TurkJBot 27(2003)71-76 ©TÜB‹TAK ResearchArticle PlantletRegenerationinAbiescilicica Carr.andAbiescilicica x Abiesnordmanniana HybridviaSomaticEmbryogenesis Boz˘enaVOOKOVÁ,AndrejKORMUT˘ ÁK InstituteofPlantGeneticsandBiotechnology,SlovakAcademyofSciences,Nitra,SlovakRepublic Received:26.12.2001 Accepted:25.10.2002 Abstract: Somaticembryogenesiswasinitiatedfromimmaturezygoticembryosof Abiescilicica Carr.anditshybridA.cilicica xA. nordmanniana.SchenkandHildebrandtmedium(SH)supplementedwith5µMbenzylaminopurinewasusedastheinitiation medium.InA.cilicica,theinitiationofembryonalsuspensormass(ESM)frequencyrangedfrom5.4to63.5%,and28.6%ofthese celllinesformedmaturesomaticembryos.InA.cilicica xA.nordmanniana,from3to27.6%ofzygoticembryosformedESM,and maturationofsomaticembryoswasobservedin34.8%lines.Forsomaticembryomaturation,Murashige,SkoogandSHmedia supplementedwith4%maltoseand10%polyethyleneglycol-4000wereused.Formaturation,80µMabscisicacidwasmost effective.Afterthreeweeksofpartialdesiccation,matureembryosgerminatedonSHmediumwith1%sucroseand1%activated charcoal,andplantletswithcotyledons,hypocotylsandradicleswereobtained. KeyWords: Conifers,maturation,germination,plantletregeneration Introduction TheinductionofsomaticembryogenesisintheAbies Thediversityandtheextentoftheworld´sforestsare Mill.genushasbeendemonstratedinfourpurespecies, declining,yetthedemandforwoodworldwideis A.alba,A.nordmanniana,A.fraseri (Pursch)Poir.andA. expectedtodoubleinthe21stcentury.Toaccommodate balsamea (L.)Mill.,(forreviewseeNorgaardand thisdemand,theproductivityoftheremainingforest Krogstrup,1995)andseveralhybrids, A.alba xA.alba, landswillhavetobeincreased,whileotherareasareset A.alba xA.nordmanniana (Gajdos˘ováetal.,1995), A. asideforconservation.Advancesinbiotechnologywill alba xA.cephalonica,A.alba xA.numidica (Salajováet acceleratetreeimprovement.Inparticular,somatic al.,1996).Despiteourknowledgeofthesomatic embryogenesisoffersnewwaysforfastermultiplication embryogenesisof Abies sp.,reportsonplant ofhigh-valueclonesforreforestation,whichwillhelpin regenerationarerare(Guevinetal.,1994;Hristoforoglu theracetoincreaseforestproductivity(Guptaetal., etal.,1995;Norgaard,1997;Salajová&Salaj,2001). 1996). Theobjectiveofthisresearchwastoinvestigatethe OneimportantconiferousspeciesinCentralEuropeis possibilityofsomaticembryogenesisinitiation,somatic Abiesalba Mill.Thisspeciesissensitivetodroughtand embryomaturationandplantletregenerationinA.cilicica otherenvironmentalstresses,andisoneofthemost anditshybrid. damagedtreespecies(Krehan,1989).Therescueof A. alba maybepossiblebymeansofintra-andinterspecific MaterialsandMethods hybridizationinordertoextenditsgeneticvariability. Abiescilicica Carr.isafastgrowingspecieswhosenatural Anartificialpollinationexperimentwascarriedoutin distributionisinAsiaMinor(Bozkufl,1987).Becauseof ArboretumMlyn˘ any,Slovakia,usingonemothertreeof itsfastgrowth,thespeciesisrecommendedfor cilicianfir( Abiescilicica Carr.)andonefathertreeof introductiontotheclimaticconditionsinSlovakia(Tokár, Caucasusfir[Abiesnordmanniana (Stev.)Spach].Female 1973).However,aspointedoutbyLapin(1973),ofno flowersof A.cilicica wereisolatedbeforeopeningtheir lessimportanceistheabilityofintroducedspeciesto