B) of age.

Leaves are important organs of the plant. Leaf area (LA) is a variable key for mostagronomic and physiological studies involving plant growth, light interception, photosynthetic
efficiency, evaporation, and responses to fertilizers and irrigation (Blanco & Folegatti, 2005).
Therefore, LA strongly influences growth and productivity. Estimating LA is a fundamental
component of crop growth models (Lizaso et al., 2003). Figure 1 showed that the M4P2 treatment tothProceedings of The 7 ACSA Conference 2011321

ginger resulted the highest value of leaf area index (LAI) at 4 months after planting. However, at 6
month old plant, the highest LAI was achieved when treated by M4P3 treatment combination.
The perennial rhizome of the ginger plant is a specialized segmented stem structure thatgrows horizontally just under the soil surface. Upright-growing shoots are produced from the tips oflateral rhizome branches. Adventitious roots and lateral growing points emerge from the nodes of
the rhizome stem. In ginger, the roots emerge from the lower rhizome sections. For commercial
purposes, ginger is grown as an annual crop, the rhizomes are harvested after seven to ninemonths when the rhizome is physiologically ripened (Wilson &Ovid, 1993).

A
200
180
160
Fresh Weight (gram)

140
120
100
80
60
40
20

M3P3

M4P0

M4P1

M4P2

M4P1

M4P2

M4P3

M4P3

M3P2

M4P0

M3P1

M3P3

M3P0

M2P3

M2P2

M2P1

M2P0

M1P3

M1P2

M1P1

M1P0

0

Treatment

B

Dry Weight (gram)

25
20
15
10
5

M3P2

M3P1

M3P0

M2P3

M2P2

M2P1

M2P0

M1P3

M1P2

M1P1

M1P0

0

Treatment
Note : each code of treatment is presented on Table 1.

Figure 2. Fresh weight (A) and Dry Weight (B) of red ginger rhizomes after 9 months old grown on various
fertilizer application.

Figure 2 showed that the highest fresh and dry weight of rhizomes produced by treatment
M1P2. M1 was soil: manure: grit (1:1:1) medium with the addition of NPK fertilizer supplement(Hyponex 20:20:20) at 1g/l given once a week (P2). While the lowest weight of rhizomes wasproduced on the M3P1 treatment, where M3 was soil: manure: grit (1:2:1) medium and P1 was aslow release NPK granules (with N:P:K ratio of 18:9:10) with the dose of 5 g/plant. This result
indicated that the slow release fertilizer (P1) provided less amount of nutrient than what ginger plantrequired during the maximum vegetative growth and generative phase.
Ginger plants, that was grown in soil containing more manures increased ginger plant
growth and rhizome production. The use of humic acid as a fertilizer could directly affectphysiological processes in plants. Besides its role as an auxin, humic acids directly influence the

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metabolic processes of plants such as respiration, synthesis of nucleic acids, and ion absorption.
The highest rhizome production of red ginger plant after 9 month growth was achieved by M1P2
treatment (174.058 gr fresh weight/ polybag and 19.75 dry weight/ polybag). In addition to that, thebest value of Harvest index (2.038) was reached by M3P0 treatment.

References
Attoe E.E. and Osodeke V.E. 2009. Effects of NPK on Growth and Yield of Ginger (Zingiber Officinale Roscoe)
in Soils of Contrasting Parent Materials of Cross River State. EJEAFChe, 8 (11): 1261-1268
Blanco, F.F., Folegatti, M.V. 2005. Estimation of leaf area for greenhouse cucumber by linear measurements
under salinity and grafting. Sci. Agric. 62, 305–309.
Kandiannan, K., Parthasarathy U., Krishnamurthy K.S., Thankamani C.K., Srinivasan V. 2009. Modelingindividual leaf area of ginger (Zingiber officinale Roscoe) using leaf length and width. Scientia
Horticulturae 120: 532–537
Lizaso, J.I., Batchelor, W.D., Westgate, M.E. 2003. A leaf area model to simulatecultivar-specific expansion andsenescence of maize leaves. Field Crops Res. (80) 1–17.
Li L., F. Chen, D. Yao, J. Wang, N. Ding, and X. Liu. 2010. Balanced Fertilization for Ginger Production. Better
Crops Vol. 94; No. 1: 25-27
Pimentel, D. 2005. Environmental and economic costs of the application of pesticides primarily in the UnitedStates. Environ. Develop. Sustain. 7, 229–252.
Watson, C.A., Atkinson, D., Gosling, P., Jackson, L.R., Rayns, F.W. 2002.Managing soil fertility in organic
farming systems.Soil Use Manage. 18, 239–247.
Wilson, H. & Ovid, A. 1993. Growth and yield responses of ginger (Zingiber officinale Roscoe) as affected byshade and fertilizer applications. J. Plant Nutrition 16(8):1539-1545.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011323

Growth Analysis of Superior Clones of
Temulawak (Curcuma xanthorrhiza Roxb.) Grown with Organic Fertilizers
Bambang Pujiasmanto* and Samanhudi
Department of Agrotechnology, Faculty of Agriculture, Sebelas Maret University
Jl. Ir. Sutami 36A, Surakarta, Indonesia 57126* Corresponding author: Tel./Fax: +62 271 637457;
bpmanto@yahoo.com

Abstract
Curcuma xanthorrhiza as a medicinal plant needs to be preserved and improved for its yield. The curcumin isvery useful for human health. This species is useful to improve the body immune system. Curcumin of many
genotypes of C. xanthorrhiza has been successfully evaluated for their potentials and its potential yield fromgenotypes throughout Java and islands for selection of superior clones. The research was done based on the
Randomized Completely Block Design (RCBD), using superior clone types as the single factor obtained from
Sragen, Jember, Sumenep, Pasuruan, Blitar, and Malang. The data were analyzed using F-test at significancelevel of 5% followed by Duncan Multiple Range Test (DMRT) at significance level of 5%. The results showedthat Malang clone had the best vegetative growth and maximum yield compared with other clones. The plantheight, leaves number, leaf width, leave area, leaf area index (LAI), dry weight of straw, fresh rhizome weightvariables of Malang clone was the highes among other clones. The maximum vegetative growth pattern of plantheight obtained by Malang clone that reached 155.13 cm, the highest fresh rhizome production of Malang clone
was 22.31 tons/ha.
Keywords: growth analysis, temulawak, Curcuma xanthorrhiza, organic fertilizer

Introduction
Temulawak (Curcuma xanthorrhiza Roxb.) is one species of medicinal plants belonging tofamily Zingiberaceae is potential to be developed, and is one of nine types of seed plants from the
Directorate General of POM as an ingredient. Utilization of this plant is varied. It is useful for
maintenance and improvement of health and treatment of disease as well as raw materials for
traditional medicines and cosmetics (Nurjannah et al., 1994). Temulawak rhizome can be used as
hepatoprotector, increase the body's immune system, anti-bacterial, anti-diabetic, anti-hepatotoxic,
anti-inflammatory, anti-oxidant, anti-tumor, diuretic, depressant, hipolipodemik, and as beverage aswell as for natural dyes (Purnomowati and Yoganingrum, 1997; Raharjo and Rostiana, 2006).
All potentials should be evaluated to determine the type of temulawak with the mostpotential to be developed in certain areas. Several types of temulawak has been successfully
evaluated its potential based on curcumin content and gowth characteristics for selection of all
types of temulawak throughout Java. Kuswanto has been elected six best types of temulawak,
including Sragen, Pasuruan, Blitar, Sumenep, Malang, and Jember.
Temulawak selected cultivars need to be tested in various production areas, in order toinvestigate their genetic capabilities grown in various areas. One of the evaluation was done byplanting selected temulawak, the superior clones was then elected to be cultivated at the production
centers of temulawak in the district of Sragen, Central Java.

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Materials and Methods
This study was started in May 2010, at Karang Pong, Kali Jambe, Sragen, Cetral Java,
located at 7° 30' LS and 110° 50' BT and altitude of 180 m above sea level. The soil type is vertisol.
The materials used in this study were temulawak seeds, consisting of six clones derived from thesuperior selection of Java.
The design of the experiment was Randomized Completely Block Design (RCBD). Originof clones was used as the single factor. The experiment had 6 standard of excellence e.i. 6 clones
obtained from Java. Each treaatment had 3 replicates. Addition of organic fertilizer was of 15
tons/ha manure.
Observation variables included plant height, number of leaves per clump, number of tillers
per hill, leaf width, leaf area, leaf area index, fresh weight of rhizomes per plant, dry weight of straw,
specific leaf area, and relative growth rate. Data were analyzed using the F test at 5% level, if there
was a significant difference, then it was followed by DMRT at the level of 5%.

Results and Discussion
Height of Plant
Malang clone temulawak observed at intervals of two weeks showed high growth rate ofplants significant different compared with other clones (Figure 1). Malang clone growth at 5 WAP
seemed to have the fastest plant height compared with five other clones. This shows the level ofadaptation of Malang clone was very high, especially during the process of adding plant height.

Figure 1. Plant height of six superior clones of temulawak (Curcuma xanthorrhiza).
Table 1. The average plant height of six superior clones of temulawak (Curcuma xanthorriza) at 19 WAP
Plant height (cm)

Clones

I

II

III

Total

Average

Sragen108.80

98.13

99.50

306.43102.13 cJember

68.38

60.63

68.25

197.2665.75 ab52.71 a74.92 bSumenep

45.00

59.38

53.75

158.13

Pasuruan

67.75

69.25

87.75

224.75Blitar

86.50

61.75

61.25

209.50146.60167.80Malang151.00465.4069.83 ab155.13 dNote: Values followed by the same letter showed no significantly difference according to DMRT at 5%.

Increased plant height of Malang clone during vegetative growth was the highest among
the clones. Sragen clone was the second having the plant height similar to Malang clone. At the lateth

Proceedings of The 7 ACSA Conference 2011325

vegetative phase (19 WAP) height of Sragen clone was only 102.13 cm, which was much lower
than that of Malang clone (155.13 cm) (Table 1). This would indicate that vertisol soil type issuitable for temulawak. Plant height among the clones was significantly different at 5% with Malangclone was the best.
Number of Leaves and Number of Tillers per Clumps
The number of leaves of the temulawak represents its during the vegetative phase. Whenthe plants reach the generative phase, the existing leaves turns to dry. The increased of leaf
number may also indicate the increase of rhozome production of temulawak. The more number ofleaves formed, the better rhizome formation during the vegetative phase of the plants. The numberof leaves produced by temulawak tuber is influenced by the number of tillers formed in a single
clump of plants. The leaves formed by the temulawak having a lot of puppies will increase thenumber of leaves corresponded to the number of puppies available. Tillers per clump of temulawak
are capable of producing 6-9 leaves which affects the increasing number of leaves in one plant.
Statistical analysis that there was no significant different among clones of temulawak interms of leaf number. Figure 2A shows that the highest number of leaves found on the Malangclone, while the Sumenep clone produced the lowest of number of leaves. The number of tillers perhill shows that the Pasuruan and Malang clones had the highest number of tillers (Fig. 2B) the
average number of tillers per hill reached 3.17. However, this amount was not significantly different
with other clones

A

B

Note: Values followed by the same letter showed no significantly difference according to DMRT at 5%.

Figure 2. Number of leaves (A) and of six clones of temulawak (Curcuma xanthorrhiza) at 19 WAP.

Pasuruan clones into clone with the highest number of suckers, is due to the number of
seedlings have buds more when compared with the five other clones. This will impact on the growthof temulawak, number of shoots that emerged after the primary buds more so it appears as a leafy
plant with a number of seedlings in a clump of more than one. The number of chicks that many of
the temulawak will have a negative effect form of competition for the acquisition of nutrients andlight. For the competition in terms of food reserves during early growth, the number of chicks that
more will tend to have stronger competition because it has only one rhizome pieces that serve as
the seed.
Leaf Width, Leaf Area and Leaf Area Index
Leaf width of all clones were not significantly difference (Figure 3A), and there was nodifference on their leaf morphology. The width of the leaf ranged from 10.96 cm to 18.00 cm. The
leaf area was significantly difference among the clones with the Malang clones was the best.
Malang clones had the highest leaf area with an area of 1,008.83 cm2, while for the lowest on
Sumenep clones with only 244.55 cm2 leaf area (Figure 3B). The leaf area index is related tophotosynthetic ability of plants. Widiastuti et al., (2004) reported that at low light intensity plants
produce larger leaves, thinner with a thin layer of epidermis. It has slight palisade tissue, wider

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spaces between cells, and more number of stomata. Meanwhile, plants that received high-lightintensity produces smaller leaves, thick, compact with fewer number of stomata, the cuticle layer
and a thicker cell wall with space between cells is smaller and hard texture of leaves.

A

B

C

Note: Values followed by the same letter showed no significantly difference according to DMRT at 5%.

Figure 3. Leaf width (A), leaf area (B) and leaf area index (C) of six clones of temulawak (Curcuma xanthorrhiza)
at 19 WAP.

Greater leaf area may be harmful, it can also be beneficial to plants. Large leaf area would
be advantageous for plants because it will enhance the ongoing process of photosynthesis. Optimal
photosynthesis caused photosynthesis product formed also increased, so plant growth will be
optimal. Irwanto (2009) revealed that the products of photosynthesis are proportional to the total
active leaf area that can perform photosynthesis. Widiastuti et al. (2004) reported that withincreased leaf area, assimilates generated will also be greater. Narrow leaf area beneficial forplants because it can reduce water loss through excessive transpiration.
Leaf area index obtained after the analysis was performed to Malang clone has a realdifference when compared with leaf area index of the other five clones. Temulawak was obtained
from leaf area index is not too large. Of the six clones of temulawak only five clones are at rates
below one. Malang clones became the most extensive high-leaf area index reached 1.13 (Figure
3C). This shows that for the Malang clones had interaction especially among the leaves of shadeplants. Meanwhile, other five clones had a smaller leaf area index, which was about 0.5. Nointeractions occur among the clones because of wide spacing used in the experiment was higher
than the leaf area produced of temulawak.
The higher of leaf area index (LAI) was beneficial. Sitompul and Guritno (1995) reveal thatthe density of the leaves is closely related populations of plants or plant spacing. The closer the
distance between plants, the higher the density between the leaves and fewer quanta of radiation(light) is to layers of lower leaves.
Biomass Dry Weight and Fresh Rhizome Weight per Plant
The statistical analysis showed that Malang clone had the highest biomass production
significantly different with other clones (Figure 4A). Dry weight of Malang clones was 168.1 g, thelowest was Pasuruan clone having 27.55 g. This indicated that with increasing leaf area oftemulawak was total plant dry weight increase corresponded to the crop yields increase.
When leaf area was higher then its dry weight, this will correspond directly to the total dry
weight of the plants. Similarly, the weight of rhizomes that produced was also high. High weight ofbiomasss showed that the assimilation process may also rearchs to maximum.
Statistical analysis of rhizome fresh weight showed that there was significantly difference
among the clones. Malang clone obtained the highest rhizome fresh weight per plant (422.57 g)(Figure 4B), meanwhile Pasuruan clones was the lowest, only reached 105.49 g. Conversion into
yield per hectare Malang clones was 22.31 tons. Other temulawak clones was reported to have lessthProceedings of The 7 ACSA Conference 2011327

then 20 tons per hectare. Therefore, Malang clone was potential to be promoted as the best clone.
This clone is best when it grows intensively on the vertisol soil.

A

B

Note: Values followed by the same letter showed no significantly difference according to DMRT at 5%.

Figure 4. Dry weight of biomass (A) and rhizome fresh weight (B) of six clones of temulawak (Curcuma
xanthorrhiza) at 19 WAP

Specific Leaf Area
Specific leaf area of six different clones showed no significantly different when they wereobserved until 17-23 WAP. At 17 WAP, Pasuruan was the best, significantly different compared withother clones, but only Sragen clone was significantly different compared with Sumenep clone at 23
WAP (Table 2).
2

Table 2. Specific leaf area of temulawak (cm )ClonesAge (WAP)
17

19

21

23

Sragen4,033.3 a3,136.7 a

8,143.3 a

8,033.3 b

Jember3,806.7 a

1,930.0 a

3,760.0 a

2,710.0 ab

Sumenep

2,556.7 a886.7 a1,620.0 a

1,556.7 a

Pasuruan

9,533.3 b

7,083.3 a

24,360.0 a

6,656.7 ab

Blitar

4,033.3 a

1,736.7 b

1,780.0 a

1,993.7 ab

Malang4,526.7 a2,863.3 a4,150.0 a

3,336.7 ab

Note: Numbers followed by same letter on the same column are not significant at different levels of multiple range tests of
Duncan's 5%.

Relative Growth Rate (RGR)
RGR on almost all clones of temulawak at the age of 19-21 weeks after planting tend to
reduce. The high reduction occurred when plants were 21 weeks old, except for Pasuruan andBlitar clones lowest RGR occurred at the age of 23 weeks after planting (Table 3). This occurs
because the plant entered generative phase. After entering the generative phase of growth, clones
Malang, Jember, Sumenep, and Sragen increased in RGR. This showed that the clones were ble toproduce a new dry matter per unit at initial dry material. Meanwhile, Pasuruan and Blitar clones was
not able to produce new dry matter per unit at initial dry material.
Table 3 shows that clones of temulawak from Malang has the highest RGR than the otherclones both at the beginning of growth or to close to maximum vegetative growth.
Decline in RGR indicated the differences in rates of photosynthesis. The process ofphotosynthesis will be disrupted if water is not enough to plants. A low rate of photosynthesis may
decrease to growth of vegetative organs, especially to the plant height. The decrease inphotosynthesis may be due to increase of stomata diffusion and non-stomata barrier. Decrease of

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RGR may also due to the slow growth of vegetative organs (leaf area, plant height, number oftillers, dry weight, and root dry weight of plants).
Table 3. Relative growth rate (RGR) of temulawak (g/week)
Age (WAP)

Clones19

21

23

Sragen2.17 ab1.37 a1.40 aJember

2.40 ab1.52 ab1.61 abSumenep

2.46 ab1.60 ab1.68 b

Pasuruan

1.75 a1.77 ab1.65 bBlitar

2.09 ab1.78 ab1.55 abMalang2.83 b

2.21 b

2.44 cNote: Numbers followed by same letter on the same column are not significant at different levels of multiple range test of
Duncan's 5%.

Conclusions
Malang superior clone had the maximum vegetative growth and yield of its fresh rhizome.
This clone also had the highest of the plant height, leaf number, leaf width, leaf area, leaf area index(LAI), dry weight of straw, rhizome fresh weight, specific leaf area, and RGR. The maximum plantheight of Malang clone was 155.13 cm, the fresh rhizomes of temulawak was 22.31 tons/ha.

References
Irwanto.

2010.
Effectofshade
ongrowthdifferences
Shoreasp.
innurseries.
http://www.geocities.com/irwantoforester/naungan_shorea.pdf. Accessed, May 27, 2010.
Nurjannah N, S Yuliani, AB Sembiring. 1994. Temulawak (Curcuma xanthorrhiza). Review of Outcomes
Research. Research Institute for Spices and Medicinal Plants. X (2): 43-57.
Purnomowati S and A Yoganingrum. 1997. Temulawak (Curcuma xanthorhiza Roxb.). Center for documentationand information science, LIPI, Jakarta. 44p.
Rahardjo M and O Rostiana. 2006. Cultivation of Rare Medicinal Plants of Purwoceng (Pimpinella pruatjan
Molkenb). Proceedings of the National Seminar and Exhibition of Medicinal Plant Development towards
Independence Family Medicine Society in Jakarta on September 7. P. 138-146.
Sitompul SM and B Guritno. 1995. Plant Growth Analysis. Gadjah Mada University Press. Yogyakarta.
Widiastuti L, Tohari, and E Sulistyaningsih. 2004. Effect of light intensity and levels dominosida on microclimate
and plant growth in pot chrysanthemum. Agrivita 11 (2) :35-42.

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Effect of Drought and Inoculation of Arbuscular Mycorrhizal Fungi in
Enhancing Productivity and Tolerance Mechanism of Grasses
PDMH Karti, a* L Abdullaha, J Nulikb, Nurhayatic, OR Nahakd , S NofyangtriaaFaculty of Animal Science, Bogor Agricultural University, Bogor, West Java. IndonesiabBPTP, Naibonat, Kupang,NTT, Indonesiac
BPT, Ciawi, West Java, IndonesiadUniversity of Timor, NTT, Indonesia*
Corresponding author: Faculty of Animal Science, Bogor Agricultural University,
Bogor, West Java. Indonesia.
Hp :+6281314227713
pancadewi_fapetipb@yahoo.com

Abstract
Drought stress considered to be one of the most important limiting abiotic factors of plant growth and yield inmany areas, and Arbuscula Mycorrhizal Fungi (AMF) symbiosis can protect host plants against its detrimental
effect. This research was conducted to study the effect of drought and inoculation of Arbuscula Mycorrhizal
Fungi on the productivity and tolerance mechanisms of Stenotaphrum secundatum and Ischaemum timurensis
grasses. This research used a completely randomized design with four treatments: D0(Control), D1 (with AMF),
D2 (drought), and D3 (drought and with AMF). Parameters observed were the soil moisture content, leaf water
potential, leaf relative water content, shoot and root dry weight, root length, proline, and soluble sugar. The data
were analyzed with analysis of variance (ANOVA) and the differences between treatments were analyzed with
Duncan range test. The results showed that drought stress significantly (P<0.05) decreased soil water content,
leaf water potential, leaf relative water content, shoot dry weight in Stenotaphrum secundatum. Drought stresssignificantly (P<0.05) decreased soil water content and leaf water potential, but enhanced proline and soluble
sugar content in Ischaemum timurensis. AMF inoculation did not affect productivity of Stenotaphrum
secundatum, but in Ischaemum timurensis enhanced proline and soluble sugar content but decreased leaf water
potential on drought stress. One of the tolerance mechanism in Ischaemum timuriensis is by increasing theproline and soluble sugar contents.
Keywords: drought, arbuscular mycorrhizal fungi, grasses, Stenotaphrum secundatum, Ischaemum timurensis

Introduction
Plants in nature are continuously exposed to several biotic and abiotic stresses, water
deprivation being one of the commonest. Soils too dry for crop production have been estimated tocover 28% of the earth's land surface (Bray, 2004). Nevertheless, plants have developed several
physiological, biochemical, and molecular mechanisms in order to cope with drought stress.
Besides the natural responses of plants against drought, it must be considered that most terrestrial
plants can establish a symbiotic association with the arbuscular mycorrhizal fungi (AMF). When the
AMF symbiosis is established the fungus receives carbon molecules from the plants, and the plants
receive nutrients (especially phosphorus) and water from the fungus (Harrison, 2005; Gosling et al.,2006). In this way, AM plants are usually more tolerant to several stresses, including drought, thannon-AM plants (Augé, 2001, 2004; Ruiz-Lozano, 2003; Ruiz-Lozano et al., 2006). This research
was conducted to study the effect of drought and inoculation of AMF to the productivity andtolerance mechanisms of Stenotaphrum secundatum and Ischaemum timurensis GrassesthProceedings of The 7 ACSA Conference 2011331

Materials and Methods
The materials used in this study were two types of grasses namely Stenotaphrum
secundatum, and Ischaemum timurensis. The two types of grasses are the result of selection about
30 forage species on preliminary research (Karti, 2010). Other materials used were fiber pots as
many as 32 units (diameter = 20, high = 100 cm), Mycofer, growing media in the form of soil andmanure, tools WP4 potentiometer, coolbox, and others.
This research used a completely randomized design with four treatments: D0 (control), D1(with AMF), D2 (drought), and D3 (drought and with AMF) and four replications. Parameters
observed were soil moisture content, leaf water potential, leaf relative water content, shoot androot dry weight, root length, proline (Bates, 1973), and soluble sugar (Dubois et al., 1956 modified
by Buysse & Merckx, 1993). The data were analyzed with analysis of variance (ANOVA) and thedifferences between treatments were analyzed with Duncan range test.

Results and Discussion
Drought stress significantly (P<0.05) decreased soil water content, leaf water potential,
leaf relative water content, shoot dry weight in Stenotaphrum secundatum (Table 1). AMF without
drought treatment did not show significant differences, but it showed significant different (P<0.05)
in drought treatment. Drought stress reduced levels of the soil led to a decrease in water
absorption. The low uptake of water caused the shoot dry weight decreased because of declining
photosynthesis process. The addition of AMF increased significantly (P<0.05) the shoot dry weight,
this is due to the ability of AMF in increasing absorption of water so that improving thephotosynthesis process.
Table 1. Effect of drought and the addition of AMF to soil moisture, leaf water content, relative water content,
shoot dry weight, and root dry weight of Stenotaphrum secundatum
Variables
Soil moisture
Leaf water potential (-Mpa)
Relatif water content (%)
Shoot dry weight (g/pot)
Root dry weight (g/pot)

Do
38.45 ± 0.46 a

Treatments
D1
D2
39.14 ± 0.59 a
23.76 ± 1.04 b

D3
22.98 ± 0.79 b-0.68 ± 0.2 b
82.28 ± 2.8 a-0.51 ± 0.6 b
87.2 ± 6.4 a-6.79 ± 1.6 a
29.69 ± 6.4 b-7.00 ± 1.7 a
34.51 ± 10.2 b

27.6 ± 3.5 a
2.5 ± 1.9

29.0 ± 4.7 a
4.8 ± 3.5

14.7 ± 2.1 c
2.9 ± 0.7

22.3 ± 2.3 ab
3.2 ± 1.0

Description: D0 (control), D1 (with AMF), D2 (drought), and D3 (drought and with AMF).

Table 2. Effect of drought and the addition of AMF to soil moisture, leaf water potential, leaf relative watercontent, root length, shoot dry weight, and root dry weight, proline and soluble sugar of Ischaemum
timuriensis
Variables
Soil moisture (%)
Leaf water potential (-Mpa)
Leaf relative water content (%)
Root lenght (cm)Shoot dry weight (g/pot)
Root dry weight (g/pot)
ProlineSoluble sugar

Doa35,52 ± 0,58b
-1,02 ± 0,19a
88,25 ± 3,50
128,0 ± 6,38
38,87 ± 9,21
5,30 ± 3,77
H
51,68 ± 7,63
LM
6,64 ± 2,43

Treatments
D1
D2
a
b
35,33 ± 1,97
23,15 ± 1,28aa
-1,36 ± 0,11
-1,46 ± 0,17
88,48 ± 0,90
86,10 ± 1,71
128,50 ± 5,80
128,25 ± 4,92
41,70 ± 18,36
29,10 ± 12,35
6,65 ± 4,20
8,30 ± 3,76
HH
43,36 ± 7,04
67,48 ± 7,43
MEF
4,78 ± 1,36
23,09 ± 2,71

D3b
24,32 ± 2,01
ab
-1,19 ± 0,27
86,78 ± 0,72
127,75 ± 11,76
30,70 ± 5,88
10,23 ± 2,99
H
59,05 ± 17,73
GH
17,24 ± 1,88

Description: D0 (control), D1 (with AMF), D2 (drought), and D3 (drought and with AMF)

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In Ischaemum timuriensis drought stress significantly (P<0.05) decreased the soil moisture
content, leaf water potential, and significantly increased (P<0.05) the proline and dissolved sugar,
but did not show significant effect in leaf relative water content, root and shoot dry weight. In
drought conditions, plants decreased leaf water potential, but with the addition of AMF the decrease
could be minimized compared to that with non-AMF. In these plants, drought treatment did notcause a change in relative leaf water content, root and shoot dry weight. This suggests that theseplants include drought resistant crops. Tolerant plants that can increase levels of proline andsoluble sugar to maintain water absorption so that the growth process can run well.
Drought resistance mechanisms in Ischaemum timuriensis is by secreting proline andsoluble sugar. In Figure 1 shows that drought stress increased proline levels in both treatments withthe addition of AMF or not. In Ischaemum timuriensis a sharp increase in proline was seen after
40th day.

Figure 1. Proline Content of Ischaemum timuriensis.

In Ischaemum timuriensis (Figure 2) shows that drought stress showed the highest
elevated levels of dissolved sugar. The addition of AMF on drought stress decreased levels of
dissolved sugar, it means that the AMF can increase the absorption of water through the hyphae.

Figure 2. Soluble Sugar of Ischaemum timuriensis.

Conclusion
Drought stress can reduce soil moisture, leaf water potential, leaf relative water content,
shoot dry weight in Stenotaphrum secundatum. AMF inoculation on drought stress can increase theshoot dry weight. On Ischaemum timuriensis drought stress can reduce soil moisture, leaf water
potential, and increasing proline and sugar dissolved. Ischaemum timuriensis is included in droughtresistant plants with resistance mechanisms through the increased secretion of proline and soluble
sugar.

thProceedings of The 7 ACSA Conference 2011333

References
Augé, RM. 2001, Water relations, drought and vesicular–arbuscular mycorrhizal symbiosis. Mycorrhiza. 11:3-42.
Auge. RM. 2004. Arbuscular mycorrhizae and soil/plant water relations. Canadian Journal of Soil
Science.84:373-381.
Bates LS. 1973. Rapid determination of free proline for water-stress studies. J Plant and Soil 39:205–207.
Buysse J, R Merckx. 1993. An improved colorimetric method to quantify sugar content of plant tissue. J
Experimental Botany 44:1627–1629.
Bray, EA. 1997. Plant responses to water deficit. Trends in Plant Science. 2:48-54.
Dubois M, KA Gilles, JK Hamilton, PA Rebers, F Smith. 1956. Colorimetric method for determination of sugars
and related substrances. Anal. Chem. 28:350–356.
Gosling, P, Hodge, A, Goodlass G, Bending GD. 2006. Arbuscular mycorrhizal fungi and organic farming.
Agriculture, Ecosystems and Environment.113:17-35.
Harrison M.J. 2005. Signaling in the arbuscular mycorrhizal symbiosis. Annual Review of Microbiology. 59:1942.
Karti PDMH. 2010. Development of drought resistant grasses and legumes through a quick selection techniquesand efficient management of aquaculture. The conference of the Research result. Research institutions
and community empowerment. Bogor Agricultural University.
Ruiz-Lozano, J.M. 2003, Arbuscular mycorrhizal symbiosis and alleviation of osmotic stress. New perspectives
for molecular studies. Mycorrhiza . 13:309-317.
Ruiz-Lozano, J.M., Porcel R., Aroca, R. 2006. Does the enhanced tolerance of arbuscular mycorrhizal plants towater deficit involve modulation of drought-induced plant genes. New Phytologist. 171:693-698.

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The Cooling Effect of Forage Crop, Kudzu (Pueraria lobata) Vine Coveringover Livestock Buildings
Takuya Koyamaa, Mika Yoshinagab, Kei-ichiro Maedaa and Akira Yamauchia*aDepartment of Biosphere Resources Science, Nagoya University, Nagoya, Japanb
Department of Architecture, Meijo University, Nagoya, Japan*
Corresponding author: Nagoya University, Chikusa, Nagoya 464-8601, Japanayama@agr.nagoya-u.ac.jp

Abstract
Heat stress during summer season can decrease livestock productivity, and cause high economic losses tolivestock farmers. Although many cooling techniques (e.g. sprinklers, fans, etc.) are available to improve thethermal environment of livestock buildings, they require high construction and energy costs. This research aims
to investigate an economical livestock buildings covering method with kudzu (Pueraria lobata), which can beused as a cooling technique during summer season and as forage for livestock after use. Kudzu is a fastgrowing, climbing and perennial vine, and its shoot can be utilized as high nutritious feed for livestock. In thisstudy, the cooling effect of the covering method with kudzu was investigated by comparing the room
temperatures (RTs) between the covered (Covered) and the non-covered fabricated house (height 2.6 m, width
3.65 m, depth 1.83 m) (Non-covered). Two-year-old kudzu seedlings were transplanted beside the south side of
the houses in May 2009 and 2010 and induced to climb the plastic nets placed over the south walls and roofs of
the houses. RTs of Covered and Non-covered and global solar radiation were measured every 5 minutes during
summer season in 2009. The covering rate and the leaf number in 18 frames (0.4 m × 0.4 m each) that were seton the south wall and roof of Covered and stem length were measured every 2 weeks in 2009. The covering rate
and the dry weight in 120 frames were measured during autumn 2010. The stem length reached around 4 mlong by July 2009. In general, the height of a standard livestock building is around 4 m. These facts showed that
two-year-old kudzu seedlings could cover the walls of a standard livestock building during summer season. The
estimated average covering rate and leaf number among 18 frames, and the RT differences between Covered
and Non-covered showed positively significant relationships (P<.001). The maximum RT difference wasobserved to be 3.44 °C, when the estimated average covering rate was 43.9 % and the estimated average leaf
number was 529 leaves m-2. The covering rate and dry weight in the frame showed a positively significant
relationship (P<.001). The maximum dry weight of 253.4 g m-2 was observed when the covering rate was
80.8%. We conclude that the kudzu covering is an effective cooling technique for livestock buildings during
summer season and is potentially capable of producing biomass to provide feed for livestock after use.
Keywords: kudzu, green wall, heat stress, livestock productivity, forage crop.

Introduction
The global average surface warming in the 21st century will range from 1.1 to 6.4 °C(IPCC, 2007). The global warming has the potential to occur adverse thermal conditions in the
livestock building, which can ultimately reduce productive performance or possibly cause deaths of
livestock (Hahn, 1995). Thus, some techniques for environmental modifications such as water
misting with spray, forced ventilation with fans or air-conditioning, shading with insulated roof orreflective coated roof are needed to maintain animal production (West, 2003). On the other hand,the employment of these techniques causes higher energy consumption, worsens global warming,and increases general costs for animal production (Nardone, 2010).
The ultimate goal of this study was to establish the economical livestock buildings covering
method with the use of kudzu (Pueraria lobata (Willd.) Ohwi) vine. That is to cover the walls androofs of livestock buildings with kudzu vine, which can be used as a cooling technique during
summer seasons and as feed for livestock after use.
thProceedings of The 7 ACSA Conference 2011335

Kudzu is a fast-growing, climbing and perennial vine. Their vines can grow 10 to 30 m in
one growing season (up to 30 cm a day), and create broad canopies (Mitich, 2000). Farmers in the
USA cultivated kudzu over a terrace to provide shade in the early 1900s (Tsugawa, 1986).
Moreover, kudzu has the wide range of utilization as forage because it is able to provide feed forsheep, swine and chickens as well as cattle (Kristen, 2011). Chemical composition and digestibility
characteristics of kudzu were equivalent to other commonly fed forage (Corley et al., 1997)
The use of external vegetation can be a useful tool for thermal regulation for buildings.
Holm (1989) showed using dynamic computer model that the leaf cover produces a constant 5 °C
cooling effect in room temperature in summer. The vegetation consistently lowered the wall surface
temperature by about 17 °C, and saved air-conditioning energy as high as 80% (Meier, 1990).
However, these studies placed little or no emphasis on the conditions of the plants.
Ip et al. (2010) analyzed the shading performance of a vertical climbing plant canopy bymeasuring number of leaf layers, area of canopy with each leaf layer and solar transmissivity, andestablished the thermal model that enables to present the shading performance of the climbing
plant canopy over its annual growing and wilting cycle. Unlike non-biological devices, the conditionsof plant sare affected by not only the characteristics of plants such as leaf area, leaf number, leaf
thickness, leaf angle, leaf movement and moisture content of leaves but also the response to theenvironmental conditions such as solar radiation, ambient temperature, humidity and soil moisture
content, which affect the shading performance, the thermal insulation and evaporative cooling
produced by transpiration.
The objectives of this paper were to investigate the cooling effect of the dynamic changesof kudzu vine covering by comparing the room temperatures (RTs) between the covered (Covered)and the non-covered container house (Non-covered), and to evaluate the dry matter production of
kudzu vine that was used for the economical livestock buildings covering method. The leaf number
and covering rate in each frame that was set on the south wall and roof of Covered, the room
temperatures of Covered and Non-covered, and environmental conditions around the houses were
determined at regular intervals during summer season. At harvest, the covering rate and dry matterweight in 120 frames were measured.

Materials and Methods
Plant materials
Hard-stems (lignified) were collected from kudzu natural stands in the Graduate School of
Bioagricultural Sciences, Nagoya University (35°09‘N, 135°58‘E) from April to May in 2008 and2009. Then, the hard-stems were cut into around 12 cm with one node and grown in the commercial
and artificial soil that is well-drained and poorly nutrient in plastic container (585mm long, 185mm
wide and 145mm deep). After cuttings were rooted and sprouted, they were transplanted to plastic
pots (25 cm in diameter and 30 cm in height) containing air-dried paddy soil and raised in a
glasshouse for one year. In winter 2008 and 2009, all stems were cut at 5cm from the soil surface,and afterwards left undisturbed. Next spring, some buds were sprouted from the rooted cuttings.
Sprouted nursery stocks (plant materials) were only used for this study.
Experimental design
Four container houses (2600mm high, 3650mm wide and 1830mm long) were set in series
from east to west on the Togo field of the Graduate School of Bioagricultural Sciences, Nagoya
University (35°06‘N, 137°04‘E) in 2009, and 5 container houses were set in 2010. One containerhouse excluding both ends was not covered with kudzu vine (Non-covered), and plant materials
were transplanted beside the other container houses in May 25, 2009 and May 24, 2010, andinduced to climb the plastic nets (mesh size: 120mm×120mm) placed on the south wall and the roofof the houses.
336thProceedings of The 7 ACSA Conference 2011

Measurements
Only two container houses excluding both ends (Non-covered and Covered) were used forevaluating the effect of the dynamic changes of kudzu vine covering on the room temperature
reduction in 2009. The houses on both ends were excluded because they received more solar
radiation than the inner houses. The stem length, the leaf number and covering rate in each frame(400mm×400mm), which was set on different heights of the south wall (650mm, 1300mm and1950mm above the soil surface) and on different locations of the roof (465mm, 915mm and
1365mm from the south edge of the roof), were measured on the 3 plants except both ends every 2
weeks from June 10 to September 29, 2009.The estimated average leaf number and covering rate
among 18 frames were calculated from the inclination of averages of these data.The room
temperatures (RTs) of Covered and Non-covered were recorded every 5 minutes with thermo
recorder (TR-72U: A＆D Co. LTD.) and global solar radiation was recorded every 5 minutes withpyranometer (MS-801: EKO INSTRUMENT CO., LTD.) from July 15 to September 22, 2009.To
evaluate the dry matter production of the economical livestock buildings covering method with useof kudzu vine, the covering rate and the dry weight in 120 frames, which were located on the south
wall and the roof of 4 containers, were measured during autumn in 2010.
Image analysis
Before taking digital images of Covered, the white-colored frames (400mm×400mm) wereset behind the plants and in front of the south walls and roofs to facilitate processing images. Digital
images, composed of 12 megapixel, were obtained with the digital camera (μ-7040: OLYMPUS
corporation). The backgrounds except the plants in the frames were cleared manually, and plants
were converted into binary image with photoshop (Adobe Photoshop CS2: Adobe). The pixels of theprocessed image were calculated with imageJ (Public domain software for image processing andanalysis in Java). Calculated pixels of the plants and the frames were used for computing thecovering rate.

Results and discussion
The stem length reached around 4 m long by July, and some vines exceeded 10 m at theend of August in 2009. In general, the height of a standard livestock building is around 4 m. These
facts showed that two-year-old kudzu seedlings could cover the walls of a standard livestockbuilding during summer season.
Fig. 2 shows that the changes in estimated average leaf number and covering rate among
18 frames. The estimated average leaf number gradually increased until the middle of July in 2009.
Afterwards, the estimated average leaf number increased rapidly and reached the peak of 530
leaves m-2 on September 10, 2009.The estimated average covering rate showed similar trend withthe estimated average leaf number, and it peaked at 45% at the final measurement on September
25.
The RTs of Non-covered were clearly influenced by ambient temperature and global solar
radiation. The RT differences between Non-covered and Covered were also influenced by ambient
temperature and global solar radiation. At the same time, the RT differences tended to gradually
increase. Therefore, the data of the RT differences were sorted and averaged out daily according to
the global solar radiation; every 0.2 kWM-2; and the processed data were used to highlight theeffect of the covering method with kudzu.
Under the global solar radiation conditions less than 0.2 kW m-2, there was a significantly
negative relationship between the RT differences, and the estimated average leaf number (P<.001)
and the estimated average covering rate (P<.001). However under the global solar radiation
conditions between 0.2 kW m-2 and 1.0 kW m-2, there were significantly positive relationships
between the RT differences, and the estimated average leaf number (P<.001) and the estimatedthProceedings of The 7 ACSA Conference 2011337

average covering rate (P<.001). The inclinations of each regression equations increased as theglobal solar radiation increased under the global solar radiation conditions between 0.2 kW m-2 and1.0 kW m-2. The maximum RT difference was observed to be 3.44 °C, when the estimated averagecovering rate was 43.9 % and the estimated average leaf number was 529 leaves m-2. The
covering rate and dry matter weight in the frame showed a positively significant relationship(P<.001). The maximum dry weight of 253.4 g m-2 was observed when the covering rate was
80.8%.
We conclude that the kudzu covering is a possible cooling technique for livestock buildings
during summer season and is potentially capable of producing biomass to provide feed for livestock
after use.

This photo was taken on September 18, 2009.

