Annex 4. Parallel Session Summary on Mutation Breeding Project

FNCA 2010 Workshops on
Mutation Breeding Project and Biofertilizer ProjectAt the beginning of Session, Dr. Artemio M. Salazar, Deputy Director of Institute of Plant
Breeding, University of the Philippines Los Baños (UPLB) delivered welcome remarks.
Session 3 Achievement and Challenges of the Project
All participating countries presented their reports on each sub-project, and the briefsummaries and discussion are as follows:

I. Following-up and Plan of Sub-Project on Insect Resistance in Orchid
Malaysia (Dr. Rusli Bin Ibrahim, Nuclear Malaysia)
Sub-Project on Insect Resistance in Orchid was terminated successfully in March, 2010. In
addition, the research work on insect resistance in orchid was reported that it useddifferent doses of gamma rays and ion-beam for irradiation, and they obtained promising
results for ion beam. PLBs were irradiated using effective doses and mutant lines throughin vitro cultures were regenerated, and it was successfully achieved for insect resistantDendrobium orchid variety through in vitro mutagenesis.
II. Conclusion and Plan of Sub-Project on Disease Resistance in Banana
Each participating country reported results and conclusion since Sub-Project on Disease
Resistance in Banana would be terminated this year. Most of countries, i.e. Malaysia, thePhilippines and Vietnam, have succeeded in produce several promising mutants tolerant tobanana diseases such as Fusarium and Banana Bunchy Top Virus (BBTV). Moreover,technology transfer for commercial use has been successfully carried out in Malaysia andthe Philippines. As for Bangladesh, which joined to the sub-project later, the protocol andscreening were established. Therefore, all FNCA participating countries recognized thisproject had accomplished fully and obtained promising results, indicating that theevaluation should be full marks 5. The summaries of reports are as follows.
Bangladesh (Dr. Md. Humayun Kabir, BAEC)

In vitro regeneration protocol of Fusarium susceptible banana cv. Sabri (AAB) wasestablished on MS medium supplemented with 5.0 mg/l BA + 2% Ads. In vitro
radiosensitivity (LD50) was determined at 35Gy. More than 2,500 plantlets weretransferred to the poly bags containing Fusarium infested soil and found heavily infested
and died after 2 to 3 months. About 1,000 plants were transferred to the field with hot spot
condition also showed symptoms of Fusarium and died within 6 to 8 months.

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Malaysia (Dr. Rusli Bin Ibrahim, Nuclear Malaysia)
Popular local banana cultivar called Berangan (Musa spp. AAA) was selected as thestarting 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 wasdetermined by 3 parameters, such as 1) Plant height (shoot length), 2) Percentage ofsurvival of irradiated explants and 3) Multiplication or Growth Rate. Based on thepercentage survival of irradiated explants, LD50 and LD100 obtained for cultivar Beranganwere 50Gy and 80Gy respectively. Using selected effective doses of 20, 30, and 40Gy, all in
vitro shoots derived from irradiated meristems were subcultured from M1V1 upto M1V5 androoted. These rooted in vitro plantlets were used to screen for tolerance/resistance against

Fusarium wilt disease. Four artificial 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 hours and later transferred to sterile sand media in the greenhouse,
2) Double tray method by inoculating in vitro plantlets that had been planted in sandmedia, 3) Nursery screening method, whereby rooted in vitro plantlets that had been
hardened for 4-8 weeks were transferred 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 hours were planted 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. Thesemutants will be further screened in the field for multi-location trials
The Philippines (Dr. Olivia P. Damasco, UPLB)
10 promising BBTV resistant mutant 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 three generations of continuous
evaluation under high BBTV infection. The preference of the aphids to the mutant lines
was evaluated using free choice or no choice feeding method. 5 mutant lines were lesspreferred by aphids. The reaction of selected mutant lines to artificial inoculation of thevirus using viruliferous aphids showed seven lines to consistently show low BBTV
incidence compared with the Lakatan control plants. Low incidences of the BBTV wereobserved in four multi-location trial sites, despite the presence of infected plants andvectors within the vicinity of trial site. Mutant lines were comparable with LK control (perplant basis) in terms of plant height, girth, total number of fruits produced, and number ofhands. Days to flowering were significant early in some mutant lines.
Vietnam (Dr. Dang Trong Luong, AGI)
Banana is widely grown in tropical regions throughout Asia and Vietnam. One of the mostserious diseases of banana is fusarium wilt, caused by Fusarium oxysporum f.sp. cubense-2-

