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Jan.2008,Volume2,No.1(SerialNo.2)JournalofLifeSciences,ISSN1934-7391,USA

    ;Primarystudyonanthercultureofbalsumpearforcallus ;andorganformation

    ;LIHuan-xiu,CHENJia,ZHENGYangxia,Zesheng,HEYan

    ;(DepartmentofHorticulture,CollegeofForestryandHorticulture,SichuanAgricultureU

    niversity,Ya’anSichuan625014,China)

    ;Abstract:Thebalsumpear(MomordicacharantiaL.)anthers ;inthemonokaryoticstageofmicrosporedevelopmentwere ;culturedinthisexperiment.DifferentPlantgrowthregulators’

    ;combinations,basemediaandcarbonsourceswerestudiedfor ;callusformationandorgandifferentiationfrombalsumpear ;anthers.Theresultshowedthatthebestmediaforcallus ;inducementwas”MS+BA0.5mg/L+NAA0.2m2/L+2.4D0.5

    ;mgfL+KT2.0me_m.with3%sugarand0.8%agar.Thebest ;mediatoinducerootsfrombalsumpearanthercalluswas: ;MS+NAA0.05mg,L+KT0.5mg/L,with3%sugarand0.8% ;agar.MOStofadventitiousrootsfromcallusweretriploid ;(2n=3x=33)

    ;Keywords:balsumpear(MomordicacharantiaL.):anther ;culture;callusinducement;organdifferentiation ;1.Introduction

    ;Balsumpear(MomordicaCharantiaL.),also

    ;namedcoldmelon,isakindofannualandrambler ;plantbelongedtomomordicaofCucurbitaceae.It ;containsrichnutritiOnandmedicinecomponentsi1.It

    ;isadoredbymanypersonsandhasbettereconomic ;benefitbydependencyandcommonplantingI21. ;Balsumpearbreedingwascarriedoutbymany

    ;researchers.Becauseofthelongtime,greatdifficulty ;andgeneticunsteadyofCucurbitaceaeplantbreeding, ;itisveryimportanttodevelopbiologictechnique ;breedingbasedoninvitroculture.Forobtainingpure ;Acknowledgments:TheauthorswouldliketothankDr. ;WANGXiaorongandDENGQunxianfortheirhelpinthe

    ;tests.ThisstudywassupportedbytheEducationBureau ;ScienceFoundationofSichuanProvince(No.20o5Ao03)and ;theSichuanAgriculturalUniversityScienceFoundation(No. ;3305).

    ;LIHuanxiu(1962),female.Ph.D.,professor.Ph.D.

    ;supervisor;researchfields:vegetablebiologicaltechnique, ;heredityandbreedingofvegetable.

    ;12

    ;doublehaploid,anthercultureisusuallyadopted[31 ;,

    ;whichcouldshortenthetimeofselectionandpure ;breedingplants.

    ;Uptonow,successfulanthercultureshappened ;mainlyonSolanaceae,Gramineae,cruciferaeplants[41. ;Homogenouslineshadbeobtainedinaveryshorttime ;byhaploidybreedingmethodsuchas

    ;microsporecultureandanthercultureofradish ;(RaphanussativusL.)andrapes(oilseed,Brassica ;napusL)15-6t.Techniquesforantherculturehadbeen ;developedandwidelyusedformostBrassicaspecies. ;Antherof15genotypesofradishhavebeenculturedin ;vitroandinducedtoundergoembryogenesisandplant ;formation.Of15genotypesevaluated,fourproduced ;embryos.Thegenotypewasthemainfactorto ;influencetheembyogenesisfromantherormicrospore ;[51

    ;.Inaddition,thegrowingconditionofdonorplants ;alsoinfluencedtheembyogenesis.Therewerefew ;reportsonsuccessfulantherculturesofCucurbitaceae ;plants,becausebetterregenerationsystemofgourd ;plantshadnotestablishednow.Balsumpearvitro ;culturemainlyconcentratedoncotyledons,hypocotyls, ;leaves,stemtipandstemwithbudnow,andonly ;obtainedcallusandinduced’fewaccidentalbuds.

    ;Thereisnotanystudyreportofbalsumpearanther ;cultureuntilnow.Theeffectsofdifferentplant ;regulators,baseculturemediaandsugarsourceon ;balsumpearanthercallusformationandorgan ;differentiationwerestudied,andhopetobeusefulin ;thefutureresearch.

