FNCA Mutation Breeding ProjectOpen Seminar

Symposium on
Radiation and Nuclear Technologies
for Crop Improvement and Productivity
in Sustainable Agriculture
Kajang, Malaysia
February 26th – 27th, 2013

FNCA / MEXT
Malaysian Nuclear Agency

Ministry of Education, Culture, Sports, Science and Technology
Forum for Nuclear Cooperation in Asia

This report was prepared by the Nuclear Safety Research Association (NSRA) as a part ofactivities on Forum for Nuclear Cooperation in Asia (FNCA), organized by MEXT.

Content
Program of Open Seminar ······································································· 1
Summaries
1. Overview······················································································· 5
1.1 Dr. Sharif Haron, MARDI, Malaysia
1.2 Dr. Sueo Machi, FNCA Coordinator of Japan

2. Mutation Breeding I ······································································ 13
2.1 Dr. Hirokazu Nakai, Sizuoka University, Japan
2.2 Dr. Rusli Bin Ibrahim, Nuclear Malaysia, Malaysia
2.3 Dr. Shu Qingyao, ZU, China
2.4 Ms. Dao Thi Thanh Bang, AGI, Vietnam

3. Mutation Breeding II ···································································· 37
3.1 Dr. Damasco P. Olivia, UPLB, The Philippines
3.2 Dr. Soeranto Human, BATAN, Indonesia
3.3 Dr. Si-Yong Kang, KAERI, Korea
3.4 Dr. Atsushi Tanaka, JAEA, Japan

4. Mutation Breeding III ··································································· 63
4.1 Mr. Suniyom Taprab, RD, Thailand
4.2 Dr. ANK. Mamun, BAEC, Bangladesh
4.3 Dr. Liu Luxiang, IAEA/RCA, China

5. Nuclear Technique forSoil Management for Sustainable Agriculture ···································· 73
5.1 Dr. Kamaruddin Bin Hashim, Nuclear Malaysia, Malaysia
5.2 Dr. Khairuddin Bin Abdul Rahim, Nuclear Malaysia, Malaysia

6. Collaboration & Technology Transfer ··············································· 81
6.1 Dr. Mohamad Osman, UiTM, Malaysia
6.2 Mr. Ragu Ponusamy, FELCRA, Malaysia
6.3 Mrs. Hayati Taib, MYAGRI, Malaysiai

ii

Program of
FNCA Open Seminar
Symposium on Radiation and Nuclear Technologies for Crop Improvement and Productivity
in Sustainable Agriculture
February 26th – 27th, 2013
Malaysian Nuclear Agency, Kajang, Malaysia

Tuesday, 26 February 2013
Time
09:00 – 09:30

AgendaRegistration
Doa Recital
Welcoming Remarks
Dr. Sueo Machi, FNCA Coordinator of Japan

09:30 – 10:30

Opening Address
YBhg. Dato’ Dr. Muhamad Lebai Juri, Director General, Malaysian
Nuclear Agency
Group photography

10:30 – 11:00

Coffee/tea break
Overview

Session 1

Chairperson: Dr Muhd Noor Muhd Yunus, FNCA Coordinator of
Malaysia/Deputy

Director

General(Research&

Technology

Development Programme), Malaysian Nuclear Agency
Strategies and Technologies in Enhancing Sustainable Agriculture
11:00 – 11:30

YBhg. Dato’ Dr. Sharif Haron,
Deputy Director General (Research),
Malaysian Agricultural Research and Development Institute (MARDI)

11:30 – 12:00

Nuclear Technique for Sustainable and Productive Agriculture
Dr. Sueo Machi, FNCA Coordinator of Japan,
Use of Agricultural Chemicals and its Impact on the Environment

12:00 – 12:30

Dr. Mohd. Khanif Yusop, Professor, Faculty of Agriculture, Universiti
Putra Malaysia (UPM)1

12:30 – 14:00
Session 2

Lunch Break
Mutation Breeding I
Chairperson: Dr. Khairuddin Abdul Rahim
World Success and Prospect of Mutation Breeding

14:00 – 14:20

Dr. Hirokazu Nakai, Project Leader of Japan,
Prof. Emeritus of Shizuoka University
Success Story of Mutation Breeding in Malaysia

14:20 – 14:40

Dr. Rusli Ibrahim, Project Leader of Malaysia,
Malaysian Nuclear Agency
Molecular Genetic Features of Induced Mutations in Rice and Soybean

14:40 – 15:00

Dr. Shu Qingyao, Project Leader of China
Professor, Zhejiang University
Success Story of Development of High Yield Rice Variety Grown in

15:00 – 15:20

VietnamMs. Dao Thi Thanh Bang,
Institute of Agriculture Genetics (AGI), Vietnam

15:20 – 15:40
Session 3

Coffee/tea Break
Mutation Breeding II
Chairperson: Dr. Rusli Ibrahim
Development of Disease Resistant Banana by Mutation Breeding in the

15:40 – 16:00

Philippines
Dr. Damasco Olivia,
University of the Philippines Los Banos (UPLB), The Philippines
Success Story of Mutation Breeding on Sorghum for Improved Drought

16:00 – 16:20

and Acid Soil Tolerance in Indonesia
Dr. Soeranto Human,
National Nuclear Energy Agency (BATAN), Indonesia
Radiation Breeding and Commercial Mutant Varieties in Korea

16:20 – 16:40

Dr. Si-Yong Kang, Project Leader of Korea
Korea Atomic Energy Research Institute (KAERI), Korea
Advanced Techniques for Mutation Breeding by Using Ion Beam

16:40 – 17:00

Dr. Atsushi Tanaka,
Japan Atomic Energy Agency (JAEA), Japan2

Wednesday, 27 February 2013
Time
Session 4

AgendaMutation Breeding III
Chairperson: Dr. Atsushi Tanaka
Success Story of Mutation Breeding in Thailand

09:00 – 09:20

Mr. Suniyom Taprab, Project Leader of Thailand
Ministry of Agriculture and Cooperatives (MOAC), Thailand
Success Story of Mutation Breeding Research on Rice in Bangladesh

09:20 – 09:40

Dr. A.N.K. Mamun, Project Leader of Bangladesh
Bangladesh Atomic Energy Commission (BAEC), Bangladesh
Towards Sustainable Development of Food & Agriculture in Asia and the

09:40 – 10:00

Pacific - Roles and Perspectives of RCA
Dr. Liu Luxiang, Lead Country Coordinator of RCA Mutation Breeding
Project, Chinese Academy of Agriculture Sciences (CAAS), China

10:00 – 10:30
Session 5

Coffee/tea break
Nuclear Technique for Soil Management for Sustainable Agriculture
Chairperson: Dr. Liu Luxiang
Development of Plant Growth Promoter and Plant Elicitor by Radiation

10:30 – 11:00

Processing of Natural Polymers
Dr. Kamaruddin Hashim, Malaysian Nuclear Agency
Bio-fertilizer Production Using Radiation Technology for Sustainable

11:00 – 11:30

Agriculture
Dr. Khairuddin Abdul Rahim, Malaysian Nuclear Agency

Session 6

Collaboration and Technology Transfer
Chairperson: Dr. Kamaruddin Hashim
Breeding of Roselle (Hibiscus sabdariffa L.) through Induced Mutations

11:30 – 12:00

Dr. Mohamad Osman,
Professor, Universiti Teknologi MARA (UiTM)
Radiation and Nuclear Technology for Crop Improvement andSustainable Agriculture

12:00 – 12:30

Mr. Ragu Ponusamy,
Federal Land Consolidation and Rehabilitation Authority (FELCRA),
Malaysia
Potential of Biofertilizer in Agroindustry: MYAGRI’s Experience in

12:30 – 13:00

Commercialization
Mrs. Hayati Taib,
Malaysian Agri Hi-Tech (MYAGRI)3

13:00 – 14:30

Lunch break

Session 7

Panel Discussion
Challenges of Application of Mutation Breeding and Nuclear techniques
for Food Security and Sustainable Agriculture
Chairman: Dr. Muhd Noor Muhd Yunus, FNCA Coordinator of Malaysia
Panel members: Invited Speakers and Experts

14:30 – 16:00

Points of Discussion
1. Contamination of environment by excess use of chemical in
agriculture
2. Importance of nuclear techniques for sustainable agriculture
3. Organic agriculture and acceptance of consumers

16:00 – 16:15

Coffee/tea break
Closing Speech

16:15 – 16:30

Dr. Muhd Noor Muhd Yunus, FNCA Coordinator of Malaysia
Dr. Sueo Machi, FNCA Coordinator of Japan4

1. Overview

1.1

Strategies and Technologies in Enhancing Sustainable Agriculture

Sharif Haron
Malaysian Agricultural Research and Development InstituteSustainable agriculture as defined by United States Department of Agriculture (USDA) is an
integrated system of plant and animal production practices having a site-specific applicationthat will, over long-term satisfy human food and fiber needs; enhance environmental quality
and the natural resource base upon which the agricultural economy depends; make the mostefficient use of non-renewable and integrate, where appropriate, natural biological cycles andcontrols; sustain the economic viability of farm operations; and enhance the quality of life forfarmers and society as a whole. The four basic criteria of sustainable agriculture are (1)ensuring basic nutritional requirements, (2) providing durable employment and decent living
to farmers, (3) maintaining productive capacity of natural resources and, (4) reducing
vulnerability of agriculture to adverse natural and socio-economic factors. Sustainableagriculture needs to fulfill the current and future needs in economy (agriculture products,
supply adequate and safe foods, economic growth), social (food security, provide employment
opportunities, culture) and environment (no pollution, mitigate climate change, conserve
biodiversity).
The challenges facing the Malaysian agriculture are daunting. The challenges are increase inpopulation and cost of production to sustain food production; pollution, reduction in
biodiversity and agro-waste utilization in maintaining environmental sustainability; lack ofsuitable land for agriculture, water scarcity and competition for use, labor unavailability;change in consumer trends such as healthy lifestyle and searching for natural products; andclimate change where there will be longer dry season, shorter rainy season with intense rain,
new pest and diseases and release of green-house gas (GHG).
Malaysia recognizes the challenges and the need to find solutions to ensure sustainability in
its agriculture endeavor. Strategies to enhance sustainable agriculture have been formulated
and these include:a) Putting policies in place such as the Food Security, Green Technology and Renewable
Energy policies,b) Ensuring wide application of good agricultural practice (GAP),c) Rational use of natural resources in environmentally and socially harmonious mannere.g. low water consumption,d) Diversification of food resources e.g. encourage consumption of food availability locally,
less dependency on certain staple food,e) Enhancing R&D and use of appropriate technologies (both preventive and curative) for
current and future needs e.g. agro waste for recycling, integrated pest management,
adaptation and mitigation to climate change including reducing GHG gas emissions,6new crop varieties and animal breeds, precision farming for optimum inputs, optimizing
land use, filling information gaps in organic farming, employing ICT tools, use of herbal
alternatives in livestock production, managing peat land, highland horticultural, rice
ecosystems and livestock productionf) Wide application of community practice,g) Greater need for capacity development and extension of expertise on the ground,h) Enhancing awareness on importance of environmental and agricultural sustainability
e.g. through early education, constant and impactful campaign.
Taking into considerations the issues and challenges in ensuring agriculture sustainability,
greater emphasis were given to develop technologies that could manage available natural
resources efficiently and maximize productivity, in anticipation of the potential scarcity inthe future. The following are some of the technologies that have been developed to enhancesustainable agriculture in Malaysia.
(1) Technologies for sustainable food production
The emphasis is to meet sufficiency level in various commodities (rice, vegetables, fruits andlivestock). This will be met only through increase yield per unit area by creating new highyielding and quality varieties and breeds, efficient cropping systems and post-harvest
handling as well as innovative production technology. In the case of rice, recently new paddy
varieties (MR 253, MR 263, MR 269) with high yield and resistant to major pest and diseases
have been developed. Two varieties under the Clearfield System, MR 220 CL1 and CL2 havebeen developed to overcome losses due to weedy rice infestation.
(2) Technologies for environmental sustainability
Malaysia is richly endowed with a myriad of natural fauna and flora that has been utilized as
genetic resources as well as biological control agents against pests and pathogen problem in
economic crops. It must also be acknowledged that abundant agricultural waste is available
and this has been turned into beneficial crop booster and bio-fertilizer. Technologies that aredeveloped to address the issue of environmental sustainability includes organic farming, zerowaste production system and energy cycling, virus-based bio-pesticides, crop diversity forbeneficial insects, tetra-cropping, bio-fertilizer based on microbial consortium, fungal based
bio-herbicides, bacteriophage based bio-herbicides, botanical pesticide, solar drying andbiogas.
Environmental-friendly virus based bio-pesticides (containing multi nucleopolyhedrosis virus
or NPV) for controlling armyworm, Spodoptera litura, are found stable at room temperature,and have longer shelf life. Whereas, fungus based bio-herbicides are developed specifically forcontrolling ‘rumput sambau’ (Echinochloa spp.) in rice.
Tetra-cropping maize with chilli has helped reduce virus disease and its vector on chilli.
Study has shown that the percentage of virus disease incidence was much lower when chilli

7(C) was planted with maize (M), okra (O), eggplant (E) in the order of MCEOMCEO comparedtotri-cropping,

MCEMCEMC,

MCOMCOMC

ordi-cropping,

MCMCMCMC

andmono-cropping, CCCCCCCC (60%, 60%, 80% and 100%, for tetra-, tri-, di- and mono-cropping,respectively).
Evaluation for potential bio-pesticide was made on selected Malaysian plants, Andrographis

paniculata (hempedu bumi), Cymbopogon spp. (serai), Pelargonium radula (jeremin), Annona
spp. (durian belanda), Citrus spp. (limau purut) and Dioscora spp. (ubi gadung). Oilformulations from Pelargonium radula and Cymbopogon citratus are found to be potential
bio-pesticidal agents against Plutella xylostella (diamond-black moth).
(3) Technologies to overcome lack of resources
Food-crop agriculture productivity is basically limited due to shortage of suitable fertile land
and as such it relies heavily on fertilizer input. Application of information andcommunication technology (ICT) via precision farming including through the use of
unmanned aerial vehicle (UAV) has helped in mapping the farming areas, define the areas
precisely for fertilizer application. Subsequently, it will help in increasing plant nutrient
uptake and reduce fertilizer wastage and water pollution. Similarly technologies likehydroponics and fertigation whether applied in open or in green house, are also practiced.
(4) Technologies for mitigation and adaptation to climate change
Climate change has become as an emerging concern to the agricultural sector needing
immediate solutions. New genotypes and breeds tolerant to multiple stresses: drought, flood,
heat, pest and diseases, will help further increase in production. This would require
substantial efforts through breeding and biotechnology based on collection, characterization,conservation and utilization of new genetic resources and also marker assisted technology(MAS). In Malaysia, for instance, new short maturing variety MR 211 with 100 days
maturation has been developed to adapt to the climate change. In addition to that, aerobic
rice production system which utilizes only half of the usual water requirement (470-650 vs.
1130-1300 mm/season) but yet giving reasonable yield (4.0 – 5.3 vs. 5-7 ton/ha) has beendeveloped as part of a strategy to climate change.
In conclusion, R&D and technologies will continue to play a crucial role in sustainableagricultural development. There is a need for enhanced collaborative research among all
parties involved to create synergies and symbiosis. New tools are required to assesssustainability, evaluate resource appropriation and production performance as well as ensure
environmental wellness. The challenge is in re-designing agro-food systems in the context ofpromoting a holistic participatory approach, extending them to farmers for adoption,
addressing the issue of self-sufficiency, and conserving our resources and environments for
future generations.

8

1.2

Nuclear Techniques for Sustainable Agriculture

Sueo Machi
Fellow, Japan Atomic Energy Agency
1. Introduction- Nuclear technology for food security“Food for all” is the main challenge of our society. We need to decrease number of people
suffering from hunger. Nuclear technology can play an important role for addressing thechallenge through soil and water management, food irradiation to reduce post-harvest loss,
sterile insect technique to reduce pesticide use, and mutation breeding for better crop yields.
Sustainability of agriculture is another challenge for next generation. Nuclear techniques canalso provide better technology which is more environmentally friendly.
Global warming continues because of increasing emission of CO2 from burning fossil fuels
due to rapid development of the world economy. The adverse effect of global warming on
agriculture is serious concern. Mutation breeding may be able to develop new varieties which
are tolerable to increased temperature.
2. Application of nuclear techniques for improving soil management for agriculture(1) Biofertilizer:
Inoculants using microorganism such as Rhizobium for fixing nitrogen from air andMycorrhizafor solubilizing phosphate can remarkably increase crop yield in the range of 20 to
70%. It has been proved that radiation sterilization of carriers of microorganism can produce
better quality inoculants and provide effective and larger scale production process.
This development has been carried out by collaboration among Asian countries under theForum of Nuclear Cooperation in Asia (FNCA) [1]. IAEA has a success story of Rhizobium
biofertilizer application in Zimbabwe for grain legumes. Application of the biofertilizer canincrease crop yield and can reduce the risk of contaminating ground water by excess use ofchemical fertilizer. In Malaysia, Indonesia and the Philippines, the radiation sterilization ofcarriers for bio-fertilizer inoculants has recently been commercially used. Development of
new mutant strains of microorganism using radiation mutation for better andmultifunctional biofertilizer is being studied.
(2) Recycle of sewage sludge to soil
Sewage sludge contains nutrient and organic matter which improves quality and fertility ofsoil. The sewage sludge, therefore, should be recycled to soil to improve quality of soil forsustainable agriculture. Since the sewage sludge is, however, contaminated by pathogens
such as salmonella and E-Coli, radiation can be effectively used to sterilize it before
application. This technology is commercially used in India and used to be used in Germany in9the past. There is a concern about heavy metals contained in the sludge. In many cities
industrial waste water is separately treated from that of households of which sewage sludge
is free of heavy metals. Recycling sewage sludge should be much more enhanced [2].
(3) Plant growth promoter (PGP) from natural polymers:
Development of production of PGP by radiation degradation of natural polymers has beenachieved by collaboration of member countries under FNCA. Oligo-chitosan produced fromcrab/shrimp shell and oligo-carageenan from seaweed by radiation degradation show
excellent effect in increasing crop yields (10-50%) for red chili, rice, tomato, potato, carrot andothers by pot and field tests in Vietnam, Malaysia, Philippines, Indonesia, Japan and
Thailand [3], [4]. In Japan oligo-chitosan has been recently commercially produced as elicitor
for flowers. The PGP from natural polymers are environmentally friendly in terms of
reducing the use of chemical fertilizer and pesticide.
(4) Super water absorbent as soil conditioner in arid region
Agriculture in arid and semi-arid region should be enhanced for better food security. Super
water absorbent (SWA) produced by radiation cross-linking and/grafting of natural polymers
such as starch can absorb large amount of water (SWA of 1 g. absorbs 400 grams of water).
The SWA is mixed in soil to retain scarce rain and irrigation water in surface soil much longer
for plant to use water efficiently in arid and semi-arid region [5]. Field tests in Vietnam prove
the increase in crop yields as shown in Table 1.
Table 1: Field Tests of SWA in Vietnam
Amount of SWA: 30kg per ha Plant Yield increase (%)
•

Corn

28.8

•

Peanut21.1•

Cotton31.1•

Coffee

23.0

•

Grape

29.8

•

Sugarcane

19.8

3. Sterile insect technique (SIT) by using radiation
Insect pests are reducing harvest of crops by 30%. In order to reduce number of pests, large
amount of chemical pesticides is used worldwide which seriously pollutes environment anddamages bio-diversity.
SIT can eradicate and/or control insect pests by the use of much reduced amount of pesticide.
For SIT puppet are irradiated to be sterile. Large numbers of sterile adult flies are released
to natural environment and mate with natural female, which lay eggs but these eggs can’t
merge. Therefore, the population of insect pest decreases generation by generation. IAEA has
well known success stories, such as eradication of tsetse flies in Zanzibar Island in Tanzania.
10Very large campaign of control of tsetse flies in Ethiopia is being implemented bycollaboration of African Union with IAEA. Several IAEA projects have been successfully
implemented for eradication or control of Mediterranean fruit flies in member states.
Japan has succeeded to eradicate melon flies in Okinawa Island around 1990 after 5 year
campaign. Now, fruits and vegetable from Okinawa can be exported to the main land ofJapan.