(FOC) with several 'races' infecting plants, some being more virulent than others. In thisstudy, we used irradiation method to generate a new mutant banana line (ABB) enhance
resistant to FOC disease. Gamma treatment had some variations: change in high, thecolour of bud, leaf, and frequency variation from 1.8% to 12.5% at cultured medium andfrom 7.5% to 15.7% at gamma mutagenic treatment formulas. At the dose of 15 or 20kGy isrevealed appropriate in experiments. The evaluation of infected disease was performed
with artificial disease on banana plant in greenhouse. Spore density at 105 spore/ml, we
had 4 methods to taking disease on banana, cultured hydroponic reveals high infected
sensitive. We have obtained 17 banana lines enhance resistance to foc disease ingreenhouse (infected rates 25%). RAPD analysis showed that mutant lines were genetic
variation comparison to original lines.

III. Country Report of Sub-Project on Composition and Quality in Rice
Each participating countries presented on the achievements of the Sub-Project on
Composition and Quality in Rice. The main objectives of the project is to obtain goodquality e.g. good amylose content, protein content, aroma etc., depend on the importanceand priority for each country. For example, Malaysia had produced some potential mutant
varieties, which are tolerance to minimal water condition and resistance to blast disease
and they will identify potential mutant lines with improvement in amylose content andaroma. Vietnam is targeting for improved quality traits such as amylose, aroma, yield andresistance to BPH. For Thailand, main objectives are protein, low phytic acid, and amylose
improvement. For the Philippines, main objectives are improvement of protein andamylose content. Japan has completed the establishment of library for amylose content ofKoshihikari and Hitomebore mutant lines. For Bangladesh, main objectives are to produce
mutant varieties, which are salt tolerance, aromatic and improvement in amylose. For
Indonesia, main objectives are improvement of amylose content and early maturity, and for
China, main objectives are improvement of amylose content for hybrid rice. It wassuggested that this sub-project should be continued for another 2 years since most of theparticipating countries have not identified the potential mutant lines with improved target
characters such as amylose, protein, aroma, etc.
The summaries of reports are as follows;
Bangladesh (Mr. Protul Kumar Roy, BAEC)
The M3 progenies derived from carbon-ion irradiated seeds of a local salt tolerant
cultivar-Ashfal were evaluated. The M3 progenies derived from either 200Gy or 40Gy doses
had been mutated as photoinsensitive, where their parent Ashfal did not flower at all. This
progenies also showed early mature characters. Seeds were collected from M3 for yield trial
in the coming aman season.
BARRI-dhan 29 was irradiated with carbon-ion beam (Doses: 30, 40 and 50Gy). 3, 4 and 2
plants were selected from 30, 40 and 50Gy irradiated population for earliness by more than
one week. In evaluation of long fine grain-aromatic line RC-48-1-2-3, observed that grain-3-

quality was long fine aromatic, better production and also early mature in nature.
Screening of some mutant lines for salinity tolerance at the seedling stage in water culture
was carried out under glass house condition. It was observed that Ashfal-350 mutant
showed tolerant and Moinamoti-350 moderately tolerant. Both are maintaining up to
reproductive stage under water culture.
China (Dr. Luo Ju, CNRRI)
Hulled seeds of Z17, local super rice cultivar, were irradiated with 7 different doses (10, 20,
40, 60, 80, 100, 120Gy) by the heavy ion beam of the TIARA, JAEA in 2010. Thesematerials with 0 doses as control were cultivated in summer of 2010. The germination rate,
survival rate, growth rate and percentage of ripening were investigated. These results
suggested that the proper irradiation doses of heavy ion beam for Z17 might be between 30
to 50Gy doses.
Indonesia (Dr. Sobrizal, BATAN)
At M6 generation, 10 semi-dwarf lines derived from irradiated KI 237 lines were selected
as promising mutant lines. The result of genetic analysis of these lines indicated that thesemi-dwarf character of these lines was controlled by a single recessive gene anddesignated as sd237-1. These lines have various amylose contents, ranging from 17.39 to
25.65%, and among these lines, 7 lines and 3 lines have white and red grain color,
respectively. Preliminary yield trial of these lines revealed that the highest yield is 7.15
t/ha for RKI 198 and followed by 7.12 t/ha for RKI 199, whereas the yield of Ciherang, anational leading variety, is only 6.52 ton/ha. The yields of other lines were not significantly
different from that of Ciherang. These lines will be subjected to advance andmulti-locations yield trials before being released as new varieties.
Japan (Dr. Minoru Nishimura, NIAS)
In addition to 12 F4 NILs of Koshihikari with low amylose content, we obtained newly 11
mutants of Koshihikari. Amylose content of these mutants varied from 0.0% to 14.0%. We
obtained 24 mutants with low amylose content from Hitomebore, second major variety inJapan by irradiation with gamma rays and an ion beam.
Malaysia (Dr. Rusli Bin Ibrahim, Nuclear Malaysia)
Mutants were induced in variety MR219 using gamma rays at 300Gy to generate superior
genotypes for minimal water requirement. The mutants were screened for water stressunder simulated non-flooded water regime under glasshouse and field conditions. Twopotential lines designated as MR219-4 and MR219-9 were selected tolerance to saturated
condition in irrigated areas and aerobic conditions with improved yield.