Figure 1. Two container houses(2600mm high, 3650mm wide and 1830mm long) excluding houses on bothends (Non-covered and Covered) were used for evaluating the effect of the dynamic changes ofkudzu vine covering on the room temperature reduction in 2009.

The estimated average leaf number and covering rate were calculated from the inclination of averages of leaf number andcovering rate among18 frames (400mm×400mm), which were located on the south wall and roof of Covered.

Figure 2. Changes in the estimated average leaf number and covering rate among 18 frames in 2009.

References
Corley, R. N., Woldeghebriel, A. and Murphy, M. R. 1997. Evaluation of the nutriative value of kudzu(Puerarialobata) as a feed for ruminants. Anim. Feed Sci. Technol. 68: 183-188
Hahn, G.L.1995. Environmental management for improved livestock performance, health and well-being. Jpn. J.
Livest. Management, 30(3): 113-127.

338

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Holm, D. 1989.Thermal improvement by means of leaf-cover on external walls –a simulation model. Energ.
Build. 14:19–30.
Ip, K., Lam, M., Miller, A. 2010. Shading performance of a vertical deciduous climbing plant canopy. Build.
Environ. 45: 81–88
IPCC (The Intergovernmental Panel on Climate Change).2007. Climate change 2007: Synthesis Report
2007..Geneva, Switzerland. Pp52.
Kristen, H. 2011. KUDZU. Am. Hist. 46 (2): 38-44
Meier, A. K. 1990.Strategic landscaping and air-conditioning savings: a literature review. Energ. Build. 15–
16:479–486.
Mitich, L. W. 2000. Intriguing world of weeds - Kudzu (Pueraria lobata (Willd.) Ohwi). Weed tech. 14: 231-235
Nardone, A., Ronchi, B., Lacetera, N., Ranieri, M.S., Bernabucci, U. 2010.Effects of climate changes on animal
production and sustainability of livestock systems. Livest.Sci.130: 57-69.
Tsugawa, H. 1986a. Cultivation and utilization of kudzu-vine (Pueraria lobata OHWI) taxonomy, geographicaldistribution, use, breeding and propagation. J. Japan.Grassl. Sci.31(4): 435-443.
West, J.W. 2003. Effects of heat stress on production in dairy cattle.J. DairySci. 86: 2131– 2144.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011339

Effect of Weed Control Management on Herbage Yield and Quality
in the Established Dwarf Napiergrass (Pennisetum purpureum Schumach)
Renny Fatmyah Utamy a, Yasuyuki Ishii b,*, Sachiko Idota b, Lizah Khairani caInterdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Japanb
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japanc
Graduate School of Agriculture, University of Miyazaki, Miyazaki, Japan*Corresponding author: Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-kibanadai Nishi,
Miyazaki 889-2192, Japan. Tel.: +81 985 58 7251; fax: +81 985 58 7251.
yishii@cc.miyazaki-u.ac.jp

Abstract
In our previous extension activity of dwarf variety of late-heading type (DL) napiergrass (Pennisetum purpureum
Schumach) to southern Kyushu, weed control management is found to be a crucial factor for obtaining good
establishment and considerable herbage dry matter (DM) yield in an established year. The objective of this study
was to examine the effect of several weed control practices, i.e. mixed sowing of annual setaria (abbreviated asS), which has no regrowth ability in stem-elongated tillers, paper-mulching (as P) and hand-weeding (as W),
compared with no-weeding (as –W) on DM yield and quality of this species for two years (Trial-1,2). Weed
control practices had a significantly (P < 0.05) positive effect on plant height, tiller density, percentage of leaf
blade and leaf area index in DL napiergrass, compared with no-weeding (S-W or –W), and paper-mulching (P or
P+S-W) had the highest yields in both trials. Setaria-sowing had a partially mitigating effect of weed damage on
growth of DL napiergrass, while additive DM gain from setaria could compensate the yield decrease in DL
napiergrass and reduce herbicide cost. Neither IVDMD nor CP content was affected by any weed control ineither trial. Thus, paper-mulching and annual setaria-sowing could be a good proposal in weed control of thisspecies.
Key words: annual setaria, dwarf napiergrass, herbage yield, paper mulch, weed control

Introduction
Based on extension activity of dwarf variety of late-heading type (DL) napiergrass(Pennisetum purpureum Schumach) to southern Kyushu, weed control management is found to bea crucial factor to obtain good establishment of this grass and achieve considerable herbage yield in
the established year (Utamy et al., 2011). Hand-weeding was a sole weed control practice, whichcaused physical and spiritual burden for farmers before the launch of herbicides. Therefore,
invention of easy and environmentally effective weed control technology has been strongly desired.
Weed invasion into the established forage crop fields is a visible sign of management
problems. Damages to forage crop production by weeds are mediated principally from loss ingrowth rate and yield, and secondarily from decline in forage quality. However, in forage crop
production, use of herbicide should be avoidable because of the negative effect on livestocks andincrease in production cost (Sakai & Kawanabe, 1981).
Weeds in the inter-row space of DL napiergrass are normally controlled by hand-mowing
machine 2‒3 times before the first defoliation of this grass. Repeated weed control is essential until
the leaf canopy is well established at the establishment. Even though close spacing is desirablefrom the weed control point of view, weeds do invade even at 50 × 50 cm of plant spacing.
Mulching at the inter- and intra-row spaces reduces weed problems by preventing the seedgermination and suppressing growth of emerged weed seedlings, resulting in facilitating soil fertility
and plant productivity (Wilson et al., 1987; Obiefuna, 1991; Salau et al., 1992). Mulching is a wellknown method for the establishment of horticulture crop such as lettuce (Moniruzzaman, 2006) and

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Proceedings of The 7 ACSA Conference 2011

tomato (Anzalone et al., 2010) and also in paddy rice field (Won et al., 2011). In the grass
cultivation, mulching is often used as living mulch or cover crop such as white clover (Deguchi et al.,2005), legume (Hiltbrunner et al., 2007) and hairy vetch (Mohammadi, 2010). However, papermulching has not been applied to DL napiergrass as weed control management.
The other way for weed control management is the oversowing of annual grass species tocompete weed at the early growth of perennial forage crops. DL napiergrass was oversown withtemperate Italian ryegrass (Lolium multiflorum Lam.) to get herbage in the spring-early summer
season (Ishii et al., 2005). In the present study, tropical annual setaria (Setaria italica cv.
Natsukanso), released from Yukijirushi Seed Co. Ltd., is utilized as once-cutting herbage with noregrowth ability if it starts stem elongation at the harvest, gives early summer growth, and should bealso ideal to suppress summer weeds at the early growth of perennial forage species (Wakamatsu,
2004).
Therefore, the objectives of this study was to examine the effect of weed control
management on dry matter yield and herbage quality in the established DL napiergrass by papermulching, oversowing of annual setaria and several time of weeding practices, compared with noweeding control in two years.

Materials and Methods
In 2008, DL napiergrass transplanted at 2 plants m-2 was imposed by three treatments (P,S+W, S-W) with three replications by Latin square design in Sumiyoshi Livestock Science Station(31°98´N, 131°46´E), University of Miyazaki (Trial-1). In 2010, DL napiergrass transplanted at 2
plants m-2 was imposed by four treatments (P+S-W, S+W, S-W, -W) with three replications by arandomized block design in Kibana Agricultural Science Station (31°83´N, 131°41´E), University ofMiyazaki (Trial-2). Growth attributes including yield, in vitro DM digestibility (IVDMD) and crude
protein (CP) content as herbage quality were determined in both trials. Efficiency of weed control
practices in plant parameters, such DM yield, IVDMD and CP content, was evaluated by thepercentage of plant parameter value in each weed control practice to that in no weed control.

Results and Discussion
Weed control management had significantly (P < 0.05) positive effects on plant density,
tiller density, percentage of leaf blade and leaf area index in DL napiergrass, compared with noweeding (S-W or –W), and paper-mulching (P or P+S-W) had the highest yields in both trials(Figure 1).
Adoption of several weeding practices such as weeding, paper-mulching and setariasowing can be assessed by the percentage of gain or loss in attributes under the particular practicerelative to those under no adoption of the practice (Table 1). Adoption of paper-mulching facilitatedto obtain largest positive gain of DM yield in DL napiergrass at two defoliations in both Trials 1 and
2, respectively. Situation was similar for the positive gain by adoption of hand-weeding, while thedegree of gain was reduced from paper-mulching (Table 1). The advantage of paper-mulching in
DM yield was closely corresponded with positive gain in plant height, tiller density and leaf areaindex through improvement in light penetration (Peltzer & Köchy, 2001) and prevention of plantdamage from weeds (TruGreen Tree and Shurub Field Guide, 2008). Positive effect of papermulching on DM yield matched with several crops such as paddy rice (Won et al., 2011), lettuce(Moniruzzaman, 2006), tomato (Anzalone et al., 2010) and turmeric (Sanyal & Dhar, 2006).
Weed control management by sowing setaria had no significant (P > 0.05) effect tosuppress weed DM yield in Trial-2 (data not shown). However, 1 g m-² DM production from setaria
reduced 4 g m-² of DM production from weeds (y = 104.553 – 0.247x, r = 0.52, P > 0.05). In severalth

Proceedings of The 7 ACSA Conference 2011341

stand establishments of perennial species, annual species are used to suppress weeds andmitigate the competition of crops from weeds. In the present study, annual setaria has
characteristics to give good growth in early summer and suppress summer weeds (Wakamatsu,
2004).

2nd

140

150

a

(A) 2008

120

120

Tiller (No. m-2)

Plant height (cm)

1st

100
80

b

a
a
b

ns

60

c

ns

40

(C) 2008

1st

90

2nd

ns

a

60

a

ns

30
20

b

a
b

0
1 Jul

1 Aug

1 Sep

1 Oct

1 Nov

0
1 Jul

1 Dec

c

1 Aug

1 Sep

S+W

80
60

a
a
ab
b

1 Nov

1 Dec

S-W

a
b
c
c

2nd

(D) 2010

a
a

120

a

b

a

90
60

b
b

c
c

b
a

40

30

20
0
1 Jul

S+W

1st

150

a
a
b
b

Tiller (No. m-2)

Plant height (cm)

100

1 Dec

2nd

a
a
a
b

(B) 2010

120

P-W

S-W

1st

140

1 Nov

Date

Date
P-W

1 Oct

1 Aug

1 Sep

1 Oct

1 Nov

1 Dec

b

b
b
b

0
1 Jul

b
1 Aug

Date
P+S-W

S+W

1 Sep

1 Oct
Date

S-W

-W

P+S-W

S+W

S-W

-W

Arrows indicate the time for the first (1st) and second (2nd) defoliation. P, interrow space was covered by paper mulch; S,
oversown with setaria; W, weeding and –W, no weed control. Symbols with different letters are significantly different among
weed control managements at each date by LSD method at 5% level. ns: P > 0.05.

Figure 1. Changes in plant height and tiller density of DL napiergrass under several weed control managementin 2008 (Trial-1) and 2010 (Trial-2).

Neither IVDMD nor CP content was affected by any weed control in either trial.
Consistently positive effect of any weed control practice on quality attributes was hardly obtained ineither trial, while decline in CP content under weeding and paper-mulching practices was common
at the second and first defoliation in Trial 1 and 2, respectively (Table 1). Living mulch with whiteclover improved plant nutrition by enhancing phosphorus uptake in maize (Deguchi et al., 2005).
The present paper-mulching and hand-weeding could not contribute to herbage quality of DL
napiergrass, possibly due to the negative correlation of DM yield with quality attributes under similar
fertilization in this species. Dwarf napiergrass cv. Mott, which has almost equivalent plant attributes
to DL napiergrass, had IVDMD and CP content at 67.5 and 13.2%, respectively (Sollenburger et al.,1988), almost corresponded with the present IVDMD at 74 and 60% at the first and second
defoliation, respectively, in Trial 1.

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Proceedings of The 7 ACSA Conference 2011

Table 1. Effect of weed control practices on efficiency in several parameters of plant total in DL napiergrassat each defoliation in 2008 and 2010Year

Defoliation

Parameter

2008(Trial 1)

1st

DM Yield
IVDMD
CP content
DM Yield
IVDMD
CP content
DM Yield
IVDMD
CP content
DM Yield
IVDMD
CP content

2nd

2010
(Trial 2)

1st

2nd

Weeding
248
4
2
7554
14
-16
74
6
-19
73
-1
-3

Practice
Paper-mulching
2466
1
-9
54091
-6
-24
275
7
-6
73
-1
9

Setaria-sowing
―
―
―
―
―
―
-4
1
11
36
0
3

DM yield, dry matter yield; IVDMD, in vitro dry matter digestibility; CP content, crude protein content.

Conclusion
Paper mulch is not common to use in DL napiergrass cultivation, while it proved to beeffective to avoid weed damage and facilitate good growth with high DM yield of this species. Costof paper mulch is 50 yen/meter (Sanyo Seishi Co. Ltd., Tottori) and setting of paper mulch is alabor-extensive weed control practice. Thus, based on the amount of natural seed bank of weeds,
paper mulch or other degradable mulching material can be applied to DL napiergrass, so as toreduce weed competition at the establishment. Annual setaria-sowing gave advantage to getherbage yield at the first defoliation of DL napiergrass, although prompt harvest time of annual
setaria should be examined in the mixed cropping with DL napiergrass.

References
Anzalone A, A Cirujeda, J Aibar, G Pardo, C Zaragoza. 2010. Effect of biodegradable mulch materials on weed
control in processing tomatoes. Weed Tech. 24: 369‒377.
Deguchi S, S Uozumi, T Tawaraya, H Kawamoto, O Tanaka. 2005. Living mulch with white clover improves
phosphorus nutrition of maize of early growth stage. Soil Sci. & Plant Nutr. 51: 573‒576.
Hilltbrunner J, P Jeanneret, M Liedgens, P Stamp, B Streit. 2007. Response of weed communities to legume
living mulches in winter wheat. Agron. & Crop Sci. 193: 93‒102.
Ishii Y, M Mukhtar, S Idota, K Fukuyama. 2005. Rotational grazing system for beef cows on DL napiergrass
pasture oversown with Italian ryegrass for 2 years after establishment. Grassl. Sci. 51: 223‒234.
Mohammadi GR. 2010. Weed control in irrigated corn by hairy vetch interseeded at different rates and times.
Weed Biol. & Manag. 10: 25‒32.
Moniruzzaman M. 2006. Effects of plant spacing and mulching on yield and profitability of lettuce (Lactuca sativaL.). J. Agric & Rural Dev. 4: 107‒111.
Obiefuna JC. 1991. The effect of crop residue mulches on the yield and production pattern of plantain (Musa
AAB). Biol. Agric. & Hort. 8: 71‒80.
Peltzer DA, M Köchy. 2001. Competitive effects of grasses and woody plants in mixed-grass prairie. J. Ecol. 89:
519‒527.
Sakai H, S Kawanabe. 1981. Ecology of grassland weed in Japan. (In) Weeds and Weed Control in Asia. Aspac,
China. Pp. 216‒239.

thProceedings of The 7 ACSA Conference 2011343

Salau A, NOA Opara, R Swennen. 1992. Effect of mulching on soil properties, growth and yield of plantain on atropical utisol in southeastern Nigeria. Soil and Tillage Research. [home page on the Internet]. [cited on 17
January 2011]. Available from URL: http://www.sciencedirect.com/science?_ob=ArticleURL&_.
Sanyal D, P Dhar. 2006. Effect of mulching, nitrogen and potassium levels on growth, yield and quality ofturmeric grown in red lateritic soil. [home page on the Internet]. [cited on 13 January 2011]. Available from
URL: http://www.actahort.org/books/769/769_17.htm.
Sollenberger LE, GM Prine, WR Ocumpaugh, WW Hanna, CS Jones Jr, SC Schank, RS Kalmbacher. 1988.
‗Mott‘ Dwarf Elephantgrass: A high quality forage for the subtropics and tropics. Circular S-356.
Agricultural Experiment Station, Institute of Food and Agricultural Sciences, University of Florida,
Gainsville, Florida, USA. Pp. 1‒18.
TruGreen Tree and Shrub Field Guide. 2008. Tree/shrub fact sheet. [home page on the Internet]. [cited on 13
January 2011]. Available from URL:
http://info.trugreen.com/ci/fattach/get/141926/1274121076/session/L2F2LzEvc2lkL0lEM3gqUmpr.
Utamy RF, Y Ishii, S Idota, N Harada, K Fukuyama. 2011. Adaptability of dwarf napiergrass under cut-and-carryand grazing systems for smallholder beef farmers in southern Kyushu, Japan. J. Warm Region. Soc. Anim.
Sci., Japan, 54: 65‒76.
Wakamatsu I. 2004. Yukijirushi Seed Co. Ltd. Bokusou to Engei. 52 (2): 13‒16. (In Japanese).
Wilson GF, R Swennen, E De Langhe. 1987. Effect of mulch and fertilizer on yield and longevity of a medium
and giant plantain and banana cultivars. Proceeding of the Third Meeting on International Cooperation for
Effective Plantain and Banana Research, 27-31 May 1985. IARPB/INIBAP. Abidjan, Cote d‘lvoire. Pp.
109‒111.
Won HY, JK Kim, WT Jeon, HS Han, JY Kim, JJ Shin. 2011. Paper Mulching Management for Weed Control
and Rice Growth in Paddy. [home page on the Internet]. [cited on 7 January 2011]. Available from URL:
http://a-c-s.confex.com.

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Proceedings of The 7 ACSA Conference 2011

Effect of Density of Mother Plants on Efficiency of Nursery Production in
Dwarf Napiergrass (Pennisetum purpureum Schumach)
Asuka Yamanoa, Yasuyuki Ishiib,*, Koutarou Moria, Renny F Utamyc, Sachiko IdotabaGraduate School of Agriculture, University of Miyazaki, Miyazaki, Japanb
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japanc
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Japan* Corresponding author: Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai Nishi,
Miyazaki 889-2192, Japan.
Tel.: +81 985 58 7251; fax: +81 985 58 7251.
yishii@cc.miyazaki-u.ac.jp

Abstract
Recently, dwarf napiergrass (Pennisetum purpureum Schumach) of late-heading type (named as DL) was
introduced widely for multi-purpose uses into Kyushu Island, Japan. Napiergrass needs to be propagatedvegetatively, while nursery production was not efficiently established yet. We examined to obtain an optimalplant density of mother plants for the efficient nursery production (per both unit land area and labor time bases).
-2
Dwarf napiergrass was transplanted at three levels (1, 4 and 8 plants m ) from cell-tray plants by Latin square
method in the University of Miyazaki (31°83´N, 131°41´E) on 2 July, 2010. Compound fertilizer containing 14%-2-1
of N, P2O5 and K2O was applied monthly at 5 g each m time (annual total 20 g) and growth attributes of plantheight and tiller density were monitored monthly. Dry matter yield (DMY) of mother plants and efficiency ofnursery production using single-node stem cutting were determined per both land area and labor time on 8
December, 2010 and wintering ability was determined on 17 May, 2011 (Trial-1). Nursery plant was grown atcell-tray bed in glasshouse during wintering from December to April and trimming effect on tillering ability ofnursery plants was checked on 13 April, 2011 (Trial-2). In Trial 1, plant height and tiller density increased with-2
-2
increasing plant density and tiller density saturated earlier at 4 and 8 plants m than at 1 plant m . Although-2
DMY and number of nursery plants per mother plant was significantly higher (P < 0.05) at 1 plant m , those per2
m were significantly higher (P < 0.05) and labor time for nursery production per cutting, significantly lower (P <-2-20.05) at 4 and 8 plants m than at 1 plant m . Wintering ability was considerably high above 93% among 3
densities. In Trial-2, emerged percentage of nursery plants was consistent around 75% among 3 densities andtrimming increased tiller number to promote quicker establishment of nursery plants in the field. Thus, 4 and 8-2plants m were concluded to be optimal for nursery production in the established year.
Keywords: dwarf napiergrass, nursery production, plant density, trimming, wintering ability.

Introduction
Napiergrass (Pennisetum purpureum Schumach) is a tropical C4 grasses and its dry matteryield was higher than other crops. Napiergrass can be used for multi-purpose uses such as feeding
to herbivores (Fukagawa et al., 2010; Utamy et al., 2011), rotational grazing use of dwarf variety(Ishii et al., 2009), feedstock for bioethanol production (Khairani et al., 2010) and phytoremediation
activity (Hamano et al., 2011) in Kyushu, Japan. Napiergrass needs to be propagated vegetatively,
while efficient nursery production is not so established that the extension of this grass species ishardly progressed. Therefore, in the present study, we examined the effect of mother plant density
on the efficiency of nursery production per both land area and labor time bases (Trial-1). In Trial-2,
we examined the effect of trimming on growth attributes of nursery plants, to obtain fasterestablishment of this grass species.

thProceedings of The 7 ACSA Conference 2011345

Materials and Methods
Dwarf variety of late-heading type (DL) napiergrass, which was overwintered on a cell-tray
bed in a glasshouse from December 2009 to April 2010, was transplanted into the field at threedensities (1, 4 and 8 plants m-2) by Latin square method in University of Miyazaki (31°83´N,
131°41´E) on 2 July, 2010. Compound fertilizer containing 14% of N, P 2O5 and K2O was applied-2-1monthly at 5 g each m time (annual total 20 g) and growth attributes of plant height and tiller
density were monitored monthly. Dry matter yield (DMY) of mother plants and efficiency of nursery
production using single-node stem cutting were determined per both land area and labor time on 8
December, 2010. Wintering ability, such as percentage of overwintered plants (POP) and regrowntiller number (RTN), was determined on 17 May, 2011 (Trial-1). Nursery plant was grown at cell-tray
bed in a glasshouse during wintering from December to April and the effect of trimming at 5 cm
above the ground on tillering ability of nursery plants was checked to determine plant height, tiller
number and dry matter weight on 13 April, 2011. Additional fertilizer was supplied at 9.8 g N m -2 on
16 April, 2010 (Trial-2).

Results and Discussion
Effect of mother plant density on growth rates of several plant attributes, dry matterproduction and nursery plant production
With the increase in mother plant density, the growth rate of plant height and especiallynumber of tillers used for nursery production tended to increase and reached the stage of maximum
tiller density earlier in early August than at the lowest density of 1 plant m-2 (Figure 1). Thereafter, it
occurred self-thinning to diminish less vigorous tillers and to decrease the tiller density at 4 and 8
plants m-2.
180

(A)

(B)

120
Tiller density (No m-2

140120
Plant height (cm

140

ns

160

100
80
60
40
20

0

100
ns

80

60
40
20
0

1-Jul

1-Aug
1 plant m-2

1-Sep

1-Oct
4 plants m-2

1-Nov

1-Dec

8 plants m-2

1-Jul

1-Aug
1 plant m-2

1-Sep

1-Oct

4 plants m-2

1-Nov

8 plants m-2

Figure 1. Changes in plant height (A) and tiller density (B) among three densities of mother plants.

Dry matter weight per plant was the highest at 1 plant m-2, followed by 4 and 8 plants m-2,
while dry matter yield per unit land area (m2) increased with the increase in mother plant density toreach the highest yield (P < 0.05) at 8 plants m-2 (Figure 2).
While number of nursery plants per mother plant was significantly higher (P < 0.05) at 1
plant m-2, those per m2 were significantly higher (P < 0.05) and labor time for nursery production per
cutting, significantly lower (P < 0.05) at 4 and 8 plants m-2 than at 1 plant m-2 (Figure 3). Even
though plant leaf area was not determined in the present study, it is estimated that optimum leaf
area index (LAI) was attained at earlier stage to enhance dry matter yield in early December with
the increase in plant density. It is suggested that the period to make internode and node with tiller
buds matured would be proportional to periods from tiller emergence as the basis for matured tiller
buds suitable for nursery production. It is suggested that even delayed tillers emerged in early2
September could be used for nursery production at 1 plant m- , which did not have enough time fortiller buds to matured (Figure 1 (B)).

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Proceedings of The 7 ACSA Conference 2011

These tiller productions also need more tillers to produce nursery plants, resulted inextending time for nursery production and increasing cost of labor power at lower densities.

3000

3000

a
DMW(g)DMW(g)

2000

b

1500

1000

a

2500

2500

a

D

b
b

500

b

0

plant

Unit
area

plant

Unit
area

plant

2000

b

1500

1000

ST

500

LB

0

a

b

ST

b

LB

plant

Unit
area

D

b

Unit
area

plant

Unit
area

plant

Unit
area

1
4
8
Plant density (plants m-2 ) and per basis

148
Plant density (plants m-2 ) and per basis

0.5

a

a

700
600

0.4

a

500

0.3

400

a

b

0.2

b

200

b

0.1

300

b

b

No. of nursery plants

Labor time（man・minute nursery plant-1 )Figure 2. Dry matter weight (DMW) of plant organs per both plant and unit area bases among three densities ofmother plants on 8 December 2010.

100

0.0

0

1

4

8

Plant density (plants m-2 )Nursery plant-1

Nursery m-2

Time

Figure 3. Nursery plant number and labor time for production per mother plant and per unit area.

Relationship between plant density of mother plants and sustainability of plants
In the present study, POP was hardly affected by plant density of mother plants and POP
overpassed 93% in all densities to show sustainability of this grass species. However, RTN per unit
land area (m2) tended to decrease consistently with the increase in mother plant density (Figure 4).
Since this phenomenon would affect negatively on maintaining tiller number for vegetative nursery
production and sustainability of mother plants, it is essential to continue examining the potential ofnursery production by these mother plants.
ns

25

100
90

20

80

15
70

10

POP（%）

Tiller density（No. m-2 ）

30

60

5

0

50
1

4

8

Plant density (plants m-2 )TNPOP

Figure 4. Percentage of overwintered plants (POP, ○) and regrown tiller density (■) on 17 May.

th

Proceedings of The 7 ACSA Conference 2011347

Effect of trimming on nursery plants
In Trial-2, emerged percentage of nursery plants was consistently 75% among 3 densities
at 2 months after planting (Figure 5). Trimming on nursery plants affected to increase tiller number
so as to promote quicker establishment of nursery plants in the field, even though plant height anddry matter weight of nursery plants decreased by the trimming practice (data not shown).

Figure 5. Changes in emerged percentage of nursery plants among three densities of mother plants during
wintering period from early December 2010 to mid-May 2011.

In conclusion, 4 and 8 plants m-2 were suggested to be optimal for nursery production in
the established year of DL napiergrass in southern Kyushu, Japan.

References
Fukagawa S, J Hirokawa, M Ookushi and Y Ishii. 2010. Fermentative quality and feed characteristics of dwarf
napiergrass (Pennisetum purpureum Schumach) silage. Japan. J. Grassl. Sci. 56: 26-33.
Hamano K, Y Ishii, A Yamano, K Mori, S Idota, DJ Kang, K Nakano, A Taniguchi and A Nishiwaki. 2011.
Development of low-cost phytoremediation techniques on heavy metal polluted soils by napiergrass
cultivation. Abstract for the 57th Meeting by the Association of the Kyushu Agricultural ResearchInstitute. Pp. 23. (In Japanese.)
Ishii Y, K Fukuyama, T Iwakiri, A Wadi and S Idota. 2009. Establishment of rotational grazing system on dwarf
napiergrass pasture oversown with Italian ryegrass by smallholder farmers of Japanese-Black breeding
beef cows in southern Kyushu, Japan. Proceedings of the 3rd Korea-China-Japan Joint Symposium on
Grassland Agriculture and Livestock Development. Pp. 218-219.
Khairani L, Y Ishii, S Idota, RF Utamy, A Yamano and A Nishiwaki. 2010. Nursery production and dry matterproductivity in six genotypes of napiergrass as biomass production in Southern Kyushu, Japan. Japan. J.
Grassl. Sci. 56 (Ext.) 14.
Utamy RF, Y Ishii, S Idota, N Harada and K Fukuyama. 2011. Adaptability of dwarf napiergrass under cut andcarry and grazing systems for smallholder beef farmers in southern Kyushu, Japan. J. Warm Region.
Soc. Anim. Sci., Japan. 54: 87-98.

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348

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Proceedings of The 7 ACSA Conference 2011

Analysis of the Major Seed Storage Protein, 13S Globulin, in Common
Buckwheat (Fagopyrum esculentum Moench)
Tomoyuki Katsube-Tanaka a,*, Nadar Khan a, Yusuke Takahashi a,Mariko NakagawaaaGraduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan* Corresponding author: Graduate School of Agriculture, Kyoto University, Kitashirakawa,
Kyoto 606-8502, Japan.
Tel.: +81-75-7536043; fax: +81-75-7536065.
tanakato@kais.kyoto-u.ac.jp

Abstract
Common buckwheat seed proteins are valuable proteins with high nutritional and biological values. The proteinsare also health-promoting proteins with many physiological functions, such as the ability to lower blood
cholesterol and to prevent accumulation of fat, constipation, mammary carcinogenesis and colon
carcinogenesis. However, the proteins are allergenic to human beings. The elimination or mitigation of theallergenic proteins is highly desired for the enhancement and potential utilization of buckwheat as a food crop.
The prevalent allergen of common buckwheat, Fag e 1, is β polypeptide of the most abundant storage protein,
13S globulin. Because the 13S globulin is composed of multiple diversified subunits, α polypeptide, thecounterpart of β polypeptide in 13S globulin subunits, was characterized in depth in this study to better
understand this allergenic protein. The 13S globulin α polypeptides were categorized into three types and were
further grouped into methionine-poor and methionine-rich subunits as major and minor types, respectively.
Besides the three known methionine-poor subunits, four new methionine-poor subunits with 0, 2, 4, and 6
tandem repeat inserts were identified. Highly polymorphic band pattern and its correlation between SDS-PAGE
and PCR analyses at a single seed level suggested that the large variation among the α polypeptides was
explained by the different lengths of tandem repeat inserts. In agreement with the fact that the tandem repeat
region is hydrophilic with many arginine residues, digestibility against trypsin, that is one of critical characteristics
for food allergen, was different between the subunits with and without tandem repeat inserts.
Keywords: 13S globulin, seed storage protein, common buckwheat (Fagopyrum esculentum Moench)

Introduction
The allergens of buckwheat seeds have been identified and characterized by several
research groups. Even though the allergens varied among patient sera. Park et al. (2000) revealed
that the 9-, 16-, 19-, and 24-kDa proteins were the most prevalent allergens and that the 30-, 43-,
and 67-kDa proteins were the least prevalent allergens. The 24-kDa protein (named Fag e 1), whichis recognized as one of the most significant allergens by researchers, is the β polypeptide of the
13S globulin. The Fag e 1-null mutant has been sought, but it has not been discovered. However,the content of a Fag e 1 has been demonstrated to change among cultivars (Maruyama-Funatsuki
et al., 2004).
Buckwheat seeds contain 8.5% to 18.9% protein. The most abundant protein is the 13S
globulin, which is salt-soluble and accounts for approximately 43% of the total seed protein. The
13S globulin is a storage protein and resembles the legumin-like seed storage protein of otherspecies, such as rice glutelin and soybean glycinin. It is considered a member of the 11S globulin
family based on its sedimentation constant, amino acid homology and similarities in biosynthetic
and accumulation processes. Like other legumin-like, seed storage proteins (i.e., rice glutelin andsoybean glycinin), the buckwheat 13S globulin is composed of multiple subunits, each of whichcontains acidic (α) and basic (β) polypeptides covalently linked by a disulfide bond.

thProceedings of The 7 ACSA Conference 2011349

The subunits of seed storage proteins show different characteristics from each other.
However, the subunit composition of buckwheat has seldom been of concern, except in somestudies by Cepkova & Dvoracek (2006), Rogl & Javornik (1996), and Bonafaccia et al. (1994). In
fact, even the differences in the subunit structures and the maximum number of subunit types are
not well known. To date, only four types of 13S globulin subunits have been identified.
In our current paper, we identified and characterized the α polypeptides of the buckwheat
13S globulin by SDS-PAGE and 2D-PAGE, coupled with immunodetection using two uniqueantibodies against the 13S globulin-related rice glutelin and soybean glycinin. Four new subunits
with various lengths of tandem repeats were confirmed by PCR analysis at a highly polymorphic
single seed level. By showing different digestibilities among the subunits, we proposed thepossibility to develop novel buckwheat plants with lowered allergenicity.

Materials and Methods
Plant materials and preparation of the 13S globulin
Flour and seeds of common buckwheat cultivar ‗Shinano-ichigo‘ were obtained from a
local milling company in the Nagano prefecture, Japan and a local nursery company in the Shiga
prefecture, Japan, respectively. Note that the commercial flour was made of uncounted number ofground seeds and was expected to show averaged and practical characteristics in terms of protein
composition. Tartary buckwheat seeds of cultivar ‗FT Rotundatiem‘ were a gift from the National
Agricultural Research Center for Kyushu Okinawa Region. The protein fraction of the 13S globulin
was extracted from either a single seed flour or commercial flour with the extraction buffer (0.035 M
potassium phosphate, pH 7.6, and 0.4 M sodium chloride). The 13S globulin was either dissolved in
the sample buffer containing 50 mM Tris, pH 6.8, 2% (w/v) SDS, 0.1% (w/v) bromophenol blue,
10% (w/v) glycerol, and 5% (v/v) 2-mercaptoethanol for SDS-PAGE analysis or dissolved in thelysis buffer containing 9.5 M urea, 2% Triton X-100, 5% 2-mercaptoethanol, and 5% Bio-Lyte 3-10
(Bio-Rad) for 2D-PAGE analysis.
SDS-PAGE, 2D-PAGE, and Western blot Analyses
SDS-PAGE was performed using 14%T acrylamide gel at a constant voltage of 200 V.
Isoelectric Focusing (IEF) was performed with IPG strips pH range 4-7 (BioRad) with a 7.5-cm
length according to the manufacturer‘s instruction. Electrophoresis in the first dimension (IEF) was
performed at 200 V for 10 min, 400 V for 10 min, 1000 V for 10 min, and 1500 V for 12 hrs. The
immunoblotting was performed according to the method mentioned in our previous report (Khan etal., 2008). Anti-rice glutelin and anti-soybean glycinin antibodies (Katsube-Tanaka et al., 2004;
Katsube et al., 1999) were used to detect the 13S globulin α polypeptides of common buckwheat.
Genomic DNA extraction and PCR Analysis
Genomic DNA extraction from the ‗Shinano-ichigo‘ single seeds and PCR analysis were
performed according to a kit (Ampdirect plus, Shimadzu, Japan). The primers used over the tandem
repeat region around 433-663 bp from the 5‘-end of the GenBank accession gene D87980 were as
follows: forward (left1), AGGATG(C/T)CCGGAGAC(A/G)T(A/T)CCA and reverse (right1),
CTAACGTTC(C/T)CATCGAGCTG for the Met-poor subunits.
Trypsin digestion
Globulin fractions extracted from the ‗Shinano-ichigo‘ buckwheat flour were incubated with
1/100 (w/w) trypsin (Promega, sequence grade) for two hours at 37°C according to the
manufacturer‘s instruction. Aliquots taken at a given times were electrophoresed and compared by
CBB staining and western blotting with the anti-glutelin antibody.

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Proceedings of The 7 ACSA Conference 2011

Results and Discussion
The 13S globulin fractions from commercial flour of common buckwheat and tartary
buckwheat seeds were resolved by SDS-PAGE, and detected by immunodetection using antiglycinin and anti-glutelin antibodies. The α polypeptides of common buckwheat cv. ‗Shinano-ichigo‘
were separated into at least 9 major bands (a2-a10) and those of tartary buckwheat were into 7
major bands (ax, a1, a6-a10) (data not shown). The α polypeptides of common buckwheat wereranged from 30 to 47 kDa according to the mobility in SDS-PAGE.
For a detailed understanding in the variations of the 13S globulin α polypeptides, 13S
globulin α polypeptides from ‗Shinano-ichigo‘ commercial flour were analyzed by 2D-PAGE using
anti-glycinin and anti-glutelin antibodies. The major 8 bands recorded on SDS-PAGE gels wereresolved into several distinct spots with different pI values and with slightly different molecularweights on 2D-PAGE gels, forming a horizontal streak (data not shown). Each horizontal streak ofspots was collectively named, instead of naming each spot individually in this study. When the spots
were examined with the anti-glycinin and anti-glutelin antibodies, most spots reacted against eitherof two antibodies with different degrees except the spot a9.

Met-poor subunit


SP

b

Mol%

kDa

pI

D87980tandem repeats (5)Met Cys0.35 1.06

αβ41.3 21.1

α β
5.2 9.1

AF152003

tandem repeats (3)0.37 1.12

37.5 21.5

5.4 9.2

D87982

tandem repeat (1)0.40 1.19

33.5 21.4

5.2 9.7

3.53 1.55

28.5 20.2

5.0 6.7

Met-rich subunit
SP



b

AY256960
Figure 1. Schematic representation of the 13S globulin subunits.

Primary structures of the known 13S globulin subunits (pro-form, α and β polypeptide withsignal peptide) of common buckwheat were compared (GenBank accession No. D87980,
AF152003, D87982, AY256960). The subunits are classified as Met-poor and Met-rich according to
the methionine content of each subunit. The Met-poor subunits have tandem repeats with different
lengths (shown as a number in brackets with the α polypeptide). Horizontal arrows denote theposition for PCR primers.
A database search and the organization of all retrieved data showed the existence of three
types of methionine scarce subunits (Met-poor subunits) and one methionine abounding subunit(Met-rich subunit) (Fig. 1). The three Met-poor subunits contain variable tandem repeat sequences.
For example, GenBank accessions D87982, AF152003, and D87980 have one, three, and fivetandem repeats respectively, whereas the Met-rich subunit of GenBank accession AY256960 has
no tandem repeat sequence. Because the number of types observed in the SDS-PAGE of the αpolypeptides were greater than the number of known subunits, PCR primers were designed atconserved positions (horizontal arrows, Fig. 1) over the region containing the tandem repeats of the
Met-poor subunit to explore novel genes. The PCR amplification with the crude genomic DNA
produced seven types of bands, which were named mp2-mp8 (data not shown). The DNAthProceedings of The 7 ACSA Conference 2011351

sequences of the representative amplified bands (mp2-mp8) were determined and aligned with theMet-poor subunits of the GenBank accessions: D87980, AF152003, and D87982. The sequence
analysis revealed that the clones for bands mp3, mp5, and mp7 correspond to D87980, AF152003,
and D87982, respectively. Additionally, the clones for bands mp2, mp4, mp6, and mp8 were fromnovel genes with 6, 4, 2, and 0 tandem repeats, respectively.
When the PCR band patterns were compared with SDS-PAGE patterns examined using
the same seeds with those for PCR analysis, the occurrence pattern of the band mp3 showed goodcorrelation with that of the SDS-PAGE band a3 (data not shown). In addition, the mp2-mp8 bands
and a2-a8 bands were observed at equally spaced intervals (data not shown). The above results
led us to hypothesize that the a2-a8 bands are correlated with mp2-mp8, respectively. In otherwords, the bands a2, a3, a4, a5, a6, a7, and a8 have 6, 5, 4, 3, 2, 1, and no tandem repeats,
respectively.
The amino acid sequences deduced from PCR amplified bands mp2-mp8 were comparedwith those of D87980, AF152003, and D87982 (Fig. 2). The alignment clearly showed the tandem
repeat regions had less diversity, except the lengths of the bands. However, it is notable that the
tandem repeat regions and their vicinities had many arginine residues, and the regions werehydrophilic according to a method used by Kyte & Doolittle (1982), indicating the regions are likely
to be exposed at the molecular surface. This feature of the tandem repeat regions strongly suggests
the regions are susceptible to trypsin.
.

mp2
D87980mp3
mp4
AF152003
mp5
mp6
D87982
mp7
mp8

.

.

.

| .

.

.

.

10
| .

.

.

.

|

.

.

.

.

20
| .

.

.

.

|

.

.

.

.

30
| .

Hydrophilic
40
.

.

.

|

.

.

.

.

|

.

Tandem repeat 1 Tandem repeat 2.

mp2
D87980mp3
mp4
AF152003
mp5
mp6
D87982
mp7
mp8

.

.

.

|

.

.

.

.

50
| .

.

.

.

|

.

.

.

.

60
|

S ES E YP QS QR D QR S R QS ES E ES S R GD QR T R QS ES E E F S R GD QR T R QS ES E E F S R GD QR T R
S E F E YP QS QR D QR S R QS ES E ES S R GD QR T R QS ES E E F S R GD QR T R QS ES E E F S R GD QR T R
S ES E YP QS QR D QP S R QS ES E E F S R GD QR T R QS ES EG F S R GD QR T R QS ES E E F S R GD QR T R
S E F E YP R F QR D QR S R QS ES E ES S R R D QR S R QS ES E ES S R GD QR S R QS ES V ES S R GD QR S R
S E F E YP R S QR D QR S R QS ES G ES S R GD QR S R QS ES E ES S R GD QR S R QS ES E E F S R G ~ ~ ~ ~ ~S E F E YP QS QR D QR S R QS ES E ES S R GD QR T R QS ES E E F S R GD QR T R QS ES E E F S R G ~ ~ ~ ~ ~S E F E YP R S ER GR H S R QS ES E ES S R GD QS S R QS ES E ES S R G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~S E F E YP QS QR GR H S R QS ES E E ES S R G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~S E F E YP QS QR GR H S R QS K T E E ES S R G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~S E F QS ES ES S R S I S ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

.

.

.

| .

.

.

.