Figure1. Co-60 irradiation facility for puppet of melon flies in Okinawa

4. Mutation breeding
Mutation breeding is one of the most important nuclear technologies for improving
agriculture in terms of yield increase and sustainability.
Radiation is the most efficient way to induced mutation of plant to improve character ofplants. In the history more than 3000 better varieties of plants were developed and registeredby using radiation mutation.
Under the FNCA project, recently, new variety of banana has been developed in thePhilippines, which is better resistant against Bunch Top Virus disease. By using this variety
farmers can save pesticide, which contributes to the protection of environment from pollution
by pesticides. The FNCA project on mutation breeding is now focusing on the development of
new rice varieties for sustainable agriculture.
5. Food irradiation
Food irradiation for food safety, quarantine for expert, and decrease in food loss by spoilage,
sprouting and insect attach is expanding worldwide in 57 countries. As shown in Table 2 theworld total amount of irradiated food for commercial purpose is about 500,000 tons andincreasing. However, in some countries including Japan public acceptance of irradiated foods
is still obstacles for expansion of food irradiation.
It should be recognized that post-harvest loss of crops is in the range of 40% in many
developing countries. Food irradiation technique should be much more used to decrease11

post-harvest loss due to spoilage, sprouting, insect and fungus attack.
Table 2: Amount of Commercial Food Irradiation
World total of irradiated foods

ca. 500,000 ton

China: Garlic, Dried vegetables, etc.

146,000 ton

Vietnam: Frozen shrimps, etc.

14,000 ton

Japan: Potatoes

8,000 ton

USA: Spices, Ground meats, Fruits

92,000 ton

Ukraine: Wheat grain

70,000 ton

Brazil: Spices, Fruits

23,000 ton

South Africa: Spices

18,000 ton

Belgium: Spices, Frozen chicken

7,000 ton

Others:ca.120,000 ton

Conclusion
Food security can be improved by increasing crop productivity by using nuclear techniques,
such as mutation breeding, food irradiation, sterile insect technique, better soil management,
and new radiation produced plant growth promoter and super water absorbent.
References[1] FNCA, http://www.fnca.mext.go.jp/english/[2] IAEA., Irradiated Sewage Sludge for Application to Cropland, IAEA-TECDOC-1317,
Vienna (2002) 238 p.
[3] http://www.fnca.mext.go.jp/english/eb/e_ws_2011_3a.pdf[4] H. Kudo, F. Yoshii and M. Tamada, “FNCA guideline on development of hydrogel andoligosaccharides by radiation processing”, JAEA-Technology 2009-050(2009)[5] N. Nagasawa, T. Yagi, T. Kume and F. Yoshii, “Radiation crosslinking of carboxymethyl
starch”, Carbohydrate Polymers, 58(2), 109-113 (2004)12

2. Mutation Breeding I

2.1

World Success and Prospect of Mutation Breeding

Hirokazu Nakai
Professor Emeritus, Former Vice President, Shizuoka University
Mutation breeding has been established as one of the most important positions of breeding
method, contributing to the welfare of human being in these past about 75 years. Studies on
induced mutations in barley were started by Stadler in 1928, following Muller (1927) who
found the usefulness of radiations to induce mutations in the experiments using fruit fly. In
these years, a lot of fruits have been obtained from both practical and fundamental points ofview through the studies on mutation breeding.
In the present paper, the fruits of mutation breeding were briefly reviewed and the prospect
or new possibilities of mutation breeding were discussed as follows. (1) Fruits of mutation
breeding in the world and Asia. (2) Development of new genes for bacterial leaf blight (BLB)
resistance through the use of thermal neutrons. (3) Breeding of rice for adaptability to low
input sustainable agriculture and resistance to BLB.
(1) Fruits of mutation breeding in the world and Asia
Total number of the mutant varieties released is at least 3212 according to FAO/IAEA Database in 2011(researched by A. Tanaka)(Fig. 1). It notes that the mutation breeding would
contribute to production of the most main crops in the world, e.g. rice, barley, wheat, maize,soybean. In addition, it is recognized from the table that Asia is the center of mutation
breeding in the world. As for mutagens used for the mutation breeding, gamma-ray is
accounted for more than 60 % of all the mutagens used. This data is reasonably thought,
because gamma-ray was convenient to be treated. It is also noted that the varieties fromion-beam (high LET), are amounting to about 6 %. This data suggest that ion-beam or highLET radiations would be useful for mutation breeding in the future.
Activities of the Mutation Breeding Project promoted by FNCA (Forum for Nuclear
Cooperation in Asia) are shown in Fig.2-Fig.5. The project was started in 2002, with thesubject “Drought tolerance in Sorghum and Soybean (2002-2006), followed by the subjects“Insect Resistance in Orchids (2003-2009), “Disease Resistance in Banana (2004-2010) and“Composition or Quality Improvement in Rice (2007-2012). We are starting new subject“Mutation Breeding of Rice for Sustainable Agriculture” for the next five years (2013-2017)(Fig. 2). The most economically important crops for Asia were treated in the sub-project of
FNCA. In orchid, mutants resistant to both mites and thrips were induced by gamma-ray andion-beam (Fig. 3). It was noted that disease resistance was gained through the use of
gamma-rays and ion-beams, respectively, against Banana Bunchy Top Virus (BBTV) andfusarium wilt disease in banana (Fig. 4). The mutants in banana and orchid were successful
in technology transfer for commercial use. It was also found effective to improve quality orcomposition in rice (Fig. 5). It was proofed that semi-dwarf mutants could be easily obtained
through induced mutations. In case of the conventional cross breeding, semi-dwarf gene
14sources of rice are very limited; a majority of the genes for the semi-dwarf varieties developedin the modern breeding of rice, e.g.” green evolution”, have been found to be derived only fromthe gene of variety, Dee-geo-woo-gen. Also, a lot of varieties of crops, fruits and flowers which
are economically valuable have been reported from Japan (Fig. 6).
(2) Development of new genes for bacterial leaf blight (BLB) resistance through use of
Thermal neutrons
BLB caused by Xanthomonas campestris pv. oryzae is one of the most serious diseases intropical Asian countries. This part is dealt with the durable or non-race-specific resistance to
BLB induced by exposure of thermal neutrons to rice seeds[1, 2, 3]. It was found that thefrequency of the resistant mutants was significantly higher in thermal neutrons andion-beam than in gamma-rays and some chemical mutagens (Fig. 7, Fig. 8) [4]. This result
suggests effectiveness of high LET radiations to be used for induction of mutants resistant todiseases. The mutants obtained were resistant to all the differential BLB races from Japanand the Philippines (Fig. 9) [3]. The M95 mutant was especially noted because of the highlevel of resistance or of complete one. The gene of M95 was estimated to be located at a part ofthe edge on the chromosome 1. This type of gene has never been found in the gene sources
concerning BLB resistance. However, the M95 mutant carrying the durable resistance gene
could not be directly used as a variety to be released, since it was of dwarf. It was confirmed
through crossing tests of M95 and the original variety, Harebare, that the genes of theresistance and the dwarf were closely linked with each other. Some recombinant plants weredetected in the F2 generation derived from the cross between M95 and Harebare, indicatingthat the M95 mutant could be easily used for the crossing.
(3) Breeding of rice for adaptability to low input sustainable agriculture (Nature Farming)and resistance to BLB
We started breeding of rice for adaptability to nature farming of typical low input sustainableagriculture without any chemicals including chemical fertilizer at Ohito experimental farmand rice fields of farmers located at 18 districts across Japan in 2004. Grain yields of several
F8 lines in the nature farming were found to be significantly higher than that of the varieties
grown in the conventional farming at every district where the selection tests were conducted(Fig. 10).
In a series of the breeding, selection experiments of the lines derived from cross of Bangladesh
varieties and recombinants carrying the M95 resistance gene were conducted in Ohito
Experimental Farm, aiming at development of varieties adaptable to nature farming andresistant to BLB(Fig. 11). As for F8 lines, some of them showed high values for the main yieldcomponents, e.g. culm length, panicle length, number of panicles comparing with the parental
varieties (Fig. 12). This result suggests possibility to develop varieties adaptable to nature
farming with durable resistance to BLB. It needs to continue the selection experiments
further at later generation.

15

(4) Discussion and conclusion for prospect of mutation breeding
The rich fruits obtained in the long history of mutation breeding have proofed the merit ofmutation breeding in comparison with the conventional cross breeding. Namely, the merits ofmutation breeding are to improve easily a target character without altering the other ones, todevelop new genes without existing in natural gene sources and to improve effectively plantsof vegetative propagation. In the three merits listed above, the second item would be more
noted for promoting breeding in future in the serious situations of lacking gene sources on aworld wide scale. We must pay attention to the usefulness of mutation breeding for developing
new gene sources. Taking disease resistance for instance, resistance gene for powdery mildew
of barley, ml-o (Freisleben and Lein, 1942) is especially noted as typical durable resistancegene [5, 6, 7].
The function of the resistance gene has not yet been destroyed by any races of the disease formore than 70 years. M95 gene for resistance to BLB developed through use of thermal
neutrons (Nakai et al., 1991) is similar to the ml-o gene. However, ml-o mutants and the M95
mutant could not be directly but indirectly used, because the gene is closely linked or
pleiotropic to dwarf or to flecking of leaves [6]. Usefulness of indirect use of the induced
mutants should be much more considered for the future mutation breeding.
We are proposing new theme, ”Mutation Breeding of Rice for Sustainable Agriculture” fornext five years in FNCA Mutation Breeding Project. There might be two approaches tocomplete the breeding purpose. One is to get the resistance to environmental stresses e.g.
diseases, insects, drought, heat, coldness and so forth. The other is to obtain the adaptability
to low input sustainable agriculture, i.e. breeding of rice varieties with ability for higher yieldin the low input conditions. The results of the author’s studies or practical trials presented inthis paper as well as the fruits obtained in the history of mutation breeding would bepromising success for these two main approaches. As for the latter approach, it was madeclear in the present studies that the adaptability to low input sustainable agriculture might
be genetically controlled [8, 9, 10]. Therefore, it can be stressed that there would be a great
possibility to improve effectively the adaptability through mutation breeding, contributing tosustainable agriculture. However, scientific data are not enough on this matter at this time,and further fundamental studies are needed.
It is concluded that mutation breeding would be promising for contribution to solve theproblems of food and environments human beings are now encountering.

16

Figure 1. Mutation breeding in the world and Asia

Figure 2. FNCA mutation breeding project

Figure 3. Insect resistance to orchid (2003-2009)

Figure 4. Disease resistance in banana (2004-2010)

Figure 5. Composition or quality in rice (2007-2012)

Figure 6. Composition or quality in rice (2007-2012)17

Figure 8. Frequency of BLB resistant M3 lines of
BLB resistant mutants in each mutagen

Figure 7. Effects of mutagens use for inductionof BLB resistant mutants

F6 lines
Figure 9. BLB resistance reactions of themutants to the differential races(Okayama, 2011)

Figure 10. Grain yield of F6 lines from thecross of Takanari x Aichinokaori

Figure 12. F8 lines from cross of Binadan 5 x
the recombinant with M95 gene for BLB
resistance

Figure 11. Breeding of rice for adaptability to low
input sustainable agriculture (Nature Farming) andfor resistance to BLB (at Ohito Farm)

18

References[1] Nakai H., Kuwahara S., Nakamura K. and Saito M., A new gene developed throughmutagenesisi for resistance of rice to bacterial leaf blight(Xanthomonas campestris pv.

Oryzae), J. Agric. Camb. 114 ; 219-224, 1990.
[2] Nakai H., Shimozawa H. and Saito M., Studies on breeding of rice for resistance tobacterial leaf blight 11. Genetic analysis for some induced mutants with the durable
resistance, Jap. Jour. Breed. 41(Supplement 1), 1991.
[3] Nakai H. and Shimozawa H., A,new gene, developed through mutagenesis with thermal
neutrons, for resistance of rice to bacterial leaf blight, Proc. of International Conference of
Evolution in Beam Applications ; 565-569, JAERI, Japan, 1992.
[4] Nakai H., Watanabe H., Kobayasi W., Kitayama S and Asai T., Studies on induce
mutations by ion-beam in plants-Induced mutants of rice resistant to bacterial leaf blight,
RIKEN Accel. Pro. Rep. 26 ; 109, 1992.
[5] Wiberg A., Mutants of barley with induced resistance to powdery mildew, Hereditas 75;
83-100,1973.
[6]Jolgensen J. H., Experience and conclusions from the work at Riso on induced mutations
for powdery mildew resistance in barley, in Induced Mutations for Disease Resistance in
Crop Plants II ; 73-87, IAEA, 1983.
[7] Jorgensen J. H., Discovery, characterization and exploitation of ml-o powdery mildew
resistance in barley, Euphytica 63 ; 141-152, 1992.
[8] Hirano K., Sugiyama T., Kosugi A., Nioh I., Asai T. and Nakai H., Relationship between
number of nitrogen-fixing rhizobacteria and growth pattern of rice varieties in the nature
farming, Breeding Science 3 ; 3-12, 2001.
[9] Hirano k., Hayatsu M., Nioh I. and Nakai H., Comparison of nitrogen-fixing bacterial flora
of

rice

rhizosphere

in

the

fieldstreated

long

term

with

agrochemicals

andnon-agrochemicals ; 155-160, 2001.
[10] Nakai H., Practical value of induced mutants of rice resistant to bacterial leaf blight, in
Plant Mutation Breeding for Crop Improvement II ; 113-127. IAEA, 1991.

19

2.2

Success Story of FNCA Mutation Breeding Project in Malaysia

Dr. Rusli Bin Ibrahim, Dr. Abdul Rahim Harun and Dr. Zaiton Binti Ahmad
Agrotechnology and Biosciences Division
Malaysian Nuclear Agency
Introduction
In conventional crop breeding, promising lines are identified only by their phenotypes. Apartfrom being time consuming, conventional breeding is also capital and labor intensive.
Innovative breeding techniques, such as the use of chemical and physical mutagens for the
induction of mutations and molecular marker-assisted breeding, are required to breakthrough.
Plant breeding requires genetic variation of useful traits for crop improvement. Often,
however, desired variation is lacking. Mutagenic agents, such as radiation and certain
chemicals, then can be used to induce mutations and generate genetic variations from which
desired mutants may be selected. Mutation induction has become a proven way of creating
variation within a crop variety. It offers the possibility of inducing desired attributes thateither cannot be found in nature or have been lost during evolution. When no gene, or genes,
for resistance to a particular disease, or for tolerance to stress, can be found in the available
gene pool, plant breeders have no obvious alternative but to attempt mutation induction.
Identification of an efficient screening approach is essential in a mutation breeding approachsince a high mutation induction rate, followed by a poor screening approach could result
inefficiency and ultimately project failure. In general, selection of a trait, such as resistance todiseases, it is recommended to screen more than 1,000 plants (sometimes more than 10,000
plants) in the field or in a greenhouse to achieve the ultimate objectives. Following selection ofpotential genotype with desired traits, it is important to verify if the selected trait is heritable.
In order to assess the heritability of a newly identified mutation, it will be necessary to
conduct multi-location trials for a minimum period of 2-3 generations.
Orchid is one of the largest families of flowering plants in the world comprising of an
estimated 20,000 to 25,000 species and untold number of hybrids of some 700 different genera(Zaharah & Rozlaily, 1991). They can be found growing at almost every latitude andstratosphere, where plants can normally grow. The range of different characteristics inshapes, sizes and colors have made orchids the plant for all types of interests and sought forby collectors. Dendrobium orchids have been the major orchid cut-flower export for Malaysia
as well as for other Southeast Asian countries like Thailand and Philippines. Thailand is the
major orchid producer with an export value of US$ 80 million in 2006, or approximately 80%of the country’s total ornamental export value. The export value of orchids from Malaysia,
which is mainly in the form of cut flowers, is estimated at RM 150 million per year,20representing approximately 40% of the total floriculture production. Japan is the main
importer for orchids from Malaysia (www.tiois.doae.go.th).
The successes of hybridization technology in producing vast number of orchid hybrids withattractive characteristics and the introduction of the in vitro technology to mass-propagate
clonal planting materials have been a tremendous boost for the orchid growing industry. Widerange of successful cultivars with attractive combinations of spray length, bud number, flower
color and form, vase life, fragrance, seasonality, and compactness have been produced throughhybridization. At the same time, tissue culture has been widely used as the standard methodof germinating seeds and propagating seedlings for the industry. Through the meristem
cloning technique, thousands of plants can be cloned and grown in a relatively short period oftime. The combinations of these two technologies have so far been very reliable in supporting
the orchid industry in the region. Commercially attractive hybrids and varieties have beenable to be mass propagated and supplied to the growers for the market.
Although new orchid hybrids are consistently released every year to meet the current demandby customers, the only problem that remains unsolved in this industry until today is the
problem of insect infestations. Insect infestations in the orchid flowers have caused a lot oflosses not only to growers but also exporters due to strict quarantine regulations. In fact, since
9th Malaysia Plan, government through Agriculture Department has put an emphasis on the
production of new cut flowers which are resistant to pests and diseases (Mohd Khuzairi 2006).
For export, sometimes the presence of even one insect can cause the whole export consignment
to be rejected by importing countries. There were cases when the whole orchid consignment
had to be shipped back to the exporting countries due to the presence of insect pests.
Unfortunately, none of the commercial varieties released so far especially cut flower varieties,
has been resistant to insect pests. Growers normally have to rely heavily on pesticides to curb
the infestation. Based on visits and communications with local commercial growers, we foundout that pesticides are applied on average twice a month, and sometimes once a week if theinfestation is serious.
Within the last ten years, banana has become an important commodity in the fruit industry
and popular varieties such as Cavendish and Berangan (local cultivar) had been widely
planted on commercial scale for both local and export markets. Banana is one of the mostimportant fruit crops grown in Malaysia. Its economic potential for export, especially to China
is enormous. Currently, banana production covers a wide spectrum of systems ranging fromthe small plot subsistance farming and local market supply to plantations producing quality
fruits for export. In the world market, Cavendish still dominates because of it’s superiority interms of fruit size and yield. However, a few popular local cultivars such as Berangan, Mas
and Rastali which have unique taste, are potential alternative to Cavendish. One of the major
set back of banana industry in Malaysia is due to low yield of local cultivars compared to21

Cavendish. They are generally tall and have some other undesirable agronomic characteristics
such as thick skin and small fruit size.
Banana cultivation possesses good potential for expansion due to strong demand as a table
fruit and also for downstream activities. Banana cultivation very much supports the current
National Agriculture Policy (NAP3 1998-2010) which stresses on maximizing land use,
increased private sector involvement, increased farmer's income and export earnings. It is
ideal for intercropping with plantation crops such as oil palm and coconut. This is importantsince more than 150,000 ha of oil palm are expected to be replaced yearly for the next few
years.
In Malaysia, the most serious constraint to the production of banana and plantains is
considered to be Fusarium wilt disease caused by Fusarium oxysporium f. sp. cubense, asoil-borne disease which affects many important cultivars of banana and plantain.

