FNCA 2006 WORKSHOP ON MUTATION BREEDING
â—?/span>Three Year Plan â—?/span>the attachmentIII â—?/span>the attachmentIV â—?/span>the attachmentV â—?/span>the attachmentVI â—?/span>the attachmentVII â—?/span>the attachmentVIII

Three Year Plan of the FNCA Project on Mutation Breeding

  FY 2005 FY 2006 FY 2007 FY 2008
Overall Schedule

The 6th FNCA Meeting
(Nov. 30 - Dec. 1, 2005, Japan)
The 7th CM
(Mar. 1 - 3 2006, Japan)

The 7th FNCA Meeting
(Autumn 2006, Malaysia)
The 8th CM
(February 2007, Japan)

The 8th FNCA Meeting
(Autumn 2007, Japan)
The 9th CM
(March 2008, Japan)

The 9th FNCA Meeting
(Autumn 2006)
The 10th CM
(March 2009, Japan)
Workshop Dec. 5-9
Malaysia		Sept. 11-15
Japan		Nov.
Korea

Sub-Project 1
"Drought Tolerance in Sorghum and Soybean"

Further selections of the tolerant lines and establishment of pure lines

 Evaluation of the promising mutant lines for drought tolerance
*Final Report		  

Sub-Project 2
"Insect Resistance in Orchid"		 [Sub-project Meeting in Thailand]
  • Irradiation of PLBs
  • Development of selection techniques(in vitro)(continue)
  • Multiplication of irradiated PLBs (three subcultures)
  • Transfer plantlets to the greenhouse for selection
    *Mid-term Review
    		• Mite
  • Development of selection techniques(in vitro) (completed)
  • Development of selectiontechniques (in vivo)(continue)
  • Transfer plantlets to the greenhouse for selections of flower color mutant(s)(continue)
  • In vitro selection for insect tolerant clones (continue)

    Thrips
  • Development of selectiontechniques (in vivo completed for seedling stage)
  • Development of selection techniques (in vitro) (continue)
  • Transfer plantlets to the greenhouse for selections of flower color mutant(s)(continue)
  • In vitro selection for insect tolerant clones (continue)
    		•  Orchid Meeting in Malaysia
  • In vivo selection for insect tolerant clones
  • Meristem cloning of flower color mutant(s)
  • Meristem cloning of tolerant mutant(s)
    *Final Report
    		•

    Sub-Project 3
    "Disease Resistance in Banana"
  • Induction of embryogenic callus from male inflorescence or other explants etc.
  • Irradiation of materials etc.
    		•  [Sub-project Meeting in the Philippines]
    <BBTV>
  • Field evaluation of material (Generation 1&2)

     <Fusarium>
  • Field Screening in hot spot (Q4-2006)
    *Mid-term Review
    		•  FY2007
    <BBTV(other diseases)>
    Greenhouse and field screening (Generation 3) (Q4-2007)

     <Fusarium>
    Greenhouse and Field screening in hot spot  (Q3-2007)    		 FY2008
    <BBTV>
    Multi location trials and performance evaluation

     <Fusarium>
    Performance evaluation
    * Final Report
    Sub-Project 4
    "Composition or Quality Crop Breeding  (tentative)"    		     FY2007
    - Exchange of breeding material and information
    - Optimization of irradiation method and mutation Induction and
    - Standardization and selection method for amylose contents    		 FY2008
    - Establishment and characterization of potential mutant lines (M1-M2)
    Mutation Breeding Database (MBDB)   FY2006
    Distribute FY2004 version on FNCA web site by semi- closed system    		 FY2007
    Distribute FY2004 version on FNCA web site by semi- closed system    		 FY2008
    Update and distribute FY2008 version on FNCA web site by semi- closed system
    Mutation Breeding Publication Database (MBPD) (draft)   Submission of publications (Continue) Submission of publications
    Build MBPD on FNCA web site     		Distribute and update MBPD on FNCA web site
    Attachment III

    Final Report for Drought Tolerance in Sorghum

    as of September 15 2006

    China, Indonesia

    Item

    Result

    1. The selection of starting breeding materials.

    - Whether it was appropriate or not?

    Sorghum is an important crop for China and Indonesia. In this project, breeding materials from the two countries have mutually been exchanged. In China, breeding materials, “Sart” and “Zhenzhu” w ere irradiate d by gamma rays in 2002; Y030 and Yuan8002 were irradiated by proton; Wusawu, Jinwu, Aisi and Yuan2B were irradiated with e lectron beams in 2003. “Sart” is a suitable one, but desirable mutants could not be obtained. Yuan 2B is also a suitable starting material because of its dual uses, either as grain sorghum or as sweet sorghum cytoplasmic male sterile (cms) line improvement. Through this project, 5 breeding materials , ME/30/9, ET/20/477, DU/20/3E, JA/30/58 and Durra , were introduced from Indonesia to China .

    In Indonesia, 5 breeding materials were introduced from China, namely Zhenzhu, Yuan 002, Yuantianza, Yuanyu 8002, and SP4 8002. Among those materials, Zhenzhu is found to be worthy for further use in breeding programs. The sorghum germplasm collections have been enhanced with these introduced materials, local and national varieties, materials introduced from ICRISAT, and some mutant lines generated from mutation breeding works.

    2. Irradiation to starting materials.

    - The determination of the methods, e.g. appropriate dose, stage of the plant, part of the organ etc.

    Gamma irradiation was used to generate mutants in China and Indonesia. In both c ountries, effective doses of g amma irradiation have been studied. In China, the optimal doses were found to be 200 - 250 Gy, while in Indonesia it was found to be 300-500 Gy. The difference might be due to the different materials treated. In China, new mutagens like proton, electron beams, and space environment were also used . For proton, appropriate dose is 300Gy under 5 MeV; for electron beams (5MeV) 30-50Gy to white seed type, 100-150 Gy to red seed type. In Indonesia, induced mutation using c arbon ion beams (220 and 320 MeV) has just been started in 2005 . Seed irradiation was conducted at TIARA, JAEA ion beam facilities and at RIKEN facilities in Japan.

    3. Selection or isolation of mutants with drought tolerance.

    - Establishment of selection method of mutants with drought tolerance.

    - Evaluation or characterization of mutants.

    S creening method s for drought tolerance used in b oth China and Indonesia were as follow s :

    A. Indirect selection:

    1. Using 25 % Polyethylene Glycol (PEG Method). This method is used in seedling stage. It can reduce water potential equivalent to natural drought condition so that water absorption by roots is affected.
    2. Using 30% solution of potassium iodide. This method is used in seedling stage. It can simulate leaf senescence and abscission equivalent to natural drought condition (Singh and Chaudhary, 1998).