70
| .

.

.

.

|

.

.

.

.

80
| .

.

.

.

|

.

.

.

.

90
| .

.

.

.

|

.

.

.

.

100
| .

Tandem repeat 3
.

.

.

|

.

.

.

.

110
| .

.

.

.

|

.

.

.

.

120
|

QS ES E E F S R GD QR T R QS ES E E F S R GD QR T R QS EG E E F S R GD QH QK I F R I R D GD V I P S P A G
QS ES E E F S R GD QR T R QS ES E E F S R G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R D GD V I P S P A G
QS ES E E F S R GD QR T R QS ES E E F S R G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F G I R D GD V I P S P A G
QS ES E ES C QG ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R D GD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R D GD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R EGD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R EGD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R EGD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D QH QK I F R I R D GD V I P S P A G~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ D EH QK V F R I R EGD V I P S P A G

Tandem repeat 4

Tandem repeat 5

Tandem repeat 6

mp2

FigureD87980 2. Deduced amino acid sequences and hydrophobicities over the tandem repeat inserts of the Met-poor
mp3
subunits.
mp4
AF152003
The amino acid sequences deduced from the PCR bands amplified for the Met-poor
mp5
mp6
subunits(mp2-mp8) were compared with that of D87980, AF152003, and D87982. The amino acids
D87982
mp7
spanning
108-212 residues from the N-terminus of the mature α polypeptides (D87980) were
mp8

aligned. The position of tandem repeats 1-6 and approximate hydrophilic area were shown by
double-headed arrows and gray color shades, respectively.
Trypsin digestions demonstrated that most of the 13S globulin α polypeptides degradedwithin two hours except for the a8 band, which is likely to have no tandem repeat (data not shown).
The intensity of the a8 band detected by anti-glutelin stayed nearly constant during the reaction,and no new, α polypeptides were detected, suggesting the degradation occurred not only at atandem repeat region but also with the entire α polypeptide sequence. Notably, the a9 band, which

352

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Proceedings of The 7 ACSA Conference 2011

seems to be derived from a Met-rich subunit having relatively higher cysteine contents, was rather
resistant to digestion according to the CBB staining (data not shown).
It has been described that resistance to digestion and processing is one of the more
important characteristics for food allergenic proteins (Bannon, 2004). Sen et al. (2002) showedpeanut allergenic protein stability upon proteinase digestion affected IgE-binding epitopes
intactness. Even though the difference in the digestibility of the β polypeptides was empirically notrevealed in this study, the tandem repeat insertion into the α polypeptides might have influenced theβ polypeptide conformation as well by a conserved inter-polypeptide disulfide bond with one of theinvolved Cys residues located 18-residues upstream from the tandem repeats. If the hypothesis iscorrect, the Met-poor subunits of the 13S globulin may have different digestibilities and distinct
potential allergenicities. Although our preliminary analysis on pepsin digestibility demonstrated little
difference among the subunits (data not shown), reducing the content of the trypsin-resistantsubunit, the a8 α polypeptide with no tandem repeats, might be useful for the development of novel
buckwheat with lowered allergenicity. Further investigation on IgE binding of buckwheat
hypersensitive patients‘ sera should be required.

Acknowledgements
The authors thank Mr. Satoru Yamaguchi for his technical assistance. Germplasms of tartary
buckwheat seeds were kindly provided by the National Agricultural Research Center for Kyushu Okinawa
Region. This work was supported in part by grants to T.K.-T. from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan for Scientific Research (C) (20580013, 2008-2010; 23580020, 2011-2013) andby a fellowship to N. K. from the Japan Society for the Promotion of Science.

References
Bannon GA. 2004. What Makes a Food Protein an Allergen? Current Allergy and Asthma Reports 4: 43–46.
Bonafaccia G, R Acquistucci and Z Luthar. 1994. Proximate chemical composition and protein characterizationof the buckwheat cultivated in Italy. Fagopyrum 14: 43–48.
Cepkova P and V Dvoracek. 2006. Seed protein polymorphism of four common buckwheat varieties registeredin the Czech Republic. Fagopyrum 23: 17–22.
Katsube T, N Kurisaka, M Ogawa, N Maruyama, R Ohtsuka, S Utsumi and F Takaiwa. 1999. Accumulation ofsoybean glycinin and its assembly with the glutelins in rice. Plant Physiology 120: 1063–1074.
Katsube-Tanaka T, JB Duldulao, Y Kimura, S Iida, T Yamaguchi, J Nakano and S Utsumi. 2004. The two
subfamilies of rice glutelin differ in both primary and higher-order structures. Biochimica et Biophysica
Acta (BBA) - Proteins & Proteomics 1699: 95–102.
Khan N, T Katsube-Tanaka, S Iida, T Yamaguchi, J Nakano and H Tsujimoto. 2008. Identification and variationof glutelin α polypeptides in the genus Oryza assessed by two dimensional electrophoresis and step-bystep immuno detection. Journal of Agricultural and Food Chemistry 56: 4955–4961.
Kyte J and RF Doolittle. 1982. A Simple Method for Displaying the Hydrophobic Character of a Protein. Journalof Molecular Biology 157: 105–132.
Maruyama-Funatsuki W, K Fujino, T Suzuki and H Funatsuki. 2004. Quantification of a major allergenic protein
in common buckwheat cultivars by an enzyme-linked immunosorbent assay (ELISA). Fagopyrum 21:
39–44.
Park JW, DB Kang, CW Kim, SH Ko, HY Yum, KE Kim, CS Hong and KY Lee. 2000. Identification andcharacterization of the major allergens of buckwheat. Allergy 55: 1035–1041.
Rogl S and B Javornik. 1996. Seed protein variation for identification of common buckwheat (Fagopyrum
esculentum Moench) cultivars. Euphytica 87: 111–117.
Sen M, R Kopper, L Pons, EC Abraham, AW Burks and GA Bannon. 2002. Protein structure plays a critical rolein peanut allergen stability and may determine immunodominant IgE-binding epitopes. The Journal of
Immunology 169: 882-887.

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354

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Isolation of soc-tuf Gene Encoding Chloroplast Elongation Factor
Tu (EF - Tu) Protein from Sugarcane (Saccharum officinarum)
Dhika Amandaa, Sony Suhandonob *School of Science and Technology, Institut Teknologi Bandung,
LABTEK XI-Genetic Laboratory, Ganeca 10, Bandung 40132, Indonesia.
Tel.: +62 22 251-1575; +62 22 250-0258; fax: +62 22 253-4107. amanda.dhika@alumni.itb.ac.id ;*Corresponding author: sony@sith.itb.ac.id

Abstract
Molecular chaperone is a molecule preventing the aggregation, denaturation, and inactivation of various proteinswhen the cells were exposed to extreme environmental conditions, such as high temperature or drought. One ofsuch proteins is an Elongation factor Tu (EF - Tu). We have isolated an elongation factor Tu gene fromsugarcane from cDNA which was heat-treated at 45°C for 16 h using reverse transcriptase PCR. The isolation
was made in two steps. The first steps using internal primer developed using tuf gene sequence from maizeavailable in the GenBank. Since partial cDNA sequence needs to be filled in, the sequence from sugarcane
which is available in KEGG genome was added into a new primer designed to make overlapped PCR. This
technique was used to clone the gene into bacterial expression vector pET-32b in order to test the soc-tuf genefunction as molecular chaperone in bacterial system. The results showed that the gene had homology of 94%
with maize tuf gene and 97% with sorghum amino acid sequence. The deduced amino acid sequence hadhomology of 92% with maize and 95% with sorghum, respectively. Gene analysis was conducted by generate
phylogenetic molecular tree. Based on the tree, Sugarcane tuf sequence was actually clustered with Sorghum
bicolor and Zea mays sequence. Thus, it had high possibility that soc-tuf gene from sugarcane having similar
motif, protein structure, and can function as molecular chaperone as tuf gene from maize.
Keywords: chloroplast protein elongation, environmental stress, heat tolerance, sugarcane (Saccharum
officinarum)

Introduction
Elongation factor Tu (EF - Tu) is a protein that involved in translational process. By binding
to GTP and aminoacyl– tRNA, EF - Tu also translocate them into ribosome A-site (Brot, 1977; Riis,
et al., 1990). EF - Tu has three domains GTP-binding protein which also has similar characteristicwith Ras proteins superfamily (Warren, et al., 2001) belongs to the family of G proteins and is highly
conserved in all organisms (Bhadula et al., 2001).
EF – Tu in organelles (chloroplast and mitochondria) has the same function as EF - 1α ineukaryotic cytosol (Miller & Wissbach, 1977). Interestingly, the organellar EF – Tu shows moresimilarities to prokaryotic EF – Tu than to eukaryotic EF - 1α (Lee, et al., 1999). Previous studies
show that chloroplast EF – Tu has 80% similarity to prokaryotic EF - Tu and has 80 – 90% similarity
to other EF – Tu from higher plants (Bhadula et al., 2001; Riis, et al., 1990). This is reflects their
origin via ancient endosymbiosis (Gray, 1992). In most of lower photosynthetic eukaryotes,
chloroplast elongation factor Tu (EF - Tu) is encoded by tuf gene in chloroplast genome (Baldauf &Palmer, 1990). While in higher plants, the genes encoding EF – Tu are located in the nucleus(Moriarty, et al., 2002; Baldauf & Palmer, 1990) and after translation, the protein is transported into
chloroplast.
In prokaryotes, EF - Tu is also involved in heat tolerance mechanism as molecularchaperones, that prevent the degradation of cellular proteins during heat – stress, increase therefolding of unfolded proteins, and increase the stability of protein (Moriarty, et al., 2002; Momcilovic& Ristic, 2004; Rao, et al., 2004 Ristic, et al., 2008). During heat shock treatments, EF–Tu

th

Proceedings of The 7 ACSA Conference 2011355

molecules are accumulated in cytoplasm and near cytoplasmic membrane of E. coli (Caldas, et al.,1998). Moreover, E. coli EF–Tu protein has ability to remain soluble and maintain its activity to 45°C
(Caldas, et al., 1998). It also binds other proteins and extends their stability up to 50°C (Caldas, etal., 1998). Previous studies showed that plants also expressed chloroplast EF–Tu protein when it
was exposed to high temperatures (Momcilovic & Ristic, 2004; 2007; Rao, et al., 2004; Ristic, et al.,
2004; 2008). Exposure to a long term heat stress increased the level of chloroplast EF–Tu protein in
mature wheat plants (Ristic et al., 2008). The level of heat-induced EF–Tu protein in heat-tolerance
wheat cultivars is greater than EF–Tu level in heat-sensitive cultivars (Ristic et al., 2008). The same
pattern was also observed in maize that heat sensitive maize line ZPL 389 did not accumulate EFTu under heat stress, while heat tolerant line ZPBL 1304 accumulate EF–Tu (Momcilovic & Ristic,
2004; 2007). Therefore, only plants, which show better tolerance to heat stress, express highamount of heat-induced EF–Tu protein. However, the evidence that chloroplast EF-Tu protein
involved in heat tolerance, so far, reported only in maize (Bhadula et al., 2001; Momcilovic & Ristic,
2004; Rao et al., 2004; Ristic et al., 2004).
In this study, EF-Tu from sugarcane (Saccharum officinarum) was used. Sugarcane is C4
plant commonly found in Southeast Asia region. Sugarcane has 3-4 times longer harvesting period
than maize. It is assumed that sugarcane has been exposed to heat stress longer than maize andhave higher temperature tolerance. The purposes of this research were to isolate soc-tuf geneencoding chloroplast elongation factor Tu (EF-Tu) from sugarcane and to investigate the genesimilarities using phylogenetic tree. Therefore, this study was conducted based on the hypotheses,
if soc-tuf gene is significantly homologous with tuf from maize or highly conserved, thus there ispossibility that the protein may function to increase heat tolerance.

Materials and Methods
Samples preparation and treatment with high temperature
In this study, sugarcane plantlets with approximately of 4-6 cm of length were used. Before
isolation, the plantlets were treated with different duration of incubation. Sugarcane plantlets wereincubated at 45°C (Ristic, et al., 2002) in incubator for 2, 4, 6, 14, and 16 h. The same treatment
was also made to control plantlets exposed at 37ºC. Sugarcane from each treatment was taken andcleaned from the attached medium. Then, 600 mg of samples were used for RNA isolation total
RNA from sugarcane was isolated by using Trizol® Solution Kit (Sigma, USA), followed by cDNA
synthesis.
Primer design and tuf gene isolation from cDNA samples
There was no publication on sugarcane tuf gene so far, thus forward primer EF-TU-S for
tuf gene amplification was designed by using maize tuf sequence. Meanwhile, maize reverse primer
DO35690 (5‘-TTACCACCCTCACGGATAGCAAACCTC-3‘) (Ristic, et al., 2004) was used to amplify
sugarcane tuf gene. It was assumed that these primers will amplify about 1370 bp of sugarcane tuf
gene. Amplification was generated using KAPA 2GTM Robust HotStart PCR system (KAPA
Biosystems, USA) and sugarcane cDNA was used as a template.
Construction of full length tuf gene using overlap-extension PCR
Based on multiple sequence alignment, the amplified sugarcane tuf gene was notcompleted. To obtain full length sequence, sugarcane sequence was aligned with sugarcane ESTfrom KEGG genome (http://www.genome.jp/kegg). Reverse primer was designed containing bothsugarcane consensus sequence and expression vector sequence. While forward primer was
designed containing both of soc-tuf partial sequence and expression vector sequence. Primers
used to generate overlap-extension PCR were, forward primer EFTU-DA.F1 and reverse primer
EFTU-DA.R1, respectively. Amplification was generated by using Phusion® Polymerase PCR
356

thProceedings of The 7 ACSA Conference 2011

system (Finnzymes, USA) with touchdown PCR. Samples were visualized using DNA
electrophoresis. Relevant DNA fragment was extracted from agarose gel and purified for further
study.
Construction of tuf gene into expression vector pET-32b using PCR-cloning
In order to investigate the ability of sugarcane tuf gene, the gene was inserted into an
expression vector pET-32b using PCR cloning (Bryksin & Matsumura, 2010). Instead of using two
type of primer (forward and reverse primer), PCR cloning only used one DNA fragment derived fromoverlap–extension PCR product containing the gene of interest and two vector-homologous
sequence at 3‘ and 5‘- end of the sequence. This vector-homologous sequence acts as a primer
and anneals to the plasmid vector to initiate the amplification.
Sequence analysis and construction of phylogenetic tree
Multiple sequence alignment was generated using CLUSTAL W in Bioedit and Geneiousalignment in Geneious 4.8.5. Nucleotide and amino acid homology were analyzed using BLASTn
and BLASTx from NCBI (http:// www.ncbi.nlm.nih.gov). Phylogenetic tree was constructed using
MEGA 5.0 software.

Results and Discussion
Nucleotide sequence homology and similarity analysis
Based on BLASTn result (Table 1), sugarcane tuf sequence had 97% homology toSorghum bicolor hypothetical protein sequence with 100% query coverage. Sorghum bicolor
hypothetical protein has not been annotated yet. However, this protein has similar characteristics
with EF-Tu proteins. In order to make the result more reliable, sugarcane tuf sequence was alignedwith maize sequence and the result showed that sugarcane sequence had 94% similarity withmaize tuf sequence encoding elongation factor Tu (EF–Tu) with 100% query coverage. E-value forthese alignments are 0, thus the result was reliable.
Table 1. BLASTn result from sugarcane EF - Tu sequence using eucaryotes database
Accession
NumberSpecies

Query
Coverage

E-value

Homology

XM_002452345.1

Sorghum bicolor hypothetical protein, mRNA

100%

0

97%

NM_001156938.1

Zea mays elongation factor Tu(LOC100284040), mRNA >gb|EU966875.1|
Zea mays clone 297889 elongation factor Tu
mRNA, complete cds

100%

0

94%

In order to predict whether the nucleotide differences within sugarcane sequence hasframe shift effect at amino acid sequence or not, sugarcane sequence must be analyzed by using
BLASTx. Based on BLASTn result, sugarcane tuf sequence had 97% homology to Sorghum bicolor
hypothetical protein sequence with 100% query coverage. The result (Table 2) showed that thedifferences within sugarcane nucleotide residues do not change amino acid sequence significantly.
Sugarcane sequence still had 95% homology with Sorghum bicolor hypothetical protein and also
92% homology with Zea mays elongation factor Tu.
In order to investigate the homology between sugarcane and procaryotes tuf sequence,
another BLASTx and BLASTn were conducted using procaryotes protein database. Based on
BLASTn result, sugarcane tuf sequence had 67% homology to Escherichia coli (acc.

thProceedings of The 7 ACSA Conference 2011357

ZP_07125171.1) elongation factor Tu sequence with 90% query coverage. Moreover, The BLASTx
result (Table 3) showed that sugarcane sequence still had 70% homology with Thermus
scotoductus SA-01 and Thermus Aquaticus elongation factor Tu, also had 69% homology with
Thermus thermophilus HB27 elongation factor Tu.
Table 2. BLASTx result from sugarcane EF - Tu sequence using eucaryotes database
Accession
Number
XP_002452390.1
NP_001149568.1

Species
Sorghum bicolor hypothetical protein
SORBIDRAFT_04g024850 >gb|EES05366.1|
Zea mays LOC100283194 >gb|ACG35888.1|
Zea mays elongation factor Tu

Query
Coverage

E-value

Homology

99%

0

95%

99%

0

92%

Table 3. BLASTx result from sugarcane EF - Tu sequence using procaryotes database
Accession
NumberSpecies

Query
Coverage

E-value

Homology

YP_004201365.1

Elongation factor Tu [Thermus scotoductus SA-01]

90%

3e-14970%

1EFT_AThe crystal structure of elongation factor EF-Tu from
Thermus Aquaticus

90%

3e-14970%

YP_005299.1

Elongation factor Tu [Thermus thermophilus HB27]

90%

1e-14869%

Based on BLAST results, it can be concluded that the sequence isolated from sugarcane
was the tuf gene coding sequence. Characterization of sugarcane tuf gene was conducted by using
various bioinformatics tools. FGENESH 2.6 from Softberry (www.softberry.com showed that soc-tuf
cDNA sequence consisted of 1404 nucleotide residue which encodes 466 amino acid.
Construction of phylogenetic tree
Phylogenetic tree (Figure 1) of tuf sequences constructed based on sample sequencealigned with the various tuf sequences which were retrieved from GenBank and analyzed using
Neighbor-Joining method with Kimura 2 parameter. The phylogenetic tree will confirm the BLASTn
result. Tentative result of the BLASTn was used to collect other sequence related to the sample
sequence, and construct the tree.
Sugarcane sequence is clustered with Sorghum bicolor with value 0.021 and Zea mays
sequence with value 0.055 and also clustered at the same group as Oryza sativa with value 0.115
and Triticum aestivum with value 0.117 which belong to Poaceae family (the scale or branch lengthrefers to the evolutionary distances used to infer the phylogenetic tree).
Based on sequence homology, several groups were formed with Thermus thermophilus asan outgroup. The homology of tuf sequence within plants (intra-clade) were higher than the interclade homology of tuf sequence. Thus, plant species formed a single cluster. While, bacterial
species also formed a single cluster. Sugarcane sequence is actually clustered with Sorghum
bicolor and Zea mays sequence at almost no nucleotide difference. It is also clustered at the samegroup as Oryza sativa and Triticum aestivum which are belong to Poaceae family. Due to soc-tuf
gene has significant homology with maize tuf, thus there is high possibility that it also has similar
motif, protein structure, and can function as molecular chaperone as tuf gene from maize in order toimprove heat tolerance abillity in organisms.

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Proceedings of The 7 ACSA Conference 2011

99
76

88
95
55
100

100
54
100
99

100
64
100

0.20

0.15

0.10

0.05

gi|24030502 Arabidopsis thaliana
Brassicaceae
gi|297791346 Arabidopsis lyrata
Solanaceae
gi|459238 Nicotiana sylvestris
Euphorbiaceae
17519750/1-1281 Cassava
Geraniaceae
gi|12830554 Pelargonium graveolens
Fabaceae
gi|292774551 Lotus japonicus
Papilionaceae
gi|18775 Glycine max
gi|241985081 Triticum aestivum
gi|6525064 Oryza sativa
Poaceaegi|226508703 Zea mays
gi|242062201 Sorghum bicolor
Saccharum officinarum
Bacillaceae
gi|255767013 Bacillus subtilis
Moraxellaceae
gi|169794206 Acinetobacter baumannii
gi|207855516 Salmonella enterica
Enterobacteriaceae
gb|AE014075.1 Escherichia coli
Thermaceae
gi|48287 Thermus thermophilus
0.00

Figure 1. Neighbor-joining tree of tuf gene sequence using Kimura 2 parameter model with Gamma distribution.

References
Baldauf, S.L., J.D. Palmer. 1990. Evolutionary transfer of the chloroplast tufA gene to the nucleus. Nature. 344:
262 - 265.
Bhadula, S.K., T.E. Elthon, J.E. Habben, T.G. Helentjaris, S. Jiao, Z. Ristic. 2001. Heat stress induced synthesisof chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line. Planta. 6:212359.
Brot, N. 1977. Molecular Mechanism of protein synthesis. (H. Weissbach, & S. Pestka, Eds.). Academic Press,
New York.
Bryksin, A.V., I. Matsumura. 2010. Overlap extension PCR cloning: a simple and reliable way to create
recombinant plasmids. BioTech. 48: 463 - 465.
Caldas, T., A. Yaagoubi, G. Richarme. 1998. Chaperone properties of bacterial elongation factor EF-Tu. Biol.
Chem. 273: 11478 - 11482.
Gray, M.W. 1992. The endosymbion hypothesis revisited. Int. Rev, Cytol. 141: 233 - 357.
Lee, J.H., J.W. Lee, Y-Y. Chung, K.H. Paek, J.S. Shin, C-H. Yun. 1999. Cloning and characterization ofchloroplast elongation factor EF-Tu cDNA of Oryza sativa L. Mol. Cells. 9 (5): 484 - 490.
Momcilovic, I., Z. Ristic. 2007. Expression of chloroplast protein synthesis elongation factor, EF-Tu, in two linesof maize with contrasting tolerance to heat stress during early stages of plant develoment. Plant Physiol.
164: 90 - 99.
Momcilovic, I., Z. Ristic. 2004. Localization and abundance of chloroplast protein synthesis elongation factor(EF-Tu) and heat stability of chloroplast stromal protein in maize. Plant Sci. 81 - 88.
Moriarty, T., R. West, G. Small, D. Rao, Z. Ristic. 2002. Heterologous expression of maize chloroplast protein
synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress. Plant Sci. 163:
1075 – 1082.
Rao, D., I. Momcilovic, S. Kobayashi, E. Callegari, Z. Ristic. 2004. Chaperone activity of recombinant maize
chloroplast protein synthesis elongation factor, EF-Tu. Eur J. Biochem. 271: 3684 - 3692.
Riis, B., S.I. Rattan, B.F. Clarck, W.C. Merrick. 1990. Eukaryotic Protein Elongation Factors. Treds Biol. Sci. 15:
420 - 424.
Ristic, Z., U. Bukovnik, I. Momcilovic, J. Fu, P.V. Prasad. 2008. Heat-induced accumulation of chloroplast
protein synthesis elongation factor, EF-Tu, in winter wheat. Journal of plant physiology. Plant Physiol.
165: 192 – 202.
Ristic, Z., K. Wilson, C. Nelsen, I. Momcilovic, S. Kobayashi, R. Meeley. 2004. A maize mutant with decreased
capacity of accumulate chloroplast protein synthesis elongation factor (EF-Tu) displays reduced
tolerance to heat stress. Plant Sci. 167: 1367 – 1374.
Warren, C., I. Lathe, B. Peer. 2001. Evolution of tuf genes: ancient duplication, differential loss, and geneconversion. FEBS Letters. 502: 113 - 116.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011359

Endophyte Microbes from Oil Palm (Elaeis guineensis) Tissues and Its
Potential as a Biocontrol for Ganoderma boninense In Vitro
Wisnu Adi Wicaksono, Rika Fithri Buana, Elizabeth Caroline Situmorang
Plant Production and Biotechnology Division,PT SMART Tbk, Bogor, Indonesia)* Corresponding author: Plant Production and Biotechnology Division, PT SMART Tbk
Jl. Raya Padjajaran No 78 FG, Bogor, Indonesia
Tel.: +6251 8310854; fax: + 6251 8310854
wisnu_adi@hotmail.com

Abstract
Endophyte microbes are microbes that colonize internal plant tissues without causing visible damage to their
host plant and they can act as promising biological control agents in oil palm. The objective of this research is toobtain endophyte microbes which produce high chitinase activity and can inhibit G. boninense growth.
Endophyte microbes are isolated from roots of oil palm planted in Padang Halaban estate, North Sumatera. Dual
culture are performed in PDA medium using G. boninense from field. Chitinase assay was measured byspectrophotometry method. Potential isolates have been identified based on ITS rDNA using ITS1 and ITS4
primers for fungi and based on 16S rDNA using 9F and 1510R for bacteria. These endophyte fungi can inhibit G.
boninense growth with inhibition ratio < 64%. T1 isolate has the highest activity of chitinase with 2.07 U/mL.
There are four potential isolates of endophyte fungi: Aspergillus, Fusarium, Hypocrea, and Trichoderma. B2.1.2
isolate of bacteria has the highest inhibition ratio (67%) but B13.10.4 isolate has the highest chitinase activity
with 3.43 U/mL. This result can be assumed that inhibition activity do not have any corelation with chitinase
activity. The identification of potential isolates showed that they are Serratia, Burkholderia, Acinetobacter, andBacillus.
Keywords: root-endophytes microbe, Ganoderma boninense, Elaeis guineensis, chitinase activity, ITS rDNA,
16S rDNA

Introduction
Basal stem rot (BSR) disease caused by Ganoderma boninense is the most destructive
disease in oil palm industries (Flood et al. 2000). Mostly, visible disease symptoms of BSR would
appear at a late stage. Development of BSR infection could happen at 6 until 12 months (Darmono,
1996). The infected oil palm would decay at basal tissue with the result that infected plant would fall
down before the economical time. At the endemic location, plant could infected less than 2 years ofplanting period. Many strategies have been conducted to control BSR included cultural, chemical
and clean clearing. Unfortunately, those strategies have not been successful, for example, the useof chemical fungicide in the field has not been proven as an effective control method (Sapak et al.,2008).
Biological control can be promising strategy to control BSR. One of microbes group which
could use for biocontrol agent is endophyte microbes. Endophyte microbes is microbes thatcolonize internal plant tissues without causing visible damage to their host plant. Recent surveys ofvarious host plants have demonstrated that endophyte microbes are ubiquitous in plant species(Shiomi et al., 2006). As internal colonisers, therefore they are more able to compete within thevascular systems with capacity to prevent infection of G. boninense.
The objective of this study was to isolate the endophyte microbes for G. boninense
biocontrol based on antagonistic characteristic and chitinase activity and also to identify endophyte
microbes based on ribosomal DNA.

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Materials and Methods
Root sampling and isolation of endophyte microbe
Oil palm roots were obtained from Padang Halaban Estate PT SMART, Tbk in North
Sumatera, Indonesia. The age of palm was 28 years with symptomless BSR (Basal Stem Rot) at
endemic areas. Random palms were sampled with the roots diameter 0.5 cm, taken about 1.0 m
away from their bases at 25 – 30 cm depth.
Root samples were surface sterilized by dipping in 5.25 % sodium hypochlorite, andsubsequently in 50, 70, and 90 % of ethanol then rinsed twice with sterilized water. Root section
was transferred to Nutrient Agar (NA) for bacteria culture and Potato Dextrose Agar (PDA) for fungi.
For bacteria were incubated at 37oC and for fungi at 28oC.
Microscopy observation of endophyte microbesa. Bacteria Gram staining. One loop of bacteria was dropped onto a slide glass and air
dried. The slide was stained with crystal violet for 1 min and then washed with alcohol, iodine for 1
min, and distilled water respectively. After that, the slide was added with safranin for one min andwashed again with distilled water to remove any staining solution. The slide was examined under abright-field microscope (Model Nikon Eclipse-50i, Japan).
b. Morphological and histological identification of fungal endophytes. Morphological
identification of fungal isolates was according to colony or hyphal morphology of the fungal culture,surface and reverse colony color, and colony texture. Histological identification of fungal
endophytes was carried out by observing the characteristics of the spores or conidia, andreproductive structures (sexual and asexual) under a bright-field microscope (Model Nikon Eclipse50i, Japan).
In vitro screening of endophyte microbe against G.boninense
The endophytes microbe isolates were screened for characteristic to inhibit G. boninese invitro by dual culture test and chitinolytic activity assay.
Dual culture test (Jinantana & Syariah 1997). A 5 mm diameter agar disc was taken from aweek-old PDA culture of G. boninense. For bacteria, agar disc with G. boninense was placed incentral PDA and 6 cm diameter filter paper with 24 hours old of endophyte bacteria was placedaround of G. boninense isolate. For fungi, agar disc with G. boninense was placed 6 cm away fromagar disc with fungi isolate. For control, agar disc Ganoderma were inoculated without endophyte
microbes. All of the antagonistic were conducted triplicate and incubated in 28 oC. The ability of theendophyte microbes to inhibit the growth of G. boninense was determined after 7 days incubation
by measuring the diameter of the G. boninense colony in control plate (R1) compare with thediameter of the G. boninense colony in plate with endophyte microbe (R2).
Chitinolytic activity assay. Bacteria and fungi isolates were inoculated to liquid chitin
medium with 0.3 % chitin (w/v) and incubated at 37 oC with orbital shaking 150 rpm. The chitinolytic
assay was measured after 1, 2, 3, 4, 5, 6, and 7 days after incubation using Spektrophotometer UVVis at 420 nm. One unit of chitinase was represented as nmol N-acetylglucosamine min-1 mL-1
protein. N-acetylglucosamine was used as a standard with variation concentration 0-200 ppm.
Identification of endophyte microbes
Isolation 16s rRNA gene for bacteria and ITS rRNA gene for fungi. Single colony of 24hours bacteria from NA was inoculated to 5 mL Luria Broth (LB) and incubated for 18 hours at 37 oC
with orbital shaking 150 rpm. The culture was centrifuged at 6000 rpm for 5 min. The pellet wasused for the isolation of DNA using Wizard Genomic (Promega) by manufacture procedure. One of
5-day-old fungi colony from PDA was inoculated into 25 mL Potato Dextrose Broth (PDB) andincubated for 5 days at room temperature. The mycelium was used for the isolation DNA using
Plant Genomic DNA (Sigma) following the manufacture‘s procedure. The 1500 bp of 16S rRNAth

Proceedings of The 7 ACSA Conference 2011361

gene for bacteria was amplified using 9F (GAG TTT GAT CCT GGC TCA G) and 1510R (GGT TAC
CTT GTT ACG ACT T) primers and 600 bp of ITS rRNA gene for fungi was amplified using ITS 1(TCC GTA GGT GAA CCT GCG G) and ITS 4 (TCC TCC GCT TAT TGA TAT GC) primers.
Polymerase chain reaction was carried out in a thermal cycler (Applied Bio system Verity) in a total
volume of 50 uL containing 2.5 mM MgCl2, 5uL of 10X Taq Buffer, 200 mM dNTPs, 50 pmoles ofeach forward and reverse primer, Dream Taq Polymerase (Fermentas), and 10 ng DNA template.
o
PCR was performed by initial denaturation at 95 C for 4 min, followed by 35 cycles of denaturationoo
at 95 C for 1 min, annealing at 55 C for 1 min, extension at 72 oC for 1 min, with final extension at
72 oC for 10 min.
Cloning and transformation. Escherichia coli DH5α cells were transformed with PCRamplified 16S rRNA genes and ITS rDNA genes ligated in pGEM easy vector plasmid (Promega)using Fermentas DNA ligation kit (Fermentas). The plasmid was transformed to E. coli DH5α using
heat shock treatment. The transformed cells (100 uL) were spread on LA plates containing X-Gal(50 ug/ml), IPTG (100 ug/ml) and ampicillin (50 ug/ml). The plates were incubated at 37oC for 16
hours to screen blue and white colonies. Positive result was confirmed by PCR using M13F and
M13R primers of white colonies.
Gene sequencing. The 16s rRNA and ITS rRNA gene was sequenced to determine thehomology with the known sequences in the NCBI database. DNA sequencing was done onFirstBase using M13 forward sequencing primer (GTAAAACGACGGCCAGT).

Results and Discussion
Seventeen of endophyte bacterias were isolated from oil palm root with chitinolytic index
ranged from 1.00 to 3.43 (data not showed). The ability of bacteria to form clear zones wasmeasured qualitatively through chitinase activity of bacteria. The chitinase enzymes produced bybacteria are able to diffuse to the media. It can be seen from the presence of clear zones on the
media. Most of the bacteria are Gram-negative (66%). According to Bell et al.,(1995), theabundance of Gram-negative endophytic bacteria in the oil palm roots was supported by population
endophytes in other plant
Endophyte fungi of T1 and T13 isolates sp. are white color colony and scattered greenish
patches become visible as the conidia are formed (Figure 1A). Microscopic of T1 and T13 isolates
showed that the hyphae are septate and hyaline. Phialides are branched, flask-shaped, andattached to the conidiophores. Conidia have round or ellipsoidal form and have green color with 3µm average diameter (Figure 1E). V2 and V3 isolates have white colony form in PDA initially, but
typically become green (Figure 1B). Phialides are branched and tapered at tips. Spherical conidia
gathered at tip of phialides in a tight, and ball-shaped (Figure 1F). A1 and A2 isolates have varies
colony color from black to pale (Figure 1C). A1 and A2 isolate showed that hyphae are septate andhyaline. Conidiophores arise from the basal foot cell found at the supporting hyphae and terminate
in a vesicle at the tip (Figure 1G). Mycelium morphology of F1 and F2 isolates are mostly white, butsometimes can be purple (Figure 1D). Some species also produce distinctly different conidia in theaerial mycelium (referred to as microconidia). Aerial mycelium is the growth of hyphae above theagar surface and often form a convex shape (Figure 1H).

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(A,E: T1 and T3 isolates; B,F: V2 and V3 isolates; C,G: A1 and A2 isolates; D, H: F1 and F2 isolates)

Figure 1. Morphological and histological of fungal endophytes.

Antagonistic test was used to measure the ability of bacteria in inhibiting the growth of
Ganoderma in vitro. Inhibition ratio of bacteria and fungi against G. boninese has different ranges.
For bacteria, B2.1.2 isolate has the highest inhibition ratio (67%), while B24.1.7 and B28.5.3 isolate
has the lowest inhibition ratio (26 %) and V3 isolate has the highest inhibition ratio (64 %) while theF2 has the lowest inhibition ratio (33 %) (Table 1). There are two possible mechanisms to inhibit
growth of G. boninense, both are antifungal compound and chitinase activity. The chitinase could
lysis cell wall of G. boninense. In addition, bacteria and fungi also capable to produce secondary
metabolites, these can inhibit the growth of pathogenic fungi. According to Sidduqque et al., (2009),
Trichoderma can produce volatile compounds with marked gas formation and capable to inhibit thegrowth of G. boninense up to 70% by dual culture method . A compound produced by Trichoderma
is known as 6 - pentyl-alpha-pyrone (6PAP) which is a secondary metabolites compound (Coney etal. 1997).
Table 1 Antagonistic potential of endophyte microbe in dual culture test
Microbe isolate
a. Bacteria
B2.1.2
B93.22.83
B28.5.3
B50.1.3
B24.1.7
B13.10.4
b. Fungi
T1
T13
V2
V3
A1
A1
F1
F2

Inhibition ratio (%)
67 a
37 b
26 d
34 c
26 d
34 c
58 b
61 ab
63 a
64 a
45 c
37 d
38 cd
33 d

Means in the same column with different alphabet(s) are significantly different (p < 0.05) according to Duncan test.

thProceedings of The 7 ACSA Conference 2011363

All microbe isolates could produce chitinase enzyme activity although with different pattern(Table 2). B13.10.4 isolate of bacteria has the highest chitinase activity (3.43 U/mL at 5 days of
incubation).Whilst, the highest enzyme activity of chitinase in fungi was T1 isolate (2.07 U /mL at 7days of incubation). Although B2.1.2 and V3 isolates have the highest inhibition ratio, they did not
produce enzyme with highest activity. According to Aktuganov (2003), chitinase activity was not
related to antifungal activity. The bacteria with high antagonist activity is not always have a highchitinase activity.
Synergism between the antifungal compounds and enzymes of bacteria can enhance therole of bacteria as bio-control pathogen (Sheri et al., 2002). Some studies suggest that the enzyme
chitinase production of the genus Trichoderma spp. is more effective than chitinase enzyme
produced by other organisms, to inhibit a various plant pathogenic fungi (Lorito et al., 1994).
Several studies are also reported that Trichoderma was capable in producing chitinase enzymes
that play a role in several fungal pathogens including Sclerotium rolfsii and Rhizoctonia solani,(Haran et al., 1996; Harman et al., 1993).
Table 2 Chitinase activity enzyme from chitinolytic microbe
Microbe isolate
a. Bacteria
B2.1.2
B93.22.83
B28.5.3
B50.1.3
B24.1.7
B13.10.4
b. Fungi
T1
T13
V2
V3
A1
A2
F1
F2

Enzyme activity (U/mL)

Time incubation (days)

1.54 c1.51 c1.44 cd
1.28 d2.08 b
3.43 a

5
6
7
5
2
5

2.07 a1.75 b
1.51 c1.16 de0.99 e0.93 e1.56 c1.30 d

7
6
2
2
6
6
6
6

Means in the same column with different alphabet(s) are significantly different (p < 0.05) according to Duncan test.

Table 3 Identification of selected microbe based on ribosomal DNA
IsolateB2.1.2
B93.22.83
B24.1.7
B13.10.4
T1
V3
A1
F1

Identity
Burkholderia sp
Serratia sp
Acinetobacter sp
Bacillus cereus
Trichoderma asperellum
Hyphocrea virens
Aspergillus sp
Fusarium oxysporum

Homology (%)
99
99
99
99
99
99
99
99

The fungi and bacteria were identified base on their ribosomal DNA. Identification of fungi
showed that there are Trichoderma, Hypocrea, Aspergillus, and Fusarium (Table 3). These four
fungi are known to be a natural bio-control of fungal pathogens (Krupke et al. 2003; Rubini et al.,2005; Adriana & Sergio 2001). Identification of potential isolates showed that they are Serratia,

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Burkholderia, Acinetobacter, and Bacillus (Table 3). These results proved that several bacteria andfungi are potential as bio-fungicide because of their high inhibition ratio and activity of chitinase.

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Aktuganov GE, Melent'ev AL, Kuz'mina LY, Galimzyanova NF, and Shirokov AV. 2003. The chitinolytic activityof Bacillus Cohn bacteria antagonistic to phytopathogenic fungi. Microbiol 72(3): 313-317
Adriana O and Sergio O. 2001. In vitro evaluation of Trichoderma and Gliocladium antagonism against thesymbiotic fungus of the leaf-cutting and Atta cephalotes. Mycopathologia 150:53-60.
Bell CR, Dickie GA, Harvey GLW and Chan FYWJ. 1995. Endophytic bacteria in grapevine. Canadian J.
Microbiol., 41: 46–53
Coney JM, Lauren DR, Poole PR, and Whitaker G. 1997. Microbial transformation of the Trichoderma metabolite
6-n pentyl-2H pyran-2- one. Nat. Prod. 60:1242-1244.
Darmono. 1996. Pendekatan bioteknologi untuk mengatasi masalah penyakit Busuk Pangkal Batang Kelapa
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Flood J, Bridge PD, and Holderness M. 2000. Ganoderma Diseases of Perennial Crops. CABI Publishing,
Wallingford. Uk 275 p
Haran S, Schickler H, Oppenheim A, and Chet I. 1996. Differential expression of Trichoderma harzianum
chitinases during mycoparasitism. Phytopathology 86:980-985.
Harman GE, Hayes CK, Lorito M, Broadway RM, Di Pietro A, Peterbauer C, and Tronsmo A. 1993. Chitinolytic
enzymes of Trichoderma harzianum: Purification of chitobiosidase and endochitinase. Phytopathology
83:313-318.
Jinantana J and Sariah M. 1997. Antagonistic effect of Malaysian isolates of Trichoderma harzianum andGliocladium Viren on Sclerotium rolfsii. Pertanika. J. Trop. Agric. Sci., 20: 35–41
Krupke AO, Castle AJ, and Rinker DL. 2003. The North American mushroom competitor Trichoderma
aggressivum f. agrressivum, produces antifungal compounds in mushroom compost that inhibit mycelial
growth of the commercial mushroom Agaricus bisporus.Mycol. Res. 107:1467-1475.
Lorito M, Hayes CK, Zoina A, Scala F, Del Sorbo G, Woo SL and Harman GE. 1994. Potential of genes andgene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides.
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Sapak Z, Meon S and Ahmad ZAM . 2008. Effect of endophytic bacteria on growth and suppression of
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-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011365

Chitinase Activities of Oil Palm Root at Early Infection of
Arbuscular Mycorrhizal Fungi
Happy Widiastuti
Indonesian Biotechnology Research Institute for Estate Crops
Jl Taman Kencana No. 1 Bogor, Indonesia 16151
Corresponding author: happywidiastuti@yahoo.com

Abstract
The activity of chitinase of oil palm root at the early of arbuscular mycorrhizal fungi (AMF) colonisation wasstudied. The results showed that early in colonisation (0-5 weeks), chitinase activity in roots increased
nevertheless eventually suppressing occurs 5-8 weeks after inoculation. Inhibition of chitinase activity is
influenced by AMF species and fertilization. Suppression of endochitinase activity of effective species is higher
than the species that are less effective, especially in the fertilization treatment. The activity of exochitinase wasdepressed in the presence of AMF colonisation. In the treatment without fertilizer, suppression of exochitinase
activity of inoculated roots with G. margarita is higher than those inoculated with A. tuberculata. Inhibition ofchitinase activities on secondary roots that occurs at the inoculation of G. margarita is higher than those
inoculated with A. tuberculata and vice versa in the primary root.
Keywords: chitinase activities, palm oil, early colonisation, arbuscular mycorrhizal fungi

Introduction
Arbuscular mycorrhizal fungi (AMF) is a fungus symbioses with the oil palm. Biochemical
processes involved in plant symbiosis with fungi has been studied. Differences in proteins bothquantitatively and qualitatively has been shown between AMF inoculated and uninoculated plants.
AMF colonization seems to be in place for increasing the size of low protein expression as shown in
tobacco (Dumas et al, 1996). The emphasis is more consistent in the chitinase activity which is also
have a role in defense plants in the early stages of AMF colonisation. Penetration of AMF in root
involves a series of changes in morphology and physiology in plants and fungi. However, as occurs
in plant-pathogen interaction mechanisms of induction and suppression associated with plant
defense is the key to the colonization of AMF and its compatibility with the host plant (GarciaGarrido & Ocampo, 2002).
The effectiveness of plant defense responses associated with the speed of the process of
introduction of specific signaling molecules called elisitor. Elisitor can be secreted by the microbesthat infect or as a result of breakdown of plant cell walls. Regulatory mechanisms of defense
responses through degradation of the molecule can be issued elisitor AMF for example byhydrolysis enzymes such as chitinase, glucanase B 1.3, kitosanase. Lambais and Mehdy (1996)
observed a suppression of activity endochitinase up to nearly twice in the AMF inoculation
treatment and the level of infection-suppressing this enzyme was higher in the effective strain AMF
compared to strains that are less effective. This research aim was to study plant defense responses
during early colonisation, especially AMF chitinase activity.