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The Philippines (Ms. Adelaida C. Barrida, PNRI)
Development of rice mutants with desirable agronomic traits, good eating quality (low tointermediate amylose content) and high protein using mutation breeding was initiated at
the Philippine Nuclear Research Institute (PNRI) as part of cooperative project with theForum for Nuclear Cooperation in Asia entitled “Composition or Quality Crop Breeding in
Rice: Grain Quality Improvement in Rice (Oryza sativa L.)”.
In M4 generation, results of amylose content analysis using iodine staining method showedthat only few lines belong to low to intermediate amylose in plants irradiated with 200 and
300Gy, while the control had intermediate to high. Slight increased in the protein content
of irradiated plants were obtained in comparison with the control.
Initial result using ion beam irradiation revealed that the best dose was 20Gy and thelethal doses were 160 and 200Gy. Earliness in the number of days to heading was observedin the M1 plants irradiated with 10 and 20Gy, and the control, however, this result needs tobe confirmed on the succeeding generations.
Thailand (Mr. Suniyom Taprab, DOR)
Seeds had been irradiated and M1 obtained in wet season 2007. M2 plants had been
selected on photoperiod-insensitive mutants. Then, M3 seed of 200 mutant lines had been
separately harvested as single hill per line. Those 200 photoperiod-insensitive mutants
were continuously screened for important agronomic traits and to be analysed for amylose
content. They were again tested for photoperiodism, tillering ability and maturity in dry
season 2009 and had been analysed for amylose content. Selected M5 mutants had beengrown in dry season 2010 and are being presently grown in wet season 2010. In 2008, wedeveloped simple technique for amylose content determination on breeding lines anddistributed to FNCA members. While amylose content was being analysed, we developed atechnique for protein analysis. Protein extraction technique reported by Iida et al.(1993)
and Tanaka et al. (2004) had been modified and tested for indica rice protein analysis.
Extracted proteins such as globulin, albumin, prolamine and glutelin from KDML105 andKoshihikari had been identified using SDS-PAGE technique (Laemmli, 1970). Low
amylose mutant lines had been achieved from KTH17 which is originally high amylose
content local variety. Various amylose content mutant lines from original low amylose
content varieties KDML105 and RD15 had been obtained. Some of those mutants
contained lower and higher amylose content than did of their wild types. Protein and its
components are being analyzed. In case of low phytic acid mutants, 2 mutant lines from
SPR1 are going to be tested for bioavailability in artificial intestine digestion. New
irradiated populations from CNT1 had been screened for low phytic acid mutants.
Vietnam (Ms. Dao Thi Thanh Bang, AGI)
Ion beam irradiation treatment was conducted with a certain number of the seed todetermine the optimal dose for experiment. From the optimal dose, 6,000 seeds of Khangdan variety was irradiated in 40Gy and 60Gy of ion beam in Japan last May 2009. Beside-5-

of that 3,000 seeds of each dose for gamma ray at 100, 150 and 200Gy for Khang dan and
Bacthom varieties. The reason to use Khang dan and Bac thom varieties for experiment isKhang dan is normal rice for common use, but Bacthom is quality rice this variety isalways give higher price for milled rice. Quality rice usually gives low yield, problem is how
to increase of yielding but still keep quality at the reasonable level. At M1 generation, 5
seeds per plant were taken in random for making M2 generation. In M2 generation, fromgamma ray as well as ion beam irradiation treatment of both Khang dan and bac thomvarieties range of segregating were observed in plant height, grain weight, maturity or
sterile. M3 generation, some interesting characteristics related to yield were chosen for
example: grain weight, short grow duration, short plant and tolerant to disease. According
to Khang dan variety, higher yield is most important then lodging character and disease
resistant. But for Bacthom variety first is also higher yield, then quality in acceptable level(still aroma) and disease resistant. Most of those characteristics will be evaluated in M4
generation. Through ion beam irradiation of Khang dan variety we recognized that thegrain weight of the seed is sensitive with ion beam.