The

disease has caused serious crop losses in Malaysia. Most of the cultivated clones originated asspontaneous variants are highly susceptible. Chemicals can be used to control many of thepests and diseases (not effective against Fusarium), but as the costs, both economically andenvironmentally, continue to rise, the need for resistant cultivars as the main component of an
integrated system for pest management, becomes imperative. A genetic approach to an
integrated pest management scenario, is environmentally sound for long term production.
Breeding for disease resistant banana cultivars using classical breeding methods remains adifficult task because of the high sterility and polyploidy of most edible cultivars besides long
generation times. Therefore, the efficiency of classical breeding may be improved byincorporating in vitro mutagenesis to generate genetic variability and biotechnologicalapproaches including molecular techniques. The use of shoot-tips, male flowers andsuspension cell cultures in mutation breeding of banana has offered several advantages overconventional in vivo techniques.
The use of ion beam and chronic gamma irradiation should be exploited fully in order toachieve the above objectives. Research activity had been focused mainly on screening andselection for mutants tolerance or resistance to Fusarium wilt disease using both popular
local cultivars and Cavendish types of banana. Therefore, the main objectives of this research
project is to use in vitro mutagenesis using gamma rays in order screen and select for

Fusarium wilt disease resistance in Pisang Berangan with improved agronomic traits such as
high yield, early flowering and short stature.
Rice is the staple food crop in Malaysia. In Peninsula Malaysia, rice production depends
largely on the irrigated lowland production system. Currently there are 241,741 ha ofirrigated rice in Peninsula Malaysia which contributes more than 85% of the national riceproduction. However, growing irrigated rice requires large amount of water. It was estimatedthat about 3,000 liters of water are required to produce 1 kg of rice. Unfortunately, there are22signs of declining water supply that threatens the sustainability of irrigated rice production.
Malaysia managed to achieve 72% self-sufficiency level in rice with the current average rice
yield of 3.7ton/ha/season. In this situation, about 28% of the local demand will have to dependon rice imports. However, exporting countries were not able to fulfill our rice requirement asthey also have to ensure enough supply for their own domestic use. Rice is the staple food andrice production consumes about 50% of the fresh water resources in Malaysia. In addition,
drought is one of the most important constraints in rice resulting in large yield losses andlimiting the average yield increase of the country.
There is an urgent need to enhance water-saving capacity or drought resistance of rice. New
varieties of rice with minimal water requirement can be developed by incorporating thewater-saving and drought resistance capacity mainly from the traditional upland to thecommercialized paddy rice cultivars. The breeding target is a high yield potential underirrigation, an acceptable grain quality, and water consumption reduced by about 50%
compared with paddy rice. In a water-limited environment, a higher level of drought
resistance and reduced yield loss by drought stress are required.
Plant breeding in conjunction with optimized soil nutrient and water management practices
is probably the most viable approach to stabilizing food production. Water shortage has
become the bottleneck of Malaysia’s food security. The development of water-saving rice
varieties to decrease water consumption in rice production is inevitably a major goal in
agriculture research. Thus, to achieve long-term food security and sustainable development in
Malaysia, ‘Water-saving or drought-resistance’ (WDR) rice varieties are urgently needed.
Several rice cultivars with a yield potential of over 10 t/ha in demonstration tests weredeveloped through conventional breeding. However, this high yield potential could not be
achieved in the large-scale production in the farmer’s fields. The main reason is the quality of
Malaysia’s paddy fields which cannot meet the growing conditions required for current
high-yielding rice. Drought is one of the most important limiting factors in more than 65% of
paddy fields in Malaysia where super rice varieties cannot perform well under drought stress.
Therefore, the development and production of drought-tolerant rice varieties, to stabilize andimprove the production levels in the low-middle-yielding fields, is needed. In recent years, thefield drought-resistance screening facility was established through mutation breeding
program and the evaluation standard was developed. Some advanced lines of droughttolerance rice varieties were identified and will be used in both molecular mapping andbreeding programmes. The objectives of this study, therefore, were to screen for advanced
mutant lines derived from gamma irradiation for high yield potential and stability underwater stress conditions.

23

Induction of insect resistance in Orchids
This project was a collaborative FNCA project involving Malaysia, Thailand, Indonesia andJapan. The objective was to jointly produce new orchid mutants that resistant/tolerant to
insect infestation. It focused on commercial hybrids or species of Dendrobium orchids; namely

Dendrobium Sonia 17 Red from Thailand, Dendrobium jayakarta from Indonesia andDendrobium mirbellianum from Malaysia. In this project, tissue culture orchid materials(protocorm-like-bodies or PLBs) were exchanged among the participated countries early inthe project. The Malaysian research team had irradiated these PLBs with two ionizing
mutagens; gamma rays and ion beams (JAEA). Following irradiation, regenerated plantlets
were randomly selected from each dose and subjected to in vitro infestation with mites andthrips, to analyse their resistance towards these pests. The main aim of in vitro infestation
was to pre-select potential mutants for secondary screening at flowering stage. Potential
insect resistant orchid mutants were then selected and subsequently planted in theglasshouse. Secondary screenings were carried out at flowering stage by challenge-infestation
with the target insects. This project, which was completed in December 2009, has successfully
generated two D. jayakarta mutants tolerant to thrips, and one D. mirbellianum tolerant toboth mite and thrips. The mutants are now being propagated to achieve large number ofclones, with hope to carry out pre-commercialization studies with a private collaborator(s) in anear future.
Multiplication work is being carried out for our mutants in the glasshouse. We hope totransfer some mutant samples to a private collaborator soon. At present, we have sign a
Non-Disclosure Agreement and Collaborative Agreement with the collaborator (Hexagon
Green Sdn Bhd) for a project on “Pre-commercialization of Mutant Orchids for Cut Flower
Industry” which was financed by Ministry of Agriculture and Agro-Based Industry, Malaysia.
This project will cover selected orchid mutants developed by Nuclear Malaysia, including ourpotential insect tolerant mutants.

Figure 1. D.jayakarta mutants tolerant to thrips24Figure 2. D. mirbeliannum mutant tolerant to both mites and thrips

Improvement of Bananas for Fusarium wilt Resistance and High Fruit Quality ThroughMutation Induction.
Popular local banana cultivar called Berangan (Musa spp. AAA) was selected as the starting
material for mutation induction using gamma rays. Radiosensitivity test was carried out by
irradiating meristem tissues obtained from suckers with a series of gamma ray doses of 0, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100Gy. Data for radiosensitivity test was determined by 3
parameters, such as 1) Plant height (shoot length), 2) Percentage of survival of irradiatedexplants and 3) Multiplication or Growth Rate. Based on the percentage survival of irradiatedexplants, LD50 and LD100 obtained for cultivar Berangan were 50Gy and 80Gy respectively.
Using selected effective doses of 20, 30, and 40Gy, all in vitro shoots derived from irradiatedmeristems were subcultured from M1V1 upto M1V5 and rooted. These rooted in vitro
plantlets were used to screen for tolerance/resistance against Fusarium wilt disease. Fourartificial screening techniques for Fusarium wilt disease had been developed, such as 1)
Dipping of in vitro shoots in Fusarium oxysporioum suspension (106 spores/ml) for 1-2 hrs
and later transferred to sterile sand media in the greenhouse, 2) Double tray method by
inoculating in vitro plantlets that had been planted in sand media, 3) Nursery screening
method, whereby rooted in vitro plantlets that had been hardened for 4-8 weeks weretransferred to media that had been pre-inoculated with Fusarium oxysporioum spore
suspension (106 spores/ml) for 2 weeks and 4) Direct field screening, whereby rooted plantlets
that had been dipped in Fusarium oxysporioum suspension (106 spores/ml) for 1-2 hrs wereplanted directly in the field. Based on the results obtained after screening of treated plants
using artificial inoculation of Fusarium oxysporioum in the field, three potential mutants had
been identified, such as 1) Tolerant/resistant mutant, 2) High yielding mutant and 3) Early
flowering mutant. These mutants were further screened in the field for multi-location trials.
Memorandum of Agreement with a commercial grower, Selamat Indah Sdn. Bhd. had beensuccessfully made for the technology transfer of tissue culture technique for micropropagationof Pisang Berangan and commercialization of selected mutants.

25

Mutation Breeding for Varietal Improvement of Irrigated Rice under Minimal Water
Conditions.
Mutants were induced in variety MR219 using gamma rays at 300 Gy to generate superior
genotypes for minimal water requirement. The mutants were screened for water stress undersimulated non-flooded water regime under glasshouse and field conditions. After several
series of selection and fixation, 12 potential lines with the required adaptive traits wererecovered at M4 generation. However, only two potential lines designated as MR219-4 andMR219-9 were selected. The lines were evaluated under replicated yield trial in KETARA
under normal flooded conditions. In KETARA, the yield of MR219-4 and MR219-9 were 4.8
t/ha and 5.5 t/ha, respectively as compared to the 5.8 t/ha of the original variety MR219. Thelines were also evaluated in MADA under saturated soil and flooded conditions. The yield of
MR219-4 was 5.9 t/ha under saturated soil condition and 7.2 t/ha under flooded condition.
For MR219-9, the yield was 6.8 t/ha and 6.1 t/ha under saturated and flooded condition,
respectively. The lines also performed satisfactorily when grown under aerobic soil conditionin MARDI Seberang Perai. Grain yield as high as 6.3 t/ha for MR219-4 and 3.4 t/ha for
MR219-9 were achieved under aerobic condition. In another development, submergencescreening was done to the same two lines and a new adaptive trait was discovered where both
MR219-4 and MR219-9 were found to be tolerant to submergence. These findings indicatedthat these two mutant lines are unique because they possess “dual” adaptations ie. flooded
and water stress regimes. The results also indicated that tolerance to submergence in rice isassociated until 8 days submergence for MR219-4 and FR13A cultivars, but mechanism in
MR219-9 cultivar is associated until 12 days submergence. These findings suggested thattolerance to submergence stress in rice may be improved by increasing the capacity ofantioxidative system.
Mutant MR219-4 was unique because it performed very well under saturated conditions in
irrigated areas and aerobic conditions (sprinkler assisted irrigation) under dryland regime. In
addition, both mutants can also tolerate submergence and therefore can be planted in floodprone rainfed areas. The superior adaptation and yield performance of mutant MR219-4
under aerobic condition was obviously a very interesting finding because its parent, MR219
has never been recommended for aerobic soil . Further research on the uniqueness of Mutant
MR219-4 should be carried out especially on the root system and water use efficiency. Basedon the overall performance, the mutants should be tested further for potential yield andadaptation under Local Verification Yield Trials in stress prone rice areas (non-granary andmarginal granary rice areas).

26

Figure 3. Mutant Line of MR219-4
Table 1: Yield and yield components of MR219-4 and MR219-9 in Muda (direct seeding method)

Yield component
Treatment

Yield (kg/ha

Saturated
FloodedMR 219 – 4
MR 219 – 9
MR 220
Water
Management x
variety6480 a6799 a6542 a6460 a6815 ans

Spikelets/p
Panicle /sq.m
anicle
503 a
500 a
447 a
512 a
517 a
ns

107 a
104 a
107 a
99 a
109 a
ns

Percent
filledgrain

1000grain wt(g)

68.2 a70.2 a73.3 a77.7 a61.7 bns

28.4 a28.3 a29.0 a28.9 a28.1 bns

Plantheight(cm)
97.5 a95.1 b97.1 a98.6 a95.4 ans

Table 2. Growth and yield performance of MR219-4 and MR219-9 in KETARA(direct seeding method).

Parameters
Panicles /plant
Plant ht
No. of tiller/hill
Percent Filled Grain
1000 grain wt(gm)Grain Length(mm)Grain width (mm)Panicle length
Yield (kg)Maturity

MR219-4

MR219-9

MR219

Difference Over parent

6.5
100.1
9
78.1
30.3
9.69
2.38
n.a
5259
110

7.4
100.4
8
87.7
30.3
9.69
2.41
n.a
6000
112

9
103.4
9
73.4
28
9.86
2.35
n.a
6444
116

lower
lower
lower
higherhigherlower
higher
n.a
lower
lower

27

2.3

Molecular Genetic Features of Induced Mutations in Rice and Soybean

Qing-Yao Shu
Institute of Nuclear Agricultural Sciences, Zhejiang University
Summary
Identification of mutated genes and molecular characterization of mutant alleles enabled
geneticists for the first time to examine the features of induced mutations, which have been
widely used in crop improvement in the past century. In my presentation, the mutated traits
induced by gamma rays will be first introduced together with their uses in rice and soybean
breeding, followed by the description of the general processes used for mutant gene
identification. The molecular features of 9 mutated genes will be elaborated together with
their effect on gene function, gene splicing and gene expression.
A thorough understanding of molecular features of both induced mutants and mutations
underlying a particular trait is essential for developing proper strategies for gene discovery
using gamma rays induced mutants. This includes three aspects: The characteristics of
induced mutant versus its parent; the overall frequency of mutations in a given mutant
genome; the nature of DNA lesions caused by gamma rays. Although these issues have notbeen fully investigated, some features have already been reported.
1. Induced Mutants and Parental Line
Mutated genes are eventually identified by gene sequence comparing between mutant and its
parent with in a targeted genomic region. Therefore, it is of paramount importance to ensurethat the mutant is originated directly from the parent through induced mutagenesis. M1plants usually have reduced pollen fertility and hence often tend to out-crossing, which can
result in genetic contamination in M2 population. It is recommended that extreme care istaken to protect M1 plants from pollens of other varieties through proper isolation whengrown in fields, and avoid other sources of mixing. However, it is practically impossible toexclude all chances of contamination, especially when the material is grown out on a large
scale in the field [4].
The selection process for mutants is indeed a process of selecting any plants that differ fromits parent variety. Therefore, the probability of selecting a contaminant as ‘mutant’ is quite
high. A selected putative ‘mutant’ needs to be verified through progeny test and subject toscrutiny using breeder’s knowledge, expertise and experience. However, such a process cannot
always lead to a scientifically correct judgment due to the potential pleiotropic effect of amutation, which can make a mutant quite different from its parent at phenotypic level.
Recently, it has recently been proven that γ rays induced mutants have microsatellite (SSR)
haplotypes highly similar to their parent. Based on the finding, it is recommended to use

28comparative SSR analysis for discriminating false mutants from true mutants [4], which havebeen nicely demonstrated in rice [5].
2. Mutation frequencies
In the past, mutation frequencies have been estimated by observing phenotypic mutants, e.g.
chlorophyll deficient, plant height, sterility etc, and expressed either by the percentage ofindividual M2 mutants or by the percentage of mutated M2 panicle rows, in which at least one
mutant is observed. Therefore, mutation frequencies vary between different traits and among
different M2 populations derived from different mutagenic treatments.
In molecular studies, the estimation of mutation frequency is performed in a different way,that is, the frequency of induced mutation is estimated by assessing DNA lesions in selected
DNA fragments of M2 plants. If the total size of assessed DNA fragment is expressed as i kilobases (kb), the total number of M2 individuals as j, and the total number of DNA lesions
identified as n, then the mutation frequency (f) is expressed as the number of mutations per
1,000 kb for ease of comparison:f = (n x 1000)/ (i x j)
This formula can be manipulated easily to determine mutation density, that is, one mutation
per a set number of DNA kilo bases.
Sato et al. [6] screened a gamma-ray-irradiated rice population by mismatch cleavage
analysis using Brassica petiole extracts. Analyses of 25 gene fragments (1.23 kb length on
average) of 2,130 M2 plants revealed six mutants. The rate of mutation induced by gamma
rays was estimated to be one mutation per 6,190 kb [6], which is about 2% of that reported in
MNU-derived rice M2 populations [7].
3. Nature of DNA lesions in induced mutants
A large amount of data on molecular features of induced mutations have become available
during the last decade; they are either from TILLING analyses of M2 (or M3 in a few cases)
plants or discovered after a mutated gene is cloned through forward genetics schemes. In
general, most mutations caused by chemical mutagens, particularly those of alkylating agents,
are base pair substitutions. However, small deletions are also infrequently identified inprogenies derived from chemical mutagenesis. In contrast to the large data available forchemical mutagenesis, molecular characterization of mutations induced by different types ofradiation remains limited. Available data indicate that mutations induced by radiations
consist of both deletions and base pair substitutions. During the past few years, we haveidentified about a dozen γ rays induced mutations in rice and identified that most of them aresmall deletions (1-6 bp), with one mutation being a ~1450 bp deletion.
Our finding in rice for γ rays induced mutations is similar to carbon ions induced mutations in
M2 plants of Arabidopsis, where majority of mutations containing 1- or 4- bp deletions [6].
29Large deletions often have significant effects on gamete development or survival and hence
the majority of them are not transmitted to M2 progeny.
4. Effect of Induced Mutations
The DNA lesions caused by induced mutation can cause various effects on gene expression
and function. In most cases gamma rays induced mutations are deletions, which can oftencause frameshift and produce truncated non-functional proteins [8-9]; In certain cases, we
also observed mutations that cause splicing changes and whole exon exclusion in cDNA [10].
We also identified a mutation that resulted from an insertion of a retrotransposon and was
down-regulated in expression.
References[1] M. C. Kharkwal and Q. Y. Shu, The role of induced mutations in world food security, Q.Y.
Shu, Ed. Induced Plant Mutations in the Genomics Era Rome, Italy: FAO, 2009.
[2] A. Krishnan, E. Guiderdoni, G. An, Y. C. Hsing, C. D. Han, M. C. Lee, S. M. Yu, N.
Upadhyaya, S. Ramachandran, Q. Zhang, V. Sundaresan, H. Hirochika, H. Leung and A.
Pereira, “Mutant resources in rice for functional genomics of the grasses”, Plant Physiol.,vol. 149, pp165-170, 2009[3] The TAIR website. [Online]. Available: http://www.arabidopsis.org/index.jsp[4] H. W. Fu, Y. F. Li, Q. Y. Shu, “A revisit of mutation induction by gamma rays in rice
(Oryza sativa L.): implications of microsatellite markers for quality control”, Mol. Breed.,vol. 22, pp 281-288, 2008.
[5] H. W. Fu, C. X. Wang, X. L. Shu, Y. F. Li, D. X. Wu, Q. Y. Shu, “Microsatellite analysis forrevealing parentage of gamma ray-induced mutants in rice (Oryza sativa L.)”, Isrl. J.

Plant Ssi., vol. 55, pp201-206, 2007.
[6] Y. Sato, K. Shirasawa, Y. Takahashi, M. Nishimura and T. Nishio, “Mutant selection fromprogeny of gamma-ray-irradiated rice by DNA heteroduplex cleavage using Brassica
petiole extract, Breed. Sci., vol. 56, pp179-183, 2006.
[7] T. Suzuki, M. Eiguchi, T. Kumamaru, H. Satoh, H. Matsusaka, K. Moriguchi, Y. Nagato
and N. Kurata, “MNU-induced mutant pools and high performance TILLING enablefinding of any gene mutation in rice”, Mol. Genet. Genomics, vol. 279, pp213-223, 2008.
[8] X. H. Xu, H. J. Zhao, ·Q. L. Liu, T. Frank, K. H. Engel, G. An, Q. Y. Shu, “Mutations of themulti-drug resistance-associated protein ABC transporter gene 5 result in reduction ofphytic acid in rice seeds”, Theor. Appl. Genet., vol. 117, pp1291–1301, 2008[9] H. J. Zhao, Q. L. Liu, X. L. Ren, D. X. Wu, Q. Y. Shu, “Gene identification andallele-specific marker development for two allelic low phytic acid mutations in rice (Oryza

sativa L.)”. Mol. Breed., vol. 22 , pp 603-612, 2008.
[10] F.J.