Materials and Methods
Planting materials used were germinated oil palm from IOPRI Medan. Germinated oil palm
was grown in sterile sand until the age of 3 months. AMF inoculation on the palm was made by the

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method of pre-nursery i.e by developing AMF colonization in P phaseoloides prior to colonisation tooil palm seedling. P. phaseoloides grown in polybags 50 x 50 cm in sized without holes containing
10 kg of sterilized Cikopomayak acid soils. Inoculum dose and optimum dose of fertilizer was basedon the optimization experiment (Widiastuti et al, 2002). Plants were maintained in a greenhouse bywatering with cooling boiled water. Harvesting was done at weeks 0, 3, 5, 8, and 11 after inoculation
by washing the roots with water taps. Observations were conducted on fresh and dry weight ofseedlings, chitinase activitieas (Lambais & Mehdy, 1996), and the percentage of AMF colonisation.
Chitinase activity was determined by counting the hydrolyzed N acetyl glucosamine using BSA asprotein standard. The two factors tested were AMF species (without AMF, A. tuberculata, and G.
margarita) and fertilizing (without and with fertilizer). The inorganic fertilizer dose of control was
based on Lubis (1992). Experimental design used was completely randomized group design withfactorial pattern.

Results and Discussion

40

18

Percentage of infection

Percentage of infection

AM fungi colonisation in oil palm root
Chemical analysis showed that the soil was very acid (pH H2O 3.8), the content of C, N,
P2O5, K2O, CaO, MgO were very low at 1.59; 0.097; 0.013; 0.01; 0.066; 0.054% and 16.23 Aldd me
100 g-1 respectively. Colonisation of AMF began weeks after inoculation in both the primary andsecondary roots. However, colonisation in secondary roots was higher than those in primary roots.
Moreover, colonisation of AM fungi in no fertilization treatment was higher than those both A.
tuberculata and G. margarita (Fig 1).
AtP0
AtP1
GmP0

16
14
12
10
8

6
4

AtP0
AtP1
GmP0
GmP1

35
30
25
20

15
10
5

2
0

0
0

3

5

8

11

Weeks after inoculation

0

3

5

8

11

Weeks after inoculation

Figure 1. Percentage of AMF colonisation in primary (left) and secondary (right) oil palm roots.

Chitinase activity in the primary root
The analysis showed that in the primary roots which were not inoculated with AMF, theactivity of exochitinase was higher compared to those of the inoculated one. Exochitinase activity
increased until the fifth week after inoculation and subsequent declined at week eight and very low
at week 11. These results indicated that the exochitinase activity was temporary and there isemphasis of exochitinase activities particularly began 8 weeks after AMF inoculation. The effect of
fertilization showed a trend that in palm roots without AMF inoculation and inoculated with G.
margarita, fertilization causes the higher activity of exochitinase compared to those not fertilized.
However, these results differed with exochitinase activity of palm roots inoculated with A.
tuberculata. The exochitinase activities of the root that are not fertilized, is higher compare to those
fertilized. Emphasis of the exochitinase activity in the inoculated roots of palm inoculated with A.
tuberculata both at without fertilization and fertilized was higher than the activity exochitinase palmroots inoculated with G. margarita.These results indicate that in the primary root, the activity of
exochitinase influenced by AMF species and the presence of fertilization.

thProceedings of The 7 ACSA Conference 2011367

Endochitinase activity was lower in primary roots compared to exochitinase activity.
Endochitinase activity in primary roots that not inoculated with AMF was higher compared to those
inoculated. Unlike to the exochitinase activity, the peak of endochitinase acitivity occurred at week 5
and declined until week 8. Activity of endochitinase of oil palm root unfertilized inoculated with A.
tuberculata is higher than those of inoculated with G. margarita. This result was in line with
exochitinase activity (Fig. 2). With the application of fertilizer, the activity endochitinase of primarypalm root inoculated with G. margarita was higher than those of the inoculated with A. tuberculata.
These results indicated that the dose of fertilizer and type of AMF affect endochitinase activity on
secondary roots.
The data of chitinase activity in general, showed that in the primary root chitinase activityof oil palm uninoculated with AMF was higher than those of inoculated with AMF. This trend
happened in the oil palm root inoculated with G. margarita, but the opposite occurred in oil palmroots inoculated with A. tuberculata.

Figure 2. Exochitinase activity (A), endochitinase (B), and total chitinase (C) of primary oil palm roots in
response to treatment.

Chitinase activity in the secondary roots
Exochitinase activity on the secondary roots of palm oil was much higher compared with
exochitinase activity in the primary root. This difference reached 10 times. Endochitinase activity in
oil palm roots without AMF inoculation was higher than those of inoculated with AMF. In thesecondary roots inoculated with A. tuberculata, endochitinase activity without fertilization was higher
than those fertilized root. These results are similar to those in primary roots. But for oil palm roots

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Exochitinase activity
nmol eq N acetyl Glu mg1h-1

inoculated with G. margarita, there was no difference of exochitinase activity between a fertilized
and unfertilized. In addition, it was shown that in fertilization oil palm root, the emphasis of chitinase
activity was slower compared to those of without fertilized.
Activity of endochitinase of secondary roots of palm was lower than exochitinase activity
on the same root. In addition, endochitinase activities of palm root that were not inoculated washigher compared to those inoculated with the AMF. In secondary oil palm roots inoculated with A.
tuberculata but unfertilized, endochitinase had a higher activity compared with those of fertilized, but
in contrary to the roots of oil palm inoculated with G. margarita. These results were similar to thoseobserved in primary roots. At the root of the fertilized palm, the activity of the endochitinase theactivity of root inoculated with G. margarita was higher compared to those inoculated with A.
tuberculata, whereas the opposite occured in the treatment without fertilization. These results werein line with that occured in the primary root. The high of endochitinase activity in roots inoculatedwith A. tuberculata followed by a higher emphasis anyway.
General description for chitinase activity in the secondary roots of palm oil showed that theactivity of chitinase in the palm oil root that were not inoculated with AMF was higher than that
inoculated with AMF (Fig 3.). In oil palm inoculated with A. tuberculata, fertilization actually
decreased the activity of chitinase in secondary roots. While palm oil inoculated with G. margarita,
there was no difference between a fertilized and unfertilized.
2500
2000

M0P0
M0P1
AtP0
AtP1
Gm P0
Gm P1

A

1500

1000
500
0
0

3

5

8

11

Week after inoculation

Figure 3. Activity of exochitinase (A), endochitinase (B), and total chitinase (C) of secondary oil palm roots in
response to treatment.
thProceedings of The 7 ACSA Conference 2011369

Possible differences in plant response to AMF colonisation in particular is evident from theactivity of chitinase can be a bookmark for the selection of the effectiveness of AMF. In the present
study demonstrated that inoculation suppressed the activity of chitinase. Suppression level was
influenced by the AMF species and doseage of fertilizer. Emphasis on treatment without fertilization
was highest in week 3 after inoculation, while the emphasis of chitinase activity on fertilization
treatment was observed 5 weeks after inoculation. Fertilization appears to slow the emphasis ofchitinase activity.
The same is true of the exochitinase activity are fertilized palm that inoculated with G.
margarita. Despite this emphasis on endochitinase activity of G. margarita inoculation lower
compared to those inoculated with A. tuberculata. Total chitinase activity in the secondary roots
either by fertilization or without fertilization showed that the inoculation of G. margarita more
suppresse than thoses the inoculation with A. tuberculata.
Changes in isoenzyme patterns and biochemical properties of several enzymes associatedwith plant defense as chitinase has been shown for tomato root colonization by AMF (Pozo et al.,1996) with the induction of new isoforms. These hydrolytic enzymes act as a defense against
pathogen attack because of its potential hydrolyze fungal cell wall polysaccharides (Pozo et al.,1996). Induction of this enzyme activity in AMF symbiosis may also be involved in the protection ofplants against pathogenic fungi (Dumas-Gaudot et al., 1996).
The present study indicated that there was an emphasis on the chitinase activity which
was influenced by inoculation with AMF, AMF species, and fertilization. Fertilization slow emphasis
chitinase activity. In addition, the data showed generally lower total chitinase activity in the presenceof fertilization compared with those not fertilized, especially in the inoculation with A. tuberculata.
While at the inoculation with G. margarita fertilization increased the chitinase activity. The samething also happened in controls. The results in this study seemed in line with Blee & Anderson(1996) suggested that P can regulate plant defense responses. The results suggested that
fertilization decreased 1.3 glucanase (occurs with β mRNA). The delay of suppression of chitinase
activity supposed to be caused by the slow of AMF colonisation in the presence of fertilization.
Emphasis on fertilization treatment to endochitinase activity in roots inoculated with A.
tuberculata is higher than those on inoculated roots of G. margarita and vice versa in the treatment
without fertilizer. In a previous study demonstrated that A. tuberculata is more effective in improving
growth and P uptake of oil palm seedlings in comparison with G. margarita (Widiastuti et al, 2002).
In addition Widiastuti et al (2005) showed that the optimum dose of inorganic fertilizer of oil palm
inoculated with G. margarita was higer compared to those inoculatred with A. tuberculata. These
results suggest that suppression of effective species to endochitinase activity is higher than those to
species that are less effective, especially in the fertilization treatment. Similar to Lambais & Mehdy(1996) the effective strain is more pressing AMF endochitinase activity compared to those that areless effective. However this only occurs at an optimal symbiosis is the application of fertilizer, while
the treatment without fertilization occurs the opposite of the less effective strain is more pressing
endochitinase activity compared to effective strain.
In this study exochitinase depressed activity in the presence of AMF colonisation,
however, there were differences in emphasis between the root level of the inoculated A. tuberculata
and G. margarita. In the treatment without fertilizer, suppression activity of inoculated roots
exochitinase of G. margarita was higher than A. tuberculata. While the result of fertilizer there was adifference in primary and secondary roots. The emphasis on secondary roots that occurs at theinoculation G. margarita was higher than A. tuberculata and vice versa in the primary root. These
results indicate that in the less effective strain, the emphasis on exochitinase activity higher
compared to the effective strains, especially on secondary roots. These results were different fromthose expressed Lambais and Mehdy (1996) who reported increased activity exochitinase up to 3
times the effective strain AMF. In this study exochitinase suppression activities occured on theeffective strain is lower than those that are less effective. The results of this study indicated that

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plants respond differently to AMF colonisation compared to pathogen infection. The mycorrhizal
plant has a defense mechanism that temporally and very weak in contrast to pathogen.

References
Blee KA, AJ Anderson. 1996. Defence-related transcript accumulation in Phaseolus vulgaris L. colonized by thearbuscular mycorrhizal fungus Glomus intraradices, Schenck & Smith. Plant Physiol. 110, 675-688.
Dumas-Gaudot, ES Slezack, B Dassi, MJ Pozo, V Gianinazzi-Pearson, and S Gianinazzi. 1996. Plant hydrolytic
enzymes (chitinases and ß-1,3-glucanases) in root reactions to pathogenic and symbiotic
microorganisms. Plant and Soil. 185, 211-221.
Garcia-Garrido, JM, and JA Ocampo. 2002. Regulation of the plant defence response in arbuscular mycorrhizal
symbiosis. J. Exp. Bot. 53, 1377-1386.
Lambais MR, and MC Mehdy. 1996. Soybean roots infected by Glomus intraradices strains differing in infectivity
exhibit differential chitinase and β-1,3-glucanase expression. New Phytol. 134, 531-538.
Lubis AU. 1992. Kelapa Sawit (Elaeis guineensis Jacq) di Indonesia. Pusat Penelitian Perkebunan Marihat
Bandar Kuala. Pematang Siantar. Sumatera Utara.
Pozo, MJ, E Dumas-Gaudot, S Slezack, C Cordier, A. Asselin, V Gianinazzi-Pearson, C Azcon-Aguilar and JM
Barea. 1996. Detection of new chitinase isoforms in arbuscular mycorrhizal tomato roots: possible
implications in protection against Phytophthora nicotianae var. parasitica. Agronomie, 16, 689-697.
Widiastuti, H. E. Guhardja, N. Soekarno, L. K. Darusman, D. H. Goenadi and S. Smith. 2002. Optimasi
simbiosis cendawan mikoriza arbuskula Acaulospora tuberculata dan Gigaspora margarita pada bibit
kelapa sawit di tanah masam. Menara Perkebunan 70, 54-62.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011371

In vitro Test of Rhizosphere Chitinolytic Bacteria as a Biocontrol
for Ganoderma boninense
Rika Fithri Buana*, Wisnu Adi Wicaksono, Elizabeth C. Situmorang
Plant Production and Biotechnology Division, PT SMART Tbk, Bogor, Indonesia* Corresponding author: Plant Production and Biotechnology Division, PT SMART tbk
Jl. Padjajaran Raya No. 78F-G, Bogor, Indonesia
Tel.: +62 251 831 0854; fax: +62 251 831 0854.
rika.fithri@gmail.com

Abstract
G. boninense is a fungal pathogen that causes basal stem rot in oil palm (Elaeis guineensis). Chitinolytic
bacteria are abundant in soil and well known as a natural biocontrol of fungal pathogen. The objective of thisresearch is to obtain rhizosphere chitinolytic bacteria as a candidate of biocontrol agent for G. boninense.
Nineteen indigenous bacteria from Padang Halaban Estate, North Sumatera, were tested for their antagonistic
properties against G. boninense by in vitro dual culture on PDA plates. Their chitinolytic activities were tested
using colloidal chitin as the substrate. Chitinase activity was determined colorimetrically by detecting the amountof N-acetylglucosamine released from colloidal chitin substrate. Crude enzyme secreted by the bacteria werecharacterized by SDS-PAGE with chitinase from Trichoderma as a control. Six of the isolates have an inhibition
activity against G. boninense growth with B3.4 showed the highest percentage inhibition ratio growth or PIRG
(58.75%). Inhibition of G. boninense growth in vitro might due to a competition for nutrients or other antifungal
compound from certain bacteria which diffuses in the agar. B3.2 showed the highest activity of chitinase (10.44
U/mL), but B3.4 has lower activity of chitinase (7.08 U/mL). It can be assumed from the result that PIRG does
not have correlation with the activity of chitinase. The results of enzyme characterization showed that B3.3, B3.2
and B3.4 has a molecular mass of 37kDA, which is a similar molecular mass with chitinase from Trichoderma
viridae
Keywords: rhizosphere bacteria, Ganoderma boninense, Elaeis guineensis, chitinase

Introduction
Basal stem rot disease (BSR) caused by Ganoderma boninense is currently a majordisease in oil palm plantations (Darmono, 1998). The disease can destroy up to 80% of the standpalms by the time when the palms are halfway through their normal economic life span (Idris, 2003).
Ganoderma boninense is a saprophytic fungus activated by favorable conditions to behave
parasitically (Statmets, 2004). Like all fungi, it has no chlorophyll and thus, lack photosynthetic
capability. Instead of manufacturing their own food, fungi absorb nutrients from either living or deadhost tissue (Haniff, 2005). the fungus typically attack already weakened oil palm as Ganoderma
seldom seriously infects undamaged trees (Paterson, 2007). The effects of Ganoderma infection on
productivity decline in palm crops have been concerned since replanting of oil palm land was beganin South-East Asia, especially in Malaysia and Indonesia (Turner, 2003).
BSR has been an endemic disease found in PT. SMART Tbk. oil palm plantation
especially in Padang Halaban, North Sumatera, Indonesia. Biological control of pathogenic fungi
provides an attractive and alternative management for fungal diseases without the negative impact
of synthetic antifungal agents that can cause environmental pollution and may induce pathogen
resistance (Haas & Défago, 2005). Plant disease control by chitinolytic bacteria has long beenreported (Sneh, 1981). Several strains of bacteria, such as Aeromonas caviae, Bacillus sp., Serratia
plymuthica, and Enterobacter agglomerans are well-known chitinolytic bacteria (Das et al., 2010).
Some Trichoderma sp. have been described as biological control agents against fungal pathogens
and widely used as biocontrol because of its mycoparasitic activity (De La Cruz, 1992). Chitinolytic
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enzymes have been considered as an important factor to control soilborne pathogens because of
their ability to degrade fungal cell walls, of which a major component is chitin (Chet, 1987).
Chitin is widely available in the soil derived from the decay and degradation of dead cells,
so that some bacteria that live on the ground are good in degrading chitin (Suryanto et al., 2005).
The aim of this study was to isolate chitinolytic bacteria from rhizosphere soils in Padang Halaban
estate and to screen their antagonistic activity against G. boninense.

Materials and Methods
Isolation of bacteria
Rhizospheric soil were collected near the root of the uninfected plant in endemic site of G.
boninense. Suspension were made by adding 10 g of soil to 100 mL sterile basic salt solution(0.85% NaCl). Ten fold serial dilutions of these suspensions were made and plated on Nutrient
Agar (NA). The colony were selected and purified with 4-way streak method on NA.
Dual-culture technique
Dual culture were made to obtain the percentage inhibition of radial growth (PIRG) of G.
boninense (Bivi, 2010). Potato Dextrose Agar (PDA) was poured onto 9 cm diameter Petri dishes.
Isolated bacteria was streaked into the PDA plate 2.5 cm from the edge of Petri dish. A 5 mm agar
disc cut from the side of an actively growing pure culture of 5-day old G. boninense placed 2.5 cm
from the edge at the opposite side of the same Petri dish. For the control plate, only G. boninense
was placed in a similar manner without bacteria on a fresh Petri dish. The experiment was made inthree replication each bacteria. The plates were incubated at 28 oC for five days. Results revealed
as mean colony growth of the causal pathogen in the presence of the bacteria and its growth on thecontrol plate (without the bacteria). The outcome of two readings was calculated into the formula for
the PIRG as below :% PIRG = R1 - R2 X 100
R1
Where, PIRG = percentage inhibition of radial growth; R1 = radial growth of G. boninense in theabsence of bacteria (control); R2 = radial growth of G. boninense in the presence of bacteria.
Chitinase assay
Chitinase activity was determined colorimetrically by detecting the amount of Nacetylglucosamine (GlcNAc) released from a colloidal chitin substrate (Reissig, 1955). Selectedisolates were inoculated into 100 ml of 10% chitin medium and incubated at 37 oC on rotary shaker
120 rpm for 5 days. Flask were removed every 24 hours and pipetted 10 mL from each mixture
were filtered through filter paper. The filtrate as a crude enzyme were pipetted 1.5 ml into 0.75 ml
phosphate buffer and 1.5 ml of 0.3% coloidal chitin in 15 ml Corning centrifuge tube. The reaction
mixture were incubated in 37 oC for 30 min, and centrifuge the mixture to separate the product fromthe remaining substrate. Pipetted 2.5 ml supernatant into 5 ml Schales reagent and 2.5 ml distilledwater. The mixture was boiled for 10 min to stop the reaction of the remain enzyme. The reaction
was measured with spectrophotometer at 420 nm. N-acetylglucosamine was used as a standard.
One unit of chitinase acivity was defined as the amount of enzyme that released 1 µmol GlcNAc or
its equivalent from colloidal chitin in 1 min (Zilda, 2006).
Protein characterization
Crude enzyme secreted by the bacteria were characterized by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) with 12 % separator gel and 4% stacking gel.
Crude supernatants protein were mixed with 2x sample buffer (1:1), boiled for 4 min, then load. The
thProceedings of The 7 ACSA Conference 2011373

gel were stained with 0,1% Coomassie brilliant blue R-250, and destained with 10% acetic acid and
20% methanol.

Results and Discussion
There are 19 indigenous bacteria isolated from rhizosphere soil in Padang Halaban.
Screening of biocontrol bacteria was carried out by dual culture. It showed that six of them have an
inhibitory activity against G. boninense growth (Table.1). B3.4 showed the highest PIRG of G.
boninense amongs other isolates, it showed 58.75% and the B3.3 is the lowest (12.20%). The
inhibition of G. boninense growth might due to the presence of chitinase or other anti-fungal
properties produced by the certain bacteria. Many publications have reported that soil bacterias arecapable in producing anti-fungal metabolites properties that inhibited mycelia growth ofphytopathogen fungi (Fernando & Linderman,1994; Sisler, 1969; Yiu-Kwok et al., 2003). Many of
the anti-fungal properties affect the physiological activities of pathogenic fungi (Vesperman, 2007).
Table 1. Antagonistic potential of soil bacteria in dual culture test
Bacterial isolates
B3.3
B14.1
B3.1.1
B3.4
B11.2
B3.2

PIRG (%)
12.20 d35.56 b22.22 c58.75 a31.11 b16.67 dMean in the same coloumn with different alphabet(s) are significantly different (P<0.05) according to DMRTBacteria B3.2 showed the highest activity of chitinase (10.44 U/mL) and the lowest was
B14.1 (7.08 U/mL) on day four of incubation. The result revealed that PIRG has no correlation withchitinase activity. Bacteria with the highest PIRG (B3.4) showed less chitinase activity than bacteriawith lowest PIRG (B3.3). This result is in accordance to Aktuganov (2003) statement that there is nocorrelation between PIRG and the ability of bacteria in producing chitinase. It can be assumed that
other anti-fungal property was responsible for the inhibitory effect in dual-culture of B14.1.
Table 2. Chitinase activity enzyme from isolated bacteria
Bacterial isolates
B3.2
B3.3
B3.4
B14.1
B3.1.1B3.2.2

Enzyme activity (U/mL)
10.44 a8.43 b
9.94 a7.08 c8.37 b
8.31 bIncubation time(Days)
444
4
4
4

Mean in the same coloumn with different alphabet(s) are significantly different (P<0.05) according to DMRT.

Crude supernatan protein secreted by six bacterial isolates were characterized. Chitinasefrom Trichoderma viridae were used as a control. The results revealed that the six isolates havedifferent banding pattern as shown in Figure 1. Chitinase from Trichoderma has a molecular massof 37 kDA (De Marco, 200), and most of chitinases have molecular mass of between 28 kDA - 43
kDA (Nielsen, 1997). Result showed that B3.2, B3.3, and 3.4 have a similar band between 30 kDA– 40 kDA, in a range with most of chitinases molecular mass. The three other isolates showed a

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similar band of 50 kDA. Singh et al, (1998) reported that chitinase from Paenibacillus sp. hasmolecular mass around 50 kDA.
kDA
150 --

60 -–50 -37—30—
25—
20—First lane represent the control of chitinase from Trichoderma viridae; Lane 1, B3.2; Lane 2, B3.3; Lane 3, B3.4; Lane 4, B14.1;
Lane 5, B3.1.1; Lane 6, B3.2.2; Lane 7, negative control.

Figure 1. SDS-PAGE analysis of total supernatan protein of isolates, stained with comassie brilliant blue.

The result showed that B3.2 was the best rhizosphere chitinolytic bacteria. The mostpromising bacteria is B3.4. It is because bacteria with more 50% PIRG can be used as bio control(Bivi, 2010). Synergism between chitinolytic and antagonistic bacteria could enhance the role ofbacteria as biocontrol pathogen. Combination of these two bacteria can be use as a biocontrol
candidate.

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Idris AS, Ismail S, Ariffin D, and Ahmad H. 2003. Control of Ganoderma-infected palm-development of pressure
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in carnation. Phytopathol 100:251-256.
Stamets P. 2004. The role of mushrooms in nature. In: Elevitch C.R (Ed.). The overstory book: cultivating
connection with trees, 2nd Edn. permanent agriculture resource, USA., pp: 74-80.
Suryanto D, Munir E, and Yurnaliza, 2005. Eksplorasi Bakteri Kitinolitik : Keragaman Genetik Gen Penyandi
Kitinase pada Berbagai Jenis Bakteri dan Pemanfaatannya.Laporan Hasil Penelitian Hibah Bersaing
Perguruan Tinggi, Universitas Sumatera Utara, Medan.
Turner PD and Gillbanks RA. 2003. Oil palm cultivation and management. The Incorporated Society of Planters
Kuala Lumpur, Malaysia.
Vespermann AM, Kai, and Piechulla B. 2007. Rhizobacterial volatiles affect the growth of fungi and Arabidpsis
thaliana. Appl. Environ. Microbiol. 73: 5639-5641.
Yiu-Kwok C, Wayne A, McCormic K, and Keith AS. 2003. Characterization of antifungal soil bacterium and its
antagonistic activities against Fusarium species. Can. J. Microbiol., 49: 253-262.
Zilda DS, Fawzya YN, and Chasanah E. 2006. Karakterisasi enzim kitosanase dari bakteri kitinolitik T5a1 yangdiisolasi dari terasi. Jurnal pascapanen dan bioteknologi kelautan dan perikanan Vol. 1 No. 1.
Zaiton S, Sariah M, and Abidin Z., 2006. Isolation and Characterisation of Microbial Endophytes from Oil Palm
Roots : Implication as Biocontrol Agents against Ganoderma. Department of Plant Protection, Faculty ofAgriculture, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia.

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The Effect of Mulch, Trichoderma, and Arbuscular Mycorrhizal Fungi (AMF)
Biofertilizer on the Growth of Oil Palm Seedlings Inoculated withGanoderma
Happy Widiastuti
Indonesian Biotechnology Research Institute for Estate Crops
Jl Taman Kencana No 1 Bogor, 16151, Indonesia
Corresponding author: happywidiastuti@yahoo.com

Abstract
A Greenhouse experiment was carried out to determine the effect of mulching, Trichoderma sp., Metharizium
anisopilae,. arbuscular mycorrhizal fungi (AMF) biofertilizer on the growth of oil palm seedlings. The experiment
was conducted in 70x80 cm sized of polybags filled with 50 kg of Latosol Ciomas soil. Mycorrhizal inoculum is in
the form of spores, hyphae and infected roots. Germinated oil palm was planted in polybag, then covered withempty fruit bunches of oil palm (EFBOP) as mulch. The dose of inorganic fertilizer was 50% for the application of
AMF biofertilizer while on the other treatment the dose of fertilizer was 100%. The experimental design wascomplete randomized design to test nine treatments: 1) without any fertilizer, 2) 100% inorganic fertilizer, 3)
EFBOP, 4) EFBOP+Trichoderma pseudokoningii+Trichoderma DT38, 5) EFBOP+T. pseudokoningii+
Trichoderma DT41, 6) EFBOP+T. pseudokoningii + Trichoderma DT38 + Metharizium anisopilae, 7) EFBOP+T.
pseudokoningii+Trichoderma DT41+Metharizium anisopilae, 8) EFBOP+T. pseudokoningii+Trichoderma DT38+
Metharizium anisopilae+AMF biofertilizer, 9) EFBOP+T. pseudokoningii+Trichoderma DT41+M. anisopilae+AMF
biofertilizer. The results showed that application of EFBOP as mulch enhanced the gowth of oil palm seedlingand combination with DT 41 and both T. pseudokoningii and Metharizium produce the similar growth with those
100% inorganic fertilizer treatment. While the addition of EFBOP+Trichoderma DT41+T. pesudokoningii
produced the highest seedling dry weight. The application of AMF biofertilizaer reduced the dose of inorganic
fertilizer yielded a similar seedling dry weight with those fertlized with 100% of inorganic fertilizer.
Key words: oil palm, mulch, empty fruit bunches oil palm, arbuscular mycorrhizal fungi, acid soil

Introduction
One method of organic matter application is to use it as mulch. Mulch is organic or
inorganic materials either natural or artificial which is placed above the soil and have a role toprotect and cover it. Some of the benefits of mulching is to minimize the occurrence of surface
runoff, increase water infiltration, protect the soil against rain, modify soil temperature, suppress
weeds, reduce the rate of dry surface soil, increase soil biological activity and modifies the level ofnutrient availability as well as to maintain or increase the availability of organic material soil (Baon,
1998). Basically the main function of mulching is to maintain the soil moisture. Water is a very
important factor in the cultivation of plants including crops. The application of mulch is notaccompanied with tillage practice. Muruganandam et al (2009) showed that the activity of enzymes
that play a role in N mineralization in no-tillage soil is significantly higher compared to conventionallycultivated soil. While White & Rice (2009) suggested that the biological activity in no-tillage soil was
detected higher compared to conventional cultivated soil.
In the microbiological aspects, organic materials addition means the addition of carbon
and energy sources to improve microbe activities. The same thing seems to occur in peat soil.
Komariah et al (1993), Nurani et al. (2007) reported that the use of microbial consortia and EFBOP
increase soil pH and base saturation and lower CEC of peat soil as well as the C/N ratio. Microbes,
in addition to functioning release nutrients or nutrient mineralization in the process of decompositionis also able to degrade toxic compounds (Sparling, 1998). However, microbe use root exudate asthProceedings of The 7 ACSA Conference 2011377

source of carbon, energy and other nutrients to support their live. Brockling et al. (2008) suggeststhat plants can regulate microbial communities through root exudates.
The application of inorganic fertilizer as well as pesticide in higher dose will disturb theactivities of soil microbes caused declining biological soil properties. These imbalances soil
properties affect the soil microbial communities to control soil borne pathogens and microbes thatplay a role in the health of the land and crops. This is supported by research conducted by Valladet. al. (2003) which proves that the application of organic fertilizer (compost) reduce plant diseaseattack. Rhizosfer manipulation seems necessary to maintain land productivity. Rousk et al (2009)
suggested that the decrease in pH will increase five times the population of fungi, and vice versa forthe bacteria. The aim of the research was to determine the effect of mulching, Trichoderma sp. , M.
anisopilae, and AMF biofertilizers on the growth of oil palm seedlings.

Materials and Methods
The experiment was conducted in a greenhouse using a 70x80 cm sized polybags filledwith 50 kg of Ciomas soil. Germinated oil palm was used as plant material and planted in polybag
and add with empty fruit bunches oil palm (EFBOP) as mulch. DT38 and DT41 isolates were
cultured in the media then transferred to PDA medium and inoculated into polybags. Mycorrhizal
inoculum is in the form of spores, hyphae and infected roots. Germinated oil palm was planted on
the polybag and the EFBOP was added as mulch. In the treatment of mycorrhizal fertilizer, thedosage of inorganic fertilizer was 50% of the recommendation dose while others were 100% of
inorganic fertilizer. The experiment was arranged in completely randomized design to test ninetreatments i.e. 1) without fertilizer, 2) 100% dose of inorganic fertilizer, 3) EFBOP, 4) EFBOP+T.
pseudokoningii+DT38, 5) EFBOP+T. pseudokoningii+DT41, 6) EFBOP+T. pseudokoningii+DT38+
Metharizium anisopilae, 7) EFBOP+T. pseudokoningii+DT41+Metharizium anisopilae, 8) EFBOP+
T. pseudokoningii+DT38+Metharizium anisopilae+AM fungi, 9) EFBOP+T. pseudokoningii DT41+
M. anisopilae+AM fungi. Each treatment was repeated 3 times. Observations made on the growth ofoil palm.

Results and Discussion
Observations of plant height showed that fertilization increased height of plants while
EFBOP solely suppressed the height growth of plant (Table 1). The application of Trichoderma sp.
and M. anisopilae and arbuscullar mycorrhizal fungi (AMF) enhanced the height of palm seedling.
Table 1. The height and leaf number of oil palm seedlings (9 months) in each treatment tested
Treatment
Withou fertilizer
100% inorganic fertilizer
EFBOP
EFBOP+Trichoderma DT38+T pseudokoningii
EFBOP+ Trichoderma DT41+T pseudokoningii
EFBOP+ Trichoderma DT38+T pseudokoningii +Meth
EFBOP+ Trichoderma DT41+T p+Meth
EFBOP+ Trichoderma DT38+T p+Meth+AMF
EFBOP+ Trichoderma DT41+T p+Meth+AMF

Height (cm)

Leave number

106 ab
119 ab
81 a
130 b
130 b
129 b
133 b
126 b
123 ab

19 a
19 a
20 a
20 a
20 a
19 a
20 a
20 a
18 a

Note: Figure (s) in each column followed by the same letter is not different according to Duncan (P < 0.05).

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Proceedings of The 7 ACSA Conference 2011

However, the application of EFBOP as mulch in term of plant height was not significantly
different compared with those were not treated (without fertilizer) and 100% inorganic fertilizer.
These results indicated that the plant height variable is not affected by EFBOP and inorganic
fertilizer application. The absence of the influence of fertilization and the addition of EFBOP as
mulch plants may caused the soil already contains enough nutrients. Inoculation treatments DT 41+ T. pseudokoningii or DT 38 + T. pseudokoningii without or in combination with M. anisopilae, andEFBOP as mulch significantly resulted in higher plant height compared to the application of EFBOP.
These results indicated that the application of Trichoderma sp. DT 38, DT 41, M. anisopilae, T.
pseudokoningii in combination with EFBOP yield positive effect on plant height. Positive influence is
probably due to the decomposition process carried out by DT 38 and DT 41, while M. anisopilae
seems to have an effect on the prevention of Oryctes.as a plant pest. The same was observed in
the treatment of DT 38 + T. pseudokoningii + plus M. anisopilae and AM fungi. AM fungi can besymbiotic with the roots of oil palm and the symbiosis can enhance nutrient uptakes, especially P
which contributes to increase plant height. In addition, the treatment was shown that application ofAMF (fertilizer 50%) showed a similar plant height with plants that 100% fertilized .These results
demonstrated that the application of AMF biofertilizer increased the efficiency of inorganic fertilizer
by 50%. The increasing inorganic fertilizer efficiency by using AMF symbiosis has also beenreported by other researchers. Some mechanisms that occur are by increasing the reach of roots,
and mineralization of organic P through the extraction phosphatase. The application of DT 41 + T.
pseudokoningii + M. anisopilae + and AMF biofertilizer was not significantly different with those of
DT 38 + T. pseudokoningii + M. anisopilae + and AM fungi in term of plant height.
Treatment of EFBOP increased the number of leaves compared to 100% inorganic
fertilizer and the unfertilizer treatment (Table 1). The application of EFBOP provides the same
number of leaves with the inoculation of DT41 or DT38 and the T pseudokoningii accompanied by
the application of M. anisopilae and AMF biofertilizer. However there is no significantly differences
between the number of leaves of all treatments tested in the blank, 100% inorganic fertilizer, or
mulching EFBOP. Interaction between AM fungi with DT 38 is better compared to the interaction of
those fungi with DT41 especially demonstrated by the number of leaves.
The fresh weight of leaves increased significantly with the addition of inorganic fertilizers
as well as giving EFBOP as mulch. Similar results were also observed in the treatment of EFBOP
addition which is accompanied by DT 38, DT 41, T. pseudokoningii, M. anisopilae and AMF
biofertilizer. It was shown that the efficiency of inorganic fertilizer can be achieved by the applicationof AMF biofertilizer combined with either DT 41 or DT 38. Similar results are also shown on thevariables fresh weight of stem. Fresh weights of oil palm seedlings stem with inorganic fertilizer ishigher than thethe blank (Table 2). Nevertheless, the fresh weight of stem between 100% inorganic
fertilizer and application of EFBOP as mulch and blank werenot significantly different. The additionof DT 41 and DT 38, which is accompanied by T. pseudokoningii and M. anisopilae results a fresh
weight of stem that were not significantly different compared to those with the addition of EFBOP as
mulch or 100% inorganic fertilizer. The application of EFBOP as mulch, AMF biofertilizer and 50%
inorganic fertilizer yield fresh weight of stem that is not significantly different to 100% inorganic
fertilizer treatment. These results indicated that the application of AMF biofertilizer improves
inorganic fertilizer efficiency as much as 50%.
Compared to the fresh weight of the stem appears that the effect of the treatments being
tested was more visible on the fresh weight of leaf variables. Application of EFBOP accompanied
by DT 41 and T. pseudokoningii addition produced the highest fresh weight of leaves. Similar
results were also observed in the treatment of EFBOP as mulch with DT 38, DT 41 or accompaniedby M. anisopilae. As for DT 38 leaf fresh weight significantly higher compared with the blank whencombined with M. anisopilae and AMF biofertilizer.

thProceedings of The 7 ACSA Conference 2011379

Table 2. The fresh weight of oil palm seedlings (9 months) in each treatment tested
Treatment
Without fertilizer
100% inorganic fertilizer
EFBOP
EFBOP+DT38+T p.
EFBOP+DT41+T p.
EFBOP+DT38+T p+Meth
EFBOP+DT41+T p+Meth
EFBOP+DT38+T p+Meth+AMF
EFBOP+DT41+T p+Meth+AMF

Leaf

Stem

450 a
850 b
863,33 b733,33 ab
1066,67 b833 b883,33 b
900 b716,67 ab

Fresh weight (g)Shoot833,33 a1433,33 ab
1583,33 ab
1683,33 ab
2000 b1566,67 ab
1750 ab
1533,33 ab
1416,67 ab

1283,33 a
2250 ab
2466,67 ab
2416,67 ab
2733,33 b
2400 ab
2633,33 ab
3433,33 ab
2133,33 ab

Root

Total

783 a
1017 ab967 ab933 ab1183 b933 ab1000 ab783 a1017 ab

2066 a3267 b3433 b3349 b3916 b3333 b3633 b4216 b3150 bNote: Figure (s) in each column followed by the same letter is not different according to Duncan (P < 0.05).

Fertilization treatments increased fresh weight of shoots as well as the addition of EFBOP
as mulch. The application of DT 41+T. pseudokoningii and DT 38+T. pseudokoningii either
accompanied by M. anisopilae or not resulted in fresh weight of shoots that are not significantly
different compared to those with 100% inorganic fertilizer and EFBOP as mulch. This result showsthat the effect of DT 41, DT 38 and M. anisopilae is not significantly different on the fresh weight ofshoots. However the application of DT 41+T. pseudokoningii and DT 38+T. pseudokoningii
accompanied by M. anisopilae and AMF biofertilizer yield shoots fresh weight equal to 100%
fertilization. These results indicated that AMF biofertilizer application increased the efficiency of
inorganic fertilizer, although this treatment may not significantly increased the fresh weight of shootsof oil palm seedlings.
Root fresh weights were not significantly different in the treatment of 100% inorganic
fertilizer, EFBOP as mulch, and EFBOP+DT 38 + T. pseudokoningii and DT 41+T. pseudokoningii
accompanied by M. anisopilae or without M. anisopilae. The same result observed in the treatmentof DT 41 or DT 38+T. pseudokoningii+M. anisopilae accompanied wth AMF biofertilizer. Other study
demonstrated that the application of DT 41 and DT 38 or AMF biofertilizer increase the root fresh
weight. The same result was observed in the total oil palm seedlings fresh weight.
Table 3. Dry weight of oil palm seedlings (9 months) in each treatment tested

Treatment
Without fertilizer
100% inorganic fertilizer
EFBOP
EFBOP+DT38+T p.
EFBOP+DT41+T p.
EFBOP+DT38+T p+Meth
EFBOP+DT41+T p+Meth
EFBOP+DT38+T p+Meth+AMF
EFBOP+DT41+T p+Meth+AMF

Leaf

Stem

123,33 a
220 a
205 a
201,67 a
225 a196,67 a
188,33 a168,33 a126,67 a

231,67 a361,67 a453,33 a386,67 a463,33 a
460 a
438,33 a
410 a
425 a

Dry weight (g)
Shoot
355 a
582 ab658 ab588 ab688 b
657 b
627 ab578 ab551 ab

Root

Total

138 a
215 ab
182 ab278 b
212 ab140 ab165 ab128 a
145 ab

493 a
797 b
840 b
866 b
900 b
797 b
792 b
726 b
698 b

Note: Figure (s) in each column followed by the same letter is not different according to Duncan (P < 0.05).