scalesusingpaperbagsasisolators.Artificialpollination intercrosswithotherspeciesofaparticularregion.Inour offemaleflowerswasperformedatthestageoftheir fieldstudiesonartificialhybridization,the A.cilicica xA. maximalreceptivityatthebeginningofMay,usingfreshly nordmanniana hybridformappearedverypromising. collectedpollenof A.nordmanniana .Exceptforthe 71 PlantletRegenerationinAbiescilicica Carr.andAbiescilicica xAbiesnordmannianaHybridviaSomaticEmbryogenesis interspecificcontrolledpollination,asmallportionof femaleflowerswereself-pollinated,servingasacontrol fortheinterspecificcrossing A.cilicica x A. nordmanniana.Theisolatorswereremovedfromfemale flowersafterpollination. Conescontainingimmatureseedsof A.cilicica Carr. fromself-pollinationaswellasfromtheinterspecific crosses A.cilicica x A.nordmanniana werecollectedat regularintervalsduringJuly–August1997.The availabilityofexplantswaslimitedbythenumberof developingmegagametophytesinacone(Tablel). Immatureseedsweresurface-sterilizedfor10minin 10%H2O2.Endospermscontainingembryos(seeFig.1) (fromJuly8toJuly24)orembryosafterexcisionfrom Fig.1. Megagametophytescontainingimmaturezygoticembryos themegagametophyte(fromAugust5toAugust26) platedoninitiationmedium. wereplatedonSHinitiationmedium(Schenk& Hildebrandt,1972)with5µMbenzylaminopurine(BA) 1)modifiedSHmediumusedinpreviousexperiments and2%sucrose.Themediumwassolidifiedwith0.3% (Vookováetal.,1977/1998),whereinthefirststepESM Phytagel.Allmediacomponentswereautoclavedat wasculturedonamediumcontaining6%lactose,10% 121°Cfor20min.Thecultureswerekeptindarknessat polyethyleneglycol-4000(PEG-4000)and40µM(±)cis- 21-23°C.Embryonalsuspensormass(ESM)proliferated trans-abscisicacid(ABA).Afteroneweekofcultivation, onamediumwith0.05%L-glutamine(GL)supplement ESMwastransferredtoamediumwith7.2%lactose, and0.1%caseinhydrolysate(CH)andweresubcultured 1%sucroseand40µMABA.Mediaweresupplemented everythreeweeks. with0.05%GLand0.01%CHandsolidifiedwith0.3% Phytagel. Maturation 2)mediumcontainedbasalsaltsandvitaminsofSH Todeterminewhetherembryogeniccelllinesrespond medium,3%maltose,10%PEG-4000,0.05%GL,40 tomaturationtreatment,allinducedcelllines A.cilicica µMABA,0.1%CHand3%Phytagel. (42lines)and A.cilicica x A.nordmanniana (23lines) weresubjectedtomaturationtreatment. Toassessthemostbeneficialmediumforsomatic embryomaturation,threecelllinesofbothA.cilicica (50, Pieceswithanapproximateweightof500mgESM 91,98)and A.cilicica xA.cephalonica (102,106,145) weretransferredto90mmplasticPetridishescontaining wereculturedonSH,GD(Gresshoff&Doy,1972)and maturationmediumindarknessat21-23°C.Petridishes modifiedMS(Murashige&Skoog,1962)media.SHand weresealedwithpolyethylenefilm.Twotypesof GDmediacontainedoriginalmacro-andmicro-elements, treatmentwereusedforsomaticembryomaturation: FeEDTAandvitamins.TheMSmediumcontained1/2 Table1. Initiationpercentageofembryogenictissuefromimmaturezygoticembryos.Thenumberofexplantsisinbrackets. Collectiondates July8 July15 July24 August5 August26 Explant Megagametophytescontaining Immatureembryos immatureembryos Species A.cilicica 63.5(75) 11.9(94) 5.4(149) 0(0) 5.7(88) A.cilicicax A.nordmanniana - 6.5(108) 3.0(108) 27.6(105) 0(101) 72 