Yuan, D.H. Zhu, Y.Y. Tan, D.K. Dong, X.J. Fu, S.L. Zhu, B.Q. Li, Q.Y. Shu,

“Identification and characterization of the soybean IPK1 ortholog of a low phytic acidmutant reveals an exon-excluding splice-site mutation:, Theor Appl Genet vol 125, pp1413-1423, 201230

2.4 Success Story of Development of High Yield and Quality Rice Variety Grown
in Vietnam
Dao Thi Thanh Bang and Le Huy Ham
Institute of Agriculture Genetics, Vietnam
1. Introduction
In Vietnam, rice plays an important role for national food security and political stability. Ricealso has a direct effect on social security because it is consumed by nearly 89 million of thetotal population and an important source of income for more than 60 million people living in
agricultural and rural areas. Rice is the country’s main crop, accounting for more than 90% of
total cereal productivity. Rice productivity in 2009 reached 38.89 million tons, about 16.89
million tons higher than in 1995 (General Statistics Office 2009), and in 2012, it is 21.7
million tons higher than in 1995 (Out look 2013). Since the 1990s, the volume of rice exports
has risen dramatically, making Viet Nam the second largest rice exporter in the world.
Nevertheless, serious concerns for food security still remain. Vietnam is one of the top 5
countries that studies reveal will be severely impacted by climate change (Ministry of Natural
Resources and Environment 2009). A rise in sea level of 1 meter will inundate about 5,000
square kilometers (km2) of the Red River Delta and 15,000 – 20,000km2 of the Mekong River
Delta, reducing total rice productivity in Vietnam by about 5 million tons. Bad harvests,
natural calamities, floods, and pests and diseases will also occur more often. Ensuring
domestic food security has thus become a national objective that requires long-term strategies
and policies, especially for the protection of the agricultural land area. Viet Nam does not only
seek to achieve domestic food security but it also plays an important role in the international
rice market, and consequently, in the food security of the international community.
After 26 years innovation politics (1986- 2012), it had a remarkable achievement due to theapplication of advanced science and technology such as the introduction of new rice varieties,
new production models, and an efficient irrigation system. Vietnam reached high speed ofincreasing in agriculture, especially in food production. Food production of Vietnam is not
only enough for domestic consumption but also for exportation. In the year of 2012, riceproduction area of whole country reached around 7.76 mil.ha, increased 117,000ha compared
to 2011. In 2012, rice productivity estimated 43.96 mil.tons increases 1.64 mil. tons compared
to 2011, and furthermore the number of rice for exportation reached 8.0 mil tons impressively.
2. Rice Situation
Despite of a decrease in rice land area, rice productivity has been rising due to rapid yield
growth. This in turn has been driven by irrigation and land development, together withtechnological change. In 1999, Viet Nam had nearly 4.5 million hectares (ha) of rice lands,
which declined to 4 million ha in 2010. However, in 2009, the rice planted area reached 7.43
million ha because local farmers owning large tracts of land planted three rice crops. As aresult, rice productivity increased from 32 million tons in 1999 to more than 38 million tons in
31

2009. It was a remarkable achievement due to the application of advanced science andtechnology such as the introduction of different rice varieties, new production models, and an
efficient irrigation system. Post harvest losses have also been reduced greatly due to themechanization of rice harvesting and drying, and soil improvement.
According to GSO (General Statistics Organization), the yields of the three crops haveincreased gradually from 1995 to 2009. In 2009, the yields of the summer–autumn andautumn–winter crops were about 4.3 – 4.7 tons/ha, while the yield of the winter–spring crop
was more than 6.1 tons/ha. In the past years, Viet Nam has significantly increased its rice
yield due to the shift from low-yield (1.5 – 2 tons/ha) to high-yield rice varieties (6.8 tons/ha),which can be grown in a shorter time (85 – 100 days/crop) and produce 2 – 3 crops/year. The
rice cultivation area has also widened because of investments in irrigation and drainage, andaluminum and salt removal. The performance and productivity of rice production in Vietnam
is increasingly within last decade (diagram 1 and 2).
Diagram 1. Rice productivity from 1878-2012 of Vietnam
42.3

45
40

38.7

38.89

2008

2009

43.7

40

35.8
32.5

35
30

Productivity 25(mil.tons) 20

22
15.9

15.9

15

9.5
10

5.5

5.2

1878

1930

6.7

4.9

10.5

10.3

1976

1980

6.1

5
0
1939

1944

1955

1960

1985

1990

1995

2000

2005

2010

2011

Year

Diagram 2. Yield performance of rice production from year 1878-2012
6
5.2 5.23 5.31

5.53 5.65

4.9

5
4.3
3.7

4
3.2

Yield
(tons/ha)

2.8

3

2.4
2.1

2

2
1.2

1.2

1.3

1.4
1.1

1
0

1878

1939

1955

1976

1985

1995
Year

32

2005

2009

2011

2012

Diagram 3. Rice production area from year 1878-2012

7.7

8

7.3

7.44

7.7

7.76

7.35

6.7

7
6
5
Area
(mil.ha) 4

7.4

5.3
4.6

4.3

4.3

5.6

5.7

6

4.8

4.6
4.1

3
2
1
0
1878 1930 1939 1944 1955 1960 1976 1980 1985 1990 1995 2000 2005 2008 2009 2010 2011 2012Year

The diagrams showed that the increasing of average yield and rice productivity in Vietnam
mostly from 2008-up to now. The main reason resulting to success of Vietnam in riceproduction is: the water management of irrigation system is making provide water for 3.45mil
ha agriculture production in which 85% rice production area, more than 90% rice production
area use new varieties and applying new technology for cultivation, especially in northVietnam in spring season.
3. Mutation Breeding in Vietnam
At present, about 15% of rice production area is covered annually by mutant varieties inVietnam. With the great achievement by the use of nuclear techniques and related
biotechniques, more than 50 mutant varieties were developed, in which most of varieties are
cereal crops, especially rice. Since the 1990s, the Agricultural Genetics Institute (AGI),
Vietnam Academy of Agricultural Sciences (VAAS), has been engaged in R&D activities andcontributed much to the technology transfer to the agricultural sector of Vietnam. Mutation
breeding is one of the major fields of institute in crop improvement, in which the biggest
accomplishments have been achieved in the development of mutation varieties for crops
production. Before 2000, objective of mutation breeding is often quantity of yield variety. Onevery interesting example from AGI is DT10 mutant variety which has been certified asnational variety in 1990 due to the high performance, good tolerant to harmful condition. Thisvariety is available in rice more than 20 years and now this is still disseminating in somespecific location. This variety covers the area around 1.0 mil. ha in agriculture production..
After success of mutant variety DT10, scientists from AGI released series of mutant varieties(17 mutant varieties) and satisfied production demand (index 1). Khangdan mutant variety
has been transferred copyright from AGI to the seed company since 2008 after it was certified
as national variety. With some advantages, the area covered with this variety is around 300
thousands ha/year in North Vietnam. In 2012, the seed company is rewarded the national
33

prize for Khangdan mutant variety due to the leading area for agriculture production of thisvariety. Recently, beside of yield varieties, scientists has been concentrating creation of newvarieties having good performance on quality (aroma, protein, amylase content, component ofquality), as well as tolerance to harmful condition of environment such as salinity, cold or hightemperature, drought, lodging variety and so on. By the year of 2008, there are several
outstanding new varieties have been certified as new varieties and ongoing projects to enlarge
mutation varieties in different provinces.
Table 1. Leading mutant varieties in rice production in north of Vietnam (duration 2008-2013)

VarietyKhangdanYear ofcertify
2008

mutantPD2

Area of

Dominant characters

production/year (ha)High yield, good tolerant to pest and

300,000

disease2010

Glutinous rice, photo sensitiveness, high

10,000yield, aroma
P6 mutant

2011

Short growth duration 85-90 days in

15,000summer season, high temperature tolerant
Nam dinh 52012

Quality, aroma, high yield

3,500

ĐB5

2008

High yield, good tolerant to pest and

5,000

diseaseĐB6

2008

High yield, good tolerant to pest and

5,000

diseaseDT39
Quelam2013

Quality,highprotein,highyield,

100

resistant to leaf blight

In the South Vietnam, mutation induction has been carried out mostly in Cuulong Rice
Research Institute and Institute of Agriculture South Science (IASS). These institutes havealso succeeded in the mutation breeding by the use of nuclear techniques. Over the past
decade, these institutes have developed eight excellent mutant varieties with high yielding,
improved quality, disease resistance, tolerance to pest and lodging resistance. Since 2008, thetotal cultivated area of these mutant varieties was more than 4 million ha in southern
Vietnam. According to the statistics from the institutes, these mutant rice varieties haveincreased income by about $374 million at the end of 2008. One of these excellent mutant
varieties (VND9s-20) became one of the top five varieties for rice production. It covered 300
thousand ha per year in south of Vietnam, due to its high yielding, good quality and tolerance
to brown plant hopper. Not only has this variety been widely cultivated in the low lands, butalso expanded to the high lands and remote mountain areas, where poor farmers are
benefiting from growing it. It is very clear that the great support from the Vietnamese
government makes the application of nuclear techniques in food and agriculture so successful.
Due to the excellent performance of the mutant rice varieties mentioned above, some of these34

mutant varieties were adopted for the national strategy program of “Eradicate hunger andalleviate poverty” in different areas, particularly for central highland region, where there aremany ethnic minorities living in remote mountain areas. Most of the eight mutant varieties
have shown out-standing performance under local production conditions. These promisingvarieties were identified in the trials with improved characters, such as high yielding,increased disease and insect resistance/tolerance, short growth duration, good quality andbetter adaptation to environment stress. Some of mutant varieties (VND 95-20, VND 99-3,
and VN121) have increased more than 30% of yield, compared to the control of the local
variety. These mutant varieties have been well accepted by local farmers and will becultivated in expanding areas.
Table 2. Economic impact of mutant rice varieties in the South Vietnam (Do KhacThinh, 2010)

Varieties

ApprovedinAnnualarea

Value
added/year
Billion VND

VND 95-20

1999

300,000

600

Wide adaptation, high quality

VND 99-3

2004

20,00040

HighCharacters

productivity,

resistanttoaluminum soil
TNDB 100

1997

30,00060

Short growth duration, high quality

OM 2717

2004

30,00060

Short growth duration, resistant tobrown plant hoppers

OM 2718

2004

30,00060

Short growth duration, resistant tobrown plant hoppers

OM 2496

2009

3000

6

High productivity, Aroma, Resistant
to Salinity

VN 121

2007

3000

6

Aroma, high production.

VN 124

2007

2000

4

Aroma, High production.

Total

-

418,000

836 = 45 mil.

-

USD
Successful story of mutation plant breeding in Vietnam is contributed from political support
by Vietnamese government for mutation plant breeding research and internationalorganization such as FAO/IAEA, through project IAEA/VIE/05/13/14 from 1997-2003. On theother hand Vietnam became official member of Forum for Nuclear Cooperation in Asia
(FNCA) in 1997. Scientists from different institutions have chance to go abroad and attend
training course, scientific symposium and workshop, or to exchange information and35

experiences with scientists working in mutation breeding.
4. Conclusion
It’s clear that mutation induction nowadays is a powerful tool for crops improvement in
Vietnam. Mutant rice varieties from institutions contribute to increase of rice performanceand productivity in recent years. This is confirmed that Vietnam Government has been
concentrating research and development of the activities on nuclear techniques in agriculture.
Beside of that farmers also benefit from scientific research activities.
References[1] Thinh Do Khac et al., 2008. Socio-Economic impacts of mutant rice varieties in Southern
Vietnam, reported at International Symposium on Induced Mutation in Plant organized by
FAO/IAEA 12-15 August, 2008-Vienna, Austria.
[2] Vietnam Agriculture newspaper 26 Dec. 2012: Rice production and exportation in Vietnam
2012.
[3] Results of the 2011 Rural, Agriculture and fishery sensus, Statistical publishinghouse-2012.
[4] Induced Plant Mutations in the genomics Era. Edited by Q.Y.Shu. 2009 “International
Symposium on induced Mutations in Plants”.
[5] Dao Thanh Bang et al. The national symposium for nuclear technology the nine
Ninhthuan, province 18-19 Aug. 2011.” Mutation breeding a powerful tool for crop
improvement”[6] Nguyen Huu Dong, Lam Quang Du, Dao Thanh Bang. Mutation: Theory and practice1997.
[7] http://www.go.vn/diendan/showthread.php?758999- Vietnam overview.

363. Mutation Breeding II

3.1 Development of Disease Resistant Banana by Mutation Breeding:Resistance Response of Selected Mutant Lines of Banana cv. Lakatan (AA)Against Banana Bunchy Top Virus (BBTV)
Olivia P. Damasco, Fe M. Dela Cueva, Ma Luz J. Sison, Eric G. Dinglasan, and Charlemagne
Alexander A. Lim
Crop Science Cluster, Institute of Plant Breeding, College of Agriculture, University of thePhilippines Los Baños
Development of BBTV resistant Lakatan through gamma-irradiation had been successfully
done as part of integrated management strategies against banana bunchy top disease(BBTD). Based on BBTV incidence in the field, % BBTV-free fruiting plants, aphid preference,and host reaction to the virus, ten mutant lines namely lines 13-30-2, 7-29-1, 22-28-2,
23-28-7, 6-30-2, 9-28-2, 9-28-3, 9-29-1, 23-30-2, and 28-30-2 were selected after four
generations of selection and evaluation. Insect preference to Lakatan mutant lines wasmonitored by artificially inoculating viruliferous banana aphids, Pentalonia nigronervosa,and counting, at weekly interval, the number of aphid per plant. Disease resistance on virus
multiplication of Lakatan mutant lines was characterized by quantifying the virus titerthrough Enzyme-Linked Immunosorbent Assay (ELISA). Results showed that not all lines
were preferred by aphids as a host for establishing a population, and generally, disease
incidence was correlated with aphid preference. Disease incidence was significantly higher(>50%) on lines that were preferred by aphids and lower (<50%) on those that were notcolonized. Virus titer was also reduced on these lines, thus reduced virus multiplication. Non
irradiated (control) Lakatan banana had comparably high population of aphid, high disease
incidence, and high virus titer. The resistance of Lakatan mutant lines to BBTD could be
attributed to non-preference of the aphid to the vector to colonize and reproduce thus
attributed to lower BBTD infection. In addition, reduced virus multiplication indicative of
low titer on mutant lines indicated a resistance response to the disease. The top five resistantmutant evaluated in multi-location sites showed low incidences of the BBTV in all
multi-location trial sites, despite the presence of infected plants and vectors in the area andwithin the vicinity of trial site. The mutant lines were differentiated from the LK control
using nine Simple Sequence Repeats (SSR) primers by the absence of 1 or 2 alleles detected
in LK control.

Keywords: gamma-irradiation, mutant Lakatan, banana bunchy top virus (BBTV) ELISA,
aphid preference, virus multiplication
Introduction
Banana (Musa sp), remains the country’s one leading fruit crop in terms of area, volume, andvalue production, and export earner. The total arable land planted with banana is more than
400,000 has (BAS, 2008). National average yield reaches to 9.4 tons/ha while corporate
plantations contribute to 40 tons/ha (Rivera, 2004). Lakatan is the most popular and38

preferred fresh table banana. Lakatan is normally grown on smallholdings in the livelihoodof many growers in the country. As such, Lakatan has a significantly higher market potential
given higher production (Aguiba, 2010).
Still, the most important biotic constraint in banana production is the occurrence and spreadof Banana Bunchy Top Disease (BBTD) caused by Banana bunchy top virus (BBTV). BBTV isspread from plant to plant by the banana aphid, Pentalonia nigronervosa, and is transmitted
in a persistent, and circulative but non-propagative manner (Hafner, 1995; Hu et. al., 1996).
Initial symptoms on BBTV-infected plants include marginal chlorosis, and dark green
streaks or dots on leaf veins, midrib and petiole. It later progresses into rosetting wherein
emerging leaves are narrower than normal with distinct marginal chlorosis and appear as“bunchy-top.” Stunted growth is observed on severely infected plants. Thus it is considered as
the most devastating virus disease of banana worldwide that can result in a 100% yield loss
since severely infected plants do not produce fruits.
While efforts are being done to come-up with a sound and effective management and control
of BBTD including eradication of infected planting materials, control of the aphid vector andwidespread use of agrochemicals; still the disease has spread dramatically over time in any
place where bananas and plantains are grown. The persistent use of these agrochemicals, inaddition, can pose serious threat to human health and environment.
BBTV resistance could not be introgressed into banana by conventional breeding methods
because edible bananas are technically not amenable to sexual breeding since they are maleand female sterile. Thus, resistance could be obtained only through biotechnology-assisted
method like mutation breeding using irradiation and chemical mutagens, somaclonalvariation, or through a direct method of in-vitro selection in the presence of stress factor.
Using gamma irradiation and in-vitro induced mutation, mutant lines and somaclonalvariants of Lakatan with varying degree of resistance and tolerance to BBTV have been
produced by the team of Damasco (2009) in IPB-CSC, CA, UPLB. The stability of BBTV
resistance in selected mutant lines has been continuously expressed in succeeding
generations. The mechanism and components of resistance of selected mutant lines against
BBTV were determined. Molecular characterization using SSR (Simple Sequence Repeats)
was also done to determine the genetic variation of irradiated lines.
Materials and Methods(1) Selection of BBTV resistant lines
Ten (10) mutant lines were selected based on the reaction on BBTV infection, preferential
studies of the insect vector, Pentalonia nigronervosa, and agronomic characteristics after
several generations of selection and evaluation in the greenhouse and the field. These lines39

were further evaluated for confirmatory studies to ascertain the stability and durability ofresistance or tolerance against BBTV infection.
(2) Resistance to P. nigronervosa: Aphid preference
Preferential studies were conducted at two different feeding procedures: by free choice
feeding or by artificial inoculation using the no choice feeding (forced feeding) method. The
aphids were allowed to colonize and multiply on the lines. Data on aphid population on the
different lines were collected at weekly interval starting from the 1st week after artificial
inoculation.
(3) Resistance to BBTV: Virus multiplication
Symptomatology, incubation period and percent incidence of BBTV on inoculated lines wereobserved and virus multiplication based on titer was quantified through Enzyme-linked
Immunosorbent Assay (ELISA). Capture and conjugate antibodies against BBrMV (Agdia)
were used in double antibody sandwich ELISA following the manufacturer’s procedure andby Clark and Adams method (1977) with some modifications. Absorbance at 405nm wasmeasured using an ELISA reader (Bio-Rad). All reactions were considered positive only if theabsorbance was equal to or above the threshold value, which is two times the averageabsorbance of the negative control.
(4) Multi-location field evaluation for disease resistance and agronomic traits
Micropropagated plantlets from top five mutant lines and LK control were established in the
farmer’s field trial sites following the recommended cultural management practices for
growing Lakatan. Monitoring for BBTV incidence and other diseases was conducted
regularly. Agronomic and yield parameters were taken at harvest.
(5) Simple Sequence Repeats (SSR) Analysis
Banana genomic DNA from each mutant line was extracted from young leaves using the
CTAB DNA extraction procedure of Doyle and Doyle (1990). A total 34 SSR banana primer
pairs were tested. All PCR reactions were performed following the published procedure.
Amplified fragments were scored for the presence (1) or absence (0) of bands in all mutant
lines.
Results and Discussion(1) Selection of BBTV resistant lines
Ten mutant lines of Lakatan (namely 28-30-2, 13-30-2, 9-28-2, 22-28-2, 9-28-3, 6-30-2, 7-29-1,
9-29-1, 23-28-7, and 23-30-2).with the best reaction on BBTV infection and aphid preference
were selected after four generations of evaluation and selection. These lines, generally, had
high percentage of fruiting plants free from BBTD at 11MAP compared to the control
Lakatan. In terms of aphid preference, initial studies on free choice feeding showed that all
the lines tested had low aphid colony count. On the other hand, BBTV incidence was40comparably lower in mutant lines ranging from 3 to 67%, in contrast to high BBTV incidence
in control Lakatan (81%).
(2) Insect vector – host relationship: Aphid preference
The aphid population taken at the peak of colonization, 3 weeks after artificial inoculation, ina free choice feeding method was significantly lower in some mutant lines (9-28-2, 9-28-3,
9-29-1 and 13-30-2) compared with control Lakatan, indicating that these lines were notpreferred by the aphids as host plants (Table 1). A very high population of aphid wasobserved on control Lakatan when no choice (forced) feeding method was utilized (Table 1).
The result of the no choice (forced) feeding experiment confirmed that mutant lines13-30-2,
9-28-2, and 9-28-3 were not preferred by the aphids. The aphid colony counts on these lines
were statistically lower than control Lakatan. In addition, lines 22-28-2 and 23-28-7 whichwere not tested with free choice feeding were likewise significantly less preferred by theaphid vector.
(3) Virus – host relationship: Symptomatology and Virus Multiplication
Table 2 presents the reaction of ten selected lines to BBTV infection under greenhousecondition. Generally, symptoms started to appear at 4 weeks after inoculation as dark green
streaks or dots on petiole extending to midrib. Marginal chlorosis and “bunchy top”
appearance having narrow and stiff leaves appeared during symptom progression. All lines,
except 7-29-1 (58%) and 23-30-2 (71%), had low incidence of BBTV compared to the Lakatan
control plants (58%) (Table 1). Line 28-30-2 (20%) had the lowest incidence of the disease incontrast to 23-20-2 (71%) with the highest incidence. In terms of incubation period, 28-30-2
and 13-30-2 had the longest period (6 weeks after inoculation) for symptom expression tovisibly express. Likewise, 13-30-2 and 22-28-2 were observed to express a delay of symptom
development which started at 5 weeks after inoculation. In virus multiplication, ELISA
result showed that all lines had the minimum OD reading lower than that of the control
plants, except line 9-29-1. On the other hand, only 13-30-2, 9-28-3, and 7-29-1 were observed
to have low virus concentration at maximum OD. Line 13-30-2 had the lowest value of 0.956.
This implies that the virus titer is lower on mutant lines compared with control Lakatan,suggesting a degree of resistance.
(4) Vector – Virus – Host Relationship
In a free choice feeding of the vector (Table 2), lines 6-30-2, 13-30-2, 23-28-7, 28-30-2 showed
low BBTD incidence (<50%) despite the preference of the vector to the host (high aphid colony
count). These results suggest that the mechanism of resistance could be due to the resistanceof the host to the virus. On the other hand, lines 9-28-2 and 7-29-1 which were less preferredby the aphids also had correspondingly low disease incidence which suggests that theresistance mechanism could be due to the less preference of aphids to the host.