The addition of 100% inorganic fertilizer increased dry weight of oil palm stems as well as
the treatment of EFBOP as mulch (Table 3). Similar result was observed in mulching treatments
EFBOP with by DT 41, DT 38 which with T. pseudokoningii and M. anisopilae and AMF biofertilizer.
Nevertheless, the results of statistical tests indicated that there is no significanly differences
between dry weight of plants fertilized with 100% treatment and other treatments. Similar results

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Proceedings of The 7 ACSA Conference 2011

were also found in variable weight of dry leaves, shoot, roots, and oil palm seedling. It seems thatthe application of EFBOP as mulch can be combined with the inoculation of Trichoderma sp, M.
anisopilae and AMF biofertilizer. Hence the application of AMF biofertilizer reduced the doseage of
inorganic fertilizer as much as 50%. The effect of treatment to Ganoderma infection is likely could
be suppressed in this experiment.

References
Baon, J. B., 1998. Konversi lengas tanah melalui pemberian bahan organik dan mulsa. Warta Pusat Penelitian
Kopi Kakao. 14(1):61-68.
Broeckling C D, A K Broz, J Bergelson, D K Manter J M Vivanco. 2008. Root exudates regulate soil fungal
community composition and diversity. Appl. Environ. Microbial. 74(3), 738-764.
Komariah, S. Prihartini, T. dan Suryadi, M.E. 1993. Aktivitas Mikroorganisme dalam Reklamasi Tanah Gambut
In: Prosiding Pertemuan Teknis Penelitian Tanah dan Agroklimat : Bidang Kesuburan dan Produktivitas
Tanah. Puslit Tanah dan Agroklimat. Bogor. Hlm. 105-113.
Murugamandam S., D. W. Israel, W. P. Robarge. 2009. Activities of nitrogen-mineralization enzymes associatedwith soil aggregate size fractions of three tillage systems. Soil Sci Soc Am J. 73, 751-759.
Nurani, D., Parmiyanti , S., Purwanta, H., Angkoso, G., Koesnandar. 2007. Increase pH of peatsoil by microbial
treatment. Internatoinal Symposium and Workshop on Tropical Peatland. Yogyakarta, 27-31 August.
Rousk, J., P C Brookes, E. Baath. 2009. Contrasting soil pH effects on fungal and bacterial growth suggest
functional redundancy in carbon mineralization. Appl. Environ. Microbiol. 75, 1589-1596.
Sparling, G.P. 1998. Soil microbial biomass, activity and nutrient cycling as indicator of soil health. In: Pankhurst,
C., Doube, B.M, Gupta, V.V.S.R. (eds). Biological Indicators of Soil Health, Wallingford: CABI
Publishing.
Vallad, G.E, L. Cooperband, and R.M Goodman. 2003. Plant foliar desease suppression mediated bycomposted forms of paper mill residuals exhibits molecular features of induced resistance.
Physiological and Molecular Plant Pathology 63 (2003) 65-77.
White, P. M., C. W. Rice. 2009. Tillage effects on microbial and Carbon dynamics during plant residue
decomposition. Soil Sci Soc Am J. 73, 138-145.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011381

The Use of RAPD Marker on Gambier‟s Breeding Program
Hamda Fauzaa*, Jamsari a, Istino Ferita a, Ade Noverta a, Azmi Dhalimi b,
Ahmad Denian c, Murdaningsih H. Karmanada

Faculty of Agriculture of Andalas University, Padang, Indonesia
Center for Agricultural Technology Assessment and Development, Bogor, IndonesiacMedicinal and Aromatic Crops Research Institute, Bogor, IndonesiadFaculty of Agriculture of Padjadjaran University, Bandung, Indonesia*Corresponding author: Faculty of Agriculture Andalas University, Kampus Limau Manih 25163
Tel: +62 751 72776; Fax.: +62 751 72702; mobile : +62 8156119287 ;hamdafauza@yahoo.comb

Abstract
Low productivity is one of the problems faced by farmers in gambier (Uncaria gambir) plants cultivation. This
problem is mainly caused by the use of low yield gambir cultivars. Although breeding effort has been initiatedsince several years ago, no significant result has been achieved until now. This is due to inadequacy ofmolecular investigations in this species. Molecular aspects are commonly related with protocol for DNA
preparation and molecular markers which are important so that it will speed up the breeding program. The
objectives of the research were to optimize the DNA isolation procedures using different protocol and type ofmaterials, to optimize DNA amplification procedures, and (3) to find out RAPD primers indicating high level ofpolymorphisms. The research was done from May to November 2009 in the Laboratory of Biotechnology and
Plant Breeding, Faculty of Agriculture, Andalas University. DNA isolation procedure and type of materials wereassessed. DNA isolation was performed using four different protocols. Type of materials tested consisted ofseven treatments. Optimizing PCR condition and RAPD primer selection delivered basic information of optimum
PCR condition. A number of 50 RAPD primers were randomly selected. The results recommended theapplication of CTAB-based method protocol, using young meristem leaves for DNA isolation.
Keywords: gambier, breeding, RAPD

Introduction
Low productivity is a major problem in gambier (Uncaria gambir) plant development. Crop
productivity ranges between 400 and 600 kg per ha (Dinas Perkebunan Sumatera Barat, 1998;
Roswita, 1990). Meanwhile the potential of this plant can reach 2100 kg per ha of dry resin(Sastrahidayat & Soemarsono, 1991). The low productivity is due to the use of low yield varieties.
Meanwhile, the use of improved varieties is one determining factor in increasing crop productivity. In
the cultivation of gambier, there is no information of using high yielding varieties.
Compared with other commodities, research and development of gambier plant breeding is
still far behind. With the use of biotechnology is expected the lag, especially in the field of plant
breeding can be pursued. Studies of molecular aspects of the basic information that will be usefulas an initial step in gambier plant breeding programs
During the previous decade, strategy for the evaluation of genetic variability was carriedout through the approach of anatomy, morphology, embryology, and physiology. This approach now
has been fasilitated with molecular techniques. The development of biotechnology in particularscience called molecular markers based on polymorphisms found in proteins or DNA, has been
widely facilitate research in disciplines such as taxonomy, ecology, genetics, and plant breeding(Weising, et al, 1995).
In the field of plant breeding, using fingerprinting techniques have been applied to various
aspects and types of plants. Various examples can be mentioned, among others, is its application
to rice. Wu and Tanksley (1993) reported the identification of allele-specific microsatellite found in

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Proceedings of The 7 ACSA Conference 2011

indica and japonica rice types. The results are then followed by the use of PCR techniques to
identify microsatelite polymorphism possessed by the upland rice and other rice cultivars. Virk et al(1995) using the RAPD technique for the distinction between the types of rice cultivation and to
identify possible duplication of cultivars. Besides the rice crop, the application of molecular markers
has also been applied to other fields. Meanwhile, for gambier plants, Fauza, et al. (2007) in early
research on diversity studies of gambier concluded that RAPD markers have a broad genetic
variability. But in this study there are several obstacles in the method of DNA isolation and RAPDPCR method technique is not stable yet, and limited use of polymorphic primers for gambier plants.
This study aimed to obtain the DNA isolation method for the gambier plant, to findinformation on material characteristics for DNA isolation, to find a method (program) appropriate in
RAPD-PCR technique for gambier plant, and to obtain primers polymorphic in plants gambiercharacterization techniques based on RAPD-PCR.

Materials and Methods
The research was done from May to November 2009 in the Laboratory of Biotechnology
and Plant Breeding, Faculty of Agriculture Andalas University. Two informations namely: DNA
isolation procedure and type of materials were assessed. Experiment of DNA isolation procedure
performed four protocols as treatments, i.e.: (1) Saghai-Maroof, et al. (1984), (2) Doyle and Doyle(1990), (3) Krizman, et al. (2006), and (4) An Michiels et al. (2003). Experiment for type of materials
applied seven treatments, namely: (1) fresh young leaf, (2) fresh old lead, (3) fresh young leaf
stored at -20ºC for one week, (4) fresh young leaf stored at 4ºC, (5) young leaf stored at room
temperature (25ºC) for one week, (6) young leaf stored with silica gel for one week (7) etiolated
fresh young leaf. Optimizing PCR condition and RAPD primer selection; these experiments
delivered basic information of optimum PCR condition that could be used in variability and pedigree
analysis based on RAPD molecular technique. Optimization had been done by applying somelevels of annealing temperature i.e.: 35°C; 37°C; 40°C; 42°C and 45°C. These annealing
temperatures will be combined with one or two cycle groups. Besides some level of DNA
concentration (5; 10; 15; 20; 25; 50; and 100 ng/reaction) and concentration of primer (5; 10; 20; 50;
and 100 pmol/reaction) applied. A number of 50 RAPD primers were randomly selected.

Results and Discussion
Optimization of DNA Isolation
Of the four isolation procedures attempted, it seems that the protocol based on Doyle andDoyle (1990) produced the highest total DNA concentration. The ratio of the intensity of DNA
fragments isolated by Doyle and Doyle (1990) showed 2-5 times higher than the standard λ DNA
used. With the DNA isolation protocol based on Saghai-Maroof et al. (1984), there was no alleged
DNA pellets obtained. While isolation by using a protocol based on Krizman et.al (2006) and basedon An Michiels et. al, (2003) for the isolation of DNA-containing plant samples sap, the results
obtained was also not optimum .
The success of protocols based on Doyle and Doyle was likely caused by the presence ofphenol compounds was mixed with Chloroform-Isoamylalcohol in one stage of isolation. Phenol isknown as a compound that is effective enough to separate the protein from cell organelles. It is
known that plant cells are very rich in polyphenols. These substances are contaminants for the
DNA isolation from gambier plant tissue. However for other species, the use of phenol during DNA
isolation process is needed (Jamsari, 2003)
Of the several types and characteristics of materials used for DNA isolation was found thatyoung leaves of gambier plants in fresh condition and young leaves stored in silica gel, were goodthProceedings of The 7 ACSA Conference 2011383

materials for the isolation of gambier plant DNA. It is characterized by the discovery of DNA pellet
after the extraction process, which in turn is also evidenced by the results of electrophoresis
analysis. While other materials did not show DNA pellet after the precipitation process.
At this stage the attempt to isolate DNA from various plant tissues by several methods was
carried out. Isolation of DNA from fresh young leaves by the CTAB method (Doyle and Doyle, 1990)
and DNA with DNA Kit produced better quality. While the isolation of DNA from young leaves thatwere stored with silica gel with CTAB method produced DNA with poorer quality than those from
fresh leaves, but by using a DNA kit produced DNA with a fairly good quality. In principle, theresulting DNA can be used to the process of PCR-RAPD amplification. Optimization results of DNA
with CTAB method are shown in Figure 1.
M

1

2

3

4

M = Lambda 50. 1,2,3 = fresh young leaves, 4 = young leaves stored in silica gel

Figure 1. The examples of optimization results of gambier plant DNA isolation.

Optimization of RAPD-PCR technique
Optimization of RAPD amplification was done by modifying the temperature and time ofthe stages of the amplification process of some methods and programs from various sources,
including: method of Williams, et al. (1990) and Ready to Go PCR Kit (RTG-PCR Kit). Based on the
results of the optimization program that will be applied in the amplification program was thatrecommended by Williams, et al. (1990), as showed in Table 1.
Table 1. RAPD PCR amplification program of gambier plant
Process
Initiation of denaturation
Denaturationi
Anneling
Extention
Final extention
Pause

Temperature (°C)

Time

96
94
36
72
72
4

2 min
30 sec
1 min
2 min
5 min
~

Total cycles
145
1

Primer Selection
To obtain a high degree of polymorphism, primers were selected by using random operon
dekamer primer technology (Almaeda, USA). As the DNA template used in the selection of thisprimer DNA pool consisting of five DNA genotype gambier based phenotypic appearance is thought
to have a distance kinship. Selections of primer for subsequent experiments based on the numberof bands (fragments) and the sharpness of the resulting ribbons. Selection was carried out on 50
types of primer RAPD method and program optimization. The primer selection was conducted at
two-phase. In the first phase used 25 primers.Three of the 25 primers, were used were for the
analysis of individual selection, the OPN-06, OPE-18 and OPY-08. Each band primer produced
between 0-5 bands.

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Table 2. List of RAPD primer and PCR product characteristics obtainedNo

PrimerName

1. OPA-11
2. OPA-12
3. OPA-15
4. OPA-19
5. OPB-01
6. OPB-06
7. OPB-09
8. OPB-11
9. OPF-08
10. OPK-06
11. OPL-13
12. OPN-19
13. OPR-11
14. OPT-16
15. OPW-11
16. OPX-01
17. OPX-04
18. OPX-07
19. OPX-09
20. OPX-15
21. OPX-19
22. OPY-04
23. OPY-20
24. OPB-17
25. OPF-05
26. OPE-18*
27. OPN-06*
28. OPY-08*

Secuences

Number offragments

CAA TCG CCG T
TCG GCG ATA GTTC CGA ACC CCAA ACG TCG G
GTT TCG CTC C
TGC TCT GCC CTGG GGG ACT CGTA GAC CCG T
GGG ATA TCG GCAC CTT TCC C
ACC GCC TGC TGTC CGT ACT GGTA GCC GTC T
GGT GAA CGC T
CTG ATG CGT G
CTG GGC ACG A
CCG CTA CCG A
GAG CGA GGC T
GGT CTG GTT G
CAG ACA AGC CTGG CAA GGC A
GGC TGC AAT G
AGC CGT GGA A
AGG GAA CGA G
CCG AAT TCC CGGA CTG CAG A
GAG ACG CAC A
AGG CAG AGC A

8
4
6
8
14
10
7
9
5
8
11
7
8
11
6
7
8
11
7
7
8
11
10
8
6
12
10
13

* The best primer on the first stage selection.

Fragment Characteristics
Fragment is quite clear, but it's rather difficult
Fragments are clear and easy

to be s c oring

to be s c oring

Fragments are clear and easy diskoring
Fragment is quite clear, but it's rather difficult

to be s c oring

Fragment is quite clear, but it's rather difficult

to be s c oring

Fragment is quite clear, but it's rather difficult

to be s c oring

Fragment is quite clear, but it's rather difficult

to be s c oring

Fragment is quite clear, but it's rather difficult
Fragments are clear and easy
to be s c oring

to be s c oring

Fragment is quite clear, but it's rather difficult

to be s c oring

Fragment is quite clear, but it's rather difficult

to be s c oring

Fragments are clear and easy

to be s c oring

Fragmen ts are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragment is quite clear, it's rather difficult

to be s c oring

Fragments a re clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

Fragments are clear and easy

to be s c oring

The second stage was the repeatation of the selection of 25 primers and the best three inthe first phase. DNA template used consisted of five different genotypes of first stage. Based on
the results of 28 RAPD primer amplification with DNA pool gambier, all tested primer products were
found in the DNA mixture. It means thar all the selected RAPD primers has the potential to be usedin the selection of individual gambier plant DNA. Products produced ranged from 4-14 fragments ofDNA. Fragments generated from the primer selection of this second stage were better than the firststage. This is due to the different DNA pool used in this experiment. Therefore RAPD primer thatcan be recommended for the analysis of kinship is the OPN-06, OPE-18 and OPY-08. More detailcan be seen in Table 2 and the results of amplification of DNA pools with 28 primers RAPD
gambier shown in Figure 2.
M

15

16 17

18

19

20

21 22

23 24

25 26

27 28

M

1

2

3

4

5

6

7

8

9

10

11

12 13

14

Description: sample mentioned in each of them according to the primary number in Table 2.

Figure 2. Amplification of gambier DNA pools with 28 primer RAPD.

thProceedings of The 7 ACSA Conference 2011385

Conclusions
The isolation of DNA for gambier generated good quality and quantity of DNA using the
CTAB method by protocol of Doyle and Doyle (1990) using young leaf tissue. Meanwhile, aprogram that should be applied for RAPD-PCR amplification was a program recommended byWilliams, et al. (1990). About 50 selected primers could be used in gambier DNA amplification, butthree primers showed a high number of bands and fragments namely: OPE-18, OPN-06 and OPY08.

References
An Michiels, W. Van den Ende, M. Tucker, L. Van Riet, and A. Van laere. 2003. Extraction of high quality
Genomic DNA from latex-containing plants. Analytical Biochemistry (315): 85-89.
Dinas Perkebunan Sumatera Barat. 1998. Statistik Perkebunan. Dinas Perkebunan Sumatera Barat. Padang.
Doyle, J.J. and J.L. Doyle. 1990. Isolation of plant DNA from fress tissue. Focus 12: 13-15.
Fauza, H., I. Ferita, Murdaningsih,H.K., N. Rostini, dan R. Setiamihardja. 2007. Variabilitas genetik tanaman
gambir berdasarkan marka RAPD. Zuriat 18 (1): 93-99.
Jamsari. 2003. Construction of High-Density Genetic and Physical Maps Around the Sex Gene M of Asparagus
officinalis L. Published Dissertation-Schriftenreihe des Institut fur Plantzenbau und zuchtung. Cristian
Alberchts Universitat zu Kiel (3) 1-171.
Krizman,M, J.Jakse, D.Baricevic, B.Javornik, and M.Prosek. 2006. Robust CTAB-activated charcoal protocol forplant DNA extraction. Acta Agriculturae Slovenica. 87:427-433
Roswita, D. 1990. Prospek tanaman gambir di Sumatera Barat. Bul. BIP Padang (01): 8-10.
Saghai-Maroof, M.A., Soliman, K.M., Sharm, P.D., Kubde, and S.A. Mulay. 1984. Ribosomal DNS spacer-length
polymorphism in barley : Mendelian inheritance, chromosomal location and population dynamic. Proc.
Natl. Acad Sci USA (81) : 8014-8018.
Sastrapradja, S., S. Dinimihardja, R. Soejono, N.W. Soetjipto, M.S. Prana. 1980. Tanaman Industri. PN Balai
Pustaka. Jakarta. 132 hal.
Weising, K., H. Nybom, K. Wolf., and W. Meyer. 1995. DNA Fingerprinting in Plant and Fungi. CRC Press, Boca
Raton. Florida. 322 p.
Williams, J.G.K., Kubelick, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V. 1980. DNA polymorphisms amplified byarbitrary primers are useful as genetic markers. Nucleic acids Res. 18:6531-6535.
Williams, J.K., A.R. Kubelik., K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified byarbitrari primers are useful as genetic markers. Nucleic Acid Research. 18(22): 6531–6535.

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386

th

Proceedings of The 7 ACSA Conference 2011

Shorea leprosula: the Most Commercial Trees to Improve“Production-Natural Forest” Productivity
Wahyudi 1) Indrawan A.2) Irdika Mansur 3) Prijanto Pamoengkas 3)
1)

Department of Forestry, Palangka Raya University
isanautama@yahoo.com
2)
PhD, Faculty of Forestry Bogor Agricultural University
3)
Faculty of Forestry, Bogor Agricultural University

Abstract
Deforestation rate in Indonesia is 1,8 million ha per year. This is due to low productivity of natural forest which is
3
-1
-1
3
-1
-1
just 0,25 m ha year(compared with teak plantation forest: 8-10 m ha year ). Decrease in naturalregeneration and forest plantation were caused by stand competition and limited of light in the forest floor.
Efforts to improve production-natural forest productivity was by applying the Selective Cutting and Strip Planting(SCSP) system. This system made optimum open area (strip) which is suitable to maximize the growth of
Dipterocarp species, especially Shorea spp as the most commercial trees in the natural tropical forest. This
research aimed to analyze and created modeling of growth and yield of Shorea leprosula plantation in the SCSP
system. The research was conducted on research plots of SCS in logged over – production natural forest of PT
Gunung Meranti forest concession, Central Kalimantan Province. Analysis of data used growth modeling foreven-aged forest. The result showed that mean annual increment of Shorea leprosula plantation at 2, 11 and 16-1-1-1
year olds were 1,06 cm year ; 1,22 cm year and 1,31 cm year in diameters, respectively. Based on even3-1
aged forest modeling, the first cycles of Shorea leprosula plantations was 32 year in the 125,14 m ha of logs
(40 cm up of diameters), therefore Shorea leprosula plantations in the SCSP system could improve the naturalforest productivity until 262,72 times. The SCSP system with Shorea leprosula plants is very applicable in thelogged over-production natural forest to improve forest productivity.
Keyword: Selective cutting and strip planting system, Shorea leprosula, growth and yield, productivity

Introduction
Indonesia is third in the world after Brazil and Zaire in wide of tropical forest regions andhas the highest biodiversity too (Whitmore, 1975; Mac Kinnon, et al., 2000). However, the conditionof natural forest resources in Indonesia tends to face degradation in the quality and quantity alongwith environmental changes nationally and also globally (Ministry of Forestry, 2008). Deforestationand degraded forest in Indonesia are caused by increasing of resident and wood requirement(Singh, et al. 1995) illegal logging, shifting cultivation, illegal mining, illegal occupation of land, forest
fire (Indrawan, 2008) conversion of forest (Saharjo, 2008), and poor forest management (Wahjono
and Anwar, 2008).
As comparison, in the year 1990‘s, logs production in Indonesia were 28 million m3 comingfrom 59,6 million ha of production forest. But in the year 2007, logs production decreased to
become 9,1 million m3 from 27,8 million ha of production forest only. Deforestation and degraded
forest will continue to happen if there isn‘t repair of production forest management system inIndonesia. Some researches of silvicultural system in Indonesia have been conducted since 1993
and applying of Selective Cutting and Strips Planting (SCSP) silvicultural system with intensive
silvicultural technique has been done limited to 25 forest concessions since 2005, using species ofDipterocarp specially Shorea spp. Shorea leprosula is one of Dipterocarp species recommended to
be developed in strips area in SCSP system. Afterwards, research on influencing of gap size andslope to increase growth and yield of Shorea spp plantation in the SCSP system is very needed tosupport this system.

thProceedings of The 7 ACSA Conference 2011387

Based on the forest function, forests in Indonesia are divided into three regions i.e.
conservation forest, protection forest and production forest. Production forest can be divided intosome forest regions, in the form of virgin forest, logged over forest, low potential forest, bushesscrub, grassland and critical land. Logged over forest and low potential forest can be managed by
Selected Cutting and Strips Planting silvicultural system using Shorea spp, especially Shorea
leprosula.
This Research aimed to compile growth and yield modeling of Shorea leprosula that isdeveloped in the strips area. Research was expected to be used by stakeholders, specially for userof SCSP system.

Methods
Research was conducted in the Permanent Sample Plots of Selective Cutting and Strips
Planting in the District of Mandau Talawang, Central Kalimantan Province that was planted in 2008
(age of 2 years), 1999 (age of 11 years), and 1994 (age of 16 years). Data were collected in 2010.
Measured parameters were diameters and high of Shorea leprosula at the aged 2 years,
11 years, and 16 years. Growth and yield of Shorea leprosula pattern was formed according to to
the increment and time (years) functions through polynomial equation (Brown, 1997; Burkhart,2003) that was:y = c1 + c2x + c3x2
Where:y: diameter (average)x: time (years)
c1,c2,c3 : coefficient

Results and Discussion
The research results of Shorea leprosula plantation in the Permanent Sample Plots of
Selective Cutting and Strips Planting that is planted in 2008 (age of 2 years), 1999 (age of 11
years), and 1994 (age of 16 years) which were collected in 2010 were shown in Table 1.
Table 1. Mean annual increment of Shorea leprosula in the PSP of SCSP at 2, 11, and 16 years old

Age (year)

Live (%)

0
2
11
16

100
84.22
61.87
61.21

MAI
Diameter (cm)
0
1.06
1.22
1.31

High (m)
0
1.40
0.94
0.82

Table 1 showed that diameter mean annual increment (MAI) of Shorea leprosula since thefirst time until 16 years old was always inreasing. MAI of Shorea leprosula was 1,36 cm/year at 16
years old with 61,21% of live. At the 16 years old, diameter and high of Shorea leprosula plants
were 21,22 cm and 13,1 m respectively. Growth and yield of Shorea leprosula was always
increasing until it achieved 30 to 40 cm in diameters (Wahyudi et.al. 2011).
Growth and yield of trees in the even-aged stand forest were different with those of
uneven-aged stand forests. Modeling must consider variation coefficient and deviation standard ofdata. There were phenomena in the field that some plants in the monocultural plantation indicateddifferences in growth level for each species. There are some species which grow very fast, fast,
slow and very slow. This phenomena is overcome by tending and thinning periodically.

388

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Proceedings of The 7 ACSA Conference 2011

Growth of plants can be assumed from time function. Diameter of plants will be getting
bigger progressively, but periodically growth must be mentioned in the model, so it needs the timeseries of data to create modeling of growth and yield. Therefore, sigmoid curve to draw the growthof yield of Shorea leprosula also needed the time series data. Modeling must accommodate all
information about species, environment, tending, increment and so on, which was made available
and trustworthy of growht and yield to create the justified modeling (Grant et al. 1997; Porte &Bartelink 2001; Vanclay 2001). Modeling can simplify the complicated calculation composed ofseveral equations at the same time with some simulation expected.

A

B

C

Figure 1. Shorea leprosula plantation in the Selective Cutting and Strips Planting system at 2 years old (A), 11
years old (B) and 16 years old (C).

Modeling using polynomial equation based on increment of Shorea leprosula and timefunctions (Brown 1997; Burkhart 2003) is as the following:
Y =0,0297x2 + 0,8208x + 0,3728 .................. R2= 86,89%
y : final diameter ; x : time (year) .
This model predicted that to achieve 50 cm up of diameter average, the time needed was 32 years
as shown in Figure 2. Correlation coefficient of this equations was 86,89%, accordingly, it indicatedthat this equation can be used to predict growth and yield of Shorea leprosula plantation on the
Selective Cutting and Strips Planting system.
Diameter (cm)60
Growth Equation of

S. leprosula

50
40
30
y = 0,0297x2 + 0,8208x + 0,3728
R2 = 0,8689

20
10
0
0

5

10

15
20
Time (year)

25

30

35

Figure 2. Growth and yield of Shorea leprosula plantation using modeling of polynomial equation.

thProceedings of The 7 ACSA Conference 2011389

Mean production of logs in PT Gunung Meranti (forest concession ) for 4 years (2007 to2010) was 22,3 m3 ha-1 of logs. Whereas, based on even-aged forest modeling, the first cycles of
Shorea leprosula plantations for 32 years, the production was 125,14 m3 ha-1 of logs (40 cm up of
diameters), therefore Shorea leprosula plantations in the SCSP system could improve the naturalforest productivity until 262,72 times. The SCSP system of Shorea leprosula is very applicable in
the logged over-production natural forest to improve forest productivity. So, Shorea leprosula ismost commercial trees to improve ―Production Natural Forest‖ productivity in the tropical forest.

Conclusion
Shorea leprosula plantation in the Selective Cutting and Strips Planting silvicultural system
can improve Production Natural Forest productivity in the tropical forest with applying the cutting
cycles as long as 32 years and predicted to produce 125,14 m3 ha-1 of logs.

References
Brown S. 1997. Estimating biomass change of tropical forest a primer. FAO Forestry Paper No.134. FAO USA.
Burkhart HE. 2003. Suggestion for choosing an appropriate level for modelling forest stand. In Amaro A, Reed
D, Soares P, editors. Modelling Forest System. CABI Publishing.
Coates KD, Philip JB. 1997. A gap-based approach for development of silvicultural system to address
ecosystem management objectives. Journal Forest Ecology and Management 99 (1997) 337-35.
Finkeldey R. 1989. An Introduction to Tropical Forest Genetic. Institute of Forest Genetics and Forest Tree
Breeding, Goettingen, Germany.
Fyllas NM, Politi PI, Galanidis A, Dimitrakopoulo PG, Arianoutsou M. 2010. Simulating regeneration andvegetation dynamics in Mediterranean Coniferous Forest. Ecology Modelling Journal. 34.
Gadow KV, Hui G. 1999. Modelling Forest Development. Kluwer Academic Publishers.
Grant WE, Pedersen EK, Marin SL. 1997. Ecology and Natural Resource Management. Systems Analysis andSimulation. John Wiley & Sons, Inc.
Kozlowski TT, Pallardy SG. 1997. Physiology of Woody Plants. Academic Press.
Porte A, Bartelink HH. 2001. Modelling mixed forest growth: a review of models for forest management. Eco.
Model. Journal.
Radonsa PJ, Koprivica MJ, Lavadinovic VS. 2003. Modelling current annual height increment of young Douglasfir stands at different site. In Amaro A, Reed D, Soares P, editors. Modelling Forest System. CABI
Publishing.
Rodriguez F, De La Rosa JA, Aunos A. 2003. Modelling the diameter at breast height growht of Populus
euramericana plantation timber in Spain. In Amaro A, Reed D, Soares P, editors. Modelling Forest
System. CABI Publishing.
Vanclay JK. 2001. Modelling Forest Growth and Yield. Applications to Mixed Tropical Forest. CABI Publishing.
Wahyudi. 2009. Selective cutting and line enrichment planting silvicultural system development on Indonesiantropical rain forest. In: GAFORN-International Summer School, Geor-August Universität Göttingen andUniversität Dresden, Germany.
Wahyudi, Indrawan, Mansur, and Pamoengkas. 2011. Growth and Yield of Plantation and Residual Trees in the
Intensified Indonesian Selective Cutting and Strips Planting System. Case Study on Kapuas District,
Central Kalimantan Province. Post-Graduate Program – Bogor Agricultural University, Bogor.

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390

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Proceedings of The 7 ACSA Conference 2011

Patogenity Test of Two Isolates of Ganoderma on Sengon Seedlings
Elis N. Herliyana a,*, Irfan K. Putra a , Darmono Taniwiryono b, Hayati Minarsih baDepartment of Silviculture, Faculty of Forestry, Bogor Agricultural University, Bogor, IndonesiabIndonesian Biotechnology Research Institute for Estate Crops (IBRIEC), Bogor, Indonesia* Corresponding author: Department of Silviculture, Faculty of Forestry, Bogor Agricultural University,
Kampus IPB Darmaga Bogor (16680), Indonesia
Fax 0251626806,
elisherliana@yahoo.com; elishe@ipb.ac.id

Abstract
Sengon tree (Paraserianthes falcataria (L.) Nielsen) currently becomes a major forest tree species widely plantedby smallholders in Indonesia. Sengon often used by farmers as component planted in agroforestry system aswell as shade trees planted in between other crops such as coffee and cacao. Ganoderma infection causebasal
stem rot disease which is becoming more prevalent and causing significant loss in sengon tree and other estate
crops. This research is based on the attack of Ganoderma tothe shade tree, sengon. Therefore, it is important to
do a specific research on the process of the inoculation of Ganoderma to prevent such attack. The aim of thisresearch is to understand the effect of the inoculation to the sengon seedlings. There are two majors in the
research i.e. non inoculation and inoculation treatments. Each treatment consisted of three observation blocks
that are considered equal and each block consisted of four plants (the sengon seedlings age were one and ahalf months) as replicates. The non inoculation treatments were all combinations of root and foodbase
treatments. Foodbase treatment itself is divided into two i.e. the sengon wood piece with varying size (3, 4, and5 cm diameters) and PDA (Potato Dextrose Agar) without inoculation of Ganoderma spp. from Ciamis
area/region (isolates of Ganoderma from lamtoro) and isolates of Ganoderma from sengon plants) derived fromthe collection of Forest Pathology Laboratory and will be called SP1 and SP2. The result showed that the control
has a better average growth compared to the inoculation treatment. The f pathogenity test shows that
Ganoderma SP2 has more pathogenic than Ganoderma SP1. the heights of seedlings that were inoculated withGanoderma SP2 are l lower than the heights of seedlingss that were inoculated with Ganoderma SP1. Root
treatments application showed that both controls and treatments blocks have better growth from sengon
seedlings using root cutting treatment. The effect on the growth of sengon seedlings showed that inoculated
seedlings in the wood with 3 centimeters diameter as a foodbase inhibit both their height and leaves growthcompared to the seedlings with 4 centimeters or 5 centimeters diameter of wood of the same foodbase.
Keywords: foodbase, Ganoderma, sengon (P. falcataria), Basal stem rot.

Introduction
Sengon tree (Paraserianthes falcataria (L.) Nielsen) currently becomes a major forest tree
species widely planted by smallholders in Indonesia. In Java, there is a total area of 400.000 ha ofcommunity forest dominated by sengon from which 895.000 m3 wood are produced annually. The
wood of this quick growing tree is processed further by paper industries to become pulp or sawed
by lumber industries as a raw material for soft wood board. Sengon often used by farmers ascomponent planted in agroforestry system as well as shade trees planted in between other crops
such as coffee, cacao and banana. Ganoderma infection is becoming more prevalent and causing
significant loss in sengon tree in Indonesia. Once the tree is infected by this cosmopolitan fungal
pathogen, sooner or later its base will become totally rotten leading to the tree dead. The disease
incidence ranges from 3% to 26%. One hundred percent disease incidence has been reported tooccur in the second generation of replanting.
This basal stem rot disease spread amongst trees primarily through roots contact. The
fungus has rigid fruiting bodies plate (conk), with or without stem, the top surface often looks shiny
waxy brown to dark brown in color, the bottom surface looks porous creamy white in color.
thProceedings of The 7 ACSA Conference 2011391

Theyoften found attached to the base of the rotten tissues near soil surface. Basidiosporesproduced from pores at bottom surface plays in important roles in the disease spread from theinfected tree to the fresh cut of the stumps. The disease symptoms look similar with those causedby heavy water stress displaying weak pale to yellowish in color of the tree canopy which is
unrecoverable and leading to leaf dryness. The disease is difficult to be controlled, because when
the symptom is observed, the infected tree is usually cannot be saved anymore by any control
means. Turner (1981) in Zakaria et al. (2004) reported that at least there are 15 species of
Ganoderma in various places in the world, which caused basal stem rot disease.
Ganoderma is a fungus causing basal stem rot disease that usually attacks the roots of thehost range. This fungus attacks is commonly found in various types Leguminoceae (Henessy and
Daly 2007), Palmae (Turner 1981 in Zakaria et al. 2005), Rubiaceae (Hindayana et al. 2002). The
authors also found Ganoderma that attack the ebony tree (Ebenaceae). This research is based on
the attack of Ganoderma to ashade tree, sengon. Therefore, it is important to do a specific researchon the process of the inoculation of Ganoderma to prevent such attack. The aim of this research isto understand how the effect of the inoculation to the sengon seedlings to find the control technique.

Methodology
This research was conducted from October 2009 - March 2010. This research was held ina greenhouse of Department of Silviculture and Forest Disease Laboratory of the Department ofSilviculture, Bogor Agricultural University.
Preparation of Equipment and Materials Research
The study was started by the preparation of sterile soil and foodbase that include a pieceof wood with diameter 3, 4 and 5cm and foodbase a PDA (Potato Dextrose Agar). The supply ofsterile soil was done by autoclaving of soil mixed media and charcoal husk compost with the ratioof 2:1:1. Autoclaving was done by the time adjacent to weaning so that the soil conditions results insteaming plastic bags are not contaminated. The PDA foodbase was prepared in two ways that are
not inoculated or inoculated.
PDAs which were not inoculated by Ganoderma, was prepared simply by making sterile
PDA medium which is then placed in polybags at weaning. The preparation of PDA inoculated
media by Ganoderma was done in conjunction with the preparation of pieces of wood that also
were infected with fungus. Isolates of Ganoderma have been cultured in a sterile culture into sterilejars containing both types of foodbase. Maturity level of culture can be seen quite well from themorphology of the fungus and spread on a PDA or timber in the jar. The uninoculated pieces,supplies quite done with skinning sengon logs that had previously been cut with a length of 5cm andvarious diameters i.e. 3, 4 and 5 cm. The pieces of wood were then boiled in a pan for a few hours
before sterilized in an autoclave.
Weaning and Maintenance
Weaning were performed in the afternoon to prevent the death of seedlings due to thestress. Weaning also performed each treatment to be easy in the preparation plant. After that,
sengon seeds will be slightly softened, about one or two days prior to adapt from the stress.
Providing treatment
There are two majors in the research i.e. non inoculation and inoculation treatments. Eachtreatment consisted of three observation blocks that are considered equal and each block consistedof four plants (the sengon seeds age were one and a half months) as replicates. The non
inoculation treatments were all combinations of root and foodbase treatments.
Foodbase treatment itself is divided into two i.e. the sengon wood pieces with varying size (3, 4,392

thProceedings of The 7 ACSA Conference 2011

and 5 cm diameters) and PDA without inoculation of Ganoderma from Ciamis area/region (isolatesof Ganoderma from lamtoro) and isolates of Ganoderma from sengon plants) derived from thecollection of Forest Pathology Laboratory and will be called SP1 and SP2. The total of treatments
are 10. Inoculation treatments were a combination of various treatments of root and foodbase which
has inoculated with Ganoderma. Types of Ganoderma were also included in a combination. The
total number of inoculation is 15 treats. Types of Ganoderma were also included in a combination.
Total for this inoculation tratement is 30 treatments.
Observation of the Treatment and Data Collection
The observation of the treatment was carried out daily with the parameters of the numberof young leaves and seeds of high accretion sengon. Data were collected on a tall sheet andanalyzed using SPSS. At the end of the study, the root shoot ratio data was taken to see the mostbalanced growth among the treatments.
Data was taken on the inoculated sengon with Ganoderma, Determination of the presenceor absence of fruiting body of Ganoderma was documented. The seeds which were harvested andcleaned were then grouped by the type of treatment and then the roots were separated from theshoots (stems). Part of the seed that is classified as part of the plant roots are located just below theformer location of the branch or branches from the first base.
After the separation of shoots and roots, the weight of each seedling roots and stems were
measured to determine the fresh weight of roots and shoots of seedlings. After the fresh weightmeasurements, parts of seeds were wrapped in a paper and dried in the oven for 24 hours at 1100
C. The second measurement was done for both roots and shoots of each seedling to calculate thedry weight of roots and shoots.
Data Analysis
The experimental design was used complete randomized design based on the assumptionthat the research conducted on a homogeneous condition. Homogeneity test is based on
experiments which only require element differentiation of treatment given. The calculation is
performed with use of SPSS, while Duncan's test was used for further testing.

Results and Discussion
In general, the result of control treatments measurement has a better growth compared tothe inoculation treatment. This result can be seen on the accretion parameter of heights andnumber of leaves. It is possible to do the observation on the treatment of the inoculation with thenegative growth tendencies. The height measurement seed, according to the regulation of The
Minister of Forestry no.3 in 2004, is the height measurement from the base of the plant seed to the
top growing spot using centimeter unit. From that definition, if the growing point is dead, then thegrowing spot underneath can replace it, so the height will be reduced.
The pathogenicity test shows that Ganoderma SP2 has more pathogenic than Ganoderma
SP1. This result can be seen on the heights of seeds that were inoculated with Ganoderma SP2 arelesser than the heights of seeds that were inoculated with Ganoderma SP1.
The root treatment application shows that both controls and treatments blocks have abetter growth than sengon seedlings using root cutting treatment. This result corresponds with
Deselina works (1999), where root cutting will produce more vigor seeds and stronger roots. This
positive result of root cutting application in inoculation treatment, reveals that infection rate of
Ganoderma is lower than the healing process as the effect of the cutting.
The effect on the size of sengon seedlings showed that inoculated seedlings in the woodwith 3 centimeters diameter as a foodbase hampered both their height and leaves growth comparedto the seedlings with 4 centimeters or 5 centimeters diameter in the same foodbase. This resultthProceedings of The 7 ACSA Conference 2011393

indicates that wood pieces with 3 centimeters diameter as a foodbase, is more optimal in spreading
Ganoderma than the 4 or 5 centimeters.
Application of foodbase type differentiation in sengon seedlings showed that wet
foodbases such as PDA is easier than dry foodbases such as wood pieces, in transmitting
Ganoderma into plant‘s roots. This can be seen from the calculation where the inoculated sengon
seedlings with the wet foodbase such as PDA, has a higher disruption than the dry one. These
results occurred in height parameter. Root sprout ratio measurement in this research shows aneffect of the Ganoderma infection on the plant. General calculation of root sprout ratio will generally
support the result using SPSS. Root cutting treatments on seedling growth sengon resulted in highseedling leaves and seedling growth better than the group without cutting the roots of sengon
seedlings (Figure 1 and 2).

Uncutting root
Cutting root
Figure 1 Comparison of the rate of increase in the child leaves the root treatment. Grey block shows theuncutting roots and red block shows the cutting roots.

Uncutting root
Cutting root

Figure 2 Comparison of the high rough at the rate of root treatment. Grey block shows the uncutting roots andred block shows the cutting roots.

Conclusion
The control treatments shows better growth compared to the inoculation treatments.
Inoculation treatments at high pathogenicity on seedlings derived from Ganoderma sengon sp2.
These were shown by the magnitude inhibition. These obtained from the average growth of the
inoculated seedlings sengon with parameters of the number of leaves and seedlings height.
Observations showed that sengon seedling growth better by cutting the roots treatments, both in thecontrols and inoculation treatments.

