ING

ICS

Joint FAO/IAEA Divisionof Nuclear Techniques

in Food and Agricultureand FAO/IAEA Agriculture and

Biotechnology Laboratory, SeibersdorfInternational Atomic Energy Agency

Vienna

INIS-XA--172

II.IJJ/

XA9952463

No. 3 June 1999

TO THE READER 1

A. STAFF 2

B. FORTHCOMING EVENTS 3

C. PAST EVENTS 4

D. STATUS OF EXISTING CO-ORDINATED

RESEARCH PROJECTS 7

E. NEW CO-ORDINATED RESEARCH PROJECTS 9

F. TECHNICAL CO-OPERATION PROJECTS 9

G. ACTIVITIES AT THE PLANT BREEDING

UNIT, SEIBERSDORF 12

H. PUBLICATIONS 15

http://www.iaea.org/programmes/nafa

http://www.fao.org/WAICENT/Agricul.htm

ISSN 1564-2 S69

3 0 - 3 9

TO THE READER

This is the third issue of the PlantBreeding and Genetics Newsletter. Sincepublishing the last issue we have hadsignificant staff changes in the PlantBreeding and Genetics Section. Dr. Leovan Zanten completed his contract with theAgency after 5 years of service as aTechnical Officer. He was the person whoalso compiled the first two issues of thePBGN. Dr. Richard Litz, after almostthree months of service, terminated histemporary assignment as a consultant onthe application of induced mutations inconnection with in vitro culture forvegetatively propagated crop improvement.The good news is that Dr. Mohan Jainjoined the Section on 2 May, filling in thepost previously occupied by Dr. BeantAhloowalia. He will be responsible, amongother duties, for our activity related to theimprovement of vegetatively propagatedcrops by induced mutations and relatedbiotechnology. He is now project officerof the FAO/IAEA/BARC Co-ordinatedResearch Project on "Cellular biology andbiotechnology including mutationtechniques for creation of new usefulbanana genotypes."

Our work in these six months hasconcentrated on planning two new CRPswhich will be initiated this year. Theevaluations of the research contract andagreement proposals for the FAO/IAEACRP on "Molecular characterization ofmutated genes controlling important traitsfor seed crop improvement" have beencompleted and 19 participants wereselected. The CRP will use inducedmutations to exploit the full power ofstructural and functional genomics for geneisolation directed at the improvement oforphan crops and allied species. It willfocus on molecular characterization of newor existing mutants affecting key

agronomic traits in model crops and usingcomparative approaches in under-utilisedcrops. We have already called for researchcontract and agreement proposals relatedto the CRP on "Mutational analysis of rootcharacters in annual food crops related toplant performance". Through this CRP weare expecting to generate a population ofroot mutants. It is foreseen that a variety ofcharacteristics of root function will bealtered in these new genetic stocksincluding root hair frequency, size anddistribution; branching pattern; rootdiameter, fraction and distribution ofvarious root types and mycorrhizaldevelopment. These genetically

characterized mutant lines will be ofparticular value for modifying plantresponses to a variety of environmentalconditions especially in marginal areas ofdeveloping countries.In addition to the daily activity related tothe organization of Research Co-ordinationMeetings, evaluation of progress reportsand technical backstopping of TechnicalCo-operation Projects, work on theestablishment of a database on successfulradiation doses for development of newmutant varieties in crop species wascontinued. Activities at the Plant BreedingUnit of the FAO/IAEA Agriculture andBiotechnology Laboratory at Seibersdorfconcentrated on the development of low-cost in vitro culture technology for inducedmutations, development an efficienttechnique for rice anther culture and onactivation of retrotransposons in rice byirradiation with gamma rays.

Miroslaw Maluszynski

A. STAFF

Joint FAO/EAEA Division of Nuclear Techniques in Food and Agriculture, IAEA,Vienna International Centre, P.O. Box 100, A-1400 Vienna, Austria.

James Dargie Director E-mail: J.Dargie@iaea.org

Manase Peter Salema Deputy Director E-mail: M.P.Salema@iaea.org

Plant Breeding and Genetics Section, IAEA, P.O. Box 100, A-1400 Vienna,Austria

Miroslaw Maluszynski Head of Section E-mail: M.Maluszynski@iaea.org

Chittranjan Bhatia Technical Officer E-mail: C.Bhatia@iaea.org

Mohan Jain Technical Officer E-mail: M.Jain@iaea.org

Karin Nichterlein Technical Officer E-mail: K.Nichterlein@iaea.org

Katayon Entekhabi Secretary E-mail: K.Entekhabi@iaea.org

Kathleen Weindl Senior Office Clerk E-mail: K.Weindl@iaea.org

FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf,Austria

Christopher Rigney Acting Head E-mail: C.Rigney@iaea.org

Plant Breeding Unit,

Javier Zapata Arias Head of Unit E-mail: F.Zapata-Arias@iaea.org

Nicolas Roux Researcher E-mail: N.Roux@iaea.org

Stefan Nielen Associate Professional Officer E-mail: S.Nielen@iaea.org

Marcela Guzman Junior Professional Officer E-mail: M.Guzman@iaea.org

Rownak Afza Researcher E-mail: R.Afza@iaea.org

Andrea Kodym Technician E-mail: A.Kodym@iaea.org

Muriel Weinreich Secretary E-mail: M.Weinreich@iaea.org

Giinther Berthold Technician E-mail: G.Berthold@iaea.org

Andreas Draganitsch Technician E-mail: A.Draganitsch@iaea.org

Franz Zwiletitsch Technician E-mail: F.Zwiletitsch@iaea.org

B. FORTHCOMING EVENTS

Research Co-ordination Meetings(RCMs)

Third and final RCM on "Improvement ofnew and traditional industrial crops byinduced mutations and relatedbiotechnology", Corvallis, Oregon, USA,2-6 August 1999.