41

Table 1. Aphid colony count on selected mutant lines 3 weeks after inoculation.

Aphid colony count
Mat/Line
selection

SymptomatologyFreechoicefeeding1

No choice(forced)feeding2

28-30-240

62*

13-30-2

22*

65*

9-28-212*
nottested
12*
27ns
14ns
1*
not
tested40**

36*bunchy top
bunchy top,rosettebunchy top

65*

23

22-28-2
9-28-36-30-2
7-29-1
9-29-123-28-7
23-20-2
LKD
control

Incubationperiod(WAI)3BBTV
incidence (%)24-6

ELISA(OD405nm)
range4
MIN

MAX

20

0.189

1.276

5-6

25

0.174

0.956

4-5

50

0.189

1.605

rosette

5

38

0.188

1.570

49*
59*
110ns
83ns

bunchy top
bunchy top
bunchy top
bunchy top

4-5
4-5
4-5
4-5

50
56
58
33

0.179
0.184
0.182
0.203

1.267
2.292
1.128
1.545

16*

bunchy top

4-5

45

0.171

1.336

76*

bunchy top

4-5

71

0.179

1.680

127

bunchy top

4-5

58

0.194

1.287

1 Data analysis using t-test at α=0.05; SD* significantly less preferred, ** more preferred, ns not significant
2 Mean values obtained from two separate trials
3Weeks after inoculation
4OD – optical density. Mean values obtained from two separate experiments given the no choice feeding method

In a no choice/forced feeding of the vector, some lines (28-30-2, 13-30-2, 22-28-2, and 23-28-7)
which were significantly less preferred by the vector (aphid colony count significantly lower
than the control) likewise showed correspondingly low BBTD incidence (<50%). On the other
hand, line 9-29-1 which was preferred by the aphid had also low BBTD incidence (32.8%). In
no choice feeding set-up, mutant lines likewise showed resistance to BBTV either throughless preference of the vector to the host or resistance of the host to the virus despite thepreference of the vector to the host.
(5) Multi-location field evaluation for disease resistance and agronomic traits
For all trial sites, low incidences of the BBTD ( were observed from planting up to 19 MAP
despite the presence of the virus and vector within the trial site and also within the vicinityof the sites. The highest percentage incidence of the BBTD was recorded at 13.3%; thediseases did not spread extensively within the trials sites and the occurrences of the disease
infection were random. The low incidence BBTD in trial sites could be due to greater numberof resistant plants (approx. 70%) in comparison with the susceptible plants (30%) planted
within the area. Furthermore, the random manner by which resistant mutant lines (withvarying resistance mechanisms) were planted could have also contributed to the low disease42

incidence in the field. The use of mutant lines coupled with the farmer’s practice of regularrouging of infected plants at least once a month provided the most effective control against
BBTV.
The BBTV resistant mutant lines were comparable with LK control in terms of plant height,
girth, total number of fruits per bunch, number of hands per bunch and number of fingers per
hand (Table 3). The number of days to flowering was significantly different for lines 13-30-2(26 says earlier), 22-28-2 (21 days earlier) and 28-30-2 (17 days earlier). Mutant lines had amean of 6-7 hands/ bunch and 17-18 fingers/hand.
(6) Simple Sequence Repeats (SSR) Analysis
The mutant lines were differentiated from the LK control using nine SSR primers (CIR 13,
CIR1113, Ma 3-90, Mb 142-2, CIR 07, CIR 09, PR 27, PR 108, PR 260 and 631a). For mostprimers (CIR 13, CIR1113, PR 27, 631a), the mutant lines were differentiated from LK
control by the absence of 1 or 2 alleles detected in LK control.
Table2. Relationship of aphid colony count and BBTD incidence as influenced
by vector feeding method in selected mutant lines.

Mat/Line

Free choice feeding of the vector

No choice/forced feeding of the vector

Aphid Colony% BBTV

Aphid Colony% BBTVCount

incidenceCount

incidence

28-30-2

40

3.1

62

19.6

13-30-2

22

40.0

65

25.0

9-28-2

12

40.0

36

50.0

22-28-2

NT

NT

65

38.4

9-28-3

12

63.6

49

50.0

6-30-2

27

50.0

59

55.6

7-29-1

14

50.0

110

57.5

9-29-1

1

66.7

83

32.8

23-28-7

NT

10.00

16

45.0

23-20-2

40

41.7

76

70.7

LKD control

23

81.2

127

58.3

selection

43

Table 3. Agronomic and yield parameters taken from BBTV resistant mutant lines(mean from multi-location trials).

Mat/line
selection

Plantheight*(cm)

Girth(cm)**

No. ofdays toflowering

Total No. offruits perbunch

No. hands/
bunch

No, offingers per
hand

9.28.2

342.6

56.1

408

123

6.6.

17

9.28.3

340.8

54.8

408

128

6.5

17

13.30.2

334.5

54.1

397***

119

6.3

17

22.28.2

347.0

55.5

402***

130

6.6

18

28.30.2

342.3

55.3

406***

130

6.5

18

LK control
t- testP=0.05

319.6

52.3

423

108

6.3

19

NS

NS

SD

NS

NS

NS

*Measurements taken at flowering ** Measurements taken 1m from the base of the pseudostem at flowering***Significantly different from LK control using t test at P=0.05

Acknowledgements
Philippine Council for Agriculture and Natural Resources Research and DevelopmentDepartment of Science and Technology (PCARRD-DOST) for research support from2007-2011.
Crop Science Cluster-Institute of Plant Breeding University of the Philippines Los Banos for
manpower support and facilities.
Philippine Nuclear Research Institute (PNRI) for radiation facilities and technology
promotion and dissemination.
Forum for Nuclear Cooperation in Asia (FNCA) for technology promotion and disseminationand support for travel and attendance to FNCA meetings.
International Atomic Energy Agency (IAEA) for earlier support for the project and technology
promotion and dissemination.
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Aguiba MM. 2010. Partnership enables RP to produce virus-resistant Lakatan banana.
http://www.mb.com.ph/node/237664/partner
BAS Selected Statistics on Agriculture 2008. http://countrystat.bas.gov.ph/
Global Invasive Species Database. 2005. Compiled by National Biological Information
Infrastructure (NBII) and IUCN/SSC Invasive Species Specialist Group (ISSG).
Hafner, G. J., Harding, R. M., & Dale, J. L. 1995. Movement and transmission of banana
bunchy top virus DNA component one in bananas. Journal of General Virology 76:
2279-2285.
Hu, J. S., Wang, M., Sether, D., Xie, W. & Leonhardt, K. W. 1996. Use of polymerase chain
reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid(Pentalonia nigronervosa). Annals of Applied Biology 128: 55-64.

44

Magnaye, L. V. 1979. Studies on the identity and relationship of the abaca and banana
bunchy top virus in the Philippines. MS Thesis, UP College of Agriculture, College,
Laguna.
Molina, A.B., Sinohin, V.G.O., dela Cueva, F.M., Esguerra, A.V., Crucido, S.S., Vida, E.,
Temanel, B.E., Anit, E.A. and Eusebio, J.E. 2009. Managing banana bunchy top virusinsmallholdings

in

the

Philippines.

http://www.actahort.org/books/828/828_39.htm

45

Acta

Hort.

(ISHS)

828:383-388.

3.2 Success Story of Mutation Breeding on Sorghum for Improved Drought andAcid Soil Tolerance in Indonesia
Soeranto Human
Center for Application of Isotope & Radiation Technology,
National Nuclear Energy Agency
Abstract
The limited factors in developing dryland farming agriculture in Indonesia are mostly related
to drought problem in the east and soil acidity in the west part of the country. The
development of dryland farming agriculture may be directed to crops that require lessagricultural inputs, have wide adaptability and with good economic values such as sorghum.
Globally, sorghum is recognized as sources of Food, Feed, Fuel and Fiber (4Fs). In Indonesia,sorghum is still regarded as a minor cop and its cultivation is limited, mostly grown by local
farmers in a specific region. Sorghum is not Indonesian origin so that the available plant
genetic variability is low. Attempts to increase sorghum genetic variation was achieved
through introducing plant materials from ICRISAT, China and Australia. These materials
were used in breeding programs by conventional, mutation and biotechnological approaches.
Through mutation breeding, a number of sorghum mutant lines were identified to havehighly tolerance to drought and soil acidity with improved yield and quality. In collaboration
with the Ministry of Agriculture, universities and private companies, some promising mutant
lines were investigated under intensive multi-location trials in several Provinces inIndonesia. In 2011, one sorghum mutant variety was released by the name of “Pahat”, anacronym of “Pangan Sehat” meaning healthy food. Meanwhile, some sorghum mutant lines
showed improved agronomic performances such as lodging resistance, early maturity, higher
sugar (brix) content and biomass production in later generations. These promising mutant
lines were kept at our sorghum germplasm collections for further breeding program towards
improvement of sorghum utilization for 4Fs. The on-going research is currently being donefor evaluating the sorghum mutant lines and released variety in farmers field in different
regions for increasing marginal land productivity. Sorghum mutation breeding was includedin the JSPS Core University Program (1999-2008), part of FNCA projects (2000-2004), andcurrently supported by IAEA through the CRP Contract No. 16947 and RAS/5/056 project.
Introduction
For agricultural purposes, arable land in Indonesia is actually dominated by dryland farming
areas. Meanwhile, wetland (paddy field) areas tend to decrease due to several reasons suchas intensive expanding of land transformation to non-agricultural purposes happened
recently. The fact that water scarcity still becomes a problem in some agricultural areas
makes dryland farming agriculture be more important and reliable for supporting
sustainable agriculture development in the country. The most significant limited factors indeveloping dryland farming agriculture in Indonesia are related to adverse conditions i.e.
46drought in the eastern part and soil acidity in the western part of the country [1]. The
development of dryland farming agriculture may be directed to include potential crops that
require less agricultural inputs (water, fertilizer etc.) with high economic values such assorghum.
Sorghum (Sorghum bicolor) is thought of being suitable crop for dry land farming agriculture
in Indonesia owing to its wide adaptability, high yielding and more tolerant to adverse
conditions than the other food crops [2]. Sorghum is a cereal crop that is usually grown under
hot and dry conditions, and globally it is used as sources of 4Fs namely Food (grain sorghum),

Feed (forage sorghum), Fuel (sweet sorghum) and Fiber (sorghum biomass). As food, sorghum
was reported to have good nutritive values with low glutein and glycemic index, andcontained the phytochemical (phenolics) that were associated with protection from and/or
treatment of chronic diseases such as heart disease, cancer, hypertension, diabetes and othermedical conditions [3].
Sorghum has a high yield potential, comparable of rice, wheat, and maize. On a field basis,yields have exceeded 11 ton/ha, with above average yields ranging from 7-9 ton/ha where
moisture is not a limiting factor. In those areas where sorghum is commonly grown, yields of
3-4 ton/ha are obtained under normal condition [4]. Sorghum is also known to have wide
adaptability, ranging from lowland, medium and highland altitude. Highest yields areusually obtained from varieties maturing in 100-120 days. Late-maturing varieties tend to beappropriate for forage crop. Available sorghum genotypes have been introduced from abroade.g. from the International Crop Research Institute for the Semi-Arid Tropics (ICRISAT).
Through plant breeding programs, some local varieties have also been released by the
Ministry of Agriculture. Further sorghum breeding, however, is needed especially to search
for and to develop genotypes tolerant to adverse conditions such as drought and soil acidity
which become the most limiting factors in dryland agricultural development in Indonesia [5].
Beside food security, Indonesia is also currently looking for alternative renewable energy
resources for ensuring energy security. Sorghum is one source of bioenergy (bioethanol) withhigh productivity. The productivity of bioethanol from sweet sorghum reached 8.419
liter/ha/year i.e. twice that of cassava (3.835 liter/ha/year) since sorghum can be planted
twice a year in Indonesia [6]. From a total of 99.50 million ha of dryland agricultural area inIndonesia, about 68.75 ha (69.1%) consisted of acid soil (ultisols) which is mainly found in
Sumatera and Kalimantan [7]. The main constraint of crop production in acid soils is
phosphor (P) deficiency and aluminium (Al) toxicity [8, 9]. Therefore, development ofsorghum cultivation in dryland farming areas requires varietes which are tolerant to suchconditions. The objective of our sorghum mutation breeding was to search for sorghum
genotypes tolerant to drought and soil acidity, with improved yield and quality for 4Fs
purposes.

47

Sorghum Mutation Breeding
Sorghum mutation breeding was conducted at the Center for Research and Development of
Isotope and Radiation Technology, National Nuclear Energy Agency (BATAN) in Jakarta,
Indonesia. Beside from ICRISAT, our sorghum breeding materials were also introduced from
India, China and Australia. Sorghum mutation breeding was included in the JSPS Core
University Program (1999-2008) and also as part of FNCA projects (2000-2004), specificallyfor screening mutants for drought tolerance.
Sorghum Durra and Zhengzu varieties from ICRISAT and China, respectively, were used as
starting breeding material in mutation breeding. The dry seeds of these sorghum varieties
were irradiated with Gamma rays emitted from Cobalt-60 source in the Gamma Chamber
4000A. The dose levels of 0-1000Gy, with increment of 100Gy, were used in the experiment of
estimating appropriate radiation doses for sorghum breeding purposes. Response of sorghum
growth in the M1 generation was studied by best-fitting curve software, and the LD-20 and
LD-50 values were determined for estimating optimal irradiation doses for sorghum breeding
purposes [10].
Some M1 plants were bulkly harvested to generate of about 4000 M2 plants. Individual plantselections based upon phenotypic performances (variations) were started in the segregating
M2 population, focusing on agronomic and yield characters which are significantly different
from the control plants. The two parental varieties and two local varieties (UPCA and Higari)
were used as control plants in the experiment. Selected plants which showed “better” traits
than the control plants were separated and registered as putative mutants for further
screening for drought and acid soil tolerance in the M3 generation.

Fig 1. Sorghum response to gamma irradiation at seedling stage (M1).

48

Screening for Drought Tolerance
Screening for drought tolerance used combination of indirect selection (PEG method) anddirect selection in the field. For the PEG method, concentration of 25 % polyethylene glycol
was used and applied in the seedling stages. According to Singh and Chaudhary, PEG could
reduce water potential equivalent to natural drought condition so that water absorption byroots could be affected [11]. A total number of 170 selected tolerant M3 plants using the PEG
method were obtained and then transplanted to the field for their seed multiplication.
Direct drought tolerance screening in the field for these selected plants was conducted indrought prone areas of Gunungkidul District, Yogyakarta Special Province during dry season
in 2002 (M4), 2003 (M5), and 2004 (M6), respectively. Sowing time was adjusted to early dry
season i.e. around March or April. Artificial irrigation was given only in early growth stage tostimulate seed germination. One month after sowing, the irrigation was stopped and theplants were entirely exposed to the natural drought condition. The growth and yield of theselected drought tolerant plants were also evaluated in the field condition under normal
season using randomized block design.
Production of total biomass, yield and its components and drought index were measured andused as criteria for selection of the tolerant mutant plants. Drought index was calculated by
dividing the grain yields under drought to that of normal condition. It measured relative
sensitivity of genotypes to water stress response with regard to grain yield production.
Through field screening and selection processes in successive generations, 10 mutant lines
were finally found and registered as highly drought tolerant. These mutant lines were B-69,
B-72, B-75, B-76, B-90, B-92, B-94, B-95, B-100 and ZH-30. Statistically, these mutant lines
had agronomic and yield performances significantly better than that of the original variety(Durra) and the control national check variety (UPCA and Higari). Some of these mutant
lines had been proposed for official reslease to the Ministry of Agriculture. In 2011 themutant line ZH-30 was released by the name of “Pahat”, an acronym of “Pangan Sehat”
meaning healthy food.

Figure 2. Seedling screening for drought

Figure 3. Tolerant plant grown in the field

tolerance (PEG method).

(B-100 mutant line).

49

Screening for Acid Soil Tolerance
Screening for acid soil tolerance was conducted indirectly in Laboratory using AlCl3 method
and also directly in the field at lands with acid soil condition. A total number of 59 selected
tolerant M3 plants using the AlCl3 method were obtained and then transplanted to the field
for their seed multiplication. Field screening and evaluation of selected M4 plants wereconducted at Taman Bogo in Lampung Province where soil condition was classified as very
acid with soil pH of 4.2 and 39% Al saturation. The parental varieties (Durra and Zhengzu)
and national varieties (Numbu and Kawali) were used as control plants.
Plant growth, grain yield, biomass, stem juice, brix content and ethanol production were
measured and used as criteria of selection for acid soil tolerance. Analysis of bioethanol was
conducted at the laboratory of Center for Starch Technology (B2TP-BPPT) at Sulusuban inCentral Lampung. Ratooning ability was also measured with regard to bioethanol production.
The experiment used randomized block design with 10 samples taken from each genotype
treatments.
Among 59 mutant lines tested in the field, a number of 15 mutants were found and identified
as highly tolerant to acid soil condition. Three of these tolerant mutants i.e. Patir-1, Patir-4
and Patir-9 are now being proposed for official release as new sweet sorghum varieties to the
Ministry of Agriculture.

ParentDurra

Mutant Lines

Figure 4. Seedling screening for tolerance to Al

Figure 5. Field screening on acid soil with

toxicity in media containing 148 μM AlCl3.

soil pH of 4.2 and 39% Al saturation.

Tolerant

Susceptible

Figure 6. Tolerat vs susceptible plants.

Figure 7. Released mutan variety “Pahat”.

50

Conclusion
Mutation breeding of sorghum has successfully produced some mutant lines that arepotential to be developed further in drought prone and acid soil areas in Indonesia. Sincesorghum can be used for food, feed, fuel and fiber sources, thus, sorghum development will
increase land productivity for sustainable agriculture and ensure food and energy security inthe country. Sorghum technology transfer to private industries has showed significant
achievements in sorghum development in Indonesia.
References[1] Hidayat, A. dan Mulyani, A. 2002. Lahan kering untuk pertanian.