References
Deselina. 1999. Respon Semai Ampupu (Eucalypthus urophylla ST Blake) Terhadap Pemotongan Akar, Input
Fosfor dan Lama Tinggal di Persemaian (skripsi). Fakultas Pertanian Universitas Bengkulu. Bengkulu
Henessy, C. dan Daly A. 2007. Ganoderma Diseases. Northern Territory Government, Plant Pathology,
Diagnostic Services. Darwin.
Hidayat, Jajat. 2002. Informasi Singkat Benih. Direktorat Perbenihan Tanaman Hutan dan Indonesia Forest
Seed Project. T. H. R. Ir. H. Juanda, Dago Pakar, Bandung.
Hindayana, Dadan et al. 2002. Musuh Alami, Hama dan Penyakit Tanaman Kakao. Direktorat Perlindungan
Perkebunan, Direktorat Jenderal Bina Produksi Perkebunan Departemen Pertanian. Jakarta.
Irwanto. 2006. Penilaian Kesehatan Hutan Tegakan Jati (Tectona grandis) dan Eucalyptus (Eucalyptus pellita)pada Kawasan Hutan WANAGAMA I. Sekolah Pasca Sarjana Universitas Gadjah Mada. Yogyakarta.

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Photita, W., Lumyong, S., Lumyong.,P., Ho, W.H., McKenzie,E.H.C. and Hyde, K.D. (2001). Fungi on Musaacuminata in Hong Kong. Fungal Diversity 6: 99-106.
Turner, P.D. 1981. Diseases and Disorders of the Oil Palm in Malaysia. Oxford University Press.
Zakaria, Latiffah; Harikrishna Kulaveraasingham, Tan Soon Guan, Faridah Abdullah dan Ho Yin Wan 2005.
Random Amplified Polymorphic DNA (RAPD) and Random Amplified Microsatellite (RAMS) of
Ganoderma from Infected Oil Palm and Coconut Stumps in Malaysia. Asia Pacific Journal of Molecular
Biology and Biotechnology Vol.13 (1):23-24.

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A PCR-based Technique for Detection Cylindrocarpon destructans,
the Causal Agent of Grapevine Black Foot Disease
Raed A. Haleema, *, Samir K. Abdullahb, Jaladet M. S. JubraelcaPlant Protection Department, Faculty of Agriculture and Forestry, University of Duhok, Iraqb
Department of Biology, Faculty of Science, University of Zakho, Iraqc
Scientific Research Center, University of Duhok, Iraq
* Corresponding author: Plant Protection Department, Faculty of Agriculture and Forestry,
University of Duhok, Iraq. Tel.: +4947504015992
raed@uod.ac

Abstract
The symptoms of Grapevine black foot disease caused by Cylindrocarpon destructans including reduction of theplant vigour with small-sized trunks, reduction in root biomass, black discolouration and brown to dark streaks inwood mainly at the base of the rootstock. According to morphological and cultural characteristics ofCylindrocarpon, all isolates from infected roots of declined plants were identified as C. destructans. PCR
technique was performed for accurate identification of C. destructans isolates. Ten isolates of the fungi wereselected from different locations. Those isolates were subjected to species-specific PCR assay. Total genomic
DNA was isolated from pure cultures of the isolates. The average DNA yields ranged between 1.5-6.7 µg/ml witha purity 1.6-1.8. The specific primers for C. destructans were used to amplify the ITS region of nuclear ribosomal
DNA (rDNA) containing ITS1, ITS2 and the intervening 5.8 rRNA genes of Cylindrocaropon. TheITS sequence
could successfully and appropriately confirmed that all isolates were correctly identified as C. destructans. This
fungus obtained from this study was reported for the first time in Iraq.
Keywords: Black foot disease, Cylindrocarpon destructans, Grapevine, PCR

Introduction
Species of Cylindrocarpon Wollenw. are common soil inhabitants that often associatedwith roots of herbaceous woody plants. Grasso and Magnano di San Lio (1975) described black
foot symptoms from nursery plants with black discoloration and gum inclusions in xylem vessels ofaffected rootstocks. Scheck et al. (1998) also described black foot symptoms as dark-brown to
black streaking in the vascular tissue of young (2–5 year-old) grapevines.
There are two negative impacts of this disease on nursery seedling production. First, these
fungi cause seedling mortality in nurseries up to 50% (Anderson et al. 1962; Anonymous, 1993).
Second, infected seedlings have a lower survival rate after out planting to reforestation sites. An
important factor compounding this problem is that in some cases the symptoms are not visible on
infected seedlings but disease can develop after transplantation.
Detection of plant pathogens directly from infected tissues has been reported for several
agricultural plant pathogens (Le´vesque et al. 1994 & O‘Gorman et al. 1994). In addition, PCR has
been used to detect soil pathogens directly from infested soil (Henson et al., 1993). This molecular
detection approache is ideally suited for the study of root rot organisms because of the difficulty inisolating and identifying some of these fungi. Hamelin et al. (1996) designed species-specificprimers (Dest1 and Dest4) to detect C. destructans from conifer seedlings. Using these primers indirect PCR assays on DNA extracted from C. destructans cultures isolated from grapevines in
Portugal, obtained a DNA fragment of 400 bp; The universal primer ITS4 and the fungus-specificprimer ITS1F were used in a first-stage fungus-specific amplification, followed by a second-stage
amplification with the primers Dest1 and Dest4 using the PCR product from stage one. This is a
simple and reliable method for detection of Cylindrocarpon spp. directly from infected grapevinesthProceedings of The 7 ACSA Conference 2011

397

(Nascimento et al., 2001).The objective of this study was develop an efficient and reliable detection
method based on PCR for the identiﬁcation and detection of C. destructans from infected seedlings.

Materials and Methods
Fungal isolation
Fungal isolation was done using two methods, i.e. isolation method from vine root andmoist chamber isolation method.
Isolation from vine roots
Small pieces of vine roots collected from five locations in Duhok governorate ( Bajelor,
Badi, College nursery, Malta nursery and Nizarke) were surface sterilized respectively by placing in
70% ethanol for 30 s, 1% NaOCl for 1 min and again in 70% ethanol for 30 s and then dried by filter
papers. Pieces of sterilized tissues were plated onto 2% potato dextrose agar (PDA) (Himedia
Laboratories Pvt. Ltd. - India) containing 0.25 mg/ml chloramphenicol. Hyphae growing out from thetissue pieces were cut and subcultured onto fresh PDA plates, and incubated at 25±2 °C (Van
Niekerk et al, 2004).
Moist chamber method
Cuttings were made from vine roots and placed in 90 mm petridishes containing sterilizedmoist filter paper. Plates were incubated at room temperature until fungal growth observed.
Propagules (spores, mycelia) were transferred to Potato-dextrose-Agar (PDA) plates. Pure culturesof each isolate were obtained by excising a hyphal tip from colony margins and plating it onto fresh
PDA.
Phenotypical characterization
All isolates were grown on PDA and MEA at 25°C in darkness or under NUV + fluorescentillumination with a 12-h photoperiod (Philips /36W) for 10 days until cultures sporulated. The
colonies were further incubated for 20 days to determine the presence or absence ofchlamydospores. The diameter of 20 chlamydospores per isolate was measured. Length and widthof 40 conidia (microconidia and one-, two-, and three-septate macroconidia) were measured.
Isolated fungi were identified based on the characters in culture and on natural substrates (Domsch
et al, 1980; Watanabe, 2002; Petit & Gubler, 2005).
DNA extraction and PCR amplification of ITS region
Ten isolates (DC1-DC10) were selected to confirm the identification by a specific primer of
the ITS region. Isolates were collected from five locations in Duhok governorate (Bajelor, Badi,
College nursery, Malta nursery and Nizarke).
Genomic DNA was extracted according to a method reported by Borges et al. (1990). The
specific primers of C. destructans ITS region (Dest1 5‘-TTGTTGCCTCGGCGGTGCCTG-3‘, Dest4
5‘-GGTTTAACGGCGTGGCCGCGCTGTT-3‘) were used to amplify the ITS region of nuclear
ribosomal DNA (rDNA), containing ITS1, ITS2 and the intervening 5.8 rRNA gene (Hamelin et al.,1996). The PCR reactions were carried out in a total volume of 25 µl, in thin-walled, 0.5 µl
Eppendorf tubes.Master mix was prepared for 12 samples of each fungus (10 isolates plus 2
control) by mixing 30 µl of 10XPCR, 30 µl of dNTPs, 24 µl forward primer, 24 µl Reverse primer, 12µl MgCL2, 4.8 µl of Taq polymerase enzyme and de-ionized distilled water was added to a final
volume of 252µl. The solution mixed and spun for 10 second in a microcentrifuge. Then, the mixture
was distributed in PCR tubes. All these steps were done on ice. Amplification was carried out in an
automated thermal cycler (Delphy 1000, Oracle Biosystems, MJ Research Inc., Watertown, MA,
USA) according to the following program: An initial denaturation at 94°C for 4 min, after which 30
398

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cycles of denaturation (1 min at 94°C), primer annealing (1 min at 58°C) and primer extension (1.5
min at 72°C) were performed (Alaniz et al., 2007). A final extension was performed at 72°C for 10
min Amplification reactions were conducted at least twice, in two separate experiments. For eachisolate, 5 µl of PCR products were mixed with 7µl loading buffer and then analyzed byelectrophoresis in 2% (w:v) agarose gels with 1xTBE buffer visualized by UV fluorescence.

Results and Discussion
Phenotypical characterization of C. destructans
Cylindrocarpon destructans (Zinssm.) Scholten, Neth. JL. PL. Path. 70 (Suppl. 2) 9 (1964).
Fig. (1) A – C. Telemorph: Neonectria radicicola (Gerlach & L. Nillsson) Mantiri and Samules
Canada J. Bot. 79: 339 (2001).
Colonies on MEA reached a diameter of 78 mm on PDA and 80 mm on MEA after 20 days
at 25°C. Colony surface slimy to felty; aerial mycelium typically sparse to felty, white to buff or ashade of brown. Colony reverse was orange to dark brown. Conidiogenous cells formed apically on
densely, irregularly branching clusters of cells borne laterally on otherwise undifferentiatedvegetative hyphae. Conidiophores 65 μm tall. Macroconidia cylindrical, mainly 4-celled, 18 – 45 (47) × 4 – 8 μm. Microconidia, cylindrical, 1-celled, 8 – 11 (-12) × 3 – 4 μm. Chlamydosporesyellowish brown, ovate to ellipsoidal, a few in a chain, 8 – 10 (-15) μm in diameter. Conidial
dimensions were in concordance with the previous identification (Petit & Gubler, 2005).

B

A

C

A) Twenty- day old colony on PDA-left, and MEA-right . B) Microconidia and macroconidia. Scale bar: 30 µm, C) Mycelia andChlamydospores, Scale bar: 15 µm.

Figure 1. Cylindrocarpon destructans.

Molecular detection of C. destructans
Genomic DNA isolation and purification
Suitable yields of genomic DNA were obtained from repeated experiments with an average
yield of 1.5-6.70 µg/ml and a purity of about (1.6-1.8) determined by spectrophotometer ratio
A260/A280. The molecular weight of DNA samples was estimated using 1% agarose gelelectrophoresis containing ‫ ג‬DNA sample as control (Fig. 2). Ratios above 2.0 correspond to RNA
contamination, while ratios below 1.6 suggest protein contamination (Sinha et al., 2001).

B

A
th

Proceedings of The 7 ACSA Conference 2011C399

M: represents unrestricted  DNA as a standard molecular weight marker. Lane1- 7: Whole Genomic DNA of some C.
destructans isolates isolated from different locations of Duhok Governorate.

Figure 2. Agarose gel electrophoresis 1% at 70 volt for 45 minutes.

Species specific primers
All isolates of C. destructans collected from different locations of Duhok Governorate were
amplified by two specific primers (Dest1, Dest4) which were designed by Hamelin et al, (1996). A
PCR fragment of about 400-bp was obtained for all of them. The agarose gel electrophoresis ofamplified products with this specific primer is shown in Figure (3). Other investigators have used thesame primers to amplify the ITS region of C. destructans (Hamelin et al., 1996; Alaniz et al., 2007).

bp

M

1

2

3

4

5

6

7

8

9

10

11

1500

1000
900
800
700
600
500
400
033

Lanes 1 (DC1)-10(DC10), C. destructans isolates. Lane 11, negative control of sterile distilled water; lane M, 1Kb Plus DNA
Ladder.

Figure 3. Agarose gel of the PCR products using primer pairs Dest1 – Dest4.

This is the first molecular detection work on C. destructans in Iraq. In this study, thedifferences in the intensity of bands were not taken in consideration; despite they may reflect thedifferences in copy number of the priming sites in the individual genome. The band intensity may
also be attributed to the difference of DNA concentration of individual isolates. This method did not
require going through all classical methods and only in a few hours the results could be obtained.
This approach is particularly well suited to soil organisms that are difficult to identify or isolate
because of the presence of other aggressive species.

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References
Alaniz, S.; M. Leon; A. Viceni; J. Garci'a-Jimenez; P. Abad-Campos and J. Armengol. 2007. Characterization of
Cylindrocarpon species associated with black foot disease of grapevine in Spain. Plant Dis. 91:11871193.
Anderson,N.,D.W.French,andD.P.Taylor.1962.Cylindrocladium root rot of conifers in Minnesota. For. Sci. 8:378–
384.
Anonymous. 1993. Insectes et maladies des arbres-Que´bec. Ministe`re des Ressources naturelles du Que´becRessources naturelles Canada. In:- Richard C. H., MG. Pierre Berube, and B. Bourassa. 1996.
Identiﬁcation of Root Rot Fungi in Nursery Seedlings by Nested Multiplex PCR. Applied andEnvironmental Microbiology. 62 (11): 4026–4031.
Borges M. I.; M. O. Azevedo; R. Bonatelli; M. S. S. Felipe and S. Astolfi-Filho. (1990). A practical method for the
preparation of total DNA from filamentous fungi. Fungal Genetics Newsletter. Volume (37). ISSN for the
online version : 1941-4765.
Domsch, K. H.; W. Gams and T. H. Anderson. 1980. Compendium of soil fungi. vol. I. Academic press. UK
Grasso, S. and G. Magnano Di San Lio. 1975. Infections of Cylindrocarpon obtusisporum on grapevines inSicily. Vitis 14: 36–39.
Hamelin, R. C.; P. Bérubé; M. Gignac and M. Bourassa .1996. Identification of root rot fungi in nursery seedlings
by nested multiplex PCR. Appl. Environ. Microbiol 62: 4026–4031.
Henson, J. M., T. Goins,W. Grey,D. E. Mathre,and M. L. Elliot. 1993.Use of polymerase chain reaction to detect
Gaeumannomyces graminis DNA in plants grown in artiﬁcially and naturally infested soil. Phytopathology
83: 283–287.
Le´vesque, C. A., T. C. Vrain, and S. H. De Boer. 1994. Development of a species-speciﬁc probe for Pythium
ultimum using ampliﬁed ribosomal DNA. Phytopathology 84:474–478.
Mantiri, F. R.; G. J. Samuels; J. E. Rahe and B. M. Honda. 2001. Phylogenetic relationship in Neonectria
species having Cylindrocarpon anamorphs inferred from mitochondrial ribosomal DNA sequences. Can.
J. Bot. 79: 334–340.
Nascimento, T.; C. Rego and H. Oliveira. 2001. Detection of Cylindrocarpon black-foot pathogens in grapevineby nested PCR. Phytopathol. Mediterr. 40: 357–361.
O‘Gorman, D., B. Xue, T. Hsiang, and P. H. Goodwin .1994. Detection of Leptosphaeria korrea with thepolymerase chain reaction and primers from the ribosomal internal transcribed spacers. Can. J. Bot.
72:342–346.
Petit, E. and W. D. Gubler. 2005. Characterization of Cylindrocarpon species, the cause of black foot disease of
grapevine in California. Plant Dis. 89:1051-1059.
Scheck, H. J.; S. J. Vasquez and W. D. Gubler. 1998. First report of black-foot disease, caused by
Cylindrocarpon obtusisporum, of grapevine in California. Plant Dis. 82: 448.
Sinha, R. P.; M. Dautz and D. P. Häder. 2001. A Simple and Efficient Method for the Quantitative Analysis of
Thymine Dimers in Cyanobacteria, Phytoplankton and Macroalgae. Acta Protozoologica 40:187 – 195.
Van Niekerk, J. M.; P. W. Crous; J. Z. Groenewald; P. H. Fourie and F. Halleen. 2004. DNA phylogeny,
morphology and pathogenicity of Botryosphaeria species on grapevines. Mycologia 96:781-798.
ndWatanabe, T. 2002. Pictorial atlas of soil and seed fungi: Morphologies of cultured fungi and key to species (2ed.) CRC Press. 486Pp.

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Photosynthetic Light Reactions in C4 Photosynthesis
Naoya Nakamuraa, Yuri Nakajima Munekagea, Akiho Yokotaa*aGraduate School of Biological Sciences, Nara Institute of Science and Technology* Corresponding author: Graduate School of Biological Sciences, Nara Institute of Science andTechnology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan
Tel.: +81 0743 72 5568; fax: +81 0743 72 5569.
yokota@bs.naist.jp

Abstract
The most productive wild plants and crops use C4 photosynthesis (Brown, 1999). C4 photosynthesis requires thecoordinated functions of two cell types in leaves, namely mesophyll (M) - and bundle-sheath (BS) - cells (Hatch,
1987). Atmospheric CO2 is initially fixed by phosphoenolpyruvate carboxylase (PEPC) in M cells. The resulting
products, C4 acids are transported into BS cells where CO2 is released by decarboxylation of C4 acids andrefixed by riburose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) functioning in the Calvin cycle (C 3cycle). Since this process increase CO2 levels at the site of RuBisCO in the BS cells, the oxygenase reaction of
RuBisCO is largely reduced and RuBisCO can achieve maximal catalytic activity of CO 2 fixation. Therefore, C4
plants have higher potential efficiencies in the use of light, water and nitrogen than C 3 plants (Long, 1999). C4
plants have evolved from ancestral C3 plants (Sage et al., 2011). In C4 plants, not only the CO2 metabolism but amanner of light reactions which is a process to produce ATP and NADPH used for CO 2 metabolism has beenchanged. However, mechanism of light reactions and its physiological roles on C4 photosynthesis are not yet
fully understood. Here, we introduce latest findings and perspectives of physiological roles of light reactions in C 4
photosynthesis.
Keywords: C4 photosynthesis, cyclic electron flow, Flaveria, Zea mays

The structure of mesophyll and bundle-sheath chloroplasts: the regulation of granaformation
Light reactions take place on thlakoid membranes in chloroplasts. The thylakoid
membranes are arranged in stacked (grana) and unstacked (stroma lamellae) in C3 plants.
Supercomplex of photosystem (PS) II and light-harvesting complex of photosystem II (LHC-II) arelocalized in grana thykoids while most of PSI and ATP synthase are localized in stroma lamellae(Albertsson, 2001). The grana stacks are more developed in low light intensity than in high lightintensity, the changes are assumed to optimize the photosynthetic efficiency in C3 plants (Ballantine
and Forde, 1970). In NADP-malic enzyme (ME) type C4 plants such as Zea mays, Sorghum bicolorand Flaveria trinervia, grana thylakoids are observed in the M chloroplasts like chloroplasts of C3
plants, but BS cells possess chloroplasts where grana thylakoids are largely decreased, as shownin Fig. 1 (Laetsch et al., 1965, Laetsch and Price, 1969, Hofer et al., 1992). Interestingly, granaformation was observed in BS chloroplasts of maize leaves at very low light intensity but wascompletely abolished at normal light intensity (Brangeon, 1973). Decrease in grana stacks of BS
chloroplasts is likely induced by high light signal and those systems would have been recruited fromC3 plants during C4 evolution.
LHC-II is suggested to be involved in grana formation by binding thylakoid membranes inC3 plants (Allen and Forsberg, 2001, Standfuss et al., 2005). LHC-II is mainly present as trimer on
grana thylakoids. It is reported that in Flaveria trinervia the amounts of LHC-II polypeptides in BS
chloroplasts is only slightly reduced compared with that in M chloroplasts (Hofer et al., 1992) and asignificant amount of LHC-II polypeptides are also detected in fractionated stroma lamellae(Munekage et al., 2010). Moreover, in maize, LHC-II trimer is identified in BS thylakoid membranesthProceedings of The 7 ACSA Conference 2011403

using Blue-Native PAGE, although BS chloroplasts of Maize are almost grana-free (Romanowska etal., 2008). These results suggest that amounts of LHC-II polypeptide and the presence of LHC-II
trimer are not sufficient for grana formation. Perhaps the additional mechanism is involved in theformation of thylakoid membranes with reduced grana in BS chloroplasts.

Development of BS cells
DevelopmentofDevelopmentofand their chloroplasts
cells andBS cellsBS
and
their
chloropalsts
their chloropalsts

F. pringlei (C3)
F. pringlei(C
F. pringlei(C33))

F. bidentis
F. bidentis(C(C) 4)
F. bidentis (C 4) 4
Fig. of1.leafLight
micrographsof leaftransverse section of Flaveria species.
Figure 1. Light micrograph‘stransverse
section of Flaveria
species.
Fig. 1. Light micrographs of leaf transverse section of Flaveria species.

Regulation of linear electron flow and cyclic electron flow around PSI in C4 photosynthesis
Linear electron flow operates to produce the both ATP and NADPH. In linear electron flow,
the electrons excised from water molecules at PSII are transferred to PSI through the cytchrome b6f
complex, and finally NADPH is produced. Protons are transported across the thylakoid membranes
from stroma side to lumen side during the process of electron transfer. This proton gradient is used
for ATP synthesis (Fig. 2). On the other hand, cyclic electron flow around PSI (CEF1) contributes toonly ATP production. In CEF1, electrons are returned from accepter side of PSI to plastoquinone
which mediate electron transport from PSII to cytchrome b6f complex (Shikanai, 2007). This electrontransport contributes to form proton gradient without NADPH production. Two CEF1 pathways havebeen identified in C3 plants.
NADPH

NADPH

ATP

NDH pathway
FQR pathway
Fd

Fd

stroma
PGR5,
PGRL1

PSI

NDH
complex

Cyt.

b6f

PQ

PSI

complexATPsynthase
Thylakoid
membrane

PC

lumen
H+

H+

H+

H+

Figure 2. Schematic model of PS1 cyclic electron transport.

Fig. 2. Schematic model of PS1 cyclic electron transport

The first pathway is called as ferredoxin-plastoquinone reductase (FQR) pathway involving
PROTON GRADIENT REGULATION 5 (PGR5) and PGRL1 (Munekage et al., 2002, Dalcorso et al.,2008). The other pathway is NAD(P)H dehydrogenase (NDH) pathway involving a plastidial NDH

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Proceedings of The 7 ACSA Conference 2011

complex composed of the eleven plastid-coded subunits (NDH-A to -K) and the over 15 nucleiencoded subunits (Peng et al., 2011). In C3 plants, the FQR pathway contributes for maintaining the
production ratio of ATP/NADPH and for photoprotection, whereas the NDH pathway functions instress resistance in C3 plants (Munekage et al., 2004, Wang et al., 2006, Munekage et al., 2008).
Localization of the NDH complex and PGR5 on stroma lamellae and distribution of PSII and PSI on
thylakoid membrane suggest that CEF1 takes place in stroma lamellae (Rumeau et al., 2005,
Munekage et al., 2010).
In C4 plants, increased CEF1 activity and decreased linear electron transport is induced. In
NADP-ME type C4 plants, BS chloroplasts of mature leaves show the inhibition of PSII activity (Wooet al., 1970). The composition of PSII complex in BS chloroplasts is investigated by many
researchers using Zea mays, Sorghum bicolor and Flaveria trinervia. Several studies are suggested
that the subunits of PSII complex, especially those of oxygen evolving complex are largely reduced
in BS chloroplasts compared with M chloroplasts (Sheen et al., 1987, Oswald et al., 1990, Hofer etal., 1992). BS chloroplasts produce only limited amounts of ATP and NADPH from liner electron
flow activity, because in this chloroplasts, PSII activity is significantly inhibited (Schuster et al.,1985). Since NADPH is supplied by decarboxylation of C4 acids by NADP-ME, only ATP is required
to be produced in BS chloroplasts of NADP-ME type. The ATPs are considered to be produced by
CEF1 activity. Significant activity of CEF1 compared with C3 plants has been reported in Zea mays,
Sorghum bicolor (Herbert et al., 1990, Asada et al., 1993). The higher amounts of NDH complex
and PGR5 in BS cell than M cell also supports activation of CEF1 in BS cell of NADP-ME type C4plants (Kubicki et al., 1996, Munekage et al., 2010). On the other hand, ATP demands are
increased in M chloroplasts in NAD-ME type C4 metabolism. In Salsola laricina and Halocharis
gossypina carrying NAD-ME type C4 photosynthesis, lower grana index and higher ratio of PSI/PSII
in M chloroplasts than BS chloroplasts are reported (Voznesenskaya et al., 1999). Moreover,subunits of NDH complex are enriched in M chloroplasts of NAD-ME type C4 plants (Takabayashi etal., 2005). Activated CEF1 likely contributes to the ATP production in C4 photosynthesis. However,for final conclusion, it is need to investigate phenotype of transgenic plants in which genes involvedin CEF1 are knocked down.

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Hatch MD. 1987. C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure.
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in mesophyll and bundle-sheath chloroplasts of the C4 plant Sorghum bicolor indicated that the complex
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Heavy Metals Concentration in Irrigation Water, Soils and Fruit Vegetables
in Coastal Area, Kota Bharu, Kelantan, Malaysia
Aweng, E. R.a , Karimah, M.a Suhaimi, O.aaFaculty of Agro Industry and Natural Resources, Universiti Malaysia Kelantan, Malaysia.
* Corresponding author: Faculty of Agro Industry and Natural Resources, Universiti Malaysia Kelantan,
Malaysia.
Tel: +06097717247; fax: +06097717232.
aweng@umk.edu.my

Abstract
This study was conducted to evaluate the levels of selected heavy metals namely Aluminium, Boron, Cadmium,
Iron, Lead, Manganese and Zinc in irrigation water, soils and selected fruit vegetables (brinjal, chilli and lady‘sfingers) which were cultivated in Kampung Badang, Pantai Cahaya Bulan in Kota Bharu, Kelantan, Malaysia.
The water used for irrigation in Kampung Badang, Pantai Cahaya Bulan had the highest concentration of Boron(3.5 ppm) followed by Manganese (1.105 ppm), Iron (0.233 ppm), Lead (0.22 ppm), Zinc (0.217 ppm),
Aluminium (0.214 ppm) and Cadmium (0.0853 ppm). However, in general, the quality of the irrigation watercomplied with the Class IV, Interim National Water Quality Standard for Malaysia, INWQS, 1985 (standard for
irrigation water) and Food and Agriculture Organization of the United Nations (FAO) Standard (1985) except
Cadmium. The irrigation water results were also compared with the Indian Standard (Awashthi, 2000), where in
general, all the measured parameters complied with the standards except Cadmium (0.01 ppm) and Plumbum(0.10 ppm). Meanwhile, the concentration of Aluminium, Iron, Boron, Zinc, Manganese, Lead and Cadmium insoils were ranged from 2.858 - 3.5ppm, 3.753 – 3.92 ppm, 3.753 – 3.92 ppm, 0.667 – 1.133 ppm, 0.253 – 0.653
ppm and 0.68 – 1.307 ppm, 0.072 – 0.181 ppm and 0.0283 – 0.0844 ppm respectively. The concentrations ofheavy metals in soils were also complied with the Indian Standard (Awashthi, 2000) and the European Union(EU) Standards (2002). In addition, the concentration of Aluminium, Iron, Boron, Zinc, Manganese, Lead andCadmium in vegetables ranged from 0.071 – 0.22 ppm, 1.28 – 2.76 ppm, 2.367 – 2.467 ppm, 0.273 – 0.32 ppm,
0.064 – 0.098 ppm, 0.0127 – 0.138 ppm and 0.0482 – 0.053 ppm respectively. All heavy metals analyzed in thevegetables also complied with the Indian Standard (Awashthi, 2000), World Health Organization (WHO)/FAO(2007) and EU Standards (2006).
Keywords: fruit vegetables, heavy metal, soil, ferum, manganese

Introduction
Contamination of the environment with toxic heavy metals has become one of the major
causes of concern for human kind. Heavy metals in surface water bodies, ground water and soils
can be either from natural or anthropogenic sources. The two basic categories of pollution areorganic and inorganic (Duke &Williams, 2008). Inorganic pollution is basically from heavy metals.
Chemical substances such as heavy metals are one of the factors which contribute toenvironmental pollution, and it was believed that it can disrupt living ecosystem (Kabata-Pendias &Pendias, 2001). Currently, anthropogenic inputs of metals exceed natural inputs due to increased
urbanization and industrialization. Industrial wastes, atmospheric deposition from crowded cities
and other domestic wastes are among the major sources of heavy metals in the surface water,
ground water and soils (Varalakshmi and Ganeshamurthy, 2010). On the other hand, heavy metals
such as Cadmium, Copper, Plumbum, Nickel, Chromium, and Mercury may be present in soil fromthe parent materials during soil formations (Plaster, 2003). Soil is a supporting layer for allorganisms in the world. The most important thing is soil acts as a medium for plant growth which
can recycle the nutrient and resources needed by plants. Soil will absorb heavy metals in thepolluted river as well as ground water and these will cause side effect for vegetable growth. As rootthProceedings of The 7 ACSA Conference 2011407

grows in the soil, it will absorb water and nutrients in solution (Plaster, 2003). Heavy metals that are
attached with soil water and soil particles will be absorbed by plant roots and accumulated invegetables (Alirzaveya et. al. 2006). Another patch way for heavy metals to get into vegetables is
via irrigation water which is contaminated by heavy metals.
This present study was aimed at finding out the levels of contamination of seven heavy
metals viz. Aluminium (Al), Boron (B), Cadmium (Cd), Iron (Fe), Lead (Pb), Manganese (Mn) andZinc (Zn) in irrigation water, soils and selected fruit vegetables in Kampung Badang, Pantai Cahaya
Bulan in Kota Bharu, Kelantan, Malaysia. A number of studies had been conducted by researchers
to identify the levels of heavy metals in vegetables from various sources. Anita et. al. (2010) stated
that, waste water irrigation led to the accumulation of heavy metals in soil and consequently into thevegetables, while, Miclean et. al. (2000) found out that the metal accumulation in vegetables grownin the vicinity of industrial sites represents a potential risk for public health. Works by Garcia et. al.
(1981) had shown that some common vegetables are capable of accumulating high levels of metals
from the soils.

Materials and methods
The study sites covered the agriculture area around Kampung Badang, Pantai Cahaya
Bulan, Kota Bharu, Kelantan, Malaysia (Figure 1). Kampung Badang, Pantai Cahaya Bulan, asuburban area located in the coastal area in the north west of Kota Bharu.

Kg Badang,PCB

Figure 1: Study Area.

Samples of the edible fruity vegetables were randomly collected from 5 x 5 m area of two
different fields. The fruit vegetables namely brinjal (Solanum melongena), chilli (Capsicum anmum)and lady‘s fingers (Hibiscus esculentus) were collected at fruiting stage or at least at 60 days after
planting. Five fruits were collected for each type of vegetables. The collected samples were storedin polyethylene bags, each sample bag for each vegetable before taking them to the laboratory forthe analysis. The soil that was used to grow the vegetables was randomly collected. Each location
comprised three samples; one sample was taken from each type of vegetable soils. In addition, ineach type of vegetable soils comprised three points, where these three point samples were mixed
as one sample with a total weight of about 50 grams. Therefore, a total of three soil samples werecollected for this study. A metal spade was used to collect the soil at 5 cm depth from the surface.
The samples were then placed in plastic bags, each sample for each bag before taking them to the

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laboratory for analysis. The source of irrigation water for this cropland was tap water from Kelantan
Water Berhad. Water samples used for irrigation were collected in a 500 ml washed polypropylene
bottle and 1 ml of concentrated acid nitric (HNO3) was added to the samples to avoid microbialactivity. For heavy metal extraction, 1g ground wet sample of plant or soil was digested in 3 ml
concentrated nitric acid (HNO3) and 1 ml of concentrated hydrochloric acid (HCl) (3+1, (v/v) ). The
mixtures were oven heated for 3 hours at 40 0C. After that, the samples were removed from theoven and let them cool for a few minutes. Then they were diluted with 150 ml de-ionized water. The
samples were then adjusted to pH 5 to 6 via acid base adjustment with hydrochloric acid andsodium hydroxide. Then, the samples were analyzed using Spectrophotometer DR5000.
Meanwhile, irrigation water analysis followed USEPA Approve for wastewater analyses andadapted from Standard Methods for the Examination of Water and Wastewater (2005) via
Spectrophotometer DR5000.

Results and discussion
The results obtained showed that in soil, the concentration of Aluminium ranged between
2.9 ppm and 3.5 ppm, where soil for growing chilli and brinjal recorded 3.5 ppm and soil for growing
lady‘s fingers recorded 2.9 ppm. The concentration of Boron was between 0.67 ppm and 1.13 ppm
and the highest recorded in soil for grow lady‘s fingers. Meanwhile, Cadmium concentration
recorded between 0.028 ppm and 0.084 and the highest rercorded in soil for growing chilli. In
addition, the concentration of Iron was between 3.75 ppm and 3.92 ppm where the highest
concentration recorded in soil for growing brinjal. The concentration of Plumbum was between
0.072 ppm and 0.181 ppm where the highest recorded in soil for growing chilli. Manganese andZinc were also measured for this study where the results showed that, the concentration ofManganese recorded was between 0.68 ppm and 1.31 ppm and Zinc recorded between 0.25 ppm
and 0.65 ppm (Figure 2, 3 and 4). All Pb, Cd and Zn values complied with the Indian Standard(Awashthi 2000) and European Union Standards, EU (2002). Generally, concentration of Iron was
the highest followed by Aluminium, Manganese, Boron, Zinc, Lead and Cadmium.
Among the heavy metals measured in three types of fruit vegetables, Boron concentration
was the highest (2.37 ppm – 2.47 ppm), where the highest concentration was recorded in brinjal
and Iron was the second highest (1.28 ppm – 2.76 ppm), and where the highest concentration wasrecorded in chilli. Meanwhile, the concentration of Cadmium ranged between 0.048 ppm and 0.053
ppm, where the highest concentration was recorded in brinjal. The concentration of Lead ranged
between 0.013 ppm and 0.138 ppm with brinjal that recorded the highest concentration. In addition,
the concentration of Zinc was ranged between 0.27 ppm and 0.32 ppm with the highest
concentration recorded in brinjal (Figures 2, 3 and 4). All Pb, Cd and Zn values complied with theIndian Standard (Awashthi 2000), WHO/FAO (2007) and Commission Regulation, EU (2006).
The concentrations of Aluminium, Boron, Cadmium, Iron, Lead, Manganese and Zinc in
irrigation water that was collected were 0.214 ppm, 3.5 ppm, 0.085 ppm, 0.233 ppm, 0.22 ppm,
1.105 ppm and 0.217 ppm respectively. Pb, and Zn values complied with the Indian Standard(Awashthi 2000), FAO (1985), and Class IV, Interim National Water Quality Standard for Malaysia(INWQS 1985) but not Cd. The concentration of Cd in irrigation water recorded 0.0853 ppm
exceeded the safe limits standard (0.01 ppm). The concentration of heavy metals in irrigation waterin sequence were B>Mn>Fe> Pb>Zn>Al>Cd.

thProceedings of The 7 ACSA Conference 2011409

Figure 2: Concentration of heavy metals in brinjal,
soil for brinjal and irrigation water.

Figure 3: Concentration of heavy metals in chilli, soil
for chilli and irrigation water.

Figure 4: Concentration of heavy metals in lady‘s fingers, soil for lady‘s fingers and irrigation water.

The results showed that, the trend of heavy metal concentrations in vegetables was similar(positive correlation) with the trend of heavy metals concentrations in irrigation water. On the other
hand, the trend of heavy metal concentrations in soil was also similar with the concentrations invegetables and irrigation water except for Aluminium (negative correlation). Meanwhile, highconcentrations of iron and manganese in soil was believed to be due to salt water intrusion (Figure
2,3 &4).

Conclusions
High concentrations of heavy metals in soil and irrigation water led to the accumulation ofheavy metals in vegetables. Heavy metal concentrations varied among the test vegetables, whichreflected the differences in their uptake capabilities and their further translocation to edible portionof the plants. The trend revealed that, high concentrations of Cd, Pb and Zn in soil and water led tohigh concentrations in vegetables, even though the concentrations in all vegetables were below thenational and international permissible limits. Irrigation water was a dominant factor to determine theconcentrations of heavy metals in vegetables compared to soil because irrigation water normally led
to the accumulation of heavy metals in soil and consequently into the vegetables. Based on the

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results, it showed that, Boron was found to be the highest in irrigation water as well as in vegetables
but not in soil. The lower concentrations of heavy metals in vegetables were believed to be due tothe good quality of irrigation water and absence of pollution in soil in the study area. It could besuggested that fruit vegetables in Kampung Badang, Pantai Cahaya Bulan were safe for
consumption.

Acknowledgements
We would like to express our deepest appreciation to the Dean of the Faculty of Agro Industry andNatural Resources of Universiti Malaysia Kelantan for his support and guidance. We are also grateful to the Vice
Chancellor of Universiti Malaysia Kelantan for his permission to publish this paper. The paper would not havebeen possible without the help from these individuals.

References
Alirzayeva, E. G., Shirvani, T. S., Yazici, M. A., Alverdiyeva, S. M., Shulurov, E. S., Ozturk, L., Ali-Zade, V. M.
and Cakmak, I. 2006. Heavy metal accumulation in Artemisia and foliaceous lichen species from the
Azerbaijan flora. Forest snow and landscape research. 80(3): 339-348.
Anita, S., Rajesh, K. S., Madhoolika, A. and Fiona, M. M. 2010. Risk assessment of heavy metal toxicity throughcontaminated vegetables from waste water irrigated area of Varanasi, India. Tropical Ecology. 51(2S):
375-387.
APHA (American Public Health Association). 2005. Standard Methods for the Examination of Water and Waste
Water. American Public Health Association, Washington, DC.
Awashthi, S.K. 2000. Prevention of Food Adulteration Act no 37 of 1954. Central and State Rules as Amended
for 1999, Ashoka Law House, New Delhi.
Committee on Food Hygiene. Houston, United States of America, ALINORM 07/30/13.
Duke, C.V.A. and Williams, C.D 2008. Soil Pollution. In: Chemistry for Environment and Earth Sciences. CRC
Press: Taylor and Francis Group.
European Union. 2002. Heavy Metals in Wastes, European Commission on Environment (http: //ec.e
uropa.eu/environment/waste/studies/pdf/heavy metals report.pdf ).
European Union. 2006. Commission regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum
levels for certain contaminants in foodstuffs. Official Journal of European Union L364/5.
FAO. 1985. Water Quality for Agriculture. Paper No. 29 (Rev. 1) UNESCO, Publication, Rome.
Garcia, W.J., Blessin, C.W., Inglett, G.E. and Kwolek, W.F. 1981. Metal Accumulation and Crop Yield for a
Variety of Edible Crops Grown in Diverse Soil Media Amended with Sewage Sludge. Environmental
Science and Technology. 15 (7): 793-804.
Interim National Water Quality Standard for Malaysia 1985. Malaysia: Department of Environment Malaysia.
Malaysia Environmental Quality Report, 2003. Interim National Water Quality Standards for Malaysia.
Department of Environment Malaysia.
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Plaster, E.J. 2003. Heavy Metals Contamination: Soil Sciences and Management. (4 ed.) Thomson Delmar
Learning.
Varalakshmi, L. R. and Ganeshamurthy, A. N., 2010. Heavy metals contamination of water bodies, soils andthvegetables in peri urban areas of Bangalore city of India. 19 . World Congress of Soil Science, Soil
Solutions for a Changing World. 1 – 6 August 2010. Brisbane, Australia.
WHO/FAO. 2007. Joint FAO/WHO Food Standard Programme Codex Alimentarius Commission 13th Session.
Report of the Thirty Eight Session of the Codex.

-- back to Table of Content --thProceedings of The 7 ACSA Conference 2011411

The Contribution of Agriculture in a Local Greenhouse Gas Turnover
Arief Sabdo Yuwono
Dept. of Civil and Environmental Engineering, Bogor Agricultural University (IPB)
PO Box 220 Bogor, Indonesia. Tel.: +62 251 8627 225
arief_sabdo_yuwono@yahoo.co.id

Abstract
The general objective of this study is to analyze the contribution of agricultural sector in a local scale
greenhouse gas turnover. It includes identification and quantification of the entire agricultural activities regarded
as greenhouse gas sources as well as those concerned as greenhouse gas sink. The study was carried out in2010 and based on a series of five years data compiled from a municipality in West Java Province as a case.
The greenhouse gas sources were categorized into four sectors covering energy sector, solid waste, agriculturaland animal husbandry sectors whereas the greenhouse gas sink was identified merely in agricultural sector. The
amount of a single greenhouse gas emission was calculated by multiplication between a source unit and its
corresponding emission factor. The total quantity of greenhouse gas emission generated by the municipality wassummation of the whole sources from the entire sectors. The amount of the whole greenhouse gas sink wassummation of the total sequestered carbon. The study result based on five years compiled data indicated thatthe average municipality greenhouse emission was in the order of 14.0 MT CO2-e per year whereas the net sink
was approximately 0.05 MT CO2-e per year. The contribution of agricultural sector was merely 0.08 % of thetotal municipality greenhouse gas sources while its contribution on the greenhouse gas sequestration (as sink)was 2.39% of the emitted greenhouse gases. It can be concluded that based on the quantitative analysis, thelocal agricultural sector is a manner to sequester atmospheric carbon.
Keywords: agricultural sector, greenhouse gas, source, sink, turnover.