Third and final RCM on "Radioactivelylabelled DNA probes for cropimprovement", Vienna, 6-8 September1999.

First RCM on "Molecular characterizationof mutated genes controlling importanttraits for seed crop improvement", Vienna,Austria, 4-8 October 1999.

Third RCM on "Cellular biology andbiotechnology including mutation techniquesfor creation of new useful banana genotypes"Colombo, Sri Lanka, 4-8 October 1999.

First RCM on "Mutational analysis of rootcharacters in annual food plants related toplant performance" is planned for the endof 1999 or the beginning of 2000.

Regional (AFRA) Training Course on"Molecular characterisation of geneticbiodiversity in traditional and neglectedcrops selected for improvement throughmutation techniques", Pretoria, SouthAfrica, 6-17 September 1999.

The purpose of this training course is totrain breeders participating in the regionalAFRA 111-18 project (RAF/5/042) invarious molecular techniques forcharacterisation of biodiversity in selected

traditional neglected crops (RAPDs, RFLP,AFLP, micro satellites, comparativemapping).

Workshop (RAF/5/035) on "Hands-onExperience on Molecular and MutationTechniques", Vienna, Austria, 13-24September, 1999.

The main objective of this workshop is totrain date palm breeders in the use ofmolecular techniques (RAPDs, AFLPs) andmutation techniques through practical skillsand hands-on experience. The participants tothis workshop are limited to only Algeria,Morocco, and Tunisia.

Seminar on "Mutation techniques andbiotechnology for tropical and subtropicalplant improvement in Asia and the PacificRegion", Los Banos, The Philippines,11-15 October 1999.

The main objective of this seminar is todiscuss with scientists of the Region thecurrent status of mutation techniques andrelated molecular genetic approaches. Theseminar will focus on the application of thesetechnologies in the areas of plant researchand crop improvement. The latestdevelopments in genetic engineering offer anumber of possibilities for the improvementof crops not previously in the main stream ofresearch. This especially applies to cropspecies of importance for tropical andsubtropical regions. Molecular genetics, incombination with mutation techniques,provides very powerful tools to enhance theproduction of local and traditional crops instress prone areas and, in this way, toimprove farm sustainability.

C. PAST EVENTS

Project Formulation Meeting of theRegional TC Project RAS/5/037 on"Mutational enhancement of geneticdiversity in rice", Bangkok, Thailand, 8-12 February 1999.

The aim of this meeting was to guiderepresentatives of countries participating inthe RAS/IAEA Project on "MutationalEnhancement of Genetic Diversity in Rice"in the preparation of a detailed programmeof activity and other project documents.Thirteen rice breeders represented elevencountries of the Region: Bangladesh,China, India, Indonesia, Korea, Malaysia,Myanmar, Pakistan, The Philippines,Thailand and Viet Nam. The meeting wasorganized at the newly established (withAgency support) Gamma IrradiationService and Nuclear Technology ResearchCentre, Kasetsart University, Bangkok.The Centre is very well organized andoffers excellent facilities for regionaltraining courses and meetings. TheInternational Rice Research Institute(IRRI) was represented by senior scientistDr. D.S. Brar.On the basis of intensive discussions adetailed plan of project activities wasformulated and necessary documentsdrafted. Also, an Agreement on Exchangeof Rice Seed Material in the form of theIAEA Project RAS/5/037 was drawn upand adopted by all participating countries.

2nd Project Co-ordination Meeting ofAFRA m-18 project on "Developmentof improved crop varieties", Bamako,Mali, 15-19 February 1999.

The meeting was jointly organised by theIAEA and the Institut Polytechnique Ruralde Formation et de Recherche Appliqueede Katibougou (EPR/IFRA) in Koulikoro.

The purpose of the meeting was toevaluate the 1997/98 programme and planand to c-ordinate the 1999/2000programme of the regional projectconsisting of three different projectcomponents. The meeting was attended bythe Project Co-ordinators from Algeria,Egypt, Ethiopia, Ghana, Libya, Madagascar,Mali, Mauritius, Morocco, Niger andTanzania, the IAEA Project Officer and aTechnical Officer from the Joint FAO/TAEADivision. Representatives of FAO, theMinistries involved in agriculture, research,education and energy participated in theopening and closing sessions of the meeting.A breeder from ICRISAT, Mali, participatedin parts of the meeting. Results of all projectcomponents and countries includingevaluation trials of sesame, safflower,cassava, cocoa, barley, sorghum, lentil anddurum wheat mutants were presented andevaluated as well as the breedingprogrammes to improve drought tolerance incereals and legumes and to improvetraditional and neglected crops. An analysisof strengths, weaknesses, opportunities andthreats (SWOT) was conducted, theprogramme for 1999/2000 activities wasdiscussed and revised and recommendationsfor further project implementation includingregional mutant germplasm exchange wereagreed upon.One cassava mutant from Ghana and eightsorghum mutants from Mali - developedunder previous Agency support - wereofficially released as improved varieties. Theofficial release of the improved sesamemutant varieties is expected in Egypt by theend of 1999. Mutants of cocoa (Ghana),barley (Libya, Tanzania), lentil (Morocco),safflower (Egypt) need further evaluation inorder to be considered for submission to theNational Variety Release Committees.Delays in implementation of mutant