Teknologi

Pengelolaan Lahan Kering. Puslitbang Tanah dan Agroklimat, Badan Litbang Pertanian.
p1-34 [in Bahasa Indonesia].
[2] Soeranto, H., Trikoesoemaningtyas, Sihono and Sungkono. 2010. Development ofsorghum tolerant to acid soil using induced mutation with gamma irradiation. Atom
Indonesia Journal Vol. 36, No. 1, April 2010, p11-15. ISSN 0126-1568.
[3] Dicko, M.H., Gruppen, H., Traore, A.S., Voragen, A.G.J., Van Berkel, W.J.H. 2006.
Phenolic compounds and related enzymes as determinants of sorghum for food use.
Biotechnology and Molecular Biology Review 1(1):21-38.
[4] House, L.R. 1985. A Guide to Sorghum Breeding. ICRISAT/FAO, 238p.
[5] Soeranto, H., Nakanishi, T., and Sihono. 2008. Developing drought tolerant sorghum
using induced mutation with gamma irradiation. Proc. of JSPS-DGHE Seminar, theUniversity of Tokyo, 28-29 Febr. 2008.
[6] Yudiarto, M.A. 2005. Pemanfaatan sorgum sebagai bahan baku bioetanol. Makalah FGD“Prospek

Sorgumuntuk

MendukungKetahanan

Pangandan

Energi”.

MENRISTEK-BATAN. Serpong, 5 Sept. 2006. [in Bahasa Indonesia].
[7] Mulyani, A., Rachman, A., Dairah, A. 2009. Penyebaran lahan masam, potensi dan
ketersediaannya untuk pengembangan pertanian. Dalam Fospat Alam. Balai Penelitian
Tanah, Bogor.
[8] Kochian, L.V., Hoekenga, O.A., Pineros, M.A. 2005. How do crop plants tolerate acid soil?
Mechanism of aluminium tolerance and phosphorous deficiency. Annu. Rev. Plant Biol.
55:459-493.
[9] Kochian, L.V., Pineros, M.A., Hoekenga, O.A. 2005. Physiology, genetics and molecularbiology of plant aluminium resistance and toxicity. Plant and Soil 274:175-195.
[10] Soeranto, H. and Sihono. 2010. Sorghum breeding for improved drought tolerance using
induced mutation with gamma irradiation. Indonesian Jurnal of Agronomy Vol. XXXVIII,
No. 2, August 2010, p 95-99. ISSN 2085-2916.
[11]Singh, B.L. and Chaudhary. 1998. The physiology of drought tolerance in field crops.
Field Crops Res. 60:41-56.

51

3.3

Radiation Breeding and Commercial Mutant Varieties in Korea

Si-Yong Kang
Radiation Breeding Research Team, Advanced Radiation Technology Institute,
Korea Atomic Energy Research Institute
Radiation technology
In the early 2000s, the Korean government decided to support fundamental and applicableresearches of radiation technologies. The Advanced Radiation Technology Institute (ARTI) of
KAERI was established to promote the research and development in the fields of radiation
applications including agricultural, industrial, environmental, food and biotechnology, radio
isotope and medicinal sciences, etc. We believe that the ARTI will significantly contribute to
the researches on radiation mutation breeding and other industry fields in the world as wellas in domestic.
Current status of mutation breeding in Korea
The mutation breeding has been achieved for new variety development, the creation of new
genetic resources, and the use of mutants for a genomics study. Before 2000 year, mutation
derived varieties in Korea was not higher than other competition countries. More than about
50 varieties have been developed by using the mutation breeding method since the mid-1960s,
and the released varieties were mostly developed (76%) by exposing to radiations. Most of thereleased mutant cultivars (74%) in Korea were food and oil seed crops, especially for
improving agronomic traits such as yield, lodging tolerance, maturity, or functional
compounds. Currently a high yield potential of cultivated crop cultivars is relatively lesspopular than before, but the expectation of value-added crops, from the farmer’s side, is inhigh demand. Accordingly, the mutation breeding program in Korea has assigned moreresources to other crop species, including some flowering and ornamental plants. Theseflowering and ornamental plants are ideal systems for a mutation breeding because theirfavored traits such as flower color or shape and plant architecture can be visually monitored
after a mutagenic treatment. Additionally, these plant species are genetically heterozygous
and often propagated vegetatively, which allows for an isolation and selection of mutants
within few generations. In recent, many researchers and breeders have been interesting on
mutation breeding techniques on major flowering and ornamental crops in Korea such asrose, chrysanthemum, lily, carnation, orchids, and clover. The potential outcomes from theprogram will be new highly valued-added cultivars which will provide greater money gains to
Korean farmers and lots of valued mutants used for a gene isolation of interest and reverse
genetics or functional genomics. From 2012, a big research project, “Golden Seed Project’ waslaunched supported from Korean Ministry of Food, Agriculture, Forest and Fisheries(MIFAFF). The main aim of the project is for the development of potential new varieties of 20
items including food and vegetable crops, animals and fishes, etc., so that rising domestic
seed industry for reducing royalty payments and seeking greater opportunities in global seed52

markets. Mutation techniques also can be contributed many research and breeding works of
the Golden Seed Project. Appropriate strategies should be implemented to complete its goals
successfully, which includes a) induction of a wide mutant spectrum, b) applications of newirradiation techniques, c) unraveling the complex genetic phenomena controlling mutant
traits, and d) development of a mass production and an intensive export system for thedeveloped cultivars.
Mutation breeding researches in KAERIThe mutation breeding in Korea has been conducted by the KAERI and some otherresearchers since mid of 1960’s. The ARTI is only one institute with essential facilities formutation breeding and irradiation in Korea. With financial support from the government(MIFAFF), a new Radiation Breeding Research Center with advanced research facilities isschedule to be completed by 2013 in ARTI, and it will play a key role in research andextension of mutation breeding technology.
Since 2000, KAERI has released 13 rice mutant varieties and distributed rice seeds tofarmers directly each year. Among them, a green-kerneled glutinous rice mutant,“Nongwonchalbyeo”

and

an

aromaticandanthocyanin

pigmented

ricemutant,“Huegseonchalbyeo”, have become very popular with farmers and consumers. In 2011, two
new rice varieties, "GoldAmi-1" and “Wonmyoung” were officially registered. “GoldAmi-1”
was developed by radiation fusion technology combining with plant tissue culture, and had
characters of 70% increased amino acid compared with the original variety "Dongan" andcheck variety "Youngan", high lysine accumulating variety. "GoldAmi-1" will be applied inbaby food and powdered milk industry. In addition, a new rice variety "Wonmyoung" withhigh quality was mutated from the original variety "Koshihikari", the most famous variety inJapan, and had characters of lodging resistance and mid-late maturing. In recent, new lines
with high tocopherol content has been selected and it will be released after official
registration.
As the outcome of recent researches at KAERI, an early mature and high-yielding mutant
soybean, “Josaengseori,” was released and two new soybean varieties (“Wonyul”, Wonhyeon”)
were applied for a variety protection right to the Korea Seed & Variety Service. Also sixteen
mutant lines lacking seed lipoxygenases and six mutant lines with a low phytic acid content
were selected and will be applied for plant variety copyright protection. In hibiscus (rose of
Sharon), six mutant varieties (“Baekseol”, “Seonnyeo”, “Daegoang”, “Ggoma”, “Changhae”
and “Dasom”) were developed through gamma ray irradiation. In particular, “Ggoma”(meaning ‘kid’) is a dwarf mutant and has received considerable attention as a ‘bonsai’ orindoor cultivation. Recently, mutation techniques have been applied to many crop species,
including some flowering and ornamental plants, medicinal and industrial plants, andvegetable plants. In recent, KAERI singly and jointly with other research organizations and53

private companies had released new mutant varieties of kenaf, orchid, rose and blackberry,etc.
A completion of genome sequencing of some model plant species left challengeable
assignment of understanding the gene functions predicted by the sequence information. A
direct approach of discovering the function of a novel gene is to use a mutant which hasaltered or eliminated function as a result of a mutagenesis. Mutants that have favored traits
for the cultivar development or distinct features for studying gene functions, therefore, areequally important. Experimental effort to apply diverse ionizing energy and irradiation
facility to induce a broad spectrum of plant mutants are now underway at KAERI.
Irradiation with an electron beam, ion beam, chronic gamma ray, and space radiation loaded
in a spaceship are of interest research topics. We assume that there will be various types ofmutants induced because of difference in ionizing ability, penetrating power, irradiation
duration, and kinds of radiation. However, only a limited information is available in ourhands so that we are planning to collect empirical data from the different radiation or
ionizing particle sources.

In order to identify gene function using mutant population,

TILLING (Targeting Induced Local Lesions IN Genomes) projects were promoted in rice.
This technique is high-throughput and reverse genetic approach and contributes in theidentification of mutation and efficient application of mutant resources. The populations of
3,000

rice

lines

were

constructedfromgamma-irradiation.

54

the

embryogenic

culturesystemand3.4

Advanced Techniques for Mutation Breeding by Using Ion Beams

Atsushi Tanaka
Quantum Beam Science Directorate, Japan Atomic Energy Agency
1. Introduction
A great deal of studies on plant mutagenesis by ionizing radiation has been carried out since
mutation was induced in maize and barley with X-rays by Stadler in 19281). Biological effectof ion beams has been also investigated and found that ion beams show high relative
biological effectiveness (RBE) of lethality, mutation, and so on compared to low linear energy
transfer (LET) radiation such as gamma-rays, X-rays and electrons2). As ion beams deposit
high energy on a target densely and locally, it is suggested that ion beams predominantly
induce single- or double-strand DNA breaks with damaged end groups whose reparability
would be low3). Therefore, it seems plausible that ion beams can frequently produce large
DNA alteration such as inversion, translocation and large deletion rather than point
mutation, resulting in producing characteristic mutants induced by ion beams. However, thecharacteristics of ion beams on the mutation induction had not been clearly elucidated yet.
On the base of the Consultative Committee for Advanced Radiation Technology in Japan, theTakasaki Ion Accelerator Advanced Radiation Application (TIARA) was established andbasic research on plant mutation by ion beams was started in 1991. For over twenty years the
biological effects of ion beams have been studied and novel mutants and varieties of crops
have been consistently and efficiently produced using mutations induced by ion beams4). At
present, plenty of research activities in Japan utilize the ion beam irradiation in several
irradiation facilities such as RIKEN RI beam factory, the Wakasa Wan Energy ResearchCenter Multi-purpose Accelerator System with Synchrotron and Tandem (W-MAST), and the
Heavy Ion Medical Accelerator in Chiba (HIMAC) of National Institute of Radiological
Sciences.
2. Irradiation methods
In TIARA, characteristics of ion beams for biological endpoints have been investigated using
Arabidopsis, rice, barley, tobacco, chrysanthemum, etc. Several kinds of energies and ionssuch as carbon (C), helium (He) and neon (Ne) were utilised in this study5). All ions weregenerated from the AVF-cyclotron in TIARA. In general, ion beams are scanned at around 70
X 70 mm, and exit the vacuum chamber through a beam window of a 30 µm titanium foil. The
sample is placed under the beam window and irradiated in the atmosphere. For example,
100-3,000 Arabidopsis or tobacco seeds are sandwiched between kapton films (8 µm
thickness) to make a seed monolayer for homogeneous irradiation. In the case of relatively
large seeds such as rice, barley or soybeam seeds, the embryo side faces the ion beams. Tissue
culture such as ornamental explants, calluses, shoot primordial in a aseptic petri dish isdirectly irradiated except that the lid of dish is exchanged to a thin film in order to decrease
the loss of ion beam energy. An irradiation sample is irradiated within 2 min.
55

3. Characteristics of mutations induced by ion beams
Mutation frequency: Mutation frequency was investigated on a gene locus basis using visible
known Arabidopsis mutant phenotypes, such as transparent testa (tt), in which the seed coatis transparent because of the lack of pigments, and glaborous (gl), in which no trichomes areproduced on leaves and stems. The average mutation frequencies of tt and gl loci induced by
C ions showed a frequency of 0.07 X 10-3 mutants/ locus, which was 20-fold higher than thoseinduced by electrons as a low LET radiation6). Koornneef et al. 7) investigated mutations
induced by EMS with a frequency of about 0.20 X 10-3 mutants/ locus. Thus, carbon ionsseemed to be about three-fold less mutagenic than EMS treatment. It is noteworthy that thehigh mutation rate or frequency by carbon ion irradiation was observed at a relatively low
dose of 150 Gy, at which virtually all plants survive.
Mutation spectrum: In order to elucidate the features of ion beams as a new mutagen, themutation spectrum induced by ion beams was compared with that induced by low LET
radiation. Nagatomi et al. 8) investigated the spectrum of mutations in flower color using
chrysanthemum cv. Taihei with pink-color petals. Most flower color mutants induced by
gamma rays were light pink, and a few were dark pink in color. By contrast, the color
spectrum of the ion beam-induced mutants shifted from pink to white, yellow and orange.
Furthermore, flower mutants induced by C ions showed complex patterns of colouration, andstriped color types, that have never been obtained by gamma-ray irradiation of this cultivar.
The mutation spectrum of flower color and flower shape of carnation was investigated by
Okamura et al. 9) investigated with carnation variety Vital, whose phenotype is spray typewith cherry pink flowers and frilly petals. Flower color mutants such as pink, white and red
were obtained by X-ray irradiation, whereas the color spectrum of the mutants obtained bycarbon ion irradiation was far wider and included pink, light pink, salmon, red, yellow,
complex and striped types (Table 1). In addition, many kinds of round shaped petals were
induced. It was suggested, therefore, that the mutation spectrum of flower color and shapeinduced by ion beams is broad and that novel mutation phenotypes can be obtained.
Table 1. Mutation spectrum of flower color in carnation

Mutation frequency (x 10-1 %)Mutagen

Light

Pink

pink

Dark

Red

SalmonCream

Yellow

MinuteComplex

Stripestriped

pink

EMS

0

5.2

0

1.0

0

0

0

0

0

3.1

Soft X-rays

1.7

8.4

0

3.4

0

0

0

0

0

0

Gamma-rays

1.7

2.6

0

1.7

0

0

0

11.3

0

0

Carbon ions

2.4

4.7

2.4

3.5

2.4

1.2

2.4

0

2.4

3.5

56

Molecular mechanisms of mutation: C ion- and electron-induced Arabidopsis mutants werecompared at molecular (DNA) level 6),10). In the case of C ions, fourteen loci out of 29
possessed intragenic point-like mutations, such as base substitutions, or deletions of several
to a hundred of bases. Fifteen out of 29 loci, however, possessed intergenic DNA
rearrangement (‘large mutations’) such as chromosomal inversions, translocations, anddeletions. In the case of electrons, nine alleles out of 12 loci had point-like mutations andthree out of 12 loci had DNA rearrangements. Sequence analysis revealed that C ion-induced
small mutations were mostly short deletions. Furthermore, analysis of chromosome
breakpoints in large mutations revealed that C ions frequently deleted small regions aroundthe breakpoints, whereas electron-irradiation often duplicated these regions. These results
could imply that different types of non-homologous end joining pathways operate in responseto the mutations induced by the two radiation types and that C ion-inducedmutations aremostly likely to result in nulls.
4. New varieties and mutants
Model plants: Several new Arabidopsis mutants, and the gene responsible for thesemutations, were identified following ion beam mutagenesis11). Ultraviolet light-B (UV-B)
resistant or sensitive mutants were obtained, and the genes responsible have been identified.
New anthocyanin-accumulating or anthocyanin-defective mutants were also obtained andsome of the genes responsible have been identified. A novel flower mutant, frill1, which has
serrated petals and sepals, and the gene responsible for this phenotype have been found. A
novel auxin mutant, the aar1-1, was also obtained. In Lotus japonicus, which is used as amodel leguminous plant, a novel hypernodulation mutant, named klavier (klv), was isolated
following irradiation with helium ions.
Crops: Ion beams have been used for inducing mutants resistant to major diseases. Mutants
resistant to bacterial leaf blight and blast disease were induced in rice12). Higher mutation
frequency was found in the ion beam treatment compared to gamma-rays or thermal
neutrons. Two mutant lines of yellow mosaic virus resistant barley were found in a screen ofca. 50,000 M2 families. By exposure of tobacco anthers to ion beams, mutants resistant topotato virus Y have been obtained. Banana mutants tolerant to black Sigatoka in vitro were
induced by carbon ions. Recently, low-nitrogen-fertilizer grown rice mutants were induced fora solution to eutrophication. Moreover, low-cadmium rice mutants were obtained from themost popular Japanese rice cultivar, Koshihikari13).
Ornamentals: Complex and stripe types of flower color have been obtained inchrysanthemum8) as described above. On the other hand, a reduced axillary flower bud
mutant that shows excellent character for labor-saving, named Aladdin, was induced bycarbon ions for the first time14). By the use of re-irradiation of this mutant using carbon ions,the ideal characters of not only a few axillary flower buds but also low temperature flowering
were also obtained. In addition to the carnation varieties9) described above, mutants of57

petunia with altered flower color and form have been induced by ion beams. In rose, mutants
with more intense flower colors or mutants in the number of petals, flower size and shape
have been obtained. Mutants induced by nitrogen or neon ions in Torenia include two groupsof flower color mutants, ones that lack genes required for color or pigment production andothers in which their expression is altered. Taking advantage of ion-beam irradiation and thenewest information of genomics in flavonoid biosynthesis, breeding glittering carnations andfragrant cyclamen with novel flower color have been succeeded 15), 16), 17).
Trees: Ion beams have also been used for generating mutants for tree breeding. Wax mutants
and chlorophyll mutants such as Xanta and Albino were obtained in the forest tree, Hinoki
cypress. For phytoremediation, shoot explants of Ficus thunbergii were irradiated withseveral kinds of ion beams in order to produce novel mutants with a high ability to mitigate
pollutants. A mutant variety, KNOX, with 40-80% greater capability to assimilate
atomospheric nitrogen dioxide has been induced18).
Microbes:

Ion-beam irradiation is also effective to obtained desired mutants of

microorganisms. One of these mutants, a new yeast variety superior in improvement of
alcohol fermentation property has been induced to produce Japanese sake.

Figure 1. Novel varieties and mutants induced by ion beams

58

From leaning to the left to the lower right: Arabidopsis UV-B resistant mutants (lower rank) ;chrysanthemum complex-color variety, "Ion-no-Seiko"; new rose-flower type carnation
variety; 'KNOX' of Ficus thunbergii; chrysanthemum variety 'Aladdin'; low-temperature
growing variety, Shizuiku 1 gou, of Musk melon; low-nitrogen-fertilizer grown rice mutant
variety from ‘Aki-no-uta’; low-cadmium rice mutant from ‘Koshihikari’; new yeast variety
improved alcohol fermentation that produces a higher amount of ethyl caproate.
5. Conclusion & Future prospect
From the number of investigations indicated above, it is plausible that the characteristics ofion beams for mutation induction induce mutants with high frequency, show a broad
spectrum of mutation phenotype, and therefore produce novel mutants.

Anothercharacteristic of ion beams for mutation induction would be to induce a minimum number ofmutations compared to other mutagens(Fig.2). As the main reason to explain thesecharacteristics of ion beams, we hypothesize that chemical mutagens, such as EMS, and low
LET ionizing radiation, such as gamma-rays and electrons, will predominantly induce manybut small modifications or DNA damage to DNA strands, resulting in several point-likemutations on the genome. In contrast, ion beams as high LET ionizing radiation will
efficiently cause not so many but large and irreparable DNA damage locally, resulting in alimited number of null mutations. Therefore, novel mutants were efficiently obtained, andmost showed distinct characteristics without detrimental characteristics, and mendelian
traits without backcrossing.
The most important question is whether mutation is randamly occurred or directed.
Nagatomi et al. 19) showed that the mutation rates of flower color of plants regenerated frompetals were higher than from leaves with both ion beams and gamma-rays. This is considered
to be because the genes for flower color in petal cells are more active than in leaf cells, which
this may lead to a higher mutation rate of regenerated plants from petals. Recently, theincrease of flower color mutation was shown by combining ion beams and high sucrose
content as pre-treatment, suggesting the possibility of directed mutation.20) If directed
mutation could be controlled in higher plants, combination of the best ion type for energy andthe best plant organ or tissue will enable the induction of objective mutation.

