TurkJBot 27(2003)243-248 ©TÜB‹TAK Review PioneeringBiotechnologicalWorksonHordeumvulgare L.cvs PerformedinCollaborationwiththe‹stanbulUniversityBiology DepartmentandtheTÜB‹TAKResearchInstituteforGenetic EngineeringandBiotechnology NerminGÖZÜKIRMIZI ‹stanbulUniversity,FacultyofScience,DepartmentofGeneticsandMolecularBiology,34459,Vezneciler,‹stanbul-TURKEY TÜB‹TAK,ResearchInstituteforGeneticEngineeringandBiotechnology,P.O.Box:21,41470,Gebze,Kocaeli- TURKEY Received:18.04.2002 Accepted:24.02.2003 Abstract: Hordeumvulgare L.(barley)isanimportantcerealcropandisalsoanexcellentmodelorganismforbiochemists, physiologists,geneticistsandmolecularbiologists. H.vulgare cvs.havebeenusedasamodelsystemforalmost30yearsatthe BiologyDepartmentof‹stanbulUniversity,‹stanbul-Turkey.Thefirststudiesonexperimentalmutagenesiswerefollowedbytiss ue culture,genetransfers,DNAmarkerapplicationsandfinallyDNAarrayswhich,progressedfurtherafterthe1990when collaborationwasestablishedwiththePlantBiotechnologygroupattheTÜB‹TAKResearchInstituteforGeneticEngineeringand BiotechnologyinGebze,Kocaeli-Turkey.ThisreviewarticleoutlinestheresultsoforiginalresearchintoTurkishbarleycultivarsand wildtypeswiththeintentionofcontributingtobarley-breedingprogrammeswithrecentbiotechnologicaltechniques. KeyWords: barley,mutation,tissueculture,genetransfer,DNAarray ‹stanbulÜniversitesiBiyolojiBölümüveTÜB‹TAKGenMühendisli¤iveBiyoteknoloji Araflt›rmaEnstitüsü‹flbirli¤iileHordeumvulgare L.cvs.’de GerçeklefltirilenÖncüBiyoteknolojikÇal›flmalar Özet: Hordeumvulgare L.(arpa)önemlibirtah›lbitkisidir,biyokimyac›lar,fizyologlar,genetikçilervemolekülerbiyologlariçinçok etkinbirmodelorganizmad›r. Hordeumvulgare cvs.yaklafl›k30y›ld›r‹stanbulÜniversitesiBiyolojiBölümü’ndekigenetiktemelli çal›flmalardamodelsistemolarakkullan›lmaktad›r.‹lkdeneyselmutasyonçal›flmalar›1990’l›y›llardaTÜB‹TAK,GenMühendisli¤i ve BiyoteknolojiAraflt›rmaEnstitüsü,BitkiBiyoteknolojisiGrubuiflbirli¤iiledokukültürü,gentransferleri,DNAmark›ruygulamalar›ve DNA“array”çal›flmalar›ilesürdürülmüfltür.BuderlememakaledeTürkkültürveyabaniarpavaryeteleriileyap›lanözgünçal›flmalar arpayetifltiricili¤inegüncelbiyoteknolojikyöntemlerlekatk›larsa¤lanmakamac›ileözetlenmifltir. AnahtarSözcükler: arpa,mutasyon,dokukültürü,genaktar›m›,molekülermark›rlar,DNA-“array” Introduction chromosomes.Moreover,ithastwo-rowedandsix- Cultivatedbarley(Hordeumvulgare L.)isthesecond- rowedtypes,accordingtospikemorphology(Bothmeret mostimportantcerealcropforTurkeyafterwheat,and al.,1991).Thegenomesizeofbarleyis5.5 picogram/haploidnucleusandisequivalentto isconsumedasfeedforlivestockand,foodforhumans 9 and,mostimportantly,isalsousedforbrewingmalts. approximately5.3x10 bp(Bennet&Smith,1976),and Barleyisalsoanexcellentmodelplantforbiochemists, 50-60%ofthegenomeconsistsofrepeatedsequences physiologists,geneticistsandmolecularbiologists (Rimpauetal.,1980).Copia-likeretrotransposonBARE- (Shewry,1992).Accordingtoworldstatistics,itis 1comprisesalmost7%ofthebarleygenome(Manninen cultivatedon53,827,895hectareswitha25,723Hg/Ha &Schulman,1993).Easeofgrowthunderlaboratory worldyieldofwhichTurkey’scontributionis3,550,000 conditionsfacilitatesthedevelopmentofmolecular hectareswithan18,592Hg/Hayield(FAO,2001).Barley