evaluation trials were mainly due to abioticstress factors during the cropping seasons.The breeding programmes under component2 (improvement of drought tolerance incereals and legumes) and component 3(improvement of traditional and neglectedcrops) are still in an initial phase of evaluationof germplasm collections and early mutantgenerations. Only programmes which havebeen initiated in the early 90's - under theprevious Agency's TC support - such asbarley in Algeria and durum wheat inMorocco have advanced mutant generationsavailable.

Completion of the CRP on Use of novelDNA fingerprinting techniques for thedetection and characterization ofgenetic variation in vegetativelypropagated crops.

This CRP was completed with thepublication of the proceedings of the finalResearch Co-ordination Meeting, Mumbai,India, 24-28 February in October 1998.The activities and achievements of the CRPcan be summarized as follows:

Various molecular marker technologieshave been used to measure geneticdiversities of vegetatively propagated cropsas shown in the individual papers presentedin this RCM. The building of this researchnetwork has permitted the transfer oftechnology and the enrichment ofknowledge and plant genetic resourcesavailable to individual projects wheretechnologies of DNA profiling [restrictionfragment length polymorphism (RFLP);randomly amplified polymorphic DNA(RAPD); simple sequence repeats (SSR);amplified fragment length polymorphism(AFLP); DNA amplification fingerprinting(DAF); and randomly amplifiedmicrosatellite polymorphism (RAMPO)]have been evaluated. Cost, convenience,

reliability and information content havebeen recognized as key criteria forselecting an appropriate profilingtechnology.The association of markers andmorphological traits led to the generationof maps in several species which allow theexploitation of alternative life cycles ofvegetatively propagated plants. Forexample, in banana one important strategyis the identification of the diploidaccessions most closely related to moderncultivars for improvement of desirabletraits in these diploids by MAS, thusrecreating polyploid, sterile andparthenocarpic cultivars. Another exampleis yam, Dioscorea spp., where mapping ofthe genome of Dioscorea tokoro, adioecious diploid species closely related tothe economically more important yamcrops, allowed the tagging of importantgenes, fostering isolation of these genes foreventual transformation of yam for geneticimprovement. In this fashion, wherelinkage analysis (detection of linkagedisequilibrium) is possible, there are notechnological barriers to extend thesestrategies to vegetatively propagated cropsof interest, and to genetically engineeragronomically important genes.

Specific achievements of this CRP

The participants in this CRP haveinvestigated various vegetativelypropagated species with differentmolecular marker systems. The details oftheir progress are presented below.

Burg, Austria - The identification andcloning of minisatellite type repetitiveelements of the oak genome supplies a newmarker system for the analysis of thegenetic diversity of oak resources.Leal, Cuba - Molecular evidence ofsomaclonal variation has been found in

sugarcane. There is possibly a role forseveral genes involved in theosmotolerance response (ATPase, delta-pyrroline-5-carboxylase reductase, osmotinand heat shock proteins).

Lagoda, France - A common basis wasestablished in which breederscommunicated their problems to moleculargeneticists. Bridging the communicationgap clarified the need for a molecularbreeding programme on banana andplantain requiring the input of a mappingand a genetic diversity analysis programme.The development of additional markerswas a prerequisite for applications outsidethe laboratory, especially PCR detectionkits. This has been achieved and will becompleted in the future by developingsequence tagged microsatellite sites(STMS) markers and sequencecharacterized amplified regions (SCARs)from AFLP.

Sangwan, France - In cassava, inducedgenetic variations have been characterizedin vitro, Using PCR-based markers (RAPDand microsatellites). The RAPD analyseswere reproducible and showed distinctpolymorphic bands.

Kahl, Germany - For yam, an intenseapplication of various DNA profilingtechniques and sequence comparisons ofchloroplast tRNA gene regions revealedgenetic diversity in various Dioscoreaspecies, various accessions of economicallyimportant yams and parents for crosses,e.g. D. tokoro. The same techniques wereemployed to clarify taxonomic problems inD. bulbifera, phylogenetic relationshipsbetween D. cayensis and D. rotundata(Guinea yam), and between these and theirputative progenitors. For the first time, adefined cross of selected parents of D.tokoro permitted profiling progeny. For

greater exploitation of the RAPDpolymorphisms, a new hybridization-basedmethod, RAMPO, was designed thatexpands the information content of a singlegel several-fold.

Parida, India - Molecular markers wereused for genetic characterization, speciesidentification, establishing phylogenetic andspecies relationships in the Indianmangrove species. Markers have also beenused to select priority areas forconservation and consolidation ofgenotypes with the capacity for toleranceto salinity.

Rao, India - Tissue cultures have beenestablished from thirteen banana genotypesbelonging to different genomic groupsincluding wild diploids. In vitro multipleshoot cultures have been irradiated and inthe early stages of field planting,regenerants have exhibited chlorophyllvariations, earliness, changes in plantheight and plant type and furtherobservations of these will follow.Molecular studies for finger printing havebeen initiated with different bananaaccessions and variants isolated fromirradiation experiments.