    B. Direct selection:

    The plants are grown directly in drought prone areas during dry season. Sowing time is usually adjusted by the end of the rainy season. Production of total biomass and yield can be used as criteria for selection.

    4. Development of promising lines by using the mutants, local adaptability test or characterization of the selected promising mutant lines.

    - The data of LAT or field trials and estimate of the ability of lines.

    China has developed 2 restoring lines ( 86002, SP84002 ) and one sweet sorghum variety ( Yuantian No.1 ) with drought tolerance . Two F1 hybrids by using these two restor ing line s were then produced. The results of yield trial and stress tolerance at different locations showed that SP84002 and Yuantian No.1 were tolerant to drought and high yielding.

    Indonesia has developed 10 drought tolerant mutant lines namely B-68, B-69, B-72, B-75, B-83, B-90, B-92, B-94, B-95, and B-100. Together with the control varieties, they are now being evaluated under multi location trials. Biomass production and grain yield were used as criteria of drought tolerance. During dry seasons, one of the mutant lines, B-100, has yield increase by 30% compared to the original variety , Durra.

    5. Availability to share promising mutant lines among the participating countries and to use them as parental lines for developing new varieties.

    In China, 2 new mutant lines developed during the project are available to share in the program.

    In Indonesia, the seeds of mutant lines B-95 and B-100 and their parent “Durra” have been sent to Chin a for further evaluation .

    6. Publication of the results or registration of developed cultivars.

    Publications related to sorghum research are listed as follows:

    1. Soeranto, H. and Nakanishi, T.M. (2003) Obtaining induced mutations of drought tolerance in sorghum. Journal Radioisotopes , Vol. 52, No. 1, p15-21.
    2. Soeranto, H. and Nakanishi, T.M. (2003) Screening sorghum mutants for drought tolerance to support sustainable agriculture development. Proceedings of the 2nd Seminar of JSPS-DGHE Core University Program in Applied Biosciences. The University of Tokyo, Japan, 15-16 February. p 166-180.
    3. Li Guiying, Gu Weibin and Keith Chapman (2004) Sweet Sorghum . Beijing: China Agricultural Science and Technology Press .
    4. Li Guiying and Su Yimin (2005) Development of a new high quality sweet sorghum-Yuantian No. 1 and its cultivation . Crops, No. 5 , p 60 (in Chinese).

    In China, a sweet sorghum mutant variety Yuantian No.1 has been officially released by National Committee of Fodder Crop Examination and Appraisal. The fresh biomass yield increased by 20%, and grain yield by 18.2%. Up to now, its planting area is ca. 1000 ha.

    7. Other result and ripple effects of the project, if there are.

    In China, trainees of IAEA/INT/5147 and IAEA/RCA/RAS5040 had opportunities to study mutation breeding by observing their experimental fields. Visiting scholars from the Philippines and Iran supported by IAEA studied mutation breeding techniques in China.

    In Indonesia, some of sorghum breeding materials have been used by university students for their studies and compan ies for the production of food, animal feed and et hanol . Sorghum cultivation can stimulate soil conservation, soil improvement, and making full use of marginal lands. In other words, it can promote a sustainable agriculture development.

    8. Future research plan

    In China, advanced sorghum lines will be further evaluated for releas e or used as new parental lines in breeding program s . Further exchange of sorghum lines with any countr y can be c onsidered. New mutant lines with high fermentable sugar content will be induced by mutation breeding.

    In Indonesia, sorghum breeding for improved quality of food, animal feed and starch and ethanol industry will be continue d . Sorghum research collaboration with national and international counterparts will be further strengthened.

    Final Report for Drought Tolerance in Soybean

    Indonesia , Malaysia, Philippines , Vietnam

    Item Result

    1. The selection of starting breeding materials.

    - Whether it was appropriate or not?

    Indonesia used cv. Guntur as breeding material to increase variability, then 2 selected mutant lines ( N o. 9 and 23) were crossed. The F1 hybrid of cv. Muria X PI-48025 was irradiated by gamma rays. Some promising mutant lines were obtained.

    Philippines used f our local varieties (PSB-Sy 4, PSB-Sy 5, BPI-Sy 4, NSIC-Sy 8) and four varieties from Vietnam ( AK- 0 6, DT-84, DT-95 and DT-96) as starting materials for mutation induction. G enetic materials were exchanged between Vietnam and the Philippines (as agreed upon in the 2002 Formulation meeting). T hese varieties were multiplied and evaluated in the Philippines . T he breeding materials w ere appropriate.

    Vie tnam u s ed 4 varieties ( PSB - Sy 4, PSB - Sy 5, BPI - Sy 4, NSIC -Sy 8) introduced from the Philippines as starting breeding materials . They, however, were not good material s because of the low thermo-tolerance and low seed yield at summer season . Vietnam need s starting materials with wide adapt a bility for temperature and different cultivat ion. In 2004 - 2005, Vietnam rece i ved 28 soybean accessions from the USA , A rg entina , China and others for sele c tion of starting material s for breeding material .

    Vietnam used 3 varieties: DT96, DT2001, D.158 as starting materials for inducing drought tolerant mutants.

    Malaysia used 3 mutant lines introduced from the Philippines (PSB-Sy 4, PSB-Sy 5, BPI-Sy 4) as the starting materials for mutation breeding program.

    2. Irradiation to starting materials.

    - The determination of the methods, eg. appropriate dose, stage of the plant, part of the organ etc.

    Indonesia identified the optimum dosage of starting materials at 150 Gy with the dose rate of 750 Gy.h -1 .

    The Philippines established a method of irradiation . Seeds of the local and introduced soybean varieties from Vietnam were irradiated with gamma rays ranging from 100 to 300 Gy. The optimum dose s were 200 and 250 Gy. This is found to be appropriate dose s.

    Vietnam had established the method: Irradiation of dry seed with optimum dose s 150, 200, 250 Gy using gamma ray was appropriate. The mutation rate in M 2 ranged between 0 . 026 - 0.037%. Control varieties ranged between 0.002 – 0.003

    3. Selection or isolation of mutants with drought tolerance.

    - Establishment of selection method of mutants with drought tolerance.

    - Evaluation or characterization of mutants.

    Indonesia and Vietnam had established the methods to increase genetic variation by the combination of hybridization and irradiation.