    ;2.Materialsandmethods

    ;Primarystudyonanthercultureofbalsumpearforca. ;1

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    ;2.1Material

    ;Theflowerbudsof’’Big—whitebalsumpe’

    ;(MomordicacharantiaL.)inYa’an,Sichuanwas

    ;selectedforcallusinducementinMay.JuneandJuly.The ;antherofflowerbudmustbeinsinglenucleusstageand ;flowerbudwere5.08.0mminlength(Fig.11.

    ;Mainreagentsandapparatus:ReagentsZeatin ;(ZT),benzyladenine(BA),kinetin(KT),IndoleAcetic ;Acid(IAA),NaphthaleneAceticAcid(NAA), ;2,4dichlorobenzeneoxygenaceticacid(2,4D),

    ;HydrolyzeCasein(HC),canesugar,agar,HgCI2 ;99.5%ethanol,aceticacid,IndoleButyricAcid(IBA), ;andwereboughtfromChengduReagentCompany,in ;Sichuan.OLYMPUSfluorescencemicroscopewas ;boughtfromChongqingYauntaiApparatusCompany.

;2.2Methods

    ;2.2.1MateriaJtreatment

    ;Theflowerbudswerepretreatedinicebox(4?)

    ;for24h.Then,theyweredippedinthe75%alcoholfor ;30s.in0.1%HgC12for7minandrinsing3to4times ;byasepsiswater.Aftertheflowerbudwasblotup ;wateronasepsisfilterpaper,theantherwascutinto4 ;to5patsandculturedonbasemediawithdifferent ;plantregulatorsforcallusformation.Theculture ;densityperbottlewas8to10antherpats.Therewere3 ;bottlespertreatmentandrepeated3times.After ;culturedindarkand33?for10days,theywere

    ;shiftedtotheconditionwithilluminationl5001xand ;26?.Theywereobservedeach5daysin20days. ;2.2.2Theeffectofdifferentplantregulatorson ;callusinducement

    ;BalsumpearantherswereculturedonMSbase ;mediumwith3%sugar.8%agaranddifferentplant ;regulators.Thecrosseddesignwith4factorsand4 ;levelswasadoptedinthetest.BAlevelwas0.5,1.0, ;1.5and2.0mg/L;NAAlevelwas0,0.05,0.1and0.2 ;mg,L;2,4Dlevelwas0,0.1,O.2and0.5mg/L;KT ;levelwas0,0.5,1.0and2.0m.

    ;2.2I3Theeflfectofdifferentbasemediaoncallus ;inducement

    ;Betterplantgrowthregulatorcombinationswere ;chosefromabovetestsandaddedintoMS,B5andN6 ;mediumindividuallyforbasemediumselectiontest. ;Theobservingmethodandtimewassameasabove. ;2.2.4Theeffectofdifferentsugarsourceon ;callusinducement

    ;Thebestplantgrowthregulatorcombinationand ;mediumwereselectedfromabovetests.Theywere ;added3%and5%canesugarandmaltoseforselecting ;bettersugarsource.

    ;2.2.5Organinducementfromanthercallus ;Thecalluswasculturedinthemediawith

    ;differentPGR(ZT2.0;BA0.5,1.O,2.0,4.0;IAA0.1; ;NAA0.05,O.2,0.5;KTO.5,1.0;ABA0.05;2,4-D ;O.05,O.5mgm)andaccessory(HC100mg/L) ;combinationsfororganinducement.Therewere3pats ;ofcallusperbattle,3battlespertreatmentandrepeat3 ;times.Allmediacontained3%canesugarand0.8% ;agar.Afterculture20days.observeandstattheresult.

    ;2.2.6Chromosomeobservationofinducedorgan ;TheadventitiOusrootsfromanthercalluswere ;pretreatedin8-hydroxidequinolinesolution(0.002 ;mol/L)for2h3h.Atierwashingcleanindistilled ;water,therootsdipinCannoyfixingsolution(ethanol: ;aceticacid=3:1)for24h.Then,theyweredispelledin

    waterbathfor15min.When ;1.0mol/LHClandin60?