Lavi, Israel - Several kinds of DNAmarkers (including minisatellites, AFLPand SSRs) were applied to both mango andavocado. Achievements include:identification of individuals, study ofgenetic relationships, identification oflinkage between some DNA markers andgenes coding for agriculturally importanttraits in avocado, and the generation of apreliminary genetic map in avocado.

Low, Malaysia - Somaclonal variationsand changes in DNA methylation weredemonstrated for the first time in rubbertrees, Hevea brasiliensis, by various DNA

fingerprinting techniques. Gammairradiation resulted in changes in DNAprofiles, but these changes werecomplicated by, and indistinguishable from,somaclonal variations.

Mignouna, Nigeria - Genetic variationamong cassava varieties was analysed withRAPD markers. The molecular taxonomyof cultivated and wild yams was establishedwith RAPD and microsatellite markers.

Terauchi, Japan - High levels of geneticdiversity have been found in naturalpopulations of wild yam suggesting theimportance of maintaining a small numberof large populations to protect geneticdiversity.

Iqbal, Pakistan - Local varieties ofsugarcane and banana were compared withRAPDs to assess their genetic diversity.

Mansvelt, Republic of South AfricaRAPD markers were used to generatefingerprints for differentiation of deciduousfruit cultivars. Gene transfer technologywas developed for deciduous fruit treesusing Agrobacterium as a vector.

Gresshoff, USA - Arbitrary primertechnology using DAF was optimized usingmini-hairpin primers, secondaryamplification of DAF products (ASAP),7M urea-10% PAGE, a 55°C annealingtemperature, and maintenance of highprimer (3 uM) and low template (1-2 ng/20 uL reaction volume) concentrations.Molecular markers were valuable foranchoring yeast artificial chromosome(YAC) and bacterial artificial chromosome(BAC) clones on molecular linkage mapsfacilitating the next step of map-basedcloning of genes induced by mutagenesis.

D. STATUS OF EXISTING CO-ORDINATED RESEARCH PROJECTS

Genetic Improvement of Underutilizedand Neglected Crops in LIFDCs throughIrradiation and Related Techniques

This CRP was initiated in 1998 with theobjective to overcome major constraints toincrease productivity of neglected andunderutilized crops by genetic improvement,in order to enhance the economic viabilityand sustain crop species diversity, and infuture to benefit small farmers. Mutationtechniques in combination withbiotechnology are applied for theimprovement of various vegetatively and seedpropagated crops: quinoa {Chenopodiumquinod), cocoyams (Colocasia esculenta,

Xanthosoma spp.), yams (Dioscorea spp.),.grain and vegetable amaranths (Amaranthusspp.) Bambara groundnut (Vignasubterranea), grasspea (Lathyrus sativa),okra (Abelmoschus esculentus), bitterpotatoes (Solanum jucepzukii, Solanumajanhuiri) and naranjilla (Solanumquitoense). The first Research Co-ordinationMeeting was held in December 1998 inVienna, Austria. At present there are 18participating institutes from Bolivia, CostaRica, Ecuador, France, Germany, Ghana,India, Indonesia, Mexico, Slovakia, SouthAfrica, Syria and Thailand including anagreement holder from IPGRI based atICARDA.

Radio-actively Labelled DNA-probes forCrop Improvement

This project, which started in 1995, has at themoment 8 participants from 4 countries. Thisproject helps to foster international co-operation to transfer modern biotechnologyto developing countries with the activeparticipation of leading laboratories. Animportant activity is the promotion of probesand primers. The third and final RCM isplanned for 6-8 September 1999 in Vienna,Austria.

Improvement of New and TraditionalIndustrial Crops by Induced Mutationsand Related Biotechnology

This CRP started in 1995 and emphasizes theapplication of induced mutations and relatedbiotechnologies to oil crop and fiber plantimprovement programmes. The programmesmainly focus on genetic improvement ofagronomic traits, resistance to biotic andabiotic stress factors and product quality inrapeseed, Indian mustard, soybean,sunflower, linseed, Cuphea, meadowfoam,evening primrose, cotton and jute. Mutationprotocols were optimized for those crops.Improved germplasm lines were developed byinduced mutations, intra- and interspecifichybridization and genetic transformation.Molecular markers for marker-assistedselection are available for some traits. Genesinvolved in fatty acid biosynthesis of Cupheacould be isolated from mutant germplasm. Atpresent there are 11 participating institutesfrom 10 countries in this project.. The thirdand final RCM will be held in August 1999 inCorvallis, Oregon, USA. (See also'Forthcoming Events')

Cellular Biology and BiotechnologyIncluding Mutation Techniques forCreation of New Useful BananaGenotypes

This CRP was initiated in 1994 with thegeneral aim to integrate radiation inducedmutations in vitro culture and moleculargenetics methods into the conventionalbreeding of banana to induce desirablevariation such as disease resistance, dwarfismand earliness, and also to promote thedevelopment of methods for large-scale andrapid multiplication of the mutants/segregants through micropropagation andsomatic embryogenesis. Plants can readily beregenerated via somatic embryogenesis forlarge-scale plant production, which is ideal forin vitro mutagenesis to the selection ofmutants with desirable agronomic traits.Flow cytometry analysis of nuclear DNAcontent in Musa showed that Musa A and Bgenomes differ in size. Since 1996, Belgiumhas become an important contributor to thisCRP. Twenty institutions world wide areinvolved. The next RCMis planned for 4-8October 1999 in Colombo, Sril Lanka.