markersfortheconstructionofgeneticmaps(Williamset isaself-pollinatingdiploidwith2n=2x=14 al.,2001).Thebarleygenomeprojectandproductionof 243 PioneeringBiotechnologicalWorksonHordeumvulgare L.cvsPerformedinCollaborationwiththe‹stanbulUniversityBiologyDepartmentandthe TÜB‹TAKResearchInstituteforGeneticEngineeringandBiotechnology barleyESTsareinprogresswithcontributionsfrom formation,spindlesplittingandnon-synchronisedphases variousorganisations(Michaleketal.,2002).Recently, intheseconddivisionetc.(Bilgeetal.,1981a,b).The thefirstfunctionalgenomicstudieswerecarriedatfor effectsofXandgammaraysonmitoticcelldivisionand stresstolerance(Öztürketal.,2002)andtissue-specific theproteincontentoftheirradiatedseedswerealso differentialexpression(Sreenivasuluetal.,2002)in investigated(Olgun,1985).Theadaptationefficiencyand barley. microyieldsofthesemutanttypeswerestudiedinthe Themainobjectiveofbarleybreedingprogrammesis frameworkof“StudiesonAgriculturalApplicationsof mainlytoincreaseyieldandgrainquality.Improvement ExperimentalMutationsInducedonNativeBarleyVariety effortsarealsoconcentratedonproducingvarieties Zafer160”TÜB‹TAK,TOAGGrantNo.162. resistanttobiotic(pathogens,fungal,viralandother Mutationstudieswerecontinuedontwoprojects,one organisms)andabioticstresses(e.g.drought,salt,cold ofwhichwassupportedbyTÜB‹TAK-TBAGGrantNo. andheat)(Dunwell,1986).Duringconventionalbreeding 515andtheotherby‹stanbulUniversityResearch programmesviahybridisationsbetweenhigh-yielding FoundationGrantNo.212/030186,ontissuecultured cultivarsandwildbarley,specifictraitsmaybe material.Intheframeworkofthefirstprojecttheeffects introgressedinback-crossingprogrammes(Nevo,1992). ofpesticideswerestudied.Theeffectsoftwocommercial Mutationbreedingisalsoimportantforwidening pesticidepreparations,2,4-dichlorophenoxyaceticacid variation.Radiationandchemicalmutagenesishavebeen isooctylester(2,4-D)andphenylmercuryacetate(PMA) usedtoincreasethenumbersandvarietiesofbarley ondifferentorganismswereinvestigated.Thesereagents whichmighthavedesirabletraits.Forexample,oneof didnotproducenumericalandstructuralchangesinthe themostpopularmaltingbarleys,“Goldenpromise”,was mitoticchromosomesof Hordeumvulgare embryo producedin1957usingradiationmutagenesis(Milne cultures(Oraleretal.,1984).Inthesecondprojectthe MarstersCo.,1970). effectsofXandgammaraysoncallicultureswere ProffessorEmineBilgeperformedthefirstbasic studied.Matureembryopartswereusedforcallus geneticexperimentsinwhichbarleywasusedatthe formationandplantregenerationwasachievedon BiologyDepartmentof‹stanbulUniversityinthe Murashige-Skoog(MS)medium(Gözük›rm›z›& frameworkoftheproject“BasicGeneticStudiesfor Ekmekçiler1987;Ar›1994). ObtainingHighQualityBarleyLines’GrantNo.162, Genetransfertechnologiesofferasuitablealternative TÜB‹TAK,TOAG.Inthisstudy,Zafer160barleyseeds forimprovingdesirablegene(s)inadirectedmanner weretreatedbeforesowingwithXandgammarays, withouttheundesirableinsertionofDNAfragments.The ethylalcohol,streptomycin,terramycin,penicillinG, establishmentofstableandregenerativetissueculture sodiumcyanideandethylmethanesulphonatesolutions. systemsisaprerequisiteforbarleytransformation. Inadditiontochlorophylldeficienttypes,large-eared, Differentexplants,immatureembryos(Breiman,1985), high-yielding,thick-stemmed,dwarfandearly-heading