B.VOOKOVÁ,A.KORMUT˘ÁK strengthMSmacroandoriginalmicro-elementsand observedfromthemegagametophytesatthemicropilar FeEDTA,modifiedvitamins;5.5µMnicoticacid,3µM end(Fig.2).Immaturezygoticembryosof A.cilicica thiamineHCl,4.9µMpyridoxinHCl,13.3µMglycineand showedarelativehighfrequencyformationofESM 0.01% myo-inositol.Allmediacontained4%maltose, rangingfrom5.4(July24–August26)to63.5%(July 10%PEG-4000,CHandGlin0.5%concentrationand 8).In A.cilicica xAbiesnordmanniana thiswasfrom3 0.3%Phytagel. (July24)to27.6%(August5)(Tablel).Toour TheeffectofABAonsomaticembryomaturationof knowledge,the63.5%frequencyofESMformationin selectedcelllineswasdeterminedbysubcultureofESM Abies hasnotbeenachievedelsewhere.Untilthenthe ontoMSmaturationmediumcontaining40and80µM highestinductionfrequency,44.6%,hadbeenreported (±)ABA. intheA.alba xA.numidica hybrid(Salajováetal.,1996). Inallmaturationtreatments,ABAwasco-autoclaved togetherwithothersubstancesinthemedia.During maturationculturesweremaintainedat21-23°C.The experimentconsistedof10replicateplasticplates(Ø60 mm),eachcontainingESMofapproximately300mg. Germinationandplantletregeneration AfterSEmaturationonthemostbeneficialmodified MSmedium,cotyledonaryembryosofthesamecelllines usedformaturationtreatmentwereusedinthe germinationexperiment.Priortogermination,the somaticembryoswereisolated,placedinPetridishes(Ø 60mm)andsubjectedtopartialdesiccationasfollows: thePetridishwasopenandplacedonmoistfilterpaper inaPetridish(Ø90mm),whichwassealedwith Fig.2. Initiationofembryogenictissueafterthreeweeksin culture. parafilm.SomaticembryosinPetridisheswerecultured indarknessat22-25°Cforthreeweeks. Thedifferenttendencywasobservedinmaturation Afterpartialdesiccation,maturesomaticembryos experimentsinwhich42celllinesof A.cilicica and23 (withatleastfourcotyledons)weretransferredto linesof A.cilicica xA.nordmanniana weretested(Table germinationmediumandculturedinthelight(16h 2).Somaticembryosinthecotyledonarystageof photoperiods)at21-23°C.Thestandardmediumfor developmentwereobservedin28.6%of A.cilicica and germinationwasSHmediumcontaining1/2 34.8%ofA.cilicica xA.nordmanniana celllines.Somatic concentrationofbasalsalts,SHvitamins,0.01% myo- embryomaturationwasobservedinbothwithlactose inositol,1%sucroseand1%activatedcharcoal(DarcoG andmaltosemedia.Itwasnotedthatmaturationon 60).Themediumwasgelledwith0.3%Phytagel.Six mediumwithlactosegaveahigherfrequencyofglobular replicationsof10embryoswerecultivatedinan (Fig.3)andtorpedo-shapedembryosthanmaltose,but erlenmayerflaskwith50mlmediumpertreatment furtherembryodevelopmentwasaberrant(abnormal). underalightintensityof110µmol.m -2 s-1 for16hper Thedurationofmaturationtreatmentwas8-10weeks. day.Germinationpercentageswereevaluatedafter40 Maturesomaticembryosobtainedonmediumwith daysofcultivation. maltosewereyellowtogreenwithcotyledons(1-6)and hypocotyls(Fig.4).Histologicalobservationofthese Statisticalevaluationofthedatawascarriedoutusing embryosshoweddifferentiationoftheradiculameristem Student’st-test. (Fig.5). Thecelllinesdifferedintheirresponsetothethree ResultsandDiscussion maturationmedia.Thenumberofglobularandmature Withinfourtosixweeksoninitiationmedium,the cotyledonaryembryospergofESMwasdifferentin