Induced Mutations for SesameImprovement

This CRP was concluded in 1998. The finalproceedings are now being edited and will beavailable as IAEA technical publication(TEC-DOC) (see issue no. 1, p. 10-11 forreport from final meeting).

Induced Mutations and other AdvancedTechnology for Production of CropMutants Suitable for EnvironmentallySustainable Agriculture

The CRP was concluded in 1998. The finalproceedings are now being edited and will beavailable as a book summarizing the results(see issue no. 2, p. 4-5 for report from finalmeeting).

Induced Mutations in Connection withBiotechnology for Crop Improvement inLatin America

The CRP was concluded in 1998. The finalproceedings are now being edited and will beavailable as an IAEA technical document(TEC-DOC) (see issue no. 2, p. 5-7 forreport from final meeting).

E. NEW CO-ORDINATED RESEARCH PROJECTS

Molecular characterization of mutatedgenes controlling important traits forseed crop improvement

Details of this CRP were published in thelast issue of this Newsletter and in PlantBreeding News (February 1999). The 1st

Research Co-ordination Meeting will beheld from 4-8 October, 1999 in Vienna.

Mutational Analysis of Root Characters inAnnual Food Crops related to PlantPerformance

Details of this CRP were published in thelast issue of this Newsletter and in PlantBreeding News (May 1999) The 1st RCMis planned for the end of 1999 or beginningof 2000, in Vienna.

F. TECHNICAL CO-OPERATION PROJECTS

Over the last three years, the Section has had technical responsibility for 43 Technical Co-operation projects. In each Newsletter we will highlight the activities and achievements in afew of our Technical Co-operation projects. The following projects are discussed in this issue:

Sorghum mutants released as improvedvarieties in Mali (MLI/5/014)

In Mali, sorghum is the second mostimportant crop and is grown on some650,00 ha, but yields only about 800 kg/ha.By irradiating locally grown well adaptedmaterial of the guinea types with 200-300Gy gamma rays, followed by selection inthe subsequent generations and fieldperformance tests over several years, plant

breeders at the Institut Polytechnique deKatibougou, Koulikoro, have released 8mutants to suit various sorghum growingregions of Mali. These have been added tothe list of cultivated varieties and species ofthe Department of Agriculture, Ministry of

Rural Development. These mutants havethe potential to yield 1000 to 1500 kg/ha,are 3.5 to 4.2 m tall (local farmers prefertall types, since the stems are used forfeeding cattle, building grain storage andshading canopies) with long panicles,resistant to lodging, some are early ripeningand others have improved resistance todrought. With names like 'Fambe','Tiedjan', 'Gnome', 'Sofin', 'Djeman','Djemanin', 'Gnoumanin', 'Sadje', true tothe West African culture, these newvarieties promise to help sustain foodproduction in the sub-Sahel of Africa. In1998, about 2000 kits of one kilo seed eachof three varieties, 'Fambe', 'Tiedjan', and'Gnome' were distributed to selectedgrowers for on-farm performance

validation. It is recognized that there is nobetter way to conserve genetic diversitythan growing local land races and varietieson farms. Since the mutant varieties havebeen derived from native material, theirrelease would also lead to conservation ofthe native sorghum germplasm as a bonusto the increased productivity. This researchhas been accomplished through the supportprovided by the Agency under variousTechnical Co-operation and CRPs duringthe past 15 years.

Rice mutant varieties released in theMekong Delta of Vietnam (VIE/5/013)

Rice is the most important food and exportcrop in Vietnam. The Mekong Delta insouthern Vietnam has the dominant role inrice production of the country with 46% ofthe total rice area. Much attention has beengiven in the past to increase rice productionon alluvial soils under irrigation and highinput conditions using high yieldingvarieties with low cooking quality.However, on saline and acid sulphate soilsof coastal areas of the south only traditionalvarieties with good cooking quality but lowyields can be grown. Therefore, the ricebreeders at Cuu Long Delta Rice ResearchInstitute in Oman, Cantho Province insouthern Vietnam initiated a riceimprovement programme to develop higheryielding varieties with good cooking qualityfor such stress-prone soils suitable forexport. They were supported under a

previous research contract to inducemutations using gamma treatment of riceseed of traditional varieties well adapted tosaline and acid sulphate soils in order toincrease production; improved mutant lineswere selected. Under a TC project theinstitute is now evaluating the mutant linesin southern provinces. Two mutant lineshave been selected for further evaluationand distribution to farmers. The mutantTNDB-100 was released in 1997. It wasdeveloped from the land race Tai NguyenDot Bien which is photoperiod sensitive,1.3-1.5 m tall, maturing in 180 - 200 dayswith grain yields of 2 - 3 t/ha. The mutantTNDB-100 is a photoperiod insensitivevariety, which matures in about 100 days.It can have grain yields of 4.5 -8 t/ha. It hasquality characteristics (long grain, lowchalkiness) that fit well in the exportmarket. Because of TNDB-100's shortergrowth cycle farmers can grow two tothree crops per year in the Mekong Delta.It has a wide adaptation to different soilconditions and performs well even on poorsoils. The rice growers and consumers likethis variety and in the 1998/99 dry seasonmore than 80,000 ha of it were grown insouthern Vietnam. Another mutant THDB,originates from mutation treatment of theland race Tep Hanh Dot Bien. It is earliermaturing (125-135 days) than the land race(200-220 days), is shorter and yields 5 to 8t/ha (land race 2 to 3 t/ha). In 1998 it wasgrown in the coastal area of the south as anautumn - winter crop - on 13,000 ha.