    Philippines used irradiation for mutation induction and screening was done both in the greenhouse and in the field by applying withdrawal irrigation method.

    Indonesia and Vietnam evaluated the mutant lines under n atural drought climat ic condition in drought-prone area.

    4. Development of promising lines by using the mutants, local adaptability test or characterization of the selected promising mutant lines.

    - The data of LAT or field trials and estimate of the ability of lines.

    Indonesia has developed 8 drought tolerant mutant lines in the M6 generations.

    Philippines has obtained p romising mutant lines for drought tolerance, earl y maturity and high yielding ability in the M3 up to M 6 generations at doses of 200 and 250 Gy. Further selections of mutant lines for drought tolerance were undertaken in the M5 and M6 generations of both the introduced varieties from Vietnam and the local ones.

    Vietnam had developed a new mutant variety of drought tolerance and high yielding, DT96 developed in 1992 which was officially released as a National Variety in 2004. Three drought tolerant lines were selected in the M3 generation . The results of local adaptability test at Hanoi A g r icultural S tation show ed that l ine 01/245 (treatment with 200 Gy) had big seed and high yielding, l ine 58/012 (treatment with 250 Gy) showed early maturity, l ine 96/26 (treatment with 150 Gy) showed toleran ce to drought and high yield.

    5. Availability to share promising mutant lines among the participating countries and to use them as parental lines for developing new varieties.

    Indonesia had distributed 4 mutant lines for field test in Malaysia and the Philippines in 2005 .

    The Philippines had developed 3 potential drought tolerance mutant lines but with limited amount of seeds to be distributed to participating countries. Seeds will be multiplied and will be available after one cropping season .

    Vietnam will share 2 mutant lines with pa rticipating countries and use them as parental lines for developing new varieties.

    Malaysia received three lines from the Philippines .

    6. Publication of the results or registration of developed cultivars.

    Indonesia has made 2 publications:

    1. Masrizal, Hari Is Mulyana, Kumala Dewi, Arwin, Ina Idayanai Rahma, and Yuliasti (2004) Usulan Pelepasan Varietas galur GH-7. P3TIR – BATAN.
    2. Masrizal, Hari Is Mulyana, Kumala Dewi, Arwin, Ina Idayanai Rahma, and Yuliasti (2006) Usulan Pelepasan Varietas galur M-220. P3TIR – BATAN.

    Philippines has 1 publication:

    1. Asencion, A.B., A.G. Lapade, A.O. Grafia , A.C. Barrida, A.M. Veluz and L.J. Marbella. 2004. Induced mutations for the improvement of Soybean ( Glycine max L.) in the Philippines . Philippine Nuclear Journal 14: 12-25.

    Malaysia has 1 publication in Malay:

    1. Norliyana Ali (2005). Penilaian prestasi tiga titisan baru kacang soya di UKM. B.Sc. Thesis, Universiti Kebangsaan Malaysia (UKM)
    Translation:
    1. Norliyana Ali (2005). Evaluation of three new mutant lines of soybean at UKM. B.Sc. Thesis, University Kebangsaan Malaysia (UKM)

    Vietnam is yet to publish paper.

    7. Other result and ripple effects of the project, if there are.

    In Indonesia :

    1. Two students of the Sriwijaya University and Bogor Agricultural University had used mutant lines to study the characters of mutant and field test.
    2. The amount of 2.2 tons breeder seeds of cv. Rajabasa have been distributed to the growers for seed production under the Department of Agriculture in 2006.

    In the Philippines :

    1. M.S. and B.S. students from the De La Salle Araneta University conducted their thesis on the effects of different doses of gamma radiation and various water regime using mutant lines.
    2. Collaboration with Bulacan National State University (BNSU) through PNRI-GIA was done

    In Vietnam :

    1. Three students from Hanoi University had opportunity to study mutation breeding by observing our activities and test fields and had completed their reports
    2. The amount of 100 tons of certified seeds of variety DT96 had been distributed to growers for commercial production
    3. Research was done in collaboration with Vietnam Atomic Energy Commission (VAEC) for seed irradiation.

    8. Future research plan

    In Indonesia , mutant line M-220 ha s been evaluated by the National Seed Evaluation C ommittee and 2 more multi-location yield trials are needed. This mutant line is expected to be released in mid 2007 . Multi location trials will also be conducted for other potential mutant lines.

    In the Philippines , d eveloped mutants will be evaluated further in other experiment stations for yield trials. The mutants will be submitted to the National Seed Industry Council (NSIC) for registration.

    Vietnam had d eveloped mutant lines that will be evaluated further in the local experiment stations for registra tion .

    Malaysia : selected mutant lines will be further evaluated at University Kebangsaan Malaysia (UKM) for their performance on drought tolerance, yield and nutritional value.

    Attachment IV

    Final Report for Drought Tolerance in Sorghum

    Project name:

    		Mutation Breeding for Drought Tolerance in Sorghum

    Project leader (country):

    		All Project Leaders (China, Indonesia)

    Date:

    		12 September, 2006

    Object of project:

    		Development of drought tolerance of sorghum
    1. Major outcome of project

      In China:

      1. A sweet sorghum mutant variety Yuantian No.1 was officially released by National Committee of Fodder Crop Examination and Appraisal. The fresh biomass yield increased by 20%, and grain yield by 18.2%. Up to now, its planting area is ca. 1000 ha.
      2. 6 lines from space-induced Yuan 8002 and gamma-irradiated Yuan 2B in China are produced. SP8002 is a new mutant restoring line with higher combining ability.
      3. A lot of individual plants with high fermentable sugar content or high biomass have been selected from F2 generation of the cross ET/20/477 (one drought tolerant mutant line from Indonesia) with sweet sorghum Roma, Rio and Rio/BJK156-1-3-1 in China.
      4. Gamma irradiation together with new mutagens like proton, electron beams, and space environment were studied for their effect on sorghum mutation induction. Optimal dose of gamma ray was found to be 200-250 Gy. For proton, appropriate dose was 300Gy under 5 MeV; for electron beams (5MeV), appropriate dose was 30-50Gy to white seed type, 100-150 Gy to red seed type.