Introduction
Global warming, or more properly, global climate change (GCC), may be the most serious
environmental challenge ever faced by mankind (Mackie and Cooper, 2009). There are many
uncertainties surrounding the issue of global warming. However, several things about thephenomenon are certain. It is known that CO2 and other greenhouse gases, such as CH4, absorb
infrared radiation by which earth loses heat. The levels of these gases have increased markedly
since about 1850 as nations have become industrialized and as forest lands and grasslands havebeen converted to agriculture (Manahan, 2000). Global warming phenomenon which is nowadays
concerned as a serious worldwide problem is essentially able to be mitigated locally based on thelocal governmental development program. The local governments can formulate action plans basedon their local greenhouse gas (GHG) mass balance and database. A local GHG turnover istherefore necessary to estimate contribution of each sector involved in emitting the GHG.
Greenhouse gases which deal with infrared-absorbing trace gases (other than water vapor) consistof carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O) and chlorofluorocarbons (CFCs). CFC
was excluded from this study case due to the lack of CFC distribution and consumption data. The
objectives of this study are to quantify the entire agricultural activities regarded as greenhouse gas
sources as well as those concerned as greenhouse gas sink and to analyze the contribution ofagricultural sector in a local scale greenhouse gas turnover.

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Materials and Methods
The study was carried out in 2010 and based on a series of five (5) years data compiled
from a municipality in West Java Province, called City of X, as a case. The greenhouse gas sourceswere categorized into four sectors covering energy sector, solid waste, agriculture and animalhusbandry sector whereas the greenhouse gas sink was identified merely in agricultural sector. The
amount of a single greenhouse gas emission was calculated by multiplication between a source unit
and its corresponding emission factor as indicated in Box 1. The total quantity of greenhouse gas
emission generated by the municipality was summation of the whole sources from the entire
sectors. The amount of the whole greenhouse gas sink was summation of the total sequesteredcarbon.
Box 1. General equation and the related emission factor for agricultural sector data source
CO2, CH4 and N2O emission from wetland or dryland paddy field:• Data source: Centre Agency for Statistics, City of X, 2005-2009• Emission factor: IPCC Guidelines for National Greenhouse Gas Inventories, 1996
• GHG mass = wetland area x plantation day x emission factor
Carbon sequestration by city re-greening program and estate crops plantation:• Data source: Centre Agency for Statistics, City of X, 2005-2009 and BPLH, City of X, 2009
• Emission factor: IPCC Guidelines for National GHG Inventories, 1996
• Carbon sequestration = area x emission factor

Greenhouse Gas Source and Sink in Agricultural Sector
Agricultural activities regarded as greenhouse gas sources and sink in The City of X
covers wetland paddy cultivation, dryland paddy cultivation, estate crops, and city re-greening
program. The amount of the absorbed carbon was estimated simply from the area of the paddy
cultivation. The similar case occurs in the estate crops as well. Therefore, the total amount of theabsorbed carbon by those plant species was calculated directly based on the area of the cultivation.
Contribution of Agricultural Sector in the Local Greenhouse Gas Turnover
The identified agricultural activities occurred in The City of X mentioned above arecompiled altogether as agricultural sector. The cultivated area by each of these activities was
identified and recorded by Centre Agency of Statistics, City of X. It was then compiled for five (5)years (2005-2009), in order to calculate the GHG emission quantity generated by agricultural
sector. Other sectors contributing greenhouse gases are energy sector, solid waste generation, andanimal husbandry. The contribution of energy sector on the total greenhouse gas production was
estimated by multiplying the amount of the energy source quantity by its corresponding emission
factor. It covers all fossil fuel consumption for the city electricity, transportation, and industry. The
amount of the generated solid waste is assumed in the order of 0.51 kg/capita/day (IPCC, 1966). By
using the total population data of the City of X, the total amount of methane emission can be
estimated. For the parameter nitrous oxide (N2O), the estimation method is almost the same as
those used in the CO2 and CH4 parameters, except for the emission factor. Global warming
potential (GWP) of CO2 is one, N2O is 21 whereas for CH4 is 310.

Results and Discussion
Estimation of the emitted CO2 from the wetland paddy field and dryland paddy field ispresented in Figure 1. It shows that in general there is tendency where the amount of emitted CO 2from the wetland paddy field is decreasing with the year. This is due to the ongoing encroachmentof the modern irrigated agricultural area that was converted to be industrial area and other landthProceedings of The 7 ACSA Conference 2011413

uses. In 2009 even, the total area of the wetland paddy field was less than a half of the total area in2005 meaning that the amount of the emitted greenhouse gas becomes less than a half as well.

(a)Figure 1. CO2 emission from wetland (a) and dryland (b) paddy field.

(b)

The amount of the sequestered GHG from dryland paddy field however, was notdecreasing in the period of 2005-2009. It showed that the CO2 sequestration from dryland paddy
field in 2009 was almost threefold of that in 2005. This was simply due to the growing area ofdryland paddy field in the City of X. The amount of methane emission from the wetland and dryland
paddy field is presented in Table 1. It revealed that according to the paddy field area, the quantity ofthe emitted methane varies directly with the total paddy area. The more paddy field area, the higher
amount of the methane emission, and vice versa. The generation of the methane in the wetland
paddy area is initiated by anaerobic condition of the inundated paddy field as it was commonly
practiced in conventional paddy cultivation system in Indonesia. As a result, methanogenic bacteria
become more active to generate methane in such kind condition. The relative contribution ofagricultural sector on total methane emission of The City X is presented in Figure 2. Agriculture
contributes merely 0.01 % of the total methane emission where the largest portion was contributedby domestic solid waste due to the characteristic of the City X as an urban area. Another main issueof an urban area is the scarcity of agricultural land. Therefore, total methane emission generated byagricultural land is consequently relatively small.
Table 1. CH4 emission from wetland paddy field
Area type

WetlandDrylandYear

Area (ha)

2005
2006
2007
2008
2009
2005
2006
2007
2008
2009

1959
1242
1364
1055
758
40
50
65
120
100

Plantation day(day/year)

Emission factor(kg/ha/day)

200
200
200
200
200
100
100
100
100
100

0.096
0.096
0.096
0.096
0.096
0.063
0.063
0.063
0.063
0.063

1
2

kg CH4

ton CO2-e

37,613
23,84626,18920,256
14,544
252.0
315.0
409.5
756.0
630.0

790
501
550
425
305
5
7
9
16
13

Data source: Centre Agency of Statistics, City of X (2005-2009)
1
IPCC Guidelines for National Greenhouse Gas Inventories, 1996
2
kgCH4 = area x plantation day x emission factor

It is estimated that approximately 5 to 20 percent methane produced and released into theatmosphere is a by-product of the anaerobic decomposition of waste. A significant source of thistype of methane production is solid waste disposal in landfills, where methanogenic bacteria break414

thProceedings of The 7 ACSA Conference 2011

down organic matter in the waste under anaerobic conditions to produce methane (Anonymous,2002). The landfilling of municipal solid waste is a significant source of atmospheric methane (CH4),
contributing to10–20% of anthropogenic methane emissions (IPCC, 2001 in Einola et al., 2008).
This is in line with the findings of Papageorgiou et al. (2009) stated that disposal of waste in landﬁlls
generates methane that has high global warming potential.
The third concerned parameter of GHG in agricultural sector is nitrous oxide (N2O) where
its theoretical emission quantity was determined by areas of the wetland and dryland paddy fieldthat were multiplied by their corresponding emission factors. Relative contribution of agriculture
sector on total nitrous oxide emission was very small, i.e. merely 0.1 percent. Agricultural sector
was not an important source of GHG from the viewpoint of the generated nitrous oxide (N2O).
Table 2. CH4 emission per sector expressed in ton CO2 equivalent (CO2-e)
SectorAverage (ton CO2-e)

Fossil fuel consumption
Domestic solid waste
Agriculture
Animal husbandry
CH4 Total emission

2,9E+05
8,2E+06
5,2E+02
6,6E+02
8,4E+06

Figure 2. Relative contribution of agricultural sector on total methane (CH4) emission.

Cumulative and yearly rate of carbon sequestration by means of city re-greening programis presented in Figure 3. The lowest part of the graph occurred in 2008 as a consequence of the regreening program that achieved 10.0 hectares merely whereas the average area of the re-greening
program normally was 23.8 hectares per year. In contrast however, the best success of the city regreening program was reached in the last year (2009) where The City of X showed theircommitment to reduce the greenhouse gas emission as re-greening area covered 41.2 hectares ina year.
Another way of carbon sequestration mode that was contributed by the city was throughestate crop plantation (Figure 3). The identified crops in The City of X were papaya, banana, durian,manioc, mango, corn, sweet potato, and others. Total area of the estate crop plantation was in therange of 3049-3849 hectares per year that is spread over the sub-city area. By opening more
plantation area, the amount of the captured atmospheric carbon dioxide would become higher.
According to de Nevers (1995), the only methods we now know to slow or stop the buildup of CO2 in
the atmosphere are to reduce the use of fossil fuels (gas, oil, coal, peat, lignite) and to stop thedeforestation of the tropical rain forests.

thProceedings of The 7 ACSA Conference 2011

415

(a)
(b)
Figure 3. Carbon sequestration by city re-greening program (a) and estate crop (b).

An important illustration of the emission profile of The City of X is an outline of the GHG
emission and the related carbon sequestration quantity. Analysis on the compiled data revealed that
agricultural sector plays a minor role in emitting greenhouse gas (GHG) as indicated in Table 3.
Percentage of the CO2 emission from agricultural sector was merely 0.17% whereas its contribution
on CH4 and N2O emission were only 0.01% and 0.06%, respectively. On the other side, however,agriculture contributes about 2.39% (Table 3) sequestration process of the emitted carbon dioxideback to the earth through photosynthesis process. It indicated that agriculture plays a significant
role in the greenhouse gas turnover in the local scale.
The quantity of the emitted methane (CH4) and nitrous oxide (N2O) were significantly
higher than the amount of emitted carbon dioxide (CO2). Such condition should be noticed carefully
since the warming effect of CH4 and N2O are much stronger than CO2. According to Jones (1992),
although increasing atmospheric CO2 is often assumed to be the major contributor to warming, thecombined effect of a number of trace gases, principally methane, nitrous oxide andchlorofluorocarbons (CFCs), though present at concentrations that are two to six orders ofmagnitude lower than CO2, can rival the effect of CO2 because, per molecule they absorb infrared
radiation much more strongly.
Table 3. Outline of the GHG emission of The City of X and the carbon sequestration
Emission/sequestration
Emission

Sequestration

416

GHG

SectorAverage quantity (2005-2009) [ton CO2-e]%

CO2

Paddy field
CO2 Total emission
CH4
Agriculture
CH4 Total emission
N2O
Agriculture
N2O Total emission
Grand total emission (source)
Average contribution of agric. sector
CO2
Sequestration by:

3.3E+03
2.0E+06
5.2E+02
8.4E+06
2.2E+03
3.6E+06
14E+06
2.0E+3

0.17
99.83
0.01
99.99
0.06
99.94
0.24
0.08

 Estate crops
 City re-greening program
Grand total sequestration (sink)

4.8E+04
1.6E+02
4.8E+04

2.38
0.01
2.39

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Proceedings of The 7 ACSA Conference 2011

Figure 4. Relative emission of The City of X based on GHG component (2005-2009).

More severe condition occurred in The City of X where GHG parameters of CH4 and N2O
were absolutely much higher than CO2 (Figure 4). As a consequence, the warming effect on theatmosphere would be much higher too. The main potential reason for such condition was that thepeople of The City of X generated solid wastes higher than the other Indonesian urban population.

References
Anonymous, 2002. Assessment of Greenhouse Gas Emissions from Solid Waste Disposal as a Result of the
Proposed New Zealand Waste Strategy. Ministry for the Environment. Reference Number 19518. New
Zealand.
de Nevers, N. 1995. Air Pollution Control Engineering. McGraw-Hill, Co. New York. Pp 506.
Einola, J.-KM., Sormunen, KM., Rintala, JA. 2008. Methane oxidation in a boreal climate in an experimental
landfill cover composed from mechanically–biologically treated waste. Sci.of the Total Environ. 407:6783.
ndJones, HG. 1992. Plants and Microclimate. A Quantitative Approach to Environmental Plant Physiology. 2 ed.
Cambridge University Press, Cambridge, UK. Pp 428.
Mackie, KR and Cooper, CD. 2009. Landfill gas emission prediction using Voronoi diagrams and importancesampling. Environ. Modeling and Software 24: 1223-1232.
th
Manahan, SE. 2000. Environmental Chemistry 7 ed. Lewis Publishers, Boca Raton, USA. Pp 898.
Papageorgiou, A., Barton, JR., and Karagiannidis, A. 2009. Assessment of the greenhouse effect impact oftechnologies used for energy recovery from municipal waste: A case for England. J. of Environ.
Management 90: 2999-3012.

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417

The Influence of Global Climate Indices on Rainfall Distribution Pattern and
Its Impact on Crop Yield in Gunung Kidul, Yogyakarta, Indonesia
Bayu DA Nugroho a,*, Hiromitsu Kannob, Ryoji Sameshima b, Hiroshi Fujii c ,Larry CM Lopezca

The United Graduate School of Agricultural Sciences, Iwate University, Japanb
National Agricultural Research Center for Tohoku Region, NARO, Japanc
Faculty of Agriculture, Yamagata University, Japan* Corresponding author: The United Graduate School of Agricultural Sciences, Ueda 3-18-34,
Morioka, Iwate, Japan 020-8550. Tel.: +81 19 643 3408; fax: +81 19 641 9296.
u0410004@iwate-u.ac.jp

Abstract
The correlation between the global climate index (SOI/SST), rainfall distribution and crop yield in rain-fed
highland areas was investigated. For this analysis, rainfall data were collected during the rainy season (OctoberMarch) for the period 1981 to 2009 in Gunung Kidul district of Yogyakarta Province, which is one of theimportant rain-fed crop producers in Indonesia. The rainfall distribution pattern found indicates that most of therainfall occurs in coastal areas, especially in the southern hilly areas, where the occurrence of the Southern
Oscillation Index (SOI) and Surface Sea Temperatures (SST) Niño.West are also highly correlated with rainfall.
Soybean yield is highly correlated with rainfall while dry land paddies, peanuts and wetland paddies yields show
a good correlation with SOI during January-February-March-April (JFMA).
Keywords: crop yield, rainfall, rain-fed area, SOI/SST

Introduction
Research on rainfall variability in Indonesia related to rain-fed agricultural areas is a criticalissue due to its relationship with El Niño and La Niña events. The delay of rainfall has a negative
effect on agricultural production in Indonesia. El Niño occurrences delayed rice harvesting inIndonesia and creates instability in food security (Harger, 1995). In 1997, rice farming in the subdistricts (Kecamatan) of Java decreased dramatically by 58% in wetland areas and 52% in dry land
areas (Irawan, 2002). The abnormal weather caused by El Niño events seriously disturbed crop
cultivation and the production of food and other commodities in the affected areas, mainly thoselocated in the rain-fed dependent highland areas.
Rain-fed highland areas are more affected by rainfall variability because of the topography
characteristics (Haylock and McBride, 2001). Rainfall variability in Indonesia correlates well with
global climate index, such as the Southern Oscillation Index (SOI) and the Sea Surface
Temperature (SST) pattern (Saji et al., 1999). At the same time, crop production in Indonesia is
influenced by rainfall variability and highland topography. Therefore, it is necessary to estimate
rainfall variability as influenced by the global climate indices (SOI and SST) in rain-fed highlandareas and its correlation with crop yields.
The aims of this study are to analyze the relationship between rainfall, global climate
indices SOI/SST and agricultural crops production based on the rainfall variability during the rainyseason from 1981-2009 in rain-fed highland areas.

Materials and Methods
Study Area
Gunung Kidul district is one of the rain-fed highland areas of Java Island, located between
7°46‘-7°09‘ S latitude and 110°21‘-110°50‘ E longitude. Based on elevation, Gunung Kidul district as

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reported by the Agricultural Service for Food Crops and Horticulture (ASFCH) is divided into threeareas of which the northern border ranges between 200-700 meters above the sea level (m); thecenter area is the lowland area with an elevation of 150-200 m, and the south area with a hilly
topography ranges from 0 to 300 m. A total of 92% of the agricultural land area in Gunung Kidul
district depends on rainfall, especially on October precipitation, as reported by the ASFCH (2006)
and the remaining 8% is irrigated. The mean annual rainfall in Gunung Kidul district is 2041
mm/year based on the record data from 1989-1998.
Secondary Data
Crops yield from 1990 to 2009 and rainfall data from 1981 to 2009 at twelve locations werecollected from the rainfall observation stations of ASFCH (2009). The SOI and SST data werecollected
from
the
Japan
Meteorology
Agency(JMA)
website(http://www.data.jma.go.jp/gmd/cpd/db/elnino/index/dattab.html) and were averaged in Niño3 (5°N5°S and 150°W-90°W), Niño.West (15°N-EQ and 130°E-150°E) and IOBW (20°N-20°S and 40°E100°E) areas, respectively. Spatial data of Gunung Kidul district with a scale of 1:25000 were takenfrom the National Coordinating Agency for Survey and Mapping (Bakosurtanal) of Indonesia (2007).
Rainfall data were the average of cumulative rainfall during the rainy season (from October
to March) from 1981 to 2009 at each station. Steps in the data analyses were as follows: first,
mapping of rainfall during the rainy season (6 months, Oct-Mar and then separately Oct-Dec (OND)
and Jan-Mar (JFM) from 1981-2009. Second, in order to analyze the distribution of rainfall in
Gunung Kidul district, a regression analysis was conducted between rainfall during the rainyseason, 6 months and 3 months (OND and JFM) from 1981 to 2009 and the averages of SOI/SST,
to identify the global climate indicators that influence rainfall. Third, correlation analysis was
conducted between the average of rainfall during the rainy season and crop yields, and finally,correlation analysis between the average of global climate indicators and crop yield during the rainyseason was conducted.
Correlation analysis between rainfall in the rainy season and crop yield, global climate
SOI/SST and crop yield was calculated only for the period 1990-2009 because of the data
availability on crop yield for this period.

Results and Discussion
Distribution of rainfall during the rainy season for the period 1981 to 2009
The analysis of rainfall distribution from 1981 to 2009 was carried out based on a six
months (Oct-Mar) and three months (OND and JFM) analysis. The distribution analysis shows thatin the southern coastal areas and in the inland western areas rainfall ranged from 1800-2200 mm,
while in the lowland central and northern areas rainfall was around 1500-1600 mm (Fig. 1). The
southern area is a mountainous area with a maximum elevation of 700 m. It caused moisture fromthe Indian Ocean carried by the wind and moving horizontally produces dense clouds along themountain ranges. These clouds cause rain in the upper region known as orographic precipitation(Roe, 2005), which is the reason why the windward side of the mountain range receives much more
precipitation than the leeward side.

th

Proceedings of The 7 ACSA Conference 2011

419

.

160
0

1800

1900

00
15

1800

1900

150
0

00
20

0
160
0
150

5

0

10 Kilometers

00
22

160
0

10

19
0
20 0
210 00
0

18
00

1700

Figure 1. Distribution rainfall map during rainy season in Gunung Kidul district 1981 – 2009.

Relationship between the averages of rainfall and SOI/SST during 1981-2009
In Correlation analysis between the averages of rainfall during the rainy season and theaverages of SOI and SST Niño.West in all sub districts, only three sub district, which has significant
correlation, Panggang (r = 0.58), Playen (r = 0.44) and Rongkop (r = 0.49) sub-districts in p<0.01
and p<0.05. Panggang and Rongkop are the southernmost sub districts in the Gunung Kidul district,which directly border with the Indian Ocean, whereas Playen sub district, located in the middle ofGunung Kidul district, is poorly influenced by the Indian Ocean. Therefore, the area along the coastshows a stronger correlation with SOI - SST Niño.West than the center area. The average rainfall
for the 9 other sub districts do not have significant correlations with the averages of SOI - SST
Niño.West, which are probably influenced by the mountainous terrain indirectly affecting thecorrelation of rainfall and SOI.
Relationship between average rainfall and crop yield
Correlation analyses between average rainfall and crop yield during JFMA (JanuaryFebruary-March-April) can be seen at Table 1.
Table 1. Correlations between the average of rainfall in rainy season and crops yield in JFMA
Dry land paddy

Maize

SoybeanPeanutWetland paddy

-0.15

-0.31

0.60**

-0.04

-0.21

Pearson correlation coefficient** Significant with p < 0.01

Based on Table 1, a strong correlation between average rainfall and crop yield during
JFMA is only for soybean yield. Therefore, from PS 2 farmers tend to grow soybean as the main
crop rather than dry land paddy in rain-fed highland areas. In the case of cassava yield, statisticalanalysis cannot be done during JFMA from 1990 until 2009, because it is not harvested during
these months. Cassava is an annual plant, which is well adapted to drought and is harvested
between July and August.
Relationship between the averages of SOI and crop yield
The relationships between the averages of SOI/SST and crop yield indicated that SOI is
correlated with crop yield (dry land paddy, r = 0.50; peanut, r = 0.55 and wetland paddy, r = 0.55),
whereas no correlation between SST Niño.West and crop yield was found (Table 2). Based on
Table 2, average SOI during JFMA corresponds well with dry land paddy, peanut and wetland

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paddy yields, and changes in the value of SOI influencing dry land paddy, peanuts and wetland
paddy yields.
Table 2. Correlation between average SOI and crops yield in JFMA
Dry land paddy

Maize

SoybeanPeanutWetland paddy

0.50*

0.28

0.25

0.55*

0.55*

Pearson correlation coefficient* Significant with p < 0.05

The usefulness of global climate index and rainfall pattern to determine suitability of crops
is needed to help farmers in their decision taking, especially in rain-fed highland areas. Our results
indicate that averages of SOI and SST Niño.West are correlated with rainfall, especially in theseaside southern hills areas.
In general, increases in the average SOI/SST Niño.West will influence the amount of
rainfall, and directly influence the cropping pattern. Our study found that increasing rainfall during
the rainy season (JFMA) from 1990 to 2009 increased soybean yield. Different results show that
averages of SOI corresponded well with dry land paddy, peanut and wetland paddy yield during
JFMA. Increases of SOI averages in JFMA by +1 will cause the increase of dry land paddy yield
during JFMA periods. The same trend can be observed for peanut and wetland paddy yield, in
JFMA.
Overall, our results show that most of the crops yield trend during the rainy season does
not correspond to global climate indices and rainfall. The main reason may be local cropping
patterns that still dominate regardless of the rainfall pattern and global climate index. Further
analysis of other rain-fed highland areas, which are different in elevation and in micro-scale (village)
by including crop yield data, is necessary.

References
Agricultural Service for Food Crops and Horticulture (ASFCH), GunungKidul district. 2006. Profil Tanaman
Pangan Kabupaten GunungKidul (In Indonesian). Pp 5 –34.
Agricultural Service for Food Crops and Horticulture (ASFCH), GunungKidul district. 2009. Rainfall database in
Gunung Kidul district. Pp 10-130.
Harger, J.R.E. 1995. Air-temperature variations and ENSO effects in Indonesia, the Phillipines and El Savador:
ENSO Patterns and Changes from 1866-1993. Atmos. Environ 29: 1919-1942.
Haylock, M. and McBride J. 2001. Spatial coherence and predictability of Indonesian wet season rainfall. J.
Climate 14: 3882-3887.
Irawan, B. 2002 Stabilization of upland agriculture under El Nino-Induced climatic risk: Impact Assessment and
Mitigation Measure in Indonesia, Economics and Social Commission for Asia and the Pacific, CGPRTCenter WORKING PAPER No. 62.
Japan Meteorological Agency, 2010 El Nino Index data, retrieved from the statistical climate service at
http://www.data.jma.go.jp/gmd/cpd/db/elnino/index/dattab.html
National Coordinating Agency for Survey and Mapping (Bakosurtanal) Republic of Indonesia. 2007 Spatial data
for Gunung Kidul district at scale 1: 25000.
Roe,G.H. 2005. Orographic Precipitation. Annu. Rev. Earth Planet. Sci 33: 645-71.
Saji,N.H., Goswami, B.N.,Vinayachandran, P.N. & Yamagata,T. 1999. A dipole mode in the tropical Indian
Ocean. Nature 401: 360-363.

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An Input-Output Analysis of the Economic Impact for Sustainable Rural
Development in Wonogiri District, Indonesia
Shintaro Kobayashi, Shigeki Yokoyama*, Satoshi Uchida, Yoshiko Iizumi
Japan International Research Center for Agricultural Scineces, Tsukuba, Japan*Corresponding author: Social Science Division, Japan International Research Center for Agricultural Scineces,
1-1 Ohwashi, Tsukuba, 305-8686, Japan.
Tel.: +81 29 838 6343; fax: +81 29 838 6342.
syokoyam@affrc.go.jp

Abstract
Wonogiri district located in Central Java is the water source of Solo River. There is a large multi-purpose
reservoir in the region. However, the reservoir is reported to be losing its function due to rapid sedimentation
caused by forest conversion and intensive farming. For sustainable resource management, assessment ofdevelopment strategies incorporating alternative agro-forest systems is required. This study, as a preparation for
the assessment of development strategies, was aimed to grasp regional industrial structure, using the inputoutput analysis. The results showed that the importance of agriculture in the region was both as an income
source especially for unskilled labor and as financial base for purchasing manufactured goods. Therefore,
environmentally sound agro-forest systems need to be proposed so that strict environmental restriction on
agriculture can be avoided.
Keywords: sustainable rural development, input-output analysis, Wonogiri.

Introduction
Wonogiri district is located in Central Java, Indonesia and is the water source of Solo River(Figure 1). There is a large multi-purpose reservoir in the region, and it has contributed to both flood
control and water utilization. However, the reservoir is reported to be losing its functions as a public
reservoir due to extremely rapid sedimentation. It is said that main reasons for the rapid
sedimentation is forest conversion and intensive farming induced by regional population increase(JICA, 2007). In order to achieve sustainable resource management in the region, assessment ofdevelopment strategies incorporating alternative agro-forest systems is required. This study, as apreparation for the assessment of development strategies, was aimed to grasp the regional
industrial structure and income multiplier effects by sector, using the input-output analysis, aneconomic analysis method.

Figure 1. Location of Wonogiri District.

thProceedings of The 7 ACSA Conference 2011423

Materials and Methods
The input-output analysis was proposed by W. Leontief (e.g. Leontief, 1986) and it wasused for projections of impacts of changes in production technology, social environment, consumer
preference, or economic policy on an economy. Based on economic statistics or economic survey,
simulation models called ―input-output model‖ were formulated. Simplified flow of the analysis was
as follows. Firstly, an input-output table which described all transactions among economic sectors
was estimated (Figure 2). Secondly, production technologies and traits of transactions wereabstracted from the table as the form of parameters called "input coefficients" (Figure 3). Lastly,using the input coefficients, transactions between demand sectors and production sectors andamong production sectors were formulated to produce simultaneous equation for simulation (Figure4). Based on the statistics of Wonogiri district, we estimated an input-output table. Tables 1, 2, and3 show sector classification of the estimated table.

DemandersIntermediate demand by producers

Total
OutputFinal demand

manufacturing services Consumer Government Investor
agriculture mining
Export
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
agriculture
200
3000
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
960
50
0
Suppliers mining(Producers) manufacturing
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
100
1000
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
services
10
2000
nnnnn
nnnnn
nnnnn
wage
400
Household
nnnnn
nnnnn
nnnnn
nnnnn : Monetary value of annual transaction
profit
200
nnnnn
nnnnn
nnnnn
: How much "mining" bought
Total Input
960: People's income from "mining": How much people consumed

Figure 2. A sample structure of an input-output table. The table is estimated based on economic statistics or
economic survey of a target region. Row vectors in the table show the amounts of supply valued atmarket prices. Column vectors show the purchase of goods and services.

Demander (Producers)
manufacturing services
agriculture mining
agriculture
nnn/NNN 200/960 nnn/NNN nnn/NNN
nnn/NNN
50/960 nnn/NNN nnn/NNN
Suppliers mining(Producers) manufacturing nnn/NNN 100/960 nnn/NNN nnn/NNN
nnn/NNN
services
10/960 nnn/NNN nnn/NNN

input coefficient matrix A
Figure 3. Abstraction of information on production technology as the form of parameters called "input coefficient" A.

DemandersIntermediate demand by producers

Final demand

Total
Outputmanufacturing services Consumer Government Investor
agriculture mining
Export
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
agriculture
200
3000
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
960
50
0
Suppliers mining(Producers) manufacturing
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
100
1000
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
nnnnn
services
10
2000
nnnnn
nnnnn
nnnnn
wage
400
Household
nnnnn
nnnnn
nnnnn
nnnnn : Monetary value of annual transaction
profit
200
nnnnn
nnnnn
nnnnn
: How much "mining" bought
Total Input
960: People's income from "mining": How much people consumed

A X  Y  X

Figure 4. Formulation of technology A, production X, and demand Y in an economy.

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Table 1. Production sectors
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19

Farm crops
Livestock and products
Forestry
Fishery
Mining and quarrying
Oil and gas manufacture
Other manufacture
Electricity supply
Gas supply
Water supply
Construction
Trade, hotel, and restaurant
Road transport
Other transport
Communication
Financial services
Real estate and business services
Public administration and social services
Personal and household services

Table 2. Demand sectors

D1
D2
D3
D4

Private consumption
Private investment
Governmental consumption
Public investment

Table 3. Value added sectors to supply primary factors to production

V1
V2
V3
V4
V5
V6

Land
Unskilled labor
Skilled labor
CapitalNatural capitalIndirect tax

In this paper, we drew skyline chart and derive income multipliers of economic activites.
Skyline chart revealed trade characteristics of target economy. Drawing method was as follows. An
input-output table could be expressed as follows:
Eq. 1
X  A X  F  E  M
where X is a column vector of total output, A is ―input coefficient matrix‖, F is a column vector ofdomestic final demand, E is an export column vector, and M is an import column vector. Solving
Eq. 1, X can be decomposed into 3 inducing factors as follows:X  ( I  A) 1  F  ( I  A) 1  E  ( I  A) 1  M

Eq. 2

where the first term of right-hand side indicates the production induced by domestic final demand F,
the second term indicated the production induced by export E, and the last term indicated thepotential domestic production to be substituted for import M. Based on Eq. 2, a skyline chart showseach factor‘s relative contributions to production on the vertical scale, with 100% was representing( I  A) 1  F . In addition, the horizontal scale showed the sectoral share in total production.

thProceedings of The 7 ACSA Conference 2011425

Income multipliers could be obtained by the following procedures. Assuming that import
was in proportion to domestic demand, Eq. 1 could be transformed as follows:~
X  A X  F  E  M (A X  F)

Eq. 3

where M~ is a diagonal matrix of import coefficients. Solving Eq. 3 for X, we obtain the following
equation.



 

1
~
~
X  I  (I  M ) A  (I  M )F  E





Eq. 4



1
~
where I  ( I  M) A is called ―Leontief inverse matrix‖ and includes information on the industrial

structure of an economy. Supposing that V~ is a diagonal matrix of value added ratio to production
1~
cost, income multiplier was column sums of a matrix V~ I  ( I  M
)A .





Results and Discussion
Based on an input-output table of Wonogiri district estimated for year 2005, we analyze
current situation of Wonogiri district. Major findings were as follows:(i) Farm crop sector was the largest income source and accounts for 46% of regional grossdomestic products (Figure 5). This implied that environmental restriction on agriculturalactivities would affect the standard of living in the region. Therefore, environmentally soundagricultural systems including agro-forestry need to be proposed so that strict environmental
restriction can be avoided.

Publicadministration
11%

Road
transport 10%
Trade, hotel,
restaurant
13%

Farm crops
46%

Figure 5. Share of income sources.

(ii)

The district exported substantial amount of Farm crops and imports manufactured products(Figure 6). This means that the district participates in a regional division of labor and aninterregional trade system. High self-sufficiency of Road transport seems to reflect the active
transportation of products including Farm crops.
(iii) Agricultural sectors had tendency to strongly induce income of unskilled labor. Trade, hotel,
and restaurant sector had also high value in income multiplier for unskilled labor (Table 4).
These facts implied that integrated activities consisting of agriculture and services such as
ecotourism was promising for pro-poor development.
This multiplier showed how much income of unskilled labor was induced by one unit of final demandfor each sector.

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20%

Self-Sufficiency

40%

60%

Import

80%

600%
550%
500%

450%
400%
350%
300%

Road
transport

Farm crops

250%
200%
150%
100%

Manufacture

Trade, hotel,
restaurant

50%

Publicadmin.

0%

Personal services

Public administration

Business services

Financial services

Communication

Other transport

Road transport

Trade, hotel, andConstruction

Water supply

Gas supply

Electricity supply

Other manufacture

Oil and gas manufacture

Mining and quarrying

Fishery

Forestry

Livestock and products

Farm crops

Figure 6. Skyline chart. Horizontal axis shows share of production and vertical axis shows self-sufficiency rate.
Table 4. Income multiplier for unskilled labor

Trade, hotel, and restaurant
Farm crops
Forestry
Personal and household services
Livestock and products
Public administration and social services
Road transport
Fishery
Construction
Other manufacture
Financial services
Communication
Mining and quarrying
Water supply
Electricity supply
Real estate and business services

0.389
0.388
0.324
0.321
0.310
0.285
0.270
0.227
0.218
0.174
0.155
0.152
0.148
0.137
0.077
0.054

Conclusions
Income source share and the skyline chart showed the importance of agriculture both as
an income source and financial base for purchasing manufactured goods. Therefore, environmental
policies to restrict agricultural activities would affect the standard of living. In order to avoid reducing
income level in the region, environmentally sound agro-forest systems need to be proposed.
Income multiplier implied that ―Trade, hotel, and restaurant‖, as well as agricultural sectors, had astrong power to induce income of unskilled labor. Therefore, integrated activities consisting ofagriculture and services such as ecotourism were promising for pro-poor development. The
remaining issues were crop-wise input-output analysis for decomposing Farm crop sector,
backward and forward linkage among sectors, and simulation analysis on income generation effects
of proposed community business models.

Acknowledgement
This work was supported by JSPS Grants-in-Aid for Scientific Research (A) 22255012.
thProceedings of The 7 ACSA Conference 2011427

References
JICA (Japan International Cooperation Agency). 2007. The Study on Countermeasures for Sedimentation in the
Wonogiri Multipurpose Dam Reservoir in the Republic of Indonesia. Japan International Cooperation
Agency.
nd
Leontief, W. 1986. Input-Output Economics, 2 . ed., Oxford Univ. Press.

-- back to Table of Content --

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Permutation Test in Evaluating the Significance of Plants in PLS-DA Modelof Jamu Ingredients
Farit Mochamad Afendi a, b*, Md. Altaf-Ul-Amin a, and Shigehiko Kanaya aaGraduate School of Information Science, Nara Institute of Science and Technology, Nara, Japanb
Department of Statistics, Bogor Agricultural University, Bogor, Indonesia* Corresponding author: Graduate School of Information Science, Nara Institute of Science and Technology,
Ikoma, Nara 630-0101, Japan.
Tel.: +81-743-72-5952; fax: +81-743-72-5953.
farit-a@is.naist.jp

Abstract
PLS-DA (Partial Least Square Discriminant Analysis) model is used to establish relationship between Jamu
ingredients, i.e. plants composition in Jamu, and Jamu efficacy to investigate which plants act as main
ingredients and which are supporting by checking the plants significance. Permutation testing is used in theinvestigation by generating the coefficients distribution under null hypothesis, i.e. the plants are not affecting
Jamu efficacy. The generation process is performed by permuting the order of the response while maintaining
the order of the predictors. The PLS-DA model then is applied to the new dataset after permutation. After
repeating this process many times, then the accumulation of the PLS-DA coefficients provides the distribution
under the null hypothesis. The proportion of the coefficients larger than or equal to the PLS-DA coefficient using
original data then serves as the p-value, which then can be compared to the significance level . By performing
this permutation process 1000 times and  = 5%, we found, over all efficacies, 231 out of 465 plants are
significant. Moreover, from literature review, among these 231 plants, the usages of 226 plants on the assigned
efficacy are supported by scientific paper.
Keywords: jamu, PLS-DA, permutation test

Introduction
Jamu is an Indonesian herbal medicine made from a mixture of several plants. So, theingredients, i.e. plants composition used, in Jamu determine the Jamu efficacy. Among theingredients of Jamu are plants used as main ingredients, which contribute primarily to its efficacy,
as well as plants used as supporting ingredients (Pramono, 2007; Redaksi Trubus, 2009).
Investigating which plants are main ingredients and which are supporting is important tocomprehensively understand the mechanism of plants used in Jamu to achieve specific efficacies.
A statistical model can be helpful in this attempt by relating plants usage in Jamu as predictors andJamu efficacy as response. Plants perform as main ingredients will have significant effect on themodel developed.
PLS-DA, a statistical model for classification and discrimination that based on Partial Least
Square Regression (PLSR) in which the dependent variable is chosen to represent classmembership (Barker and Rayens, 2003), is suitable for this analysis regarding that large number ofplants are used in Jamu and; on the other hand, Jamu efficacy are in categorical scale. Hence, the
PLS-DA coefficients can be used to explore the relationship between plants and Jamu efficacy.
Furthermore, the absence of significance testing on PLS-DA coefficients is replaced by permutation
testing, which generated the coefficients distribution under null hypothesis through resampling of
the existing data (Good, 2005).

thProceedings of The 7 ACSA Conference 2011429

Materials and Methods
DatasetThe data used in the present study are the commercial Jamu registered at The National
Agency for Drug and Food Control (NA-DFC) of Indonesia. The data contain 3138 Jamu and in total
they use 465 plants. Each Jamu is classified into one of nine efficacy categories, namely: (1) urinary
related problems (URI), (2) disorders of appetite (DOA), (3) disorders of mood and behavior (DMB),(4) gastrointestinal disorders (GST), (4) female reproductive organ problems (FML), (6)musculoskeletal and connective tissue disorders (MSC), (7) pain/inflammation (PIN), (8) respiratory
disease (RSP), and (9) wounds and skin infections (WND).
In the present study, the plants usage in each Jamu provides the predictors in PLS-DA
model while the Jamu efficacy serves as the responses. Let Xij (i = 1, 2, …, n; j = 1, 2, …, m; n =3138; m = 465) denotes the status of plant j usage in Jamu i, where Xij = 1 if Jamu i use plant j and0 otherwise, and let Yik (k = 1, 2, …, 9) denotes the efficacy status of Jamu i, where Yik = 1 if Jamu i
has efficacy in category k and 0 otherwise. Note that k Yik = 1 meaning that each Jamu isclassified into one efficacy category only.
PLS-DA Modeling
The details of the PLS-DA modeling are as follows (Barker and Rayens, 2003; Wold et al,2001). Let T (n x c) is a matrix of the underlying factors of X and is obtained by maximizing its
covariance with the corresponding matrix of the underlying factors of Y, that is
T = XW(1)
where W (m x c) is a matrix of weight, and c is the number of factors extracted. Matrix T, multiplied
by matrix of X–loadings P (m x c), is a good summaries of X
X = TPt + E(2)
so that the X–residuals E (n x m) is small. In addition, matrix T also a good predictors of Y
Y = TQt + F(3)
where Q (9 x c) is matrix of Y–loadings. The Y–residuals F (n x 9) express the deviation between
the observed and the predicted responses.
Substituting Eq. (1) into Eq. (3) we obtain multiple regression model of PLSDA
Y = XWQt + F = XB + F(4)where the PLS-DA coefficient matrix B (m x 9) is calculated as
B = WQt(5)
Note that each plant has a set of coefficient containing 9 values, one for each efficacy.
Permutation Test in PLS-DA Model
Permutation testing is a resampling method intended to provide underlying distribution oftest statistic under null hypothesis, which then can be used to calculate p value. Unlike theconventional statistical testing, which assuming the null distribution follows some theoretical
distribution, the permutation testing provides the null distribution by an empirical distribution which
generated through resampling of the data sample at hand. The idea of permutation testing in PLSDA is illustrated in Figure 3. The details of the steps are as follows.
Step 1. Resampling of Jamu data.
In this step, new Jamu data set under null hypothesis, i.e. plants are not affecting the Jamu
efficacy, is generated by resampling the existing Jamu data set. The resampling process isperformed by permuting the order of the response randomly while maintaining the order ofthe predictors. This process ensures the relationship between the predictors and the
response, if any, in the original Jamu data set is destroyed. Thus, the result from thisresampling is the new Jamu data set under null hypothesis. Let
denotes the new
response obtained from the permutation process.
Step 2. PLS-DA modeling on the new jamu data set.