Current Operational Projects are:

BGD/5/019COL/5/017COS/5/021COS/5/023CPR/5/010CPR/5/011CPR/5/013ECU/5/020ELS/5/008

Extension services to farmers on promising mutant varietiesMutation breeding of plantain and riceRadioactive probes for plant disease diagnosisImproved mutant varieties of rice and bananaInduced mutations for improvement of riceImprovement of cotton and rapeseed through induced mutationsInduced mutations to improve rice qualityResistance to disease in cacao and babacoImprovement of potato through in vitro mutation breeding

10

ETH/5/011GHA/5/026GUA/5/012INS/5/026INS/5/027IRA/5/007IRQ/5/011IRQ/5/015KEN/5/021MAG/5/008MAK/5/004MAL/5/021MLI/5/014MON/5/009MYA/5/008PAK/5/033PAK/5/035PAK/5/039PER/5/024PHI/5/027RAF/5/029RAF/5/035RAF/5/042RAS/5/037RLA/5/035SRL/5/030SUD/5/023THA/5/045URT/5/020VEN/5/018VIE/5/013VIE/5/014ZAM/5/020

Improvement of tef through mutation breedingImprovement of cassava through mutation breedingMutations and biotechnology for crop improvementMutation breeding of bananasMutation breeding of ornamental plantsMutation techniques for crop improvementNuclear techniques in cereal productionInduction of mutations in crops through in vitro cultureImproved drought resistance of crops by induced mutationsMutation techniques and biotechnology for rice and cassavaMutation and doubled haploid techniques to improve wheatMutation breeding and biotechnology for plant improvementField performance of selected mutants of sorghum and riceNuclear techniques to improve production of wheat and legumesMutation breeding in grain legumesDevelopment of leaf curl tolerant varieties of cottonDevelopment of salt tolerant varieties of basmati ricePest resistant chickpea through induced mutationIntroduction of barley and other native crop mutant cultivarsMutation breeding of priority agricultural cropsNuclear techniques in plant breeding and biotechnology (AFRA X)Control of bayoud disease in date palmDevelopment of improved crop varieties (AFRA III-18)Mutational enhancement for genetic diversity in rice (RCA)Evaluation of cereal crop mutants (ARCAL XXIA)Mutation breeding in bananas and plantainsImproving cotton and sugar cane crops (phase II)Radiation induced mutations for bean and chrysanthemumImproving productivity of basic food crops in TanzaniaGenetic improvement of fruits and pepperImprovement of basic food crops through induced mutationsRice mutant varieties for saline landImprovement of beans through mutation breeding

11

G. ACTIVITIES AT THE PLANT BREEDING UNIT, SEIBERSDORF

Development of low-cost tissue culturetechnology for induced mutations'

Mutation induction combined with in vitrotechniques can provide useful tools toenhance genetic variability for theimprovement of agronomic traits such asearliness in fruiting, pest and diseaseresistance, yield and quality. Tissue culturetechniques such as shoot-tip culturefacilitate the isolation of homohistonts inmutation induction studies and in theselection and multiplication process ofimproved genotypes.• A study was conducted to reduce

expenses in tissue culture systems byusing sunlight instead of artificial lightduring culture incubation and readilyavailable cheap sugars and gellingagents in the medium.

• Musa cv. 'Grande Naine', our modelplant, was micropropagated undervarious conditions. The experimentswere carried out during twoexperimental periods (during July toSeptember 1997 and April 1998 toJanuary 1999). Three different locations- a growth chamber, a sunlit room and agreenhouse were available for cultureincubation. In the growth chamber, thecultures were kept at 25 °C with aphotoperiod of 16 hours at 65 umol m"2

s"1 provided by cool-white fluorescenttubes. In the sunlit room without airconditioning, the boxes were placed infront of a window facing west. Undernatural light conditions plants wereexposed to temperatures from 16 - 43°C, PPFD (Photosynthetic Photon FluxDensity) of up to 860 umol m"2 s'1 andphotoperiod ranged from 8-16 hours.

•• In both experimental years, the number

of shoots produced was always highestin the greenhouse followed by the sunlit

room and was lowest in the growthchamber. Plant quality was satisfactoryin all three locations but under naturallight conditions the plants were lighterin colour and had a bigger leaf size thanin the growth chamber. During summer1997 when average PPFD were around200 umol m'2 s'1, browning of leavesand loss of turgor were observed in thegreenhouse, but in any caseacclimatisation was not affected. Insummer 1998 additional shadingcovered the cultures, reducing averagePPFD below 100 umol m"2 s'1 . Nobrowning occurred, thus showing thatby managing light intensity problemswith plant quality could be overcome.

• A wide selection of commercial sugarsfrom different countries was tested. Thesugars varied in their colour (white todark brown) and were produced fromsugar beet or sugar cane. All white andlight brown sugars were found equalsubstitutes for tissue culture gradesucrose. Dark brown sugars were foundto be of limited value, if electricalconductivity exceeded 400 uS/cm.