      In Indonesia:

      1. From irradiation of Durra, 10 drought tolerant mutants were obtained in Indonesia. These lines produce biomass (stem and leaves) and grains significantly higher than the parent Durra and the national check variety (UPCA and Higari) in the dry season. From irradiation of Zhenzhu, a promising mutant "Zh-30"was obtained which is found to be highly tolerant to drought and promising to be developed further as a white grain sorghum.
      2. The appropriate gamma-ray dose for sorghum mutation was found to be 300-350 Gy, and the selection of mutants for drought tolerance has been done by indirect methods (PEG and KI methods) and direct method in the field.
      3. Sorghum germplasm collection has been established by some desirable mutant lines, local and national varieties, exchanged materials from China and introduced accessions from ICRISAT.
      4. Good sorghum research collaboration with some national and international counterparts and the end users has been established.
      5. A private company (LIPPO Enterprises) has identified some sorghum mutant lines to be good for food, animal feed, starch and ethanol industries.

      Publications:

      1. Soeranto, H. and Nakanishi, T.M. (2003). Obtaining induced mutations of drought tolerance in sorghum. Journal Radioisotopes, Vol. 52, No. 1, p15-21.
      2. Soeranto, H. and Nakanishi, T.M. (2004). Screening sorghum mutants for drought tolerance to support sustainable agriculture development. Proceedings of the 2nd Seminar of JSPS-DGHE Core University Program in Applied Biosciences. The University of Tokyo, Japan, 15-16 February. p166-180.
      3. Li Guiying, Gu Weibin and Keith Chapman. (2004). Sweet Sorghum. Beijing: China Agricultural Science and Technology Press.
      4. Li Guiying and Su Yimin (2005). Development of a new high quality sweet sorghum-Yuantian No. 1 and its cultivation. Crops, No. 5, p60 (in Chinese).
    2. Evaluation indicators for above outcome
      Socio-economic impact
      (Application or Achievement of project object) : 5 point
      Scientific impact (Basic technology or Activity): 4 point
    3. Project outlook

      □Continuation □ Change/Revision ■Termination

      The reason:
      The project has achieved its objectives by developing potential mutant lines to be released as new varieties for drought tolerance or new materials for future breeding programs.

    4. Ripple effect to other fields or Benefits to end-users

      In China:

      1. Trainees of IAEA/INT/5147 and IAEA/RCA/RAS5040 had opportunities to study mutation breeding by observing their experimental fields.
      2. Visiting scholars from the Philippines and Iran supported by IAEA studied mutation breeding techniques in China.

      In Indonesia:

      1. Some of sorghum breeding materials have been used by university students and researchers from different institutions for their research studies.
      2. Private companies (LIPPO Enterprises and Great Giant Pineapple Co.) are still investigating the sorghum mutant lines to be used for food, animal feed, and materials for industries (starch and ethanol industries).In addition, sorghum will also be developed for helping soil conservation in the pineapple plantation in Lampung Province.
      3. Some local farmers were trying to use the sorghum mutant lines for making traditional food such as "Sogi" and "Ladu".
    5. Opinion about the project (problems, ideas, remarks, proposal)
      1. Training courses for young scientists are needed for sorghum research and development.
      2. The financial support to research group from government should be increased.
      3. Some assistance from FNCA is needed, especially for irradiation services on sorghum research or mutation breeding in sorghum.
      4. Exchange of sorghum germplasm among participating countries should be further enhanced.
      5. Exchanges of expertise between China and Indonesia (with financial support from FNCA)

    Final Report for Drought Tolerance in Soybean

    Project name: Mutation Breeding for Drought Tolerance in Soybean
    Project leader (country): All Project Leaders (Indonesia, Vietnam, the Philippines, Malaysia)
    Date: 12 September 2006
    Object of project: Development of drought tolerance of soybean.
    1. Major outcome of project:

      In Indonesia

      Eight mutant lines have been developed by means of pure line selection in M6 generations for drought tolerance.

      In the Philippines

      Three promising mutant lines with drought tolerance and high yielding ability have been obtained in the M3 up to M6 generations at doses of 200 and 250 Gy [of 200and 250 Gy irradiation] . Further selections of mutant lines were undertaken in the M5 and M6 generations of both the introduced varieties from Vietnam and the local ones.

      In Vietnam

      A new mutant variety of drought tolerance and high yielding, DT96 developed in 1992 was officially released as a National Variety in 2004. Three drought tolerant lines were selected in the M3 generation. The results of local adaptability test at Hanoi Agricultural Station showed that line 01/245 (treatment with 200 Gy) had big seed and high yield line 58/012 (treatment with 250 Gy) showed early maturity, line 96/26 (treatment with 150 Gy) showed tolerance to drought and high yield.

      Publications:

      Indonesia has made 2 publications in Indonesian:

      1. Masrizal, Hari Is Mulyana, Kumala Dewi, Arwin, Ina Idayanai Rahma, and Yuliasti (2004) Usulan Pelepasan Varietas galur GH-7. P3TIR – BATAN.
      2. Masrizal, Hari Is Mulyana, Kumala Dewi, Arwin, Ina Idayanai Rahma, and Yuliasti (2006) Usulan Pelepasan Varietas galur M-220. P3TIR – BATAN.

      Malaysia has 1 publication in Malay:

      • Norliyana Ali. 2005. Penilaian prestasi tiga titisan baru kacang soya di UKM. BSc. Thesis University Kebangsaan Malaysia Bangi Malaysia .

      Philippines has 1 publication:

      • Asencion, A.B., A.G. Lapade, A.O. Grafia , A.C. Barrida, A.M. Veluz and L.J. Marbella. 2004. Induced mutations for the improvement of Soybean ( Glycine max L.) in the Philippines . Philippine Nuclear Journal 14: 12-25.

      Vietnam has 1 publication in Vietnamese:

      • Mai Quang Vinh et al. Drough-tolerant and high-quality soybean variety DT96 suitable for mountaineous areas of North Vietnam . Proceeding of workshop North Vietnam Moutaineous Provinces. YenBai city, 2/2004. Pp.196 – 204 (On Vietnamese, Summary in English)

      Mutant registration:

      • A mutant line GH-7 generated from cv. Guntur has been released as cv. Rajabasa in 2004 in Indonesia .
      • In Indonesia , mutant line M-220 has been evaluated by the National Seed Evaluation Committee and 2 more multi-location yield trials are needed. This mutant line is expected to be released in mid 2007.
      • In Vietnam , DT 96 was officially released as a national variety in 2004.
    2. Evaluation indicators for above outcome
      Socio-economic impact
      (Application or Achievement of project object) : 5 point
      Scientific impact (Basic technology or Activity): 3 point
    3. Project outlook

      □Continuation □ Change/Revision ■Termination

      The reason:
      The project has achieved its objectives by developing potential mutant lines to be released as new varieties for drought tolerance or new materials for future breeding programs.