IV. Ion-beam Induced Mutation Breeding of Rice
This research activity was started in January 2009 and most of FNCA participating
countries had produced promising results with irradiation of ion beam, which depended on
their materials and experimental conditions. Some countries have problem with the seedgermination after treatment, especially for Bangladesh and Malaysia. As for Vietnam, they
achieved good result in germination but not so promising result at field condition. Most of
the members suggested to repeat the experiment by irradiating the seeds with ion beam fornext year in 2011. Dr. Atsushi Tanaka, Japan Atomic Energy Agency (JAEA) discussed theappropriate population size for irradiation treatment and suggested the minimal numberof initial cells to be irradiated at least 5,000 in order to achieve high frequency of mutation.
The protocol for selection of seeds after irradiation was also discussed and it was suggested
to harvest 5 seeds from 5 panicles from each M1 plant.
Most of participating countries had completed the experiment at M2- M3 generation and forthe year 2011, potential mutant lines with improved quality traits such as amylose will be
evaluated. Finally, it was suggested that this activity should be continued at least foranother 2 years in order to produce promising results.

Session 4 Possible Cooperation with IAEA/RCADr Luxiang Liu, Project Lead Country Coordinator of RCA/IAEA project RAS5045, China,
Chinese Academy of Agricultural Sciences (CAAS), reported on the regional activities andmain achievements of the RCA project. The summary of report is as follows ;There are 14 member states participating in this RCA Project. The overall objectives are todevelop and transfer methodologies for induction and identification of mutated genes
contributing to improved quality and stress tolerance and develop improved breeding-6-materials using molecular marker-assisted selection. For 2007-2010, RCA had conducted 2
regional meetings and 3 regional training courses and published training manuals andworking

document.

For

2010,

the

major

achievements

of

RCA

includenewtechniques/methods established and improved including mutation detection platform using
EMAIL and TILLING methods, development of new mutated populations and mutant lines.
33 new mutant varieties were officially released under this project by the end of September
2010.

Possible Synergy FNCA/RCA
Dr. Sueo Machi, FNCA Coordinator of Japan, suggested the possible FNCA/RCA synergy
on mutation breeding. All participants agreed following points ;a. FNCA Project of Mutation Breeding to be continued to develop mutant varieties of ricefor quality improvement. The project studies could be supported by moleculartechniques that have been focused in the RCA project RAS 5045 in the Member States.
b. FNCA project results on ion-beam induced mutation breeding could be used by the new
RCA project on Mutation Breeding for the development of new mutant varieties by
sharing of experience and knowledge.
c. In near future, workshop of Mutation Breeding Project of FNCA should be held inparallel with workshop of Mutation Breeding project of RCA in order to have joint
session for one or two days to share views, information and experience to enhancesynergy of the two projects.
It was also agreed that FNCA and RCA would go as planned and communicate with
scientists within each country.
Session 5 Future Plan and Meeting Report
I. Activity Plan for Mutation Breeding of Rice : Quality, Dwarf, Selection Method ofTargeted Mutants;
Dr. Nishimura presented an example of future activities of Mutation Breeding Project inJapan. Japan needs 2 more years to complete the project. For other members, Bangladesh
joined the project, so they need 2 more years to complete. All members need to do analysis
on grain quality in 2011 and probably finalize the report in 2012. The members agreed tocontinue mutation breeding by sharing of facilities of ion beam and other mutagenesis inother aspects. For this project, members suggest two more years to finalize the report.
II. Possible Application of Mutation Breeding for Sustainable Agriculture in Asia
The 3 points to be discussed for the breeding purposes of rice and mutagenesis to be usedwere proposed by Dr. Hirokazu Nakai, Project Leader of Japan, as follows:a. Resistance to various environmental stresses e.g. soil salinity, minimal water input,
various diseases (bacterial leaf blight/sheath etc.), drought, high and low temperature,
heavy fluctuation of weather, etc.
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b. Adaptability to the conditions of the organic farming (or low input sustainableagriculture)c. Mutagenic effect, including that of ion-beam.
Referring to the above proposals, the future plan of Mutation Breeding Project was discussed.
The direction of the above proposals was generally accepted. It was concluded as follows ;1) The projects already started, e.g. quality of rice, content of protein, etc. may be continued
for another one year.
2) Trials of mutation breeding for resistance to environmental stresses need to bechallenged depending on the situations of each country.
3) As for mutagens to be used, every country should share irradiation facilities formutagenesis treatments of plant materials.

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