    ;theroottipsshownmilkwhiteandotherpartsshown ;transparency,therootswerewashedanddyedby ;Kabaofuchsin/1.Theroottipswerepressedand ;observed.Thteecalluspatsinthesamemediumwere ;chosenandthreerootsinthesamecalluspatwere ;observed.Thechromosomenumberwascountedin ;rootnp.

    ;2.2.7Observationandanalysismethodoftestresult ;Thedatawerecountedeach5daysin20days,The ;dataon20dayswereanalyzedbyDuncanandTukey ;(LSD,a,b=0.05).

    ;Callusinducedrate(%)=(thenumberofinduced ;callus/thenumberofculturedantherpats)×10o%. ;

    ;P

    ;——

    ;r

    ;

    ;imarystudyonanthercultureofbalsumpearforcallusandorganformation

    ;Theadventitiousrootdifferentiationrate=(the ;callusnumberinducedroots/thenumberofcalluspats) ;×lOO%.

    ;Theaverageadventitiousrootnumber=(induced ;totaIrootnumber/thecallusnumberinducedroots) ;×10o%.

    ;3.Results

    ;3.1TheeffectofdifferentPGRcornbinationon ;callusformationfrombalsumpearanther

    ;Tablelshowedthattreatment4wasthebestone ;f0rcallusformation.whichhadthehighestcallus ;formationrateandsignificantlyhigherthantreatment1, ;2.9and15.Thelowestcallusformationratewas ;treatment9.ThebestcallusformationmediumwasBA ;0.5mg/L+NAA0.2mg几十2,4D0.5mete+KT2.0

    ;mg/L.Calluswasyellow&greenonthemedium(Fig. ;2.

    ;Table1Theeffec~ofdifierentPGRcombinationonanthercallusformation

;TreatmentNo./PGRcombination(mg,L)Callusformationrate(%)

    ;1.MS+BA0.5

    ;2.MA+BA0.5+NAA0.05+2.4D0.1+KT0.5

    ;3.MA+BA0.5+NAA0.1+2.4D0.2+KT1.0

    ;4.MA+BA0.5+NAA0.2+2.4D0.5+K1,2.0

    ;5.MA+BA1.o+2.4D0.1+KT1.0

    ;6.MA+BA1.O+NAA0.05+2.4Dol2+K1,2.0

    ;7.MA+BA1.(NAAO.1+2.4D0.5

    ;8.MA+BA1.(NAA0.2+KTO.5

    ;9.MA+BA1.5+2.4D0.2+K1,2.O

    ;1O.MA+BA1.5+NAA0.05+2.4D0.5

    ;11.MA+BA1.5+NAA0.1+KT0.5

    ;l2.MA+BA1.5+NAA0.2+2.4D0.1+KT1.0

    ;l3.MA+BA2.o+2.4D0.5+KT0.5

    ;14.MA+BA2.O+NAA0.O5+KT1.O

    ;l5.MA+BA2.0+NAAO.1+2.4Do.1+K.O

    ;l6.MA+BA2.0+NAA0.2+2.4D0.2

    ;l0.14a

    ;l0.40a

    ;53.07ab

    ;89.8lb

    ;55.25ab

    ;46l36ab

    ;72.15ab

    ;52.06ab

    ;3.O3a

    ;63.77ab

    ;33.83ab

    ;51.40ab

    ;36.88ab

    ;56.75ab

    ;28.12a

    ;47.7Oab

    ;Note:Theabovedatawereanalyzedafterturningoverjustincredibleof%turnoverpositive

    profound.

    ;3.2Theeffectofdifferentbasemediaoncallus ;inducementfrombalsumpearanther ;Thecallusformationratesoftreatment18and28 ;weresignificantlyhigherthanothertreatments.The

    ;resultshownthatMS+BA0.5+NAA0.2mg/L+2,4D

    ;0.5me/t,+KT2.0mg/LandN6+BA0.15+NAA0.05 ;mg/L+2,4D0.5mg/Lwerebettermediaforanther ;callusformation.Callushadgrowthpoweronthese ;media(Fig.34).InthesamePGRcombination,MS

    ;wasbetterthanB5forcallusformation(Table2). ;Table2Theeffectsofdifferentbasicmediaforanthercallusformation

    ;Pmarystudyonanthercultureofbalsumpearfo

    ;rcallusandor

    ;ganformation

    ;3.3Theeffectofdifferentcarbonsourceon ;callusinducementfrombalsumpearanther ;