    4. Ripple effect to other fields or Benefits to end-users
      In Indonesia 2.2 tons of cv. Rajabasa seeds have been distributed to the certified growers for seed multiplication under the Department of Agriculture.
      In the Philippines , collaboration with Bulacan National State University (BNSU) through PNRI-GIA was done. This institution will provide experimental area for large population to screen the developed mutant lines.
      In Vietnam 100 tons of certified seeds of cv. DT96 had been distributed to growers for commercial production. Up to now, DT96 planting area is ca 3000 ha in 2005.

    5. Opinion about the project (problems, ideas, remarks, proposal)
      The adequate screening or selection method for drought tolerance of mutants is needed.
      The technical assistance to research group from FNCA is proposed by some countries.
    Attachment V

    Midterm Report for Disease Resistance in Banana

    1. The selection of Starting breeding Materials.

  • Whether it was appropriate or not.

    • <Fusarium wilt>
    Indonesia : Cv Berangan (AAA), Race 4
    Malaysia : Cv Berangan (AAA), Race 4
    Vietnam : Cv Tay (ABB), Race 1

    <Banana Bunchy top (Nematodes, Sigatoka) >
    Philippines : Cv Lakatan (AA/AAA)

    2. The determination of culture methodologies

  • Tissue culture of clones, and Isolation and multiplication of selected mutant clones.
    		•  - Explant and medium
    Indonesia : shoot tips (meristem) from male buds/ MS +3 mg/l BAP +0.5 mg/l IAA
    Malaysia : shoot tips (meristem)
           Cell suspension MS + 5mg/l BAP
    Vietnam : shoot tips/ MS + 4mg/l BAP + 0.5 mg/l NAA
    Philippines : proliferating shoot cultures/ MS + 5 mg/l BAP

    3. Irradiation to Starting Materials.

  • The determination of the methodologies, e.g. optimum dose, radiation sensitivity, etc.
    		•

    a) Radiosensitivity test / no. of plants/dose
    Indonesia : 0-80 Gy, 10 shoot tips/dose
    Malaysia : 0-100Gy, 100 meristems, 5 replicates
    Vietnam : 0, 10, 20, 30, 40, 50Gy; 10 shoot tips/dose
    Philippines : 0, 5, 10, 20, 25, 30, 40, 60, 80, 100Gy; 50 clumps/dose

    b) Radiation dose
    Indonesia : LD 50 – 40 Gy, LD 100 - 80 Gy
           Optimum dose; 10-20Gy
    Malaysia : LD 50 – 50 Gy, LD 100 - 80 Gy
           Optimum dose; 20, 30, 40 Gy
    Vietnam : LD 50 – 30 Gy, LD 100 - 60 Gy
           Optimum dose; 20 Gy
    Philippines : LD 50 – 20-25 Gy, LD 100 - 80 Gy
           Optimum dose; 20 and 25 Gy
    Dose rate

    4. Selection of Mutants Clones with Disease Resistance.

  • Establishment of screening technique.

    5. Testing and Evaluation of Selected Mutants in the field.

  • The data of field trials and estimate of their ability.
    		•

    [ Indonesia ]
    greenhouse screening: Soil from ‘hot spot' are placed in plastic pots and 10 day-old tissue culture plants are planted (1 plant per pot), evaluate for 3 months.

    Field screening: Those that survived in the greenhouse are planted in the ‘hot spot's. No. of plants; 489

    [ Malaysia ]
    Screening:

    1. Dipping method- 1-2 hrs soaking in spore suspension (10 6 spores/ml)
    2. Double tray method; hardened plants for 4 weeks are planted in sterile sand media in tray and Fusarium spores are poured in the first tray.
    3. Plants are hardened for 4-8 weeks in individual polybags and transfer in tray with coir dust containing Fusarium for 2 weeks. Evaluation is done 4-6 weeks after planting.

    Field screening: Those that survived from the above screening method are transferred to hot spot. Those resistant plants are multiplied and transferred back to hot spot for three generations.

    [ Vietnam ]
    Greenhouse screening:

    1. Hydrophonic; 1 week
    2. Dipping method: 24 hrs soaking plants in spore suspension (10 5 spores/ml)

    No. of plants; 500 soaked in container at the same time

    1. Field screening: hot spot
      Condition of ‘hot spot': more than 50 % infection of the susceptible check
    2. Maximum number of plants to be tested for in vitro: 5,000 plants
    3. Parameters for evaluation: 1) yellowing of the leaves/ browning of the pseudostem
    4. ELISA test: Indexing virus using ELISA test;

    for BBTV compulsory
    for CMV and BBrMV optional

    [ Philippines ]
    Greenhouse screening: Tissue-cultured plants are planted in plastic pots and inoculated with viruliferous aphids (10 aphids/ plant). Evaluation is done 1 to 9 months after inoculation. ELISA test is done on plants without BBTV symptoms. No. of lines; more than 6,000 plants were screened and 114 were selected without BBTV symptoms.

    Field screening: Those plants with negative ELISA are multiplied by tissue culture and planted in the field under high disease pressure for further evaluation. Evaluation will be done for 3 generations either in the field or farmers' field. No. of lines; 32 (Generation 1) and 10 (Generation 2). No. of plants per line; 30. All the mutant lines are maintained in tissue culture.

    6. Publication of the results (Reports etc.)

    Internal report, seminar and conferences, publication in journals, public patent
    Indonesia :

    1. Ishak and Ita Dwimahyani (2005) Evaluation of banana mutant lines growth and tolerance to Fusarium f.sp cubense (FOC), Stigma Vol. XIII (1) pp:26-29
    2. Ishak, Sasanti, W. and Yulidar (2004) Cloning and propagation of banana mutant line for selection purposes (Technical report 2004, CAIRT)
    3. Ishak, Sasanti, W. Yulidar (2005) Cloning and propagation of banana mutant lines for selection purposes (Technical report 2005, CAIRT)

    7. Other result and ripple effects of the project, if there are.

    Tissue culture using meristem-six months
    Suspension cell cultures- 12 months

    8. Opinion about the project (problems, ideas, remarks, proposal, etc.)

    Difficulty in finding and building hot spot
    Limited budget ( Indonesia , Malaysia , Philippines , Vietnam )
    National program of banana is ending ( Malaysia )