matureembryos(Lupotto,1984),apicalmeristems(Chen mutantswereobtainedinM1 andsucceedinggenerations &Smith1975),anthers(Kao&Horn1982), ofthetreatedmaterial.Asaresultofseedirradiation microspores(Köhler&Wenzel1985),cellsuspensions with16,000raddosesofX-rays,amutantbarleycalled (Kott&Kasha1984)andprotoplasts(Lazzeri&Lörz KA/14wasobtained.Theearshapeofthismutant 1990)havebeenusedforthispurpose. resembledthatofthehoodedtype,and,thenumberof In1987,underagrantfromNATO-TU-BIOTECHI, tillersandtheyieldwerehigherthanthecontrol.After No.842insubproject1.2.2entitled“CallusInduction, artificialpollinationofZafer160femaleswith1000rad PlantRegenerationandChromosomalVariationsin gammairradiatedpollen,short-stemmedandearly Barley’calluscultureswereinducedonmatureembryo mutantsappearedintheF 2 generation.Theirheading mesocotylexplantsinZafer160barley.Thecallus timewas23daysearlierthanthatofthecontrol.Meiosis inductionratiowas54%inMSmediumsupplemented wasstudiedintheanthers,andthefollowing with1mg/l2,4-dichlorophenoxyaceticacid(2,4-D). abnormalitieswereobservedinthetreatedmaterial: Aftertransferat22,45,360and540daysofcultureto breakingandstickingtogetherofthechromosomes, MSmedium,containinglowerconcentrationsofor chromatinbridges,translocations,micronucleus lacking2,4-D,onlythe45-day-old-callusshowedsomatic 244 N.GÖZÜKIRMIZI embryogenesis(Fig.1).Abnormalitiesinboththe forthehealthygerminationoftransformedplantsfrom numberandstructureofchromosomesincreasedwith matureelectroporatedembryos.Genetransfer theageofthecalli.Thisphenomenonmightberelatedto performedon3-day-oldculturesresultedinthehighest thelossofregenerationabilityin540-day-oldcalli.In germinationfrequencies.Transgenesiswasconfirmedby vitroregeneratedplantletsgaverisetonormal-looking PCRandSouthernhybridisationanalyses(Gürel& plantsaftertheirtransfertosoil.Regeneratedplantshad Gözük›rm›z›,2003). thenormaldiploidchromosomenumberintheirroottips Avarietyofmolecularmarkershavebecomeavailable (Gözük›rm›z›etal.,1990).Antherandmicrospore inrecentyears(Mohansetal.,1997;Guptaetal.,1999), culturesofthesamevarietywerealsoestablished(Ar›et andeffortsarealsobeingmadetoidentifythemost al.,1992). efficientandcost-effectivemarkersthatcanbeusedby Planttransformationwasachievedusingthe practicingplantbreeders.Inadditiontotheiruseinplant electrophoresisofgerminatingseeds(Ahokas,1989)or breeding,molecularmarkershavebeenputtoseveral theincubationofembryosinaDNAsolution(Töpferet otheruses,includinggenomemapping(Kleinhofsetal., al.,1989),PEGandelectroporation-mediatedprotoplast 1993;Hanetal.,1993),DNAfingerprinting(Faccioliet transformation(Junkeretal.,1987;Teerietal.,1989), al.,1999)andthestudyofgeneticdiversity(Baumetal., microsporeselectroporation(Joersboetal.,1990), 1997). particulebombardment(Wan&Lemaux1994),macro- In1992,theplantbiotechnologygroupinTÜB‹TAK injections(Mendeletal.,1990)andmicro-injections startedinvestigatingmolecularmarkersusingRAPD (Olsen,1991)inbarley. techniques.Tissuecultureregeneratedplantletswere testedforstability(Gözük›rm›z›etal.,1992),methods ThePlantBiotechnologyGroupwasorganisedin weredevelopedforhybridselectionfromwildlinesand