• Starch at concentrations that supportedplant growth gave problems in handling.Satisfactory results were obtained with amixture of corn or potato starch (6 %)combined with gelrite (0.05 %) or agar(0.1%).

• Under our experimental conditions, theuse of natural light, commercial sugarand starch/gelrite would be 90 %cheaper than the use of artificial lighting,tissue culture grade sucrose and gelrite.

Effect of spikelet position on rice antherculture efficiency

The potential of anthers from different partsof the panicle to induce callus was

12

investigated with the japonica rice varietyTaipei 309. Our results showed that thecallusing abilities of anthers from differentspikelet positions were significantlydifferent. After plating 4483, 4496, 4348anthers from the basal, middle and top parts,the percentage of anthers forming calli was20% in the basal part, 12% in the middlepart and 8% in the top part. The anthers ofbasal parts containing pollen at alluninucleate stages, including early, middleand late, showed higher callus inductionfrequency than those from middle and topparts. The green plantlet regenerationfrequencies of top, middle and basalspikelets were around 18% in all threecases. From our results it would appear thatanthers from the basal part of the panicleshould be used in anther culture of rice inorder to obtain higher efficiencies, andthereby optimise the usefulness of thistechnique in rice breeding programmes.

Activation of retrotransposable elementsin rice and induction of insertionalmutagenesis as a tool for cloning genes ofinterest

This investigation was focused on the effectof mutagenic treatment on retrotransposonactivation in rice. The Tos 17retrotransposon known to be activated byprotoplast culture was isolated from riceand used as a DNA probe in Southernhybridizations. By analysing rice plantsderived from radiosensitivity tests it wasshown that the induction of newretrotransposon integrations was possible byy-irradiation, but it was a relatively rareevent. The number of new copies in thegenome was very low compared to the copynumber after protoplast culture. This is ofgreat advantage for the aim of tagging agene by insertional mutagenesis because theidentification of an affected gene will beeasier as with multiple new integrations. Ina molecular analysis of rice gametoclonal

variants using again Tos 17 as a probe, arestriction fragment length polimorphism(RFLP) was detected. One fragment thatappeared in the controls did not appear inthe gametoclones. The DNA methylationstatus of these plants was checked and itwas proved that the missing band was notdue to changes in the methylation pattern.

Development of in vitro culture protocolsin tef (Eragrostis tef ZUCC.)

Fig. 1 Androgenesis in the variety Dz-Cr-37. Seeds from donor plants irradiated with400 Gy.

We studied the effect of radiation on callusinduction and green plant regeneration usingdifferent in vitro culture techniques. Seeds,anthers, spikelets, and young leaves wereused as explants. Different induction andregeneration media were evaluated toestablish the best culture conditions.

Some of the results are summarized below:

• Highest calli induction and green plantregeneration efficiency was observed forspikelets and anthers from donor plantsirradiated with 400 Gy.

• Calli induction was higher for anthersand spikelets plated in semi-solid N6(calli induction medium) medium with

13

maltose as carbohydrate source insteadof sucrose.

No significant difference was found amongthe three MS regeneration media that weretested, but the highest production of singleshoots was observed in the regenerationmedium containing 200 mgl"1 Myo-inositol,1 mgl"1 BAP, 0.5 mgl"1 NAA and 0.45%,agarose Green plant regenerationefficiencies for androgenesis responsereached values between 16-35%.• Production of multiple shoots per calliwas observed in almost all the treatments.On average 28 plants per calli wereproduced, with a maximum of 183 singleshoots.DH1 green plants were transferred to thegreen house and harvested during March-April (1999) and will be send to Ethiopiafor field studies. Some plants in the greenhouse showed plant height reduction.

Gametoclonal variation in the varietyPokkali

Anther culture response of two indicaphotosensitive varieties Nona Bokra andPokkali was evaluated. Even though salttolerant, due to their plant height (140-169cm) and low yield, they are not suitable forgrowing in areas with saline soils. From125 DH green plants regenerated fromPokkali, three gametoclonal variants wereidentified in the first generation (labelled as.101, 112 and 113) showing plant heightreduction and photoperiod insensitivity (Fig.2). Self pollinated panicles were singleharvested and grown in pots for evaluationof the DH2 generation. All DH lines wereplanted randomly. Only the threegametoclones with quantitative alterationsin comparison with the control populationwere studied further.Uniformity of major characters amonggametoclones showed on average 45% inplant height reduction. The number ofproductive tillers per plant, number of

grains per panicle, and fertility did not showimportant variations, and othermorphological characters such as grainshape, size and cariopside color remainedthe same.RAPD analysis was performed in 18 DH2gametoclonal lines, in 3 normal doubled-haploid plants from Pokkali and in a seedderived plant from Nona Bokra and Pokkali.

Fig. 2: Gametoclonal variants of Pokkali

Polymorphisms were found among thegametoclonal lines, including both loss ofparental bands, and the appearance of novelnon-parental bands. There were found cleardifferences with 5 of 21 primers tested.

A new method to monitor cytochimeradissociation through three differentpropagation techniques in Musa spp.