    9. Collaboration with other institution

    Malaysia ; University of Malaya , private company
    Indonesia : Andalas University , private company
    Vietnam ; Vegetable and Fruit Research Institute, private company
    Philippines ; PNRI, IAEA, PCARRD, banana farmers

    10. End Users

    Malaysia ; Farmers (1 ha) cooperative and private plantation for banana (more than 40 ha)
    Indonesia ; Traditional farmers (0.5 – 2 ha)
    Vietnam ; Small farmers (less than 1 ha)
    Philippines ; Small farmers (less than 5 ha)

    Attachment VI

    Sub-project on "Insect Resistance in Orchid"

    The main objective for this project is to produce insect resistant orchids. Three orchid varieties are used in this study namely, Dendrobium Sonia "No. 17 Red" ( Thailand ), D. mirbelliannum ( Malaysia ) and D. jayakarta ( Indonesia ). Protocorm-like bodies (PLBs) of these varieties have already been exchanged among the participating countries early in the project. In addition to these varieties, Thailand also uses D. Sonia "Earsakul" for this project.

    Present status:

    Indonesia

    1. Three to 6 months after gamma irradiation of D. Jayakarta at the doses of 0, 40, 80, 160, 320, 640 and 1280 Gy, the LD50 obtained for the 3 types of explants were as follows;
      A.LD50 for PLBs was 40 Gy (3 months)
      B.LD50 for plantlets was 240 Gy (3 months)
      C.LD50 for young shoots was 60 Gy (6 months)
    2. The optimum growth medium for PLBs of D. Sonia "No17 Red" was VW medium + coconut water + activated charcoal.

    Malaysia

    1. Malaysia uses three approaches to obtain insect resistant orchids, namely; in vitro mutagenesis using both ion beam and gamma irradiations, genetic transformation with Agrobacterium tumefaciens carrying a chitinase gene, and in vitro / in vivo insect screening on irradiated as well as transgenic orchids.
    2. Using ion beam irradiation, PLBs of both D. mirbelliannum and D. jayakarta have been successfully regenerated into plantlets and acclimatized in the glasshouse. Plantlets of D. mirbelliannum have been inoculated with mites using an in vitro screening method. Putative tolerant plants have been obtained and planted in the glasshouse for further screening.
    3. Using gamma irradiation, explants from all three varieties of Dendrobium have been regenerated into plantlets. D. mirbelliannum and D. jayakarta plantlets have been transferred to the glasshouse for in vivo screening with mites. D. Sonia "No 17 Red" plantlets will be transferred to the glasshouse once enough number of plantlets are obtained.
    4. Through genetic transformation, putative transgenic plants have been obtained as confirmed by antibiotic selection, GUS analysis and polymerase chain reaction (PCR). These plantlets have been transferred to glasshouse and further screening will be carried out once the transgenic glasshouse is available.
    5. Presently, in vivo (glasshouse) selection for insect resistance was carried out by growing the regenerated plants in the glasshouse without the application of pesticides and fungcides.

    Thailand

    1. LD50 analysis of D. Sonia "Earsakul" at 3 growth stages; PLBs, young plantlets and back bulbs, were carried out. The optimum doses for gamma irradiation for each growth stage were obtained.
    2. Natural infestation of thrips on chronic gamma irradiated seedlings of D. Sonia "No. 17 Red" and D. Sonia "Earsakul" were investigated. Some treated plantlets showed less infestation symptoms by thrips. These plantlets will be further analysed for thrips resistance at the flowering stage.

    Future Plan:

    The following work will be carried out to complete the project;

    Indonesia

    1. Mass rearing of thrips for screening of irradiated plants
    2. Mass propagation of irradiated plantlets for inoculation with thrips
    3. Propagation and maintenance of irradiated plants until the flowering stage for screening of both insect resistance and flower colours.

    Malaysia

    1. In vitro screening of plantlets of D. jayakarta & D. Sonia "No 17 Red" with mites.
    2. In vitro screening of transgenic plantlets
    3. In vivo screening of putative mite tolerant plantlets. Challenge-inoculation with mites will be carried out once the contained glasshouse is ready in order to avoid cross-contamination with other plants in the glasshouse.
    4. Propagation of irradiated plants until the flowering stage in the glasshouse for screening of flower colours.

    Thailand

    1. Selection for thrips resistance for irradiated plants at the flowering stage.
    2. Selection for other characters such as flower colours, size and morphology.
    3. Meristem cloning of selected mutants for large scale propagation and testing to confirm the results for thrips resistance and other characteristics.
    4. For thrips resistant mutants, challenge-inoculation with thrips will be conducted in order to select for the most resistant lines using the standard protocol for insect resistance analysis recommended by FNCA orchid project leaders at the FNCA 2006 Mutation Breeding Workshop.

    Agreement among participating countries

    Following discussion on the project, all participants have agreed on the following points;

    1. A standard protocol for insect resistance analysis on the irradiated orchids will be performed in all participating countries.
    2. Indonesia will provide the rearing technique of thrips to Thailand .
    Attachment VII

    Proposal for the new sub-project
    on "Composition or Quality Crop Breeding"

    Country: China, Indonesia, Japan, Korea, Malaysia, Thailand, Vietnam (Philippines) (Bangladesh)
    1. Objective Crop
      Rice
    2. Intended Composition/Quality
      Amylose content, Protein, Fiber content, Vitamin A, Anthocyanin, Resistant starch, Phytin
    3. Working Periods
      FY2007-2011
    4. Working Plan
      Research project:
      1. Creation of materials for breeding amylose library of primary rice varieties
        1. Mutation Induction by using irradiation of seeds or any other materials (tissue culture)
        2. Screen for amylose and/or other characters related to quality
        3. Evaluation of mutants for desirable characters
        4. Developing desirable lines or varieties
      2. Collaboration
        1. Exchange of seeds
        2. Exchange of tissue culture materials
        3. Exchange of released mutants
        4. Exchange of released varieties
        5. Exchange of Information:
          - Methodology for Irradiation
          - Methodology for Experiments
          - Screening methodology
        6. Fellowship Training Program:
          - Scientific Attachment
          - Workshop Training Program
          - Experts
        7. Irradiation Service
      Japan will provide service of ion beam in TIARA and chronic gamma-ray
      irradiation in gamma field.
      Malaysia will provide service of chronic gamma-ray irradiation in gamma green house. Korea will provide service of chronic gamma-ray irradiation with gamma phytotron.
    1. Backgrounds
      Low amylose : Low a m ylose content is a key determinant of eating quality of rice. Typical characteristics are: (1) a strong sticky texture; (2) a tendency to remain soft when cooled; and (3) exhibit a glossy appearance when cooked or processed as cake or cookies. Because of these characteristics, low amylose rice is utilized as a raw material for ordinary cooking; mixed cooking with non-glutinous rice, cooked rice balls, chilled sushi. The Wx allele that characterize low amylose rice has been differenciated into a Wx-a or Wx-b allele depending on the amount of expressed Wx protein by the locus. The Wx-a gene is distributed in indica rice and the Wx-b gene is mainly distributed in japonica rice ( Sano , Theor. Appl. Genet. 1984, 68, 467-473 ). There is additional research that has recently revealed that amylose content may be controlled by a group of isogenes at the Wx locus (Blligh et al. 1995; Ayres et al. 1997; Shu Q ingyao, 1999). "Milky Queen" is a mutant rice cultivar that possesses a missense base change mutation ( Wx-mq ) that was induced by MNU treatment (Sato et al., Breed. Sci. 2002, 52, 131-135). There are several reports on additional low amylose genes that are complementary and non-allelic to the wx locus. Among these are du-1 and du-2 mutations which induce similar effects on the splicing of Wx-b transcript during the formation of endosperm tissue (Issiki et al., Plant J., 2000, 23, 451-460).