59

Figure 2. Characteristics of ion-beam induced mutation in higher plants
6. References
1) STADLER, L. J., Mutations in barley induced by X-rays and radium, Science 68 (1928)
186-1872) BLAKELY, E.A., Cell inactivation by heavy charged particles, Radiat.Environ.Biophys. 31(1992) 181-196
3) GOODHEAD, D.T., Molecular and cell models of biological effects of heavy ion radiation,
Radiat.Environ.Biophys. 34 (1995) 67-72
4) TANAKA, A. et al, Studies on biological effects of ion beams on lethality, molecular natureof mutation, mutation rate, and spectrum of mutation phenotype for mutation breeding inhigher plants. J.Radiat.Res. 51 (2010) 223-233
5) TANAKA, A & HASE, Y., Applications to biotechnology: Ion-beam breeding of plants, in
Charged Particle and Photon Interactions with Matter: edited by Y. Hatano, Y.
Katsumura and A. Mozumder, CRC Press, 6000 Broken Sound Parkway, 270 Madison
Avenue, New York, New York 10016. (2011) pp943-957
6) SHIKAZONO, N., et al., Analysis of mutations induced by carbon ions in Arabidopsis

thaliana, Journal of Experimental Botany 56 (2005) 587-596
7) KOORNNEEF, M., et al., EMS- and radiation-induced mutation frequencies at individualloci in Arabidopsis thaliana (L.) Heynh. Mutat. Res. 93 (1982) 109-123

608) NAGATOMI, S., et al., Mutation induction on chrysanthemum plants regenerated from invitro cultured explants irradiated with C ion beam, TIARA annual report 1995 5 (1996)
50-52
9) OKAMURA, M., et al., Wide variety of flower-color and -shape mutants regenerated fromleaf cultures irradiated with ion beams, Nucl. Inst. and Meth. in Phys. Res. B 206 (2003)
574-578
10) SHIKAZONO, N., et al., Mutation rate and novel tt mutants of Arabidopsis thaliana
induced by carbon ions, Genetics 163 (2003) 1449-1455
11) TANAKA, A., Mutation induction by ion beams in Arabidopsis, Gamma Field Symposia
38 (1999) 19-28
12) NAKAI, H., et al., Studies on induced mutations by ion beam in plants, JAERI-Review 19(1995) 34-36
13) ISHIKAWA, S., et al., Ion-beam irradiation, gene identification, and marker-assisted
breeding in the development of low-cadmium rice, PNAS 109 (2012) 19166-19171
14) UENO, K., et al., Effects of ion beam irradiation on chrysanthemum leaf discs andsweetpotato callus, JAERI-Review 2002-035 (2002) 44-46
15) OKAMURA, M., et al., Breeding glittering carnations by an efficient mutagenesis system,
Plant Biotechnology 29 (2012) 209–214
16) ISHIZAKA, H., et al. Production of novel flower color mutants from the fragrant cyclamen(Cyclamen persicum_C. purpurascens) by ion-beam irradiation, Plant Biotechnology 29(2012) 201–208
17) HASE, Y., et al., Development of an efficient mutagenesis technique using ion beams:
Toward more controlled mutation breeding, Plant Biotechnology 29 (2012) 193–200
18) TAKAHASHI, M., et al., Mutants of Ficus pumila produced by ion beam irradiation with
an improved ability to uptake and assimilate atmospheric nitrogen dioxide, International
Journal of Phytoremediation, 14 (2011) 275-281
19) NAGATOMI, S., et al., Chrysanthemum mutants regenerated from in vitro explants
irradiated with 12C5+ ion beam. Institute of Radiation Breeding, Technical News No.60(1997)
20) HASE, Y., et al., Efficient induction of flower-color mutants by ion beam irradiation inpetunia seedlings treated with high sucrose concentration. Plant Biotechnology 27 (2010)99-10361

62

4. Mutation Breeding III

4.1

Success Story of Mutation Breeding in Thailand

Suniyom Taprab
Rice Department, Ministry of Agriculture and Cooperatives, Thailand
Mutation breeding through irradiation and chemical application had been continuously donein Thailand for long period of time. Various species of plants such as ornamental plants,
vegetables, field crops and rice were induced mutation for various purposes. In Thailand,
small nuclear reactor supplying irradiation from Cobalt 60 and Cesium 137 sources had beenutilized for mutation breeding. With restricted amount of budget, nuclear scientists andmutation breeders, mutation breeding in Thailand was not widely supported but it createdgreat impact on rice breeding. Three rice varieties derived from mutation breeding in theyears 1977 1978 and 1981 are still widely being cultivating at present. So, this paper will
mostly tell success story of mutation breeding on rice.
In 1959, Thai government by Rice Department released the best aromatic rice, namely Khao
Dawk Mali 105 (KDML105), derived from local variety Khao Dawk Mali via pure line
selection. KDML105 is non-glutinous rice, photoperiod sensitive with medium duration of
maturity. It has very good grain quality in according to long grain quality standard. It haslong grain (7.5 mm long brown rice) with slender shape, transparent (without chalkiness) andshiny polished grain and also yielded high milling quality (Figure 1). Its cooking quality had
been widely accepted by consumers in various countries. Its distinct aroma (odor) with softand slightly sticky cooked rice had been well known in world rice market. So, more than ahalf of rice cultivated area in Thailand had been occupied by KDML105 (Figure 2) since it
had advantages in tolerance to acidity, salinity and moderately tolerant to drought. Its
disadvantages are susceptible to diseases and insect pests, low yield and could be cultivatedonly one crop per year. With high grain quality, KDML105 is premier exported rice ofThailand and domestically widely used to develop high grain quality varieties and abiotic
stresses tolerant varieties.

Figure 1. KDML105 milled rice

Figure 2. Rice cultivated area in wet season 201164

In 1965, KDML105 had been induced mutation by 20 Kilorad gamma irradiation of Cobalt
60 source. Its mutant had been tested and selected by conventional pedigree selection
method. Many derivatives obtained from this program. One promising glutinous mutant had
been released in 1977 namely RD6. One year later, 1978, an earlier matured mutant wasalso released namely RD15.
RD6 had very good grain physical properties similar to its original KDML105 (Figure 3). It
also gives very cooking quality with soft sticky without paste-like and aromatic cooked rice.
More than 80 percent of glutinous rice cultivated area in Thailand is being occupied by RD6.
RD15 is very similar to KDML105 in all characteristics except its earlier matured (Figure 4).
RD15 has 7-10 days earlier in maturity as compared to KDML105, it is more suitable forsemi-arid area and some area with water shortage. Since it has good grain quality witharoma, RD15 is another premier exported aromatic rice of Thailand.

Figure 3. Grains of RD6

Figure4. Grains of RD15

In 1969, Thailand released the first semi-dwarf and photoperiod insensitive variety namely
RD1. This variety caused very big change in rice cultivation in Thailand. Since it isphotoperiod insensitive with semi-dwarf plant height, it has good response to high productive
inputs and provides high yield in all growing seasons through year round. Thailand can
produced more paddy and extend exporting since then. Many modern varieties had beendeveloped after introduction of RD1. This is another green evolution in Thailand. When RD1
was being recommended to the farmers, it had also been induced mutation in 1969. It had
been irradiated with fast neutron of 1 Kilo Rad dosage. Mutants had been tested and selectedby conventional pedigree selection method until 1981. New glutinous semi-dwarf type andphotoperiod insensitive variety, namely RD10 had been released (Figure 5). RD10 had been avariety utilized to increase glutinous rice production in Thailand until present and had also
been exported.

65

Figure 5. Grains of RD10
Even though Thailand has limited resources, but mutation rice breeding is still being
conducted. Good grain quality mutants are expected to achieve from local photoperiod
sensitive varieties. Photoperiod insensitive aromatic mutant is particular breeding goal.
Meanwhile, other properties of grain quality are being induced in high yielding varieties. For
example, less chalkiness is being screened from mutants of high chalky variety, RD25. Somemutants of high yielding varieties are being screened for low phytic acid content. Fromcollaborated project of FNCA, mutants of KDML105 and RD15 are being screened for various
amylose content and low protein content. Isogenic lines of photoperiod insensitive mutants
with various amylose content that retain aroma were obtained (Fig 6a, 6b). This is a big
advantage for genetic study on aromatic rice and new opportunity for Thailand to produce
various products of aromatic rice to the world market.

Figure 6a. Amylose content of KDML105 mutants

66

Figure 6b. Amylose content of RD15 mutants

4.2

Success Story of Mutation Breeding Research on Rice in Bangladesh

A. N. K. Mamun
Plant Biotechnology and Genetic Engineering Division
Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission
Bangladesh has so far release nine improved varieties of rice using induced mutation
technique. Bangladesh Institute of Nuclear Agriculture has play a vital role to produce andrelease these new varieties. Two varieties called Iratom-24 and Iratom-38 were developed byirradiating the seeds of ‘IR 8’ and released by national seed board of Bangladesh (NSB) in
1974 for Boro (December-May) and Aus (March-June) seasons. The mutated characteristics ofthese varieties are short crop duration, resistant against bacterial leaf blight (BLB) andmedium fine grain size. ‘Binasail’ is also developed by irradiating the seeds of ‘Naizersail’ andreleased in 1987 for Aman (July-December) season with mutated characteristics like long
panicles, more number of grains, medium fine grains with higher weight, early maturity withphoto-insensitivity and higher grain and straw yields. ‘Binadhan-4’, ‘Binadhan-5’ and‘Binadhan-6’ were developed by irradiating F2 seeds of parents ‘BR-4’and ‘Iratom-38’ andreleased 1998, ‘Binadhan-4’ for Aman and ‘Binadhan-5’ and ‘Binadhan-6’ for Boro season.
‘Binadhan-7’ was developed from M3 seeds of the cv ‘Tai Nguen’ of Vietnum. This variety was
release in 2007 for Aman season with earliness, long fine grain and high yield. Early mature
character of this variety helps to escape drought and insect attach during flowering anddrought stages. Moreover it helps increase cropping intensity by facilitating timely
cultivation of following winter crops. ‘Binadhan-8’ was released in 2010, Crop duration
130-135 days, Salinity tolerance, Seedling stage: 12-14 dS/m, Mature stage: 10-12 dS/m,
Suitable for cultivation in both Boro and Aman season. ‘Binadhan-9 was developed byhybridizing between the local cv. ‘Kalozira’ and an exotic mutant line Y-1281. It was released
in 2012 for Aman season, and also can be grown in Boro season. Unlike the parent ‘Kalozira’it has short duration, long and slender grains with slight aroma. Using recent ion bean
irradiation technique we has already developed two mutant lines that can be grown after
harvest mustard or rapeseed during the first week of February to first week of March and canbe harvested after 121-134 days. These new developed mutant lines produce high yield andrelatively fine grains.

674.3 Towards Sustainable Development of Food & Agriculture in Asia and thePacific — Significant Roles of IAEA / RCA Projects
Luxiang Liu
Lead Country Coordinator, IAEA / RCA Project RAS/5/056
National Center of Space Mutagenesis for Crop Improvement, Institute of Crop Science,
Chinese Academy of Agricultural Sciences
1. Overview of RCA
The Regional Cooperative Agreement for Research, Development and Training (RCA) is anintergovernmental agreement established in 1972 under the auspices of the International
Atomic Energy Agency (IAEA). It is open to the participation of any Member State of the
IAEA in the area of South Asia, South East Asia and the Pacific or the Far East. A Member
State from one of these regions may become a party to this Agreement by notifying its
acceptance thereof to the Director General of IAEA. There are now eighteen Government
Parties to the RCA, i.e., Australia, Bangladesh, China, India, Indonesia, Japan, Korea,
Malaysia, Mongolia, Myanmar, Nepal, New Zealand, Pakistan, Philippines, Singapore, Sri
Lanka, Thailand and Vietnam.
The vision of RCA is to strive to become a respected regional resource community in nuclear
science and technology, competent and cost effective in providing high impact solutions on asustainable basis to socio-economic development problems for identified end-users within the
Region and the Member States. During the past 40 years, more than 100 RCA technicalcooperation projects in Agriculture including the field of plant mutation breeding weresuccessfully organized and implemented through the appropriate national institutions in the
RCA Member States.
2. Global Food & Agriculture Situation
There are at least 1 billion hungry people worldwide in 2009, and the Asia and the Pacificregion leads the biggest estimated regional distribution of hunger. Price volatility makes both
smallholder farmers and poor consumers increasingly vulnerable to poverty. The developingworld's poor are experiencing the effects of higher commodity prices, and declining
agricultural productivity growth is exacerbating the problem.
Lifting a billion people out of poverty and feeding an extra 2.3 billion by 2050 will require
increasing cereal production by 70%, doubling the output of developing countries.
Accelerating development of agriculture to continually increase productivity should be thefinal approach to end the poverty.
3. Inputs for Nuclear Agriculture through RCA
Since the establishment of the RCA, many problems in the agricultural sector have been68

effectively addressed using nuclear technologies. There are totally seventeen RCA projects inthe agricultural sector have been implemented by far with focus on four main areas, i.e.,
plant mutation breeding, animal health &production, food irradiation and soils and land use.
Through these projects, more than 780 young scientists were trained, more than 640 seniorscientists were appointed to participate in the coordinating meeting or workshop, more than
110 expert missions or scientific visit were implemented for specific technique issue.
4. The Impacts of RCA Projects on Plant Mutation Breeding
Mutation breeding techniques have played very significant roles in combating poverty andfood security problems by developing new mutant germplasm and mutant varieties. By theend of 2009, 1877 mutant varieties, which accounts for more than 60% of the total number ofmutant varieties in the world, have been released or approved for cultivation in the Asianand Pacific region. Such great success of mutation breeding is partially contributable to theprevious RCA projects on mutation breeding, which have provided training and techniques in
the Member States and organized research and development activities in enhancing crop
genetic diversity, development of new techniques and germplasm, and facilitated theexchange of information and genetic materials.
(1) Increased Mutant Germplasm Exchange and Utilization
In the Projects RAS5037 and RAS5045, one of the major task is to exploit direct or indirect
use of mutant varieties/lines in other countries than its origin, through germplasm exchange
and evaluation among participating countries. This has been carried out through RMMTs for
rice, soybean, mungbean, sesame, groundnut, wheat and sorghum. Mutant lines were further
demonstrated in farmers’ fields and national yield trials when they have the potential to bereleased as a new variety in testing countries, or were used in breeding programs as a donor
variety if they had useful characteristics. There are totally 135 mutant varieties/lines of rice,wheat, soybean, mungbean, groundnut and sesame were exchanged and evaluated in morethan 80 locations among the 14 participating Member States. At least 15 non-native mutant
varieties were identified for further yield trial for commercial release.
Through the systematic trials in Thailand, one soybean mutant variety from Vietnam (DT84)
and one from Korea (Bangsakong) consistently outperformed (higher yield or early maturity)
local check varieties. The counterparts have thus proposed to officially release these two lines
as a new variety for farmers in rice based cropping systems in the upper northern part andlower northern part of Thailand, respectively. Ten groundnut varieties plus local controls
were tested in Indonesia, Philippines, Sri Lanka and Vietnam. All five countries except
Vietnam have identified promising lines which have been recommended for either variety
release or inclusion in national new variety tests. In Sri Lanka, for example, after
demonstration

trialson

farmers’

land,

four

varieties,

i.e.

B/30/12/10,

Kidang,Binschinabadam 2 and Karisma Serene appeared to be very promising and will berecommended for official release. It was reported that some of the exchanged mutant
69germplasm in the RCA Member States have already been used as parents in their respective
breeding programs, which potentially can lead to the development of even more new varieties
in the coming years.
Based on the mutant germplasm exchange network, an autonomous non-governmental
professional association on mutagenesis study was established with the name of Asia andOceania Association of Plant Mutagenesis (AOAPM).
(2) New Mutant Germplasm Developed
Hundreds of well characterized stable mutants of wide range of crops and for importantagronomic traits such as improved yield, resistance to diseases, protein, oil, starch and sugar
content Here are a few examples of significance in future crop improvement: Mutants
resistant to soybean crinkle leaf disease (Thailand); soybean mutants with enhanced
tolerance to salinity (India); soybean mutant lines with reduced phytic acid content (China);
rice mutant MR219-4 with drought tolerance( Malaysia).
In other crops, various mutants have also been developed, e.g. a large collection of mungbean
and groundnut mutants have been developed in Thailand and India, respectively. Individual
mutant lines are developed in all participating institutes, e.g. drought tolerant mutant lines
were developed in China and Indonesia, respectively for wheat and sorghum; no-shattering
sesame lines and easy-cooking soybean lines in Korea; early maturing groundnut in Sri
Lanka; and virus resistant mungbean lines in Pakistan.
(3) New Mutant Varieties Released
70 more new mutant varieties have been developed and disseminated in the RCA Member
States through RCA projects. Taking an example of the number of released mutant varieties
under the RCA project RAS5045:
In 2007 a total of 15 mutant varieties where released in: Bangladesh 1 rice variety;
China 1 rice and 4 wheat varieties, India 1 each of groundnut, sunflower, soybean,
mustard, pigeon pea and cowpea and 2 mungbean varieties; Indonesia 1 rice
variety.



In 2008, 9 mutant varieties were released in: China 1 wheat variety; India 1
groundnut variety; Indonesia 1 each of rice, soybean & cotton varieties; andVietnam 4 rice varieties.



In 2009, 6 mutant varieties in: China 1 rice variety; India 1 variety each ofgroundnut and pigeon pea; Indonesia 2 rice varieties; and Vietnam 1 rice variety.



In 2010, 7 mutant varieties where released in: Australia 1 barley variety; China 2
wheat varieties; India 1 mungbean variety; Indonesia 1 each of rice soybean
varieties; and Sri Lanka 1 tomato variety.



In 2011, 2 mutant varieties of sorghum and rice had been released in Indonesia as
70

at the time of the meeting.
The dissemination areas of officially released mutant verities under the RCA Project
RAS5045 were about 118 mha, with a benefit increase of about 227 millions USD for farmers
in the participating MSs.
5. Plant Mutation Breeding In CAAS
Mutation technique has become one of the most fruitful and widely used methods for crop
improvement in China, which have played a very important role in China's crop production,
especially food crop production. The total number of mutant varieties officially released, by
December 2011, accumulated to 810 including 45 crops and ornamental species. With the best
fit cultivation practices, the maximum area in total per year for mutant varieties cultivation
was over 9 million hectares, which accounted for about 1/10 of the total planting area forrelevant species in the past 20 years in China.
Protocols for crop mutation induction by spaceflight environment, energetic heavy ion beams,
fast neutrons and gamma rays irradiation have been established and applied for mutationinduction. Doubled haploids based in vitro mutagenesis and screening on salt tolerance were
optimized. A TILLING platform for wheat mutant gene discovery and lab screening
methodologies for rice quality characters were set up. More than 20 mutant varieties in rice,wheat, corn and vegetables were developed and officially released by the national orprovincial authority in CAAS in the past ten years. The total extension areas of officiallyreleased mutant verities since 2007 were about 700,000ha, with a benefit increase of about 75
millions USD for farmers in China.
6. Conclusions
Nuclear techniques can enhance biodiversity, improve crop cultivars and increase farmer’s
income. Routine utilization of induced mutations for crop improvement have made great
achievements especially through RCA. Induced mutants are not GMOs, as the mutant genes
have been changed from their own exist genes.