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Proceedings of The 7 ACSA Conference 2011

PLS-DA model is performed on the new Jamu data set obtained from Step 1. The matrix X
and provide the predictors and responses, respectively. The coefficient matrix obtained
is denoted by .
Step 3. Accumulation of PLS-DA coefficients
After all permutation rounds R (in the present study we performed R = 1000 times), the
PLS-DA coefficient in each round r is accumulated into coefficient distribution C, which isthe distribution of PLS-DA coefficient under null hypothesis. Let Cjk,r denotes the coefficientof plant j on efficacy k at permutation round r.
After the distribution of PLS-DA coefficient under null hypothesis is obtained, the p value intesting the effect of plant j on efficacy k is calculated as the following. Note that the hypothesis to betested is.
H0: bjk  0Thus, the p value is calculated asvsH1: bjk > 0.
(6)

where

is an identity function that equal to 1 if the argument is fulfilled and equal to0 otherwise. Basically, the p value is the proportion of the coefficients larger than or equal to the
PLS-DA coefficient using original data. The null hypothesis is rejected if the p value is smaller than
the significance level .
Determination of iteration number R

r=0

Step 1:Creating new dataset by permuting the order of the response while
maintaining the order of the predictors

Step 2:a. PLS-DA modeling on new dataset
b. Calculating PLS-DA coefficients

Increment of r

Yes

rR
No

Step 3:

Accumulation of PLS-DA coefficients

Figure 3. The schematic diagram of the permutation steps used in the present study.

thProceedings of The 7 ACSA Conference 2011431

Results and Discussion
In the present study, in selecting the number of components used in PLS-DA, we
performed 5-folds cross validation as follows. The Jamu data set are splitted randomly into five sets.
In each round, we combined four sets as training set and the other one as testing set. We apply
PLS-DA with number of components q on training set and use the model to predict the responses of
the testing set. The PLS-DA is applied once more by selecting another four sets as training set andthe other one as testing set. After each set is once selected as testing set, the whole process isrepeated using number of components q + 1.
Prediction Error Sum of Square (PRESS) using number of components q for efficacy groupk is calculated as:

wheredenotes the prediction of the efficacy k for Jamu i using the PLS-DA modelobtained without Jamu i, i.e. i is within the testing data, and using number of components q. The
plot of this statistic against number of component q is shown in Figure 4. The plots are almost
constant starting from q = 10 onward for all nine efficacy groups. Thus, the number of componentsof PLS-DA model for the original Jamu data set (and also for all 1000 permutation rounds) is set to
10. Analyzing PLSDA using 10 components on the original Jamu data set, we obtain the percent
variation accounted for predictors and responses are equal to 5.5% and 40.5%, respectively.

Figure 4. PRESS plot of 5–fold cross–validation.

Figure 5 gives illustration of the coefficient distribution under null hypothesis obtained fromthe permutation process. In this illustration, both plants are evaluated due to their usage for Jamu
that useful for efficacy URI. The mean of the two distributions (and also for all other distributions)are very close to 0, as expected, indicating that the distributions are generated under null
hypothesis. The normal curves were sketched on both distributions to show that not all permutation
distributions can be approached with normal distribution. This result supports the p value calculation
using empirical distribution as formulated in Eq (6). Using significance level  = 5%, we canconclude that Phellodendron chinense is significantly affecting the Jamu efficacy URI, whereas
Foeniculum vulgare is not.

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Proceedings of The 7 ACSA Conference 2011

The results of the significancy testing of all plants used in each efficacy are shown in Table
1. Note that one plant may be used for more than one efficacy. From the testing, we observed 234
plants (50.3% among all 465 plants) showing no significant status for all 9 efficacies; whereas theother 231 plants have significant status which comprise of 189 plants (40.6%) are significant only
for 1 efficacy, 38 plants (8.2%) are significant for 2 efficacies, and the other 4 plants (0.9%) aresignificant for 3 efficacies.
Besides testing the plants usage statistically, furthermore, we also check from scientificpaper the usage of significant plants on their corresponding efficacy. The results are shown in Table
1. We obtained that most of the testing results are supported by scientific paper.

Figure 5. Illustration of the coefficient distribution under null hypothesis obtained using permutation process.
Table 1. The number of significant plants resulting from permutation testing and their support status fromscientific papers

Efficacy

Significant plants

Number of plants used in thecorresponding efficacy

Total

80
148
47
290
182
270
183
105
120

20
21
12
26
40
40
39
36
43

URI
DOA
DMB
GST
FML
MSC
PIN
RSP
WND

Support from scientific paper
19
21
11
26
39
39
38
35
43

(95.0%)(100.0%)(91.7%)(100.0%)(97.5%)(97.5%)(97.4%)(97.2%)(100.0%)Conclusion
PLS-DA model is used to establish relationship between plants composition in Jamu andJamu efficacy to investigate which plants act as main ingredients and which are supporting by
checking the plants significance. Permutation testing is used in the testing by generating thecoefficients distribution under null hypothesis from which p value is calculated and then comparedwith significance level . By performing this permutation process 1000 times and  = 5%, we found,thProceedings of The 7 ACSA Conference 2011433

over all efficacies, 231 out of 465 plants are significant. Moreover, from literature review, among
these 231 plants, the usages of 226 plants on the assigned efficacy are supported by scientificpaper.

References
Barker M, and W Rayens. 2003 Partial least squares for discrimination. J. Chemometrics. 17: 166-173
rd
Good PI. 2005. Permutation, Parametric and Bootstrap Tests of Hypotheses, 3 ed. Springer, New YorkPramono S. 2007. Jamu in Indonesian Daily Life and Industry. Institute of Natural Medicine, University of
Toyama, Toyama
Redaksi Trubus. 2009. Herbal Indonesia Berkhasiat: Bukti Ilmiah dan Cara Racik. PT Trubus Swadaya, Depok
Wold S, M Sjöström, and L Eriksson. 2001. PLS-regression: a basic tool of chemometrics. Chemometrics andIntelligent Laboratory Systems, 58: 109-130-- back to Table of Content --

434

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Proceedings of The 7 ACSA Conference 2011

Index of Authors

A Junaedi, 175
Abdul Munif, 315
Ade Noverta, 380
Agus Rachmat, 91
Agus Sutanto, 291
Ahmad Denian, 380
Ahmad Ghozi Mansyuri, 225
Akihiko Kamoshita, 19
Akihiro Fujii, 239
Akihiro Kishikawa, 235
Akiho Yokota, 1, 296, 401
Akira Miyazaki, 107
Akira Yamauchi, 51,153, 334
Alhassan I Zakaria, 29
Ali Asgar, 249, 269
Alia Astuti, 220
Amol Dahal, 56
Anischan Gani, 72
Arief Sabdo Yuwono, 410
Aries Kusumawati, 225
Asadi, 180, 209
Asma Sembiring, 274
Asuka Yamano, 343
Atsushi Kato, 296
Atsushi Nakagawa, 14Atsushi Yoshimura, 34
Azmi Dhalimi, 380
Baiq Nurul Hidayah, 282
Bambang Pujiasmanto, 324
Bambang Sapto Purwoko, 39, 45, 80, 85
Bayu D A Nugroho, 416
Bernadetta Rina Hastilestari, 91
Boonrat Jongdee, 61
Candra Catur Nugroho, 141
Catur Hermanto, 291
Chairunas, 72
Chiharu Hongo, 96
Chiharu Sone, 24
Chihiro Shimada, 14
Chika Nakashima, 14
Chojiro Kojima, 14
D Sagala, 175
Darman M Arsyad 180, 209
Darmono Taniwiryono, 389
Deni D Sulaeman, 80

thProceedings of The 7 ACSA Conference 2011

Desta Wirnas, 80, 85
Dewi Indriyani Roslim, 130
Dewi Sukma, 291
Dhika Amanda, 353
Dina RR, 287
Dwi Praptomo Sudjatmiko, 282
Eiji Tanesaka, 235
Elis N Herliyana, 389
Elizabeth Caroline Situmorang, 370, 358
Elly Kesumawati, 66
Ence Darmo Jaya Supena, 220
Endang Yuniastuti, 244
ER Aweng, 405
Eri Sofiari, 274
Eriko Hayashi, 153
Erita Hayati, 66
Ernawati Giri R, 287
Fadlilla Dewi Rakhmawaty, 13
Farit Mochamad Afendi, 427
Futoshi Kato, 125, 252, 262
Futoshi Sasaki, 125, 252
Gunardi Sigit, 96
H Aswidinnoor, 39
Hai Yan, 196
Hamda Fauza, 380
Handarto, 96
Happy Widiastuti, 364, 375
Haruka Yamaguchi, 215
Hayati Minarsih, 389
Hayuru Yoshida, 279
Heni Purnamawati, 225
Heni Safitri, 80, 85
Hiroaki Shirakawa, 96
Hiroki Iwamoto, 96
Hiroki Suto, 279
Hiromitsu Kanno, 416
Hiroshi Ehara, 5, 159
Hiroshi Fujii, 416
Hirotake Itoh, 230
Hiroyuki Tsuji, 14
Hitoshi Naito, 5, 159
Hitoshi Sato, 230
Hozumi Yoshida, 230
Ima MZ, 287
Indrastuti A Rumanti, 39435

Indrawan A, 385
Irdika Mansur, 385
Irfan K Putra, 389
Irfan Martiansyah, 130
Iskandar Lubis, 225
Istino Ferita, 380
Iswari S Dewi, 39, 45, 80, 85
Izuru Ohki, 14
J Nulik, Nurhayati, 331
Jaladet MS Jubrael, 395
Jamsari, 380
Jing Ju, 107
Jonathan M Niones, 51
Jose MA Ornai, 85
Julie M Warren, 282
Jun-Ichi Sakagami, 24, 29
Kalimatul Jumro, 185
Kazuo Oki, 96
Kei Kajisa, 119
Keiichi Hayashi, 29
Kei-ichiro Maeda, 334
Keisuke Katsura, 191, 205
Keita Nabeya, 165
Keitaro Watanabe, 76
Ken-ichiro Taoka, 14
Kentaro Kawaguchi, 147
Khairun Nisa, 72
Kinasih Prayuni, 91
Kinya Akashi, 296
Kiyoshi Fujii, 153
Ko Shimamoto, 14
Kohtaro Isek,i 61
Koki Homma, 61, 96, 101, 191
Kokoro Hayashi, 14
Koshi Yoshida, 96
Koutarou Mori, 343
Kyoko Furuita, 14
Kyoko Takahashi, 279
L Abdullah, 331
Lailatul B, 287
Larry C Purcell, 191
Larry CM Lopez, 416
Lia Hadiawati, 282
Lizah Khairani, 339
M Ghulamahdi, 175
M Karimah, 405
Maya Melati, 175 185
Makara Ouk, 19
Makie Kokubun, 196

Malem McLeod, 72
Manabu Watanabe, 165
Mari Iwaya-Inoue, 215
Mariko Nakagawa, 348
Marlin Sefrila, 137
Masao Sato, 34
Masayasu Maki, 96
Masayuki Murai, 56
Mathias Fosu, 29
Md. Altaf-Ul-Amin, 427
Michihiko Fujii, 113
Michio Kawasaki, 279
Midori Yamaguchi, 14
Miftahudin, 130
Mika Yoshinaga, 334
Mikio Sekinuma, 125, 252
Mitsuo Kambayashi, 96
Mitsuo Kuwada, 101
Mitsuo Saito, 239
Mochamad Arief Soleh, 196
Morio Matsuzaki, 305
Motonobu Yoshida, 235
Muhafaz Zulusfitri, 66
Muhamad Syukur, 80
Muhammad Syafii, 45
Munandar, 137
Munif Ghulamahdi, 225
Murdaningsih H Karmana, 380
Mutsumi Akama, 165
Nadar Khan, 348
Naoto Inoue, 125, 252, 262
Naoya Nakamura, 401
Naoyoshi Kawano, 24
Narasimham Upadyayula, 11
Nguyen TB Yen, 19
Nurul Khumaida, 141
Ohn Mar Khin, 34
OR Nahak, 331
Ornprapa Anugoolprasert, 159
Osuhaini, 405
Paul PS Teng, 15
PDMH Karti, 331
Peter Dawson, 282
Peter Slavich, 72
Prijanto Pamoengkas, 385
Putri DU, 287
PV Vara Prasad, 2
R Kirana, 256
Raed A Haleem, 395

436

Proceedings of The 7 ACSA Conference 2011th

Randall L Nelson, 191
Renih Hayati, 137
Renny Fatmyah Utamy, 343, 339
Rika Fithri Buana, 358, 370
Rita Harni, 315
Ritsuko Hara, 96, 101
Rochadi Abdulhadi, 310
Rudiyanto, 319
Ryohta Shinohara, 113
Ryoji Sameshima, 196, 416
Ryuji Yoshida, 76
S Nofyangtri, 331
Sabaruddin, 137
Sachiko Idota, 339, 343
Sahuri, 175
Saki Hoshiyasu, 296
Samanhudi, 324
Samir K Abdullah, 395
Sandra Arifin Aziz, 175, 200
Satoko Yasumoto, 305
Satomi Sakazono, 169
Satoshi Nakamura, 165, 239
Satoshi Shimamura, 169
Satoshi Uchida, 421
Satya Nugroho, 91
Sayuri Kajihara, 169
Seiji Ishihara, 113
Seishi Yoshida, 262
Seito Hosokawa, 279
Shao-Hui Zheng, 215
Shigehiko Kanaya, 427
Shigeki Yokoyama, 119, 421
Shigeru Shigeoka, 3
Shina kinoshita, 159
Shintaro Kobayashi, 421
Shinya Kasajima, 230
Shojiro Tamaki, 14
Sintho Wahyuning Ardie, 141
Siswanto, 315
Somchai Chakhatrakan, 159
Sony Suhandono, 287, 353
Sri Astuti Rais, 225
Sri Ayu Dwi Lestari, 200
Sri Rahayu, 300, 310
ST Rahayu, 249, 256
Stanis Malangen, 56
Sudarsono, 91, 291
Sudjudi, 282
Sufardi, 72
thProceedings of The 7 ACSA Conference 2011

Suharsono, 220
Susanti Indriya Wati, 244
Susumu Arima, 215
Suwarto, 141
Taiki Yoshihira, 191
Takako Tsuji, 153
Takashi Hashimoto, 12
Takashi Yuasa, 215
Takayuki Nakano, 235
Takehiro Natori, 125
Takeshi Akaoka, 56
Takumi Iwakura, 56
Takuya Koyama, 334
Tamao Hatta, 29
Tantono Subagyo, 13
Tatik Chikmawati, 130
Tatsuhiko Shiraiwa, 101
Tatsuhiko Shiraiwa, 96, 191, 205
Tatsuro Miyazaki, 119
Teiji Nakamura, 165
Terry Hill, 282
Tetsushi Yoshida, 107, 153
Tomohiro Konno, 196
Tomohito Hayashi, 153
Tomoko Yanase, 14
Tomoyuki Katsube-Tanaka, 348
Tong Li-Tao, 34
Tony Liwang, 17Toru Hata, 56
Toshiaki Kokubo, 107
Toshihiro Mochizuki, 34, 169
Tri Muji Ermayanti, 319
U Sumpena, 249
Wahyudi, 385
Wikanya Prathumyot, 5Wisnu Adi Wicaksono, 358, 370
Y Puspitasari, 175
Yahaya Inusah, 29
Yasuhiro Tsujimoto, 29
Yasuo Nakamaru, 230
Yasuyuki Ishii, 339, 343
Yekti Asih Purwestri, 14
Yohei Kawasaki, 191, 205
Yoshiaki Inukai, 51
Yoshihiro Hirooka, 101
Yoshihiro Hirooka, 96
Yoshiko Iizumi, 421
Yoshinori Yamamoto, 107
Youji Nitta, 165
437

Yu Tanaka, 205
Yudiwanti WE Kusumo, 225
Yuji Araki, 19
Yuji Matsue, 34
Yuka Ogaki, 14
Yukari Terakado, 305
Yukiyo Yamamoto, 29

Yulong Wang, 107
Yuri Nakajima Munekage, 401
Yurie Okada, 153
Yushi Ishibashi, 215
Yusuke Goto, 165, 239
Yusuke Takahashi, 348
Zaitun, 72

438

Proceedings of The 7 ACSA Conference 2011th

List of Organizing Committee

ChairmanVice Chairman I
Vice Chairman II
Secretary
Treasurer
Secretariat

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

Funding
Scientific Program

Seminar Program

LogisticthSony Suharsono (IPB)
Miftahuddin (IPB)
Bambang Purwantara (BIOTROP)
Komang G Wiryawan (IPB)
Utut Widyastuti (IPB)
Noor Faiqoh Mardatin (IPB)
Dewi Suryani (IndoBIC)
Desi Maryanti (PT Paqar)
Herry Kristanto (Monsanto)
Elisabeth Maria (PT Sentra Biosains Dinamika)
Muhammad Yunus (Balitbiogen, Balitbangtan)
Antonius Suwanto (IPB)
Satya Nugroho (LIPI)
Karden Mulya (Balitbiogen, Balitbangtan)
Ence Darmo Jaya Supena (IPB)
Trimuji Ermayanti (LIPI)
Hayati Minarsih (Balit Bioteknologi Perkebunan)
Saptowo J Pardal (Balitbiogen, Balitbangtan)
Ahmad Junaedi (IPB)
Trikoesoemoeningtyas (IPB)
Syamsul Komar (IndoBIC)

Proceedings of The 7 ACSA Conference 2011

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Proceedings of The 7 ACSA Conference 2011

List of ParticipantsNo

Name of Participant

Institution

1.

Abdul Munif

Faculty of Agriculture, Bogor Agricultural University,
Indonesia

2.

Agus Rachmat

Research Center For Biotechnology,
Indonesian Institute of Sciences, Indonesia

3.

Agus Sutanto

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

4.

Ahmad Anwar Ismail

University of Malaysia, Kelantan, Malaysia

5.

Ahmad Rifqi Fauzi

Department of Agronomy, Faculty of Agriculture,
Universitas Negeri Papua, Indonesia

6.

Akihiro Fujii

Graduate School of Agriculture, Tohoku Univ., Miyagi,
Japan

7.

Akihiro Suzuki

Saga University, Japan

8.

Akiho Yokota

Nara Institut of Science and Technology, Japan

9.

Akira Yamauchi

10.

Ali Asgar

11.

Alia Astuti

Department of Biology, Bogor Agricultural University,
Indonesia

12.

Anggayudha Ananda Rasa

Bandung Institute of Technology (ITB), Indonesia
anggayudhaa@gmail.com

13.

Anggiani Nasution

Indonesian Center for Rice Research, ICRR,
Sukamandi, Indonesia

14.

Anugoolprasert Ornprapa

Mie University, Japan

15.

Ardha Apriyanto

School of Life and Sciences,
Bandung Institute of Technology, Bandung, Indonesia

16.

Ari Susilowati

Department of Biology, Faculty of Mathematics andNatural Sciences, University of Sebelas Maret.
Jl. Ir. Sutami No. 36A, Kentingan, Surakarta,
Indonesia 57126

17.

Arief Sabdo Yuwono

Dept. of Civil and Environmental Engineering (SIL),
Bogor Agricultural University, Bogor, Indonesia

18.

Arum Sekar Wulandari

Faculty of Forestry, Bogor Agricultural University,
IndonesiathProceedings of The 7 ACSA Conference 2011

Graduate School of Bioagricultural Science,
Nagoya University, Nagoya 464-8601, Japanayama@agr.nagoya-u.ac.jp
Indonesian Vegetables Research Institute
Jl. Tangkuban Perahu No. 517 Lembang 40791
Tel.(022)2786245, PO BOX 1586 Bandung, Indonesia
asgar1957@yahoo.com

441

19.

Asma Sembiring

Indonesian Vegetable Research Institute (BALITSA)
Jl.Tangkuban Perahu No.517 Lembang,
Bandung-40395, Indonesia
Tel : +62 22786245; fax : 62 22786416
asma_h_1999@yahoo.com

20.

Asmini Budiani

Indonesian Biotechnology Research Institute for
Estate Crops, Bogor, ndonesia

21.

Astri S

Perkindo/Perkin Elmer

22.

Atsushi Kato

Laboratory of Plant Development and MolecularMorphogenesis,
Graduate School of Biological Sciences,
Nara Institute of Science and Technology (NAIST),
Nara 630-0101, Japan

23.

Aweng a Mr. /l Eh Rak

University of Malaysia Kelantan (UMK), Malaysia

24.

Baiq Nurul Hidayah

25.

Bambang Pujiasmanto

26.

Bambang Sapto Purwoko

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor Indonesia

27.

Bayu Dwi Apri Nugroho

The United Graduate School of Agricultural Sciences,
Ueda 3-18-34, Morioka, Iwate, Japan 020-8550.
Tel.: +81 19 643 3408; fax: +81 19 641 9296;
u0410004@iwate-u.ac.jp

28.

Cailin Lei

Japan International Research Center for Agricultural
Sciences (JIRCAS)

29.

Cassandra

Monash University, Australia

30.

Cecep

Bogor Agricultural University, Indonesia

31.

Chengyun Li

Japan International Research Center for Agricultural
Sciences (JIRCAS)

32.

Chisato Sugiyama

Graduate school of Agriculture,
Tokyo University of Agriculture and Technology,
Fuchu, Tokyo183-8509, Japan
Tel: +81 42 367 5672; Fax: +81 42 367 5671;
50011531016@st.tuat.ac.jp

33.

Darman M Arsyad

Central Institute for Agricultural Technology
Assessment and Development, Bogor, Indonesia

34.

Deddy

Crop Life Indonesia

35.

Dedeh Hartati

Bogor Agricultural University, Indonesia

442

Assessment Institute for Agricultural Technology(AIAT)
Nusa Tenggara Barat, Indonesia
Department of Agrotechnology,
Faculty of Agriculture, Sebelas Maret University,
Surakarta, IndonesiathProceedings of The 7 ACSA Conference 2011

36.

Delima H A

Center of Agricultural Research and Development,
Ministry of Agriculture, Bogor, Indonesia

37.

Dewi Indriyani Roslim

Department of Biology, Bogor Agricultural University,
Bogor, Indonesia

38.

Dewi Sukma

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor Indonesia

39.

Dhika Amanda

School of Science and Technology,
Bandung Institute of Technology, Bandung, Indonesia

40.

Dian Catur P

PT. BISI International Tbk.

41.

Dwi Praptomo

42.

Dwinita W Utami

43.

Elis Nina Herliyana

Bogor Agricultural University, Indonesia

44.

Elly Kesumawati

Faculty of Agriculture, University of Syiah Kuala,
Banda Aceh, Indonesia

45.

Endang Yuniastuti

Faculty of Agriculture, Sebelas Maret University,
Indonesia

46.

Endro Gunawan

Center of Agricultural Research and Development,
Ministry of Agriculture, Bogor, Indonesia

47.

Eri Maai

Graduate School of Bioagricultural Sciences,
Nagoya University, Aichi, Japan

48.

Eries Dyah Mustikarini

49.

Etsushi Kumagai

50.

Etti Swasti

Faculty of Agriculture, Andalas University. Padang,Indonesia

51.

Eva Erdayani

Research Center for Biotechnology, Indonesian
Institute of Science, Cibinong, Indonesia

52.

Fadhillah

PT Pandu

53.

Fadlila Dewi R

Syngenta

Farit Mochamad Affendi

Graduate School of Information Science,
Nara Institute of Science and Technology, Ikoma,
Nara 630-0101, Japan.
Tel.: +81-743-72-5952; fax: +81-743-72-5953;
farit-a@is.naist.jp

54.

th

Proceedings of The 7 ACSA Conference 2011

Assessment Institute for Agricultural Technology(AIAT)
Nusa Tenggara Barat, Indonesia
The Agency of Crop Biodiversity and Genetic
Resources,
Center of Agricultural Research and Development,
Ministry of Agriculture, Bogor, Indonesia

Jurusan Agroteknologi FPPB, Universitas Bangka
Belitung. Balunijuk, Merawang, Bangka
Kepulauan Bangka Belitung, Indonesia
NARO Tohoku Agricultural Research Center, 4Akahira, Shimokuriyagawa, Morioka, Iwate 020-0198,
Japan

443

The United Graduated School of Agricultural
Sciences,
Kagoshima University, Japan
Faculty of Agriculture, Shinshu University. 8304
Minamiminowa, Nagano 399-4598, Japan.
Tel.: +81 265 77 1409 ; fax: +81 265 77 1409;
fkato@shinshu-u.ac.jp
Interdisciplinary Graduate School of Science andTechnology, Shinshu University, Minamiminowa,
Japan
Faculty of Agriculture, University of Sebelas Maret(UNS), Surakarta
Jl. Ir. Sutami No.36A Kentingan Surakarta (57126),
Indonesia
Tel/Fax: (0274) 637457; hadi_hpt@yahoo.com

55.

Fatichin56.

Futoshi Kato

57.

Futoshi Sasaki

58.

Hadiwiyono

59.

Hamda Fauza

Faculty of Agriculture, Andalas University,
Kampus Limau Manis 25163 Padang, Indonesia

60.

Happy Widiastuti

Indonesian Biotechnology Research Institute for
Estate Crops, Bogor, Indonesia

61.

Haruna Tanaka

Mie University, Japan62.

Hassan Y. Al Ayied

Natural Resources and Environment ResearchInstitute (NRERI)

63.

Hayati Minarsih

Indonesian Biotechnological Research Institute for
Estate Crops, Bogor, Indonesia

64.

Hiroshi Ehara

Graduate School of Bioresources, Mie University
Japan

65.

Hiroshi Miyake

Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi,
464-8601, Japan.

66.

Hiroyuki Shimono

Faculty of Agriculture, Iwate University,
3-18-8, Ueda, Iwate, 020-8550, Japan.
Tel.: +81 19 621 6146; fax: +81 19 621 6146;
shimn@iwate-u.ac.jp

67.

Hiroyuki Tsuji

Laboratory of Plant Molecular Genetics,
Nara Institute of Science and Technology, Japan

68.

Indrastuti Apri RumantiIndonesian Center for Rice Research (BB Padi)
Jl Raya 9 Sukamandi, Indonesia

69.

Inez HS Loedin

Research Center for Biotechnology, Indonesian
Institute of Science, Bogor, Indonesia
And
IRRI Manila, Philippine

70.

Iskandar Lubis

Department of Agronomy and Horticulture, Bogor
Agricultural University, Bogor, Indonesia

71.

Iswari S Dewi

ICABIOGRAD
Jl. Tentara Pelajar no 10A Bogor, Indonesia
iswari.dewi01@gmail.com

72.

Jinbin Li

Japan International Research Center for Agricultural
Sciences (JIRCAS)

444

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Proceedings of The 7 ACSA Conference 2011

73.

Jonathan Niones

Nagoya University, Japan

74.

Jose Maria AO

Bogor Agricutural University, Indonesia

75.

Jun-ichi Sakagami

Japan International Research Center for Agricultural
Sciences (JIRCAS)

76.

Kazunobu Toriyama

Japan International Research Center for Agricultural
Sciences (JIRCAS)

77.

Keisuke Katsura

Graduate School of Agriculture, Kyoto University,
Japan

78.

Kentaro Kawaguchi

National Agriculture and Food Research Organization(NARO)

79.

Khay Sathya

Japan International Research Center for Agricultural
Sciences (JIRCAS)

80.

Kohtaro Iseki

Graduate School of Agriculture, Kyoto University,
Kyoto, Japan

81.

Koki Homma

Graduate School of Agriculture, Kyoto University,
Kyoto, Japan82.

Kulvinder S Gill

Department of Crop and Soil Science, Washington
State University

83.

Kuniyuki Saitoh

Graduate School of Natural Science and Technology,
Okayama University, Okayama, Japan

84.

Kustiariyah

Faculty of Fisheries & Marine Sciences,
Bogor Agricultural University, Indonesia

85.

Lailatul Badriyah

School of Life and Sciences,
Bandung Institute of Technology, Bandung, Indonesia

86.

Latifah K Darusman

Biofarmaka, Bogor Agricultural University, Indonesia

87.

Lia Hadiawati

Assessment Institute for Agricultural Technology(AIAT)
Nusa Tenggara Barat, Indonesia

88.

Loida M Perez

Japan International Research Center for Agricultural
Sciences (JIRCAS)89.

Mai Goto

Graduate School of Agriculture,
Tokyo University of Agriculture and Technology,
Fuchu, Tokyo, Japan

90.

Maya Melati

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

91.

Md. Ansar Ali

Japan International Research Center for Agricultural
Sciences (JIRCAS)

92.

Michihiko Fujii

Faculty of Education, Shizuoka University, JapanthProceedings of The 7 ACSA Conference 2011

445

93.

Michio Kawasaki

Faculty of Agriculture and Life Science,
Hirosaki University, Aomori, Japan94.

Miftahudin

Department of Biology, Bogor Agricultural University,
Bogor, Indonesia

95.

Mikio Nakazono

Graduate School of Bioagricultural Sciences,
Nagoya University, Japan

96.

Mikio Sekimuna

Interdisciplinary Graduate school of Science andTechnology,
Shinshu University, Japan

97.

Mituhiro Obara

Japan International Research Center for Agricultural
Sciences (JIRCAS)

98.

Mochamad Arief Soleh

Graduate School of Agricultural Science,
Tohoku University, Japan

99.

Motonobu Yoshida

Dept of Agricultural Science, Kinki University,
Nakamachi 3327-204, Nara, Japan

100.

Muhammad Addip Novianto

BMKG

101.

Muhammad Syafii

Bogor Agricultural University, Bogor, Indonesia

102.

Muhammad Yunus

The Agency of Crop Biodiversity and Genetic
Resources,
Center of Agricultural Research and Development,
Ministry of Agriculture, Bogor, Indonesia

103.

Munif Ghulamahdi

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

104.

Nagao Hayashi

Japan International Research Center for Agricultural
Sciences (JIRCAS)

105.

Naomi Asagi

Ibaraki University, Japan106.

Naoya Nakamura

Graduate school of Biological Sciences,
Nara Institute of Science and Technology (NAIST),
Ikoma, Nara 630-0101, Japan

107.

Narasimham Upadyayula

Monsanto Singapore Co (Pte) Ltd Singapore

108.

Nguyen Thi Bich Yen

Asian Natural Environmental Science Center,
The University of Tokyo, Japan

109.

Nguyen Thi LangJapan International Research Center for Agricultural
Sciences (JIRCAS)

110.

Nguyen Thi Thanh Thuy

Japan International Research Center for Agricultural
Sciences (JIRCAS)

111.

Nguyen Van Kien

Division of Genebank Management,
Plant Resources Center
An Khanh, Hoai Duc, Ha Noi, Vietnam

112.

Niken TM Pratiwi

Faculty of Fisheries & Marine Sciences,
Bogor Agricultural University

446

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Proceedings of The 7 ACSA Conference 2011

113.

Niken Trisnaningrum

PT. BISI International Tbk.

114.

Nishe Fransiska

Bogor Agricutural University, Indonesia

115.

Nobuhiro Noda

Mie university
nobu0witch3@yahoo.co.jp
514-8507 Mie, Tsu-shi, 1577 Kurimamachiya-cho

116.

Nurliani Bermawie

BALITTRO Bogor, Indonesia

117.

Nurul Khumaida

Department of Agronomy and Horticulture,
Bogor Agricultural University, Indonesia

118.

Ohn Mar Khin

Faculty of Agriculture, Kyushu Univ., Fukuoka, Japan

119.

Panca Dewi Manu Hara Karti

Faculty of Animal Husbandry,
Bogor Agricultural University, Bogor Indonesia

120.

Paul PS Teng

National Institute of Education,
Nanyang Technological University, SIngapore

121.

Phoumi Inthapaya

Japan International Research Center for Agricultural
Sciences (JIRCAS)

122.

Poonsak Mekwatanakarn

Japan International Research Center for Agricultural
Sciences (JIRCAS)

123.

PV Vara Prasad

Department of Agronomy, Kansas State University,
Manhattan, Kansas 66506, USA

124.

Raed A Haleem

University of Duhok (UoD),
Faculty of Agriculture and Forestry,
Kurdistan region, Iraq

125.

Rahmat H

CENTRAS, LPPM, Bogor Agricultural University,
Indonesia

126.

Randy Hautea

SEAsia Center Director and ISAAA Global
CoordinatorInternational Service for the Acquisition of Agribiotech Applications (ISAAA)

127.

Renih Hayati

Fac. of Agriculture, Sriwijaya University, Palembang,Indonesia

128.

Renny Fatmyah Utamy

Faculty of Agriculture, University of Miyazaki, Japan

129.

Retno Lestari

Universitas Indonesia

130.

Rika Fithri Nurani B

PT SMART Tbk.

131.

Rika Raffiudin

Department of Biology, Bogor Agricultural University,
Bogor, Indonesia

132.

Rinda Kirana

Institution Vegetables Research (IVEGRI), IndonesiathProceedings of The 7 ACSA Conference 2011

447

133.

Ritsuko Hara

Graduate School of Agriculture, Kyoto University,
Japan

134.

Rudiyanto

Research Center for Biotechnology , Indonesian
Institute of Science, Bogor, Indonesia

135.

Rujito Agus Suwignyo

Faculty of Agriculture, Sriwijaya University,
Palembang, Indonesia

136.

Ruth Martha Winnie

Bogor Agricultural University, Bogor, Indonesia

137.

Samanhudi

Department of Agrotechnology,
Faculty of Agriculture, Sebelas Maret University
Jl. Ir. Sutami 36A, Surakarta, Indonesia 57126

138.

Sandra Arifin Aziz

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

139.

SantosoIndonesian Center for Rice Research,
Jln Raya 9, Sukamandi, Subang 41256, West Java,
Indonesia

140.

Sara

PT ITS
Biomass Production and Processing Research Team,
Production Systems Research Area,
National Agricultural Research Center Tsukuba,
Japan
School of Food, Agricultural and Environmental
Sciences,
Miyagi University, Sendai, Miyagi, Japan
Department of Agronomy and Horticulture,
Faculty of Agriculture,
Bogor Agricultural University, Bogor, Indonesia

141.

Satoko Yasumoto

142.

Satoshi Nakamura

143.

Satriyas Ilyas

144.

Shao-Hui Zheng

Coastal Bioenvironment Center, Saga University,
Karatsu, Saga 840-0021, Japan

145.

Shigeki Yokoyama

Japan International Research Center for Agricultural
Sciences (JIRCAS)

146.

Shigeru Shigeoka

Department of Advanced Bioscience,
Faculty of Agriculture, Kinki University, Japan

147.

Shinsuke Kasa

Graduate School of Bioresourches andBioenvironmental Science, Kyushu University, Japan

148.

Shintaro Kobayashi

Japan International Research Center for Agricultural
Sciences, Tsukuba, Japan

149.

Shinya Kasajima

Faculty of Bioindustry, Tokyo University of Agriculture,
Hokkaido, Japan

150.

Shiro Mitsuya

Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Japan151.

Shunsuke Adachi

Tokyo University of Agriculture and Technology,
Japan

152.

Sintho Wahyuning Ardie

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

448

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Proceedings of The 7 ACSA Conference 2011

153.

Sobir

Bogor Agricultural University

154.

Sony Suhandono

School of Life and Sciences, Bandung Institute ofTechnology, Bandung, Indonesia

155.

Sri Rahayu

Bogor Botanical Gardens,
Indonesian Institute of Sciences, Bogor, Indonesia

156.

Stanis Malangen

United Graduate School of Ehime University,
Matsuyama, Ehime, 790-8566, Japan

157.

Suharsono

Department of Biology, Bogor Agricultural University,
Bogor, Indonesia

158.

Sulandjari

Faculty of Agriculture , Sebelas Maret University,
Surakarta, Indonesia

159.

Suliman Ali Al-Khateeb

Environment and natural resources Department,
King Faisal University, Kingdom of Saudi Arabia.
P.O.Box 420, Alhofof 31982.
skhateeb@kfu.edu.sa

160.

Supijatno

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

161.

Susumu Arima

Faculty of Agriculture, Saga University,
Saga 840-8502, Japan

162.

Suwarni Tri Rahayu

Institution Vegetables Research (IVEGRI) Indonesia

163.

SuwarnoIndonesian Center for Rice Research,
Jln Raya 9, Sukamandi, Subang 41256, West Java,
Indonesia

164.

Tadashi Hirasawa

Tokyo University of Agriculture and Technology,
Japan

165.

Tadashi Sato

Japan International Research Center for Agricultural
Sciences (JIRCAS)

166.

Taiichiro Ookawa

Tokyo University of Agriculture and Technology,
Japan

167.

Takao Oi

Graduate School of Bioagricultural Sciences,
Nagoya University, Aich, Japan

168.

Takashi Homma

The College of Agricuture, Ibaraki University,
3-21-1, Ami, Ibaraki 300-0393, Japan
Tel: +81 29 888 8556; fax: +81 29 888 8556
homma@mx.ibaraki.ac.jp

169.

Takasi Hashimoto

Graduate School of Biological Science,
Nara Institute of Science and Technology, Japan

170.

Takehiro Mukouyama

Tokyo University of Agriculture and Technology,
Japan

171.

Takeshi Kano

Japan International Research Center for Agricultural
Sciences (JIRCAS)th

Proceedings of The 7 ACSA Conference 2011

449

172.

Takunari Kono

Graduate school of Biological Sciences,
Nara Institute of Science and Technology (NAIST),
Ikoma, Nara 630-0101, Japan173.

Takuya Koyama

Department of Biosphere Resources Science,
Nagoya University, Nagoya, Japan

174.

Tantono Subagyo

Syngenta Indonesia, Perkantoran Hijau Arkadia,
Tower C, 8th fl, Jakarta

175.

Tati Kristianti

School of Science and Technology,
Bandung Institute of Technology, Bandung, Indonesia

176.

Thelma F Padolina

Japan International Research Center for Agricultural
Sciences (JIRCAS)

177.

Titi Rahayu

USA Embassy, Jakarta

178.

Tito Deski

PBS, IFPRI

179.

Tohru Kobata

Faculty of Life and Environmental Science,
Shimane University, 1060 Nishikawatsu-cho,
Matsue, 690-8504, Japan180.

Tomohito Hayasi

Graduate School of Bioagricultural Sciences Nagoya
University, Nagoya, Japan

181.

Tomomi AbikoJapan International Research Center for Agricultural
Sciences (JIRCAS)

182.

Tomoyuki Katsube-Tanaka

Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Kyoto 606-8502, Japan183.

Tony Liwang

Plant Production and Biotechnology Division,
PT SMART Tbk, BII Plaza Tower 2, 10th floor.
Jl. MH Thamrin 51 Jakarta, Indonesia

184.

Toshiaki Kokubo

United Graduate School of Agricultural Sciences,
Ehime University, Japan

185.

Toshihiro Mochizuki

Faculty of Agriculture, Kyushu University, Japan186.

Toshiki Yamamoto

Mie University, Japan187.

Totok Hesterianoto

Faculty of Fisheries & Marine Sciences,
Bogor Agricultural University, Indonesia

188.

Tri Lestari

University of Bangka Belitung, Bangka Belitung,Indonesia

189.

Tri Muji Ermayanti

Research Center For Biotechnology, Indonesian
Institute of Science, Bogor, Indonesia

190.

Tsutomu Ishimaru

Japan International Research Center for Agricultural
Sciences (JIRCAS)

191.

Utut Widyastuti

Department of Biology, Bogor Agricultural University,
Bogor, Indonesia

450

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Proceedings of The 7 ACSA Conference 2011

192.

WahyudiDepartment of Forestry, Palangka Raya University,
Palangka Raya, Indonesia

193.

Wisnu Adi Wicaksono

PT SMART Tbk

194.

Woro Umayi Ananda

Syngenta

195.

Xin Zhao

The United Graduated School of Agricultural
Sciences,
Kagoshima University, Japan

196.

Yasuhiro Tsujimoto

Japan International Research Center for Agricultural
Sciences (JIRCAS)

197.

Yasumoto Satoko

Biomass Production and Processing Research Team,
Production Systems Research Area,
National Agricultural Research Center Tsukuba,
Japan

198.

Yasuyuki Ishii

Faculty of Agriculture, University of Miyazaki, Japan

199.

Yohei Kawasaki

Graduate School of Agriculture, Kyoto University,
Japan

200.

Yohei Koide

Japan International Research Center for Agricultural
Sciences (JIRCAS)

201.

Yoshihiro Nishihara

Faculty of Agriculture, Saga University, Saga, Japan202.

Yoshimichi Fukuta

Japan International Research Center for Agricultural
Sciences (JIRCAS)

203.

Youji Nitta

Ibaraki University 300-0393 Japan

204.

Yudiwanti Wahyu E Kusumo

Department of Agronomy and Horticulture,
Bogor Agricultural University, Bogor, Indonesia

205.

Yukari Isobe

The Colledge of Agriculture, Ibaraki University, Ami,
Ibaraki, Japan

206.

Yushi Ishibashi

Crop Science Lab., Faculty of Agriculture, Kyushu
University, Hakozaki, Fukuoka 812-8581, Japan

207.

ZaitunFaculty of Agriculture, University of Syiah Kuala,
Banda Aceh, IndonesiathProceedings of The 7 ACSA Conference 2011

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