      Numerous low amylose content mutant s are conserved in the national genebank s in China , Japan , Korea , Malaysia and Vietnam . As a consequence, future research priorities should focus on the following: (1) screening and enhancing the germplasm of low amylose mutants; (2) further elucidating the molecular mechanism (s) relevant to the low amylose mutation; (3) complementation studies of other genes, independent of the wx locus, that have effects on low-amylose content; and (4) developing high quality functional rice cultivars for special use by pyramiding low amylose genes and additional genes associated with various rice quality factors.
    2. Objectives
      Low amylose : To breed promising varieties having low amylose content
    3. Expected Outputs
      Low amylose
      In China: Obtaining 3-5 mutants expressing low amylose content following irradiation treatment , (2) agronomic and performance evaluation of these 3-5 mutant lines; (3) elucidate the genetics of these mutants with a fine mapping approach; and (4) d eveloping 2-3 low amylose content varieties.

      In Indonesia , (1) the development of Indonesian elite rice varieties with improved grain quality; (2) studies comparing the grain quality as well as yield improvements of elite varieties (IR64, IR36 and Diah Suci) are conducted using u nique germplasms.

      In Japan , (1) develop an amylose mutant library with amylose-content gradients of approximately 2% for Koshihikari; and (2) develop about ten low-amylose mutant lines for the other leading varieties in Japan .

      In Korea , develop new rice cultivar s exhibiting low and high amylose content.

      In Malaysia, develop Improved selections of "Pongsu seribu, one or two varieties having long grain size, low amylose content, unique aromatic properties, and other important agronomic traits.

      In Vietnam , (1) develop about 10 superior mutant lines exhibiting low amylose contents ranging from 24-25% to 18-21%; (2) develop lines exhibiting elevated protein content with a grain phenotype and additional elite characteristics that are suitable for local markets. Comparative examples could be cv. "Khang Dan", "cv. Q5", etc.
    Attachment VIII