References[1] Uma Lele, Food Security for a Billion Poor, Science, 2010, 327, no. 5973, p. 155426[2] Guo Hui-Jun, Jin Wen-Kui, Zhao Lin-Shu, Zhao Shi-Rong, Zhao Hong-Bing, and Liu
Lu-Xiang，Mutagenic Effects of Different Factors in Spaceflight Environment of Shijian-8
Satellite in Wheat, Acta Agronomica Sinica 2010, 36(5): 764−770.
[3] Liu L-X, Guo H-J, Zhao L-S, Li J-H, Gu J-Y, Zhao S-R, Wang J. Current status and outlook
perspectives of induced mutations for plant improvement. Journal of Nuclear Agricultural

Sciences, 2009, 23(6): 1001-1007.
[4] Liu L.X., Guo H., Zhao L., Gu J., Zhao S. Achievements in the past twenty years andperspective outlook of crop space breeding in China. Journal of Nuclear Agricultural

Sciences, 2007, 21(6), 589-592.
71

[5] IAEA, Report of the Final Coordination Meeting of the IAEA/RAC TC project RAS/5/045“Improvement of Crop Quality and Stress Tolerance for Sustainable Crop Production Using
Mutation Techniques and Biotechnology”, Bangkok, 21-25 March 201172

5. Nuclear Technique for Soil Management
for Sustainable Agriculture

5.1 Development of Plant Growth Promoter and Plant Elicitor by Radiation
Processing of Natural Polymers
Kamaruddin Hashim
Malaysian Nuclear Agency
1. Pilot Scale Production of Oligochitosan
Pilot plant production of oligochitosan was carry out using the existing facility of gamma
vulcanize rubber latex available at MINT Technology Park of Malaysian Nuclear Agency.
Firstly, chitosan powder was irradiated by gamma for 50 – 75kGy irradiation dose prior todissolution in lactic acid to reduce it molecular weight. Pre-irradiated chitosan powder (3%)
was dissolved in 2%lactic acid solution and 0.1% to 0.3% hydrogen peroxide prior toirradiation at 12kGy by continuous flow gamma irradiation. The solution is then neutralizedwith sodium hydroxide to pH 6-8. The molecular weight of oligochitosan produce will be inrange of 10kDalton to 15kDalton depends on the initial molecular weight of chitosan powder.
Minimum production of oligochitosan by this system is 2.2 ton per run with concentration
20,000ppm. This process is more efficient and cost effective in comparison with othertechnique such as enzymatic and acidic hydrolysis degradation. Radiation degradation
oligochitosan has a potential to be used as plant elicitor and plant growth promoter for many
type of plants – vegetables, perennial crops and others. Protocol on the production ofoligochitosan has been established.
2. Application of oligochitosan as plant growth promoter and elicitor
2.1 Field test on rice
Field test using oligochitosan produce by pilot plant was performed on rice as plant growthpromoter and elicitor in collaboration with FELCRA Bhd, government owned company inagriculture business, Sg. Burung Agriculture Department and Malaysian Agriculture
Research Development Institution (MARDI). Field test with FELCRA Bhd was implemented
form August 2008 until January 2011 for 4 season rice plantation. However, field test withthe agriculture department is only for 2 seasons from January 2011 until December 2011.
The germination study on rice seed indicate that seedling period is shortened to 10-12 days
compare to normal practice 15-17 days using commercial chemical germination product.
Where, rice seed was soaked with 100ppm oligochitosan and the plant sprayed 3 times with
40ppm oligochitosan during the seedling period. During the germination period, rice seed ofoligochitosan treatment does not required nutrient compare to chemicals based treatment
need additional nutrient to support the growth. Result of field test at FELCRA Bhd show thatseed treatment and spray with 100ppm oligochitosan increase rice yield up to 26% compare tocontrol without oligochitosan. Field test at agriculture department give only up to 15%
increase in yield by spray 40ppm oligochitosan.

Blast study at MARDI shown thatoligochitosan has elicitation effects on paddy plants against blast attack. Laboratory study
74effect of oligochitosan on growth of Pyricularia oryzae has shown that oligochitosan 40ppm
enough to prevent the growth. The field trial results also shown that oligochitosan which isenvironmental friendly product, can replace chemical fungicide commonly use by the farmers
and at the same time give significant increase in yield. Protocol on application ofoligochitosan on rice has being established.
2.2 Field test on agarwood plant
Interest on Agarwood plant is due to its resin which being commercially explore for perfume
production. Oligochitosan is used to replace using chemical pesticide in grown the plant.
Study on oligochitosan as plant elicitor, the result from field trial on agarwood plant shown
less disease infection and the survival rate of the plant also increase. Early stage of growth
up to 2 years for agarwood plant, the plant can easily infected by fungus and insect. By
spraying oligochitosan every 1-2 week with minimum concentration 80 – 100pmm, the plantis being protected from infection. Result reveal that about 58% increase in height anddiameter of the plant when foliar spray with oligochitosan. Field test on agarwood plant
shown that oligochitosan improve growth and has elicitor effect on the plant.
2.3 Other application of oligochitosan
Tissue culture study on banana and pineapple indicate that the existence of oligochitosan in
the media increase the growth of the tissue plant. In the case of banana, 2ml of 10000g/mol
oligochitosan increase the weight more than 3 times compare to control without oligochitosan.
For the pineapple, 40ppm oligochitosan in the media is enough to triple the growth of thepineapple tissue culture plant. Testimonial of farmers on the application of oligochitosan asplant growth promoter shown that oligochitosan sprayed on the trunk of jack fruit plantincrease fruit production.

75

5.2
Biofertilizer production using radiation technology for sustainableagriculture
Khairuddin Abdul Rahim, Phua Choo Kwai Hoe, Ahmad Nazrul Abd Wahid, and Pauline
Liew Woan Ying
Division of Agrotechnology and Biosciences, Malaysian Nuclear Agency
The landscape of Malaysian agriculture is dominated by plantation crops of oil palm andrubber, in particular, and followed by rice, fruit trees, vegetables and herbal plants. Malaysia
has a history of heavy usage of chemical fertilizers (220 kg/ha), which was normallyassociated with high productivity as compared to average Asian countries (80 kg/ha). About
90% of the chemical fertilizers are imported, thus making the country’s agroindustry
vulnerable, generating an issue of national security. Nitrogenous fertilizers are the mostused; for example, urea is used at 70 Mt/yr @ RM 1500/t. 450,000 MT are N-based Fertilizers(urea & ammonium sulphate/chloride/nitrate). About 60% of oil palm cost of production is on
fertilizers. For comparison purposes FIVE kg of Bioorganic fertilizer is able to supply thesame amount of nutrients as ONE kg of N-based fertilizer. Thus, if we were to totally replace
N-based fertilizers, a total of 7,250,000 MT Bioorganic fertilizers needs to be produced andreadily available. Currently that capacity is far from attainable. Government subsidised
chemical fertilizers for farmers also contribute to the favourable adoption of chemicalfertilizers in normal agricultural practice.
With the present concerns on crop quality, food safety, and environmental safety there isincreasing awareness on biofertilizers among the agricultural industry, even the large
plantation companies. Several of them are beginning to be actively pursuing the ‘green’’approach to complement their usage of chemical fertilizer. For example Felda, Sime Darby
and Felcra are embarking on their own biofertilizer production, especially for oil palm. Theirinterest in biofertilizer is partly due to increasing cost of chemical fertilizers, particularly
urea, and partly to awareness on green technology for crop production. It is estimated thatthe market size for bioorganic fertilizer in Malaysia is RM 638.87 million/yr.
The main issue is sustainable agriculture. Malaysia is facing infertile soil due to loss of top
soil and years of planting on same soil in addition to increasing pest and diseases. The oilpalm industry, for example, faces pressure to become more sustainable, especially to achieve
the Sustainable Oil Certificate and Roundtable of Sustainable Oil Palm. An importantelement to consider is mitigating climate changes, where the climate pattern suitable forplanting, weeding and fertilizing hardly exist, hence providing greater stress to overallplantation management.
Nuclear technology is central to the activities, through the use of gamma irradiation forsterilisation of various carriers and the use of isotopic tracers to evaluate the efficiency of76

uptake of certain major nutrients, especially N, through the usage of biofertilizers. For the
future, the use of ionising radiation for mutagenesis of biofertilizer microorganisms is
proposed. Figure 1 the flow chart of a typical biofertilizer production utilising gamma
irradaiation for sterilisation of carrier or substrate.
BIOFERTILIZER PRODUCTION
EFB
POME

Carrier Production
Packing

Mixing35-50 kGy
Gamma-irradiation
MINTec-Sinagama

H2O
STERILE
CONDITION

Quality
Assurance

Inoculum Production
Gamma-irradiated

bags

Field
Experiments/
Ready for Use

Mother culture
Purity test

Incubation
28°C ± 2

Batch Culture

Inoculation

Sources: Khairuddin Abdul Rahim et al. (2004, 2005, 2006);
10
Zakry Fitri Abdul Aziz et al. (2005; 2006)

Figure 1. Flow Chart of Biofertilizer Production

Most recent research at the university and research institutions utilised the gamma
irradiation facility at MINTec-SINAGAMA, Malaysian Nuclear Agency for sterilisation ofsubstrates or carriers. These substrates from compost of empty fruit bunches of oil palm, peatand vermiculite can be packed in plastic bags and sterilised in bulk. MYAGRI, a biofertilizer
company in Malaysia has been sending their vermiculite carriers for sterilisation for several
years now, and indicated the suitability of the technology for it commercial activities, which
include close vicinity to MINTec-SINAGAMA. Figure 2 shows the gamma irradiation
facilities at Nuclear Malaysia.
Gamma
sterilisation
process

SINAGAMA Irradiation Plant
Cobalt-60 Gamma Irradiator

Source: Prof. Dr Zulkifli

Gammacell

11

Figure 2. Gamma Irradiation facilities at Nuclear Malaysia

77

The opportunities lie ahead of the Malaysian biofertilizer industry.
•

Less reliance on imported chemical fertilizers (many from fossil fuel based sources) –cost savings•

Protection of biological assets from plant diseases– healthier plants result in higher
yield, longer lifespan and higher productivity•

Established high value green and biotechnology products and services have goodpotential in other palm oil producing countries (Indonesia, PNG, Western Africa) –Malaysia as hub for ‘bio and green’ technologies•

Large fertilizer production and customized blends allows for niche productdevelopment – good potential for development of organic food industry•

Demand for treated organic fertilizers in Japan, Korea, Europe and the USA

The is much need on INNOVATION in formulation (e.g. multifunctionality); exploration of
biodiversity; mutagenesis of microorganisms; on methods of application e.g. Biofertilizer +Oligochitosan), Biofertilizer + Biochar or without the use of expensive or cumbersome
carriers at all. We have to be innovative in the ‘positioning” of biofertilizer in Malaysia
agroindustry, for example thorugh collaboration with companies for transfer of technology
and commercialization; providing incentives to biofertilizer companies in support of ‘GreenTechnology’ for food safety and societal wellbeing; proposal for government subsidy of
biofertilizers to farmers, as given to chemical fertilizers; and enhance promotional activities.
There for policy support, for instance on gulation on QC/standards of biofertilizer products.
As well as continued scientific and technical support from groups like the Forum for Nuclear
Cooperation in Asia (FNCA) – Biofertilizer Project (www.fnca.mext.go.jp).
There has been inroads of biofertilizer in the Malaysian agroindustry. However there is stillmuch to be done, to ensure the small successes be escalated to bigger ones in the future. The
following need to be considered:•

Awareness in agroindustry; market needs; demand driven; issues of food safety andsecurity.

•

Vibrant R&D in research institutes, universities and agribioproduct companies•

Avenues of commercialisation of R&D products.

•

Supportive government policies – green technology, bioeconomy initiatives•

Availability of advanced manufacturing technologies, including biotechnology andirradiation technology; this will lead to production of quality products•

Receptiveness of agroindustry to advanced technologies.

•

Effective promotional programmes.

For biofertilizers to have good reputation versatility is important, especially in terms offunction, effectiveness, ease of application and affordability.

78

Acknowledgement
1. MOSTI
2. Malaysian Nuclear Agency
3. FNCA
4. Universiti Putra Malaysia
5. MYAGRI Group of Companies
6. Inno Integrasi
7. Fellow researchers and proponents of biofertilizer.

79

80

6. Collaboration & Technology Transfer

6.1

Induced Mutation Breeding of Roselle (Hibiscus sabdariffa L.)§O. MOHAMAD1*, M. ZAINAL2, and M. MUHSIN3
1 Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA
2 Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM)
3 UKM-MTDC Symbiosis Programme, Universiti Kebangsaan Malaysia (UKM)

Extended Abstract
Roselle (Hibiscus sabdariffa L.) is a tropical annual plant species in Malvaceae family, which
originated from West Africa, predominantly from Sudan. It is a multi-purpose plant species
used for a myriad of purposes, from health to other uses in different countries spanning from
Sudan, West Indies, Mexico, Germany, India, Malaysia, Indonesia, Thailand to China. It has
a naturally satisfying sour taste and considerably unique nutritional characteristics, inparticular its high contents of vitamin C and anthocyanins. Roselle was introduced into
Malaysia in early 1990s. Then its commercial planting was first promoted by the Departmentof Agriculture in Terengganu in 1993 in collaboration with Universiti Malaya and MARDI.
The present total planted area is still small; however, its planting has now spread to manystates with total planted area of about 100 hectares annually. At present, there are two mainvarieties available to growers, namely UMKL-1 (aka Terengganu variety) and Acc. 21 (aka
Arab variety). The latter variety yields higher, both in terms of fruits and calyces. These twovarieties are mainly cultivated to produce pro-health juice for their high contents of vitamin
C and anthocyanins from freshly harvested calyces. The calyces are also processed into sweet
pickle, tea, and jam to some extent.
The roselle industry has faced many ups and downs like a yo-yo. Today, there are about 18
companies still involved in the production, processing and marketing of roselle products forthe local markets. It is estimated the total market value of the Malaysian roselle industry to
be approximately RM10-15 million, with 65-80% staying with the processors. The domestic
market consumes 500 tons of roselle calyces annually, of which over 80% are processed asconcentrate and drink. The current annual export value of fresh calyces is estimated at
RM2.5 million, mainly to Australia. Currently, it is estimated there are 25,000 domestic
consumers.
Worldwide, crop improvement efforts through conventional breeding have been little
attempted since hybridization is very difficult due its cleistogamous nature of reproduction,although there have been reports of considerably low natural outcrossing. Based on
cytological observations (2n=72) and trait segregation patterns, roselle is believed to be atetraploid species. Therefore, its segregating populations would need longer time for fixation
if compared to diploid species. To overcome this reproductive obstacle, mutation breeding
programme was initiated in UKM in 1999 in collaboration with Nuclear Malaysia (thenknown as MINT). In 2006, new work on mutation breeding was applied on two main varieties;
82however, only Arab variety responded favourably to radiosensitivity tests. These tests, basedon height of 2-week-old seedlings, determined the LD50 value of 80 Gy. Seeds of Arab variety
were then treated with gamma radiation from 60Co source using LD50 dose at UKM to produce
M1 generation. Selection and evaluation were done from M2 through M6 generations,alternating and overlapping between UKM, Bangi, Selangor and TFirdauce, Tasek Gelugor,
Penang in order to achieve rapid generation advance. Six potential selections identified andfurther evaluated. As a result of this mutation breeding programme, UKM launched in 2009
three of these stable selections as new varieties. The three new varieties are named UKMR-1,
UKMR-2 and UKMR-3. They are easily differentiated by their calyx colour, namely bright red,dark red and light green, respectively. To date, UKMR-3 has been approved for registration by
Department of Agriculture under the Plant Protection of New Plant Varieties Act 2004.
From then onwards, the development of the three new roselle varieties has enabled the roselle
industry to move forward, particularly in terms of providing additional choices for growers andthe industry. Together with the two main varieties, they have pushed roselle to gain wider
acceptance by the growers and consumers. Research done in UKM had also discovered thatsome roselle selections contain high contents of hydroxycitric acid (HCA), a potent
anti-obesity compound found in many nutritional supplements for managing body weight. To
date, three patent applications related with roselle have been filed.
The three new varieties exhibited significant differences for plant characteristics compared toits parent Arab variety and also the control Terengganu variety, showing better performancefor four parameters. These varieties have a number of their own special characteristics but ingeneral, they have overall features of having shorter maturity, medium plant size and reduced
plant height compared to their parent Arab variety. The leaf colour is generally green except for
UKMR-2 which possesses purplish-red pigmentation in its stems and leaves. The fruit shape is
attractive and somewhat different from their parent variety, but the fruits of UKMR-2 closely
resemble that of the parent. These three new varieties are superior to both Arab andTerengganu varieties in terms of plant height (shorter and more lodging resistant) and shorter
crop cycles of approximately 4 - 4½ months (i.e. these three varieties mature earlier by morethan one month). To ensure their genetic uniformity, the mother plants of these varieties aremaintained vegetatively such as through microcutting in soilless polythene mist-propagationchamber, while field planting of these varieties will use seeds harvested from the uniform
mother plants.
While roselle has been known to have been introduced into the country many decades ago, theactual crop improvement work on roselle in Malaysia is considered quite recent and has taken
quite a ‘long’ journey, where it was first introduced by Universiti Malaya in 1990s and afterwards
promoted by Department of Agriculture and MARDI, its breeding work was initiated andaccomplished in UKM, Bangi in 1999-2010, later continued in IIUM, Kuantan 2010-2012, andpresently it will continue its journey further at Universiti Teknologi MARA (UiTM).
83

6.2

R&D Activities in FELCRA

Ragu Ponusamy
Head of R & D, Federal Land Consolidation and Rehabilitation Authority, Malaysia
FELCRA, (Federal Land Consolidation and Rehabilitation Authority) was established IN
1966 with the objective of poverty eradication and improving the living standards of the ruralpoor. FELCRA achieves by developing land and cultivating with oil palm, rubber, rice andother profitable crops. To date FELCRA has successfully developed 220,000 ha. of land with arecord of 95,000 families as beneficiaries. FELCRA R&D activities are currently focused onyield improvement strategies such as Breeding and Tissue culture techniques for productionof superior planting materials besides conducting trials on other crops of commercial value.
Radiation and Nuclear technology has great potential particularly in FELCRA’s Rice sector
and short term crop like banana, stevia etc. As the Research and Development Department in
FELCRA is still in its infancy, FELCRA is open to new technologies which would ultimately
result in better yields and improved living conditions of the rural poor.
FELCRA is willing to participate in collaborative research efforts particularly in the area ofAgronomy, Biotechnology, Soil fertility and Green technology.

Keywords: Poverty eradication, collaboration, yield improvement.

84

6.3 Potential of Bio-fertilizer in Agro-industry:
Commercialization

MYAGRI’S Experience in

Hayati Md TaibGroup Managing Director, MYAGRI Group of Companies, MALAYSIAMYAGRI is a group of biotechnology companies dedicated to providing more efficient,effective and environmentally friendly solutions to challenges faced by today’s agriculturaland environmental management. MYAGRI principal activities consist of identifying, isolating,and propagating beneficial micro-organisms which are then formulated into ready-to-use
products with specific and efficient delivery systems. These products are aimed at improving
crop productivity, land rehabilitation, bio-remediation of wastes and long term sustainabilityof agriculture, environment and living.
MYAGRI’s range of premium biotechnology products include MYCOgold® - a root enhancer,
TRICHOgold® - a plant defence booster, MYCOblend® - a semi-organic fertilizer, aGricare®
BIO-ORGANIK - premium grade compost, D’CompTM Bio-plus – a bio-organic decomposerand METAXORB® - a bio-pesticide.
MYAGRI’s Bio-Organic program is a green waste management solution that allows the
Plantation and Agriculture Industry to seriously look at Nutrient Recycling for all of their
bio-resources, and reduce or complement the application of chemical or inorganic products.
The program assists the Oil Palm Plantation to convert all Green Wastes available at the Oil
Palm Mill such as Empty Fruit Bunch (EFB), Mesocarp Fiber, Decanter Cake and Palm Oil
Mill Effluent (POME) Sludge. These materials are treated and composted until they become
mature organic material. They are later enriched with MYAGRI's beneficial microbes to
produce Bio-Organic fertilizer. One of the microbes used in this program is a bacillus based
formulation, trade named aGricare® Organic N, that has been developed in collaboration
with Malaysian Nuclear Agency in 2010.
MYAGRI is committed to continuing R&D and commercialization of beneficial microbes andgreen biosciences that will improve the current agricultural practices; and welcomes
collaboration with local and international organizations.

Keywords: Bio-fertilizer, Biowaste conversion, beneficial microbes, yield improvement8586