Genetic improvement methods based onmutation techniques are particularlyimportant for Musa species where there islimited sexual reproduction that couldgenerate genetic variation. However, as inother biotechnology approaches such asgenetic transformation, in-vitro mutationinduction techniques applied to

14

improvement of Musa spp. has a majorlimitation: the treatment of multicellularmeristems with mutagenic agents (physicalor chemical) results in a high degree ofchimeras. The chances of a mutated cellgrowing out into a sector or layer andmanifesting itself depend on its positionwithin the apex as well as its growth rate ascompared with the surrounding (non-mutated) cells. It is often suggested topropagate into the fourth vegetative cycle,MiV4, in order to dissociate chimeras butfundamental studies have not been carriedout to verify this postulation. It is difficultto monitor mutated cells, but genomicmutations can easily be induced usingcolchicine and detected using flowcytometry.Our study consisted of inducing ploidychimerism (mixoploidy) and assessingchimeric dissociation by the fourthvegetative generation after propagatingthrough three different propagation systems(shoot-tip culture, multi-apexing culture andcorm slide culture).The results showed that the greatestpercentage of cytochimera dissociation ofmixoploid shoots was achieved with themulti-apexing technique followed by corm

slide culture technique and shoot-tippropagation technique respectively.Presumably a combination of highproliferation rate and production ofadventitious buds could explain why themulti-apexing technique favours chimerismdissociation.Whether it was applied during the firstpropagation cycles or after 5 propagationcycles, the multi-apexing techniqueincreased the proportion of cytochimeradissociation. To a less extend after 5subcultures since probably the selectedmixoploid shoots were already stabilisedinto periclinal chimeras and thus moredifficult to dissociate.We could reduce significantly thepercentage of cytochimera after 3subcultures but not completely. Thus,another propagation system based on singlecell origin is still needed to speed up theselection procedure. This study has shownthat by mixoploidy induction and flowcytometry detection, it would be possible toverify the unicellular (or multicellular) originof any propagation system

H. PUBLICATIONS

Working Material: "Evaluation of Cereal Crop Mutants". Report on the Second WorkingGroup Meeting on "Rice Multilocation Trials", Uruguay, 23-27 March, 1998.

Working Material: "Mutational Enhancement of Genetic Diversity in Rice" Report of theProject Formulation Meeting held in Bangkok, Thailand, 8-12 February 1999.

Working Material: Proceedings of the Co-ordination Meeting of Project AFRA III-18"Development of Improved Crop Varieties", held in Bamako, Mali, 15-19 February 1999.

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P L E A S E C O M P L E T E T H I S R E G I S T R A T I O N F O R M A N D S E N D I T T O T H E P L A N TB R E E D I N G A N D G E N E T I C S S E C T I O N A T T H E F O L L O W I N G A D D R E S S :

W A G R A M E R S T R A S S E 5 , P . O . B O X 100 , A - 1 4 0 0 V I E N N A , A U S T R I AT E L E F A X : (+43-1 ) 2 6 0 0 7 , T E L E P H O N E : (+43-1) 2 6 0 0

N E W C R O P V A R I E T Y D E V E L O P E D T H R O U G H M U T A T I O N I N D U C T I O N O R B YC R O S S I N G W I T H I N D U C E D M U T A N T S

A . Lat in n a m e of species:

I I I I I I I I I 1 I I I I I I 1 I I I I I I I I I I I I I I 1 I I I I

Eng l i sh n a m e : I I I I I I I I I I I 1 I I I 1 I

B . N a m e of n e w var ie ty (cult ivar):

I I I I I I I I I I I I I I I I I I I I I I I

C. Y e a r of re lease from breeder : I I I 1 |

D . P lace and D a t e of official approval:

E . Parent variety( ies) - if n e w variety results from a cross wi th mutant , indicate w h i c h is themutant :

mutant

1- I I I I I I I I I I I I I I I 1 I I I I I y e s / no

2 . I I I I I I I I I I I I I I I I I I I I I y e s / no

3 . I I I I I I I I I I I I I I I I I I I I I y e s / no

F. M a i n i m p r o v e d characters of variety (indicate if character is derived from mutat ion or not ) :

mutat ion der ived

1- I I I I I I I I I I I I I I I I I I I I I y e s / n o

2 . I I I I I I I I I I I I M 1 I I I I I I y e s / no

3 . I I I I I I I I I I I I I I I I I I I I I y e s / no

G. K i n d ( s ) o f mutagen ic treatment: I I I I I I I I I I I I I I I I I I I I

H . D o s e s ( s ) and/or concentrat ion(s) : | I I I I I I I I I I

I. Y e a r of mutagen ic treatment: | I I [ I

J. H o w w a s the var ie ty bred:

K . N a m e ( s ) of breeder(s ) and institute(s):

I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I 1 I I I I I I I I I I I I I I I 1 I

a d d r e s s : I I I I I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I I I I I I

I I I I I 1 I I I I I I I I I I I I I I I

L . E x t e n t of acceptance by growers :

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- Commercial value:

- Hectares of cultivation:

- Other:

M. References (published articles, official documents, etc.):

N. Name of person contributing this information:

THANK YOU FOR YOUR KIND COLLABORATION !

17

Plant Breeding and Genetics Newsletter

Joint FAO/IAEA Division of Nuclear Techniques in Food and Agricultureand FAO/IAEA Agriculture and Biotechnology Laboratory, Siebersdorf

International Atomic Energy AgencyWagramer Strasse 5, P. 0. Box 100

A-1400 Vienna, Austria

Printed by the IAEA in ViennaJuly 1999

99-02287