    MUTAION BREEDING PUBLICATION DATABASE

    List Number Country Year Crop Author Title Institution Source Note Key words 1
    1 Japan 1998 Japanese pear T. Masuda, T. Yoshioka, T. Sanada, K. Kotobuki, M. Nagata, M. Uchida, K. Inoue, K. Murata, K. Kitagawa, and A. Yoshida A new Japanese pear cultivar 'Osa Gold' Institute of Radiation Breeding (IRB), Japan; Tottori Holticulture Experiment Station Bull. Natl. Inst. Agrobiol. Resour. 12: 1-11 in Japanese with English summary and captions of Fig. and Tables  
    2 Japan 1988 Japanese pear T. Sanada Selection of resistant mutation to black spot disease of Japanese pear by using host-specific toxin IRB, Japan Japan. J. Breed. 38: 198-204 English  
    3 Japan 1988 Japanese pear T. Sanada, T. Nishida, and F. Ikeda Resistant to black spot disease of Japanese pear 'Nijisseiki' induced by gamma rays IRB, Japan J. Japan. Soc. Hort. Sci. 57(2): 159-166 English  
    4 Japan 1994 Japanese pear K. Murata, K. Kitagawa, Masuda, K. Inoue, K. Kotobuki, M. Uchida, M. Nagara, T. Yoshioka, H. Tabira, H. Watanabe, A. Yoshida Selection of mutant Japanese pear resistance to black spot disease by acute irradiation of Gamma-rays Tottori Horticulture Experiment Station; IRB, Japan J. Japan. Soc. Hort. Sci. 62(4): 701-706 in Japanese with English summary and captions of Fig. and Tables  
    5 Japan 1992 Japanese pear K. Kotobuki, T. Sanada, T. Nishida, H. Fujita, and F. Ikeda Gold Nijisseiki', a new Japanese pear mutant cultivar resistant to black spot disease induced by chronic irradiation of gamma-rays IRB, Japan Bull. Natl. Inst. Agrobiol. Resour. 7: 105-120 in Japanese with English summary and captions of Fig. and Tables  
    6 Japan 1994 Japanese pear T. Sanada, K. Sagisaka, J. Soejima, T. Moriguchi, S. Teramoto, and K. Kotobuki Inheritance of intermediate resistance to black spot disease in an induced Japanese pear mutant, 'Gold Nijisseiki' Furuit Tree Research Station, Japan J. Japan. Soc. Hort. Sci. 62(4): 689-693 English  
    7 Japan 1999 Japanese pear T. Masuda, T. Yoshioka, K. Kotobuki, and T. Sanada Selection of mutants highly resistant to black spot disease in Japanese pear 'Gold Nijisseiki' by irradiation of gamma-rays IRB, Japan Bull. Natl. Inst. Agrobiol. Resour. 13: 135-144 English  
    8 Japan 2006 soybean Y. Takagi and T. Anai Development of novel fatty acid composition in soybean oil by induced mutation Saga Univ. Oleo Science 6(4): 195-203 in Japanese with English summary and captions of Fig. and Tables fatty acid
    9 Japan 1969 soybean Y. Takagi The second type of gamma-ray sensitive gene RS2 in soybean Glycine max (L.) Merrill IRB Gamma Field Symposia 8: 83-94 Gamma Field Symposia  
    10 Japan 1998 soybean Y. Takagi, S. M. Rahman, and T. Anai Construction of novel fatty acid composition in soybean oil by induced mutation Saga Univ. Gamma Field Symposia 37: 17-28 Gamma Field Symposia fatty acid
    11 Japan 1999 soybean S. M. Rahman, T. Kinoshita, T. Anai, and Y Takagi Genetic relationships between loci for palmitate contents in soybean mutants Saga Univ. The Journal of Heredity 90(3): 423-428 copyright fatty acid
    12 Japan 1998 soybean S. M. Rahman, T. Kinoshita, T. Anai, S. Arima, and Y Takagi Genetic relationships of soybean mutants for different linoleic acid contents Saga Univ. Crop Sci. 38: 702-706 copyright fatty acid
    13 Japan 1998 soybean T. Kinoshita, S. M. Rahman, T. Anai, and Y. Takagi Inter-locus between genes controlling palmitic acid contents in soybean Saga Univ. Breeding Science 48: 377-381 English fatty acid
    14 Japan 1997 soybean S. M. Rahaman and T. Takagi Inheritance of reduced linolenic acid content in soybean seed oil Saga Univ. Theor. Appl. Genet. 94: 299-302 copyright fatty acid
    15 Japan 1997 soybean S. M. Rahman, Y. Takagi, ane T. Kinoshita Genetic control of high stearic acid content in seed oil of two soybean mutants Saga Univ. Theor. Appl. Genet. 95: 772-776 copyright fatty acid
    16 Japan 1996 soybean S. M. Rahman, Y. Takagi, ane T. Kumamaru Low linolenate sources at the FAN locus in soybean lines M-5 and IL-8 Saga Univ. Breeding Science 46: 155-158 English fatty acid
    17 Japan 1996 soybean Y. Takagi and S. M. Rahman Inheritance of high oleic acid content in the seed oil of soybean mutant M23 Saga Univ. Theor. Appl. Genet. 92: 179-182 copyright fatty acid
    18 Japan 1996 soybean S. M. Rahman, Y. Takagi, and T. Kinoshita Genetic analysis of palmitic acid contents using two soybean mutants, J3 and J10 Saga Univ. Breeding Science 46: 343-347 English fatty acid
    19 Japan 1996 soybean S. M. Rahman, Y. Takagi, and T. Kinoshita Genetic control of high oleic acid content in the seed oil of two soybean mutants Saga Univ. Crop Sci. 36: 1125-1128 copyright fatty acid
    20 Japan 1995 soybean Y. Takagi and S. M. Rahman Variation of different fatty acids in mutants in comparison with natural soybean varieties Saga Univ. Bull. Fac. Agr. Saga Univ. 79: 23-27 English fatty acid
    21 Japan 1995 soybean S. M. Rahman, Y. Takagi, K. Miyamoto, and T. Kinoshita High stearic acid soybean mutant induced by X-ray irradiation Saga Univ. Biosci. Biotech. Biochem. 59(5) 922-923 English fatty acid
    22 Japan 1994 soybean S. M. Rahman, Y. Takagi, K. Miyamoto, and T. Kawakita Inheritance of low linolenic acid content in soybean mutant line M-5 Saga Univ. Breeding Science 44: 379-382 English fatty acid
    23 Japan 1994 soybean S. M. Rahman, Y. Takagi, and S. Towata Inheritance of high linoleic acid content in the soybean mutant line B739 Saga Univ. Japan. J. Breed. 44: 267-271 English fatty acid
    24 Japan 1999 soybean Y. Takagi, S. M. Rahman, and T. Anai, S. K. Wasala, T. Kinoshita, and M. Khalekuzzaman Development of reduced linolenate soy mutant by re-irradiation and its genetic analysis Saga Univ. Breeding Science 49: 1-5 English fatty acid
    25 Japan 1989 soybean Y. Takagi, A. B. M. M. Hossain, T. Yanagita, and S. Kusaba High linolenic acid mutant in soybean induced by X-ray irradiation Saga Univ. Japan. J. Breed. 39: 403-409 English fatty acid
    26 Japan 1990 soybean Y. Takagi, A. B. M. M. Hossain, T. Yanagita, T. Matsueda, and A. Murayama Linolenic acid content in soybean improved by X-ray irradiation Saga Univ. Agric. Biol. Chem. 54(7): 1735-1738 English fatty acid
    27 Japan 2001 soybean S. M. Rahman, T. Kinoshita, T. Anai, and Y. Takagi Combining ability in loci for high oleic and low linolenic acids in soybean Saga Univ. Crop Sci. 41: 26-29 copyright  
    28 Japan 1993 rice S. Iida, T. Takano, Z. Ikezawa, and T. Nishio Evaluation of a rice mutant as a material of hypoallergenic rice IRB Japan. J. Breed. 43: 389-394 English hypoallergenic rice
    29 Japan 2005 rice M. Nishimura, M. Kusaba, K. Miyahara, T. Nishio, S. Iida, T. Imbe, and H. Sato New rice varieties with low levels of easy-to-digest protein, 'LGC-Katsu' and 'LGC-Jun' IRB Breeding Science 55(1):103-105 English low protein
    30 Japan 2006 rice Y. Sato, K. Shirasawa, Y. Takahashi, M. Nishimura, and T. Nishio Mutant selection from progeny of gamma-ray-irradiated rice by DNA heteroduplex cleavage using petiole extract. Tsukuba Univ. Breeding Science 56: 179-183 English TILLING
    31 Japan 2003 rice M. Kusaba, K. Miyahara, S. Iida, H. Fukuoka, T. Takano, H. Sassa, M. Nishimura, and T. Nishio Low glutelin content1: a dominant Mutation That suppresses the glutelin mutagene family via RNA silencing in Rice. IRB The Plant Cell 15: 1455-1467 copyright RNAi
    32 Japan 1993 rice S. Iida, E. Amano, and T. Nishio A rice (Oryza sativa L..) mutant having a low content of glutelin and a high content of prolamine IRB Theor. Appl. Genet. 87: 374-378 copyright low content of glutelin
    33 Japan 1993 rice T. Nishio and S. Iida Mutants having a low content of 16-kDa allergic protein in rice (Oryza sativa L.) IRB Theor. Appl. Genet. 87: 317-321 copyright low allergenic protein
    34 Japan 1997 rice S. Iida, M. Kusaba, and T. Nishio Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes IRB Theor. Appl. Genet. 94: 177-183 copyright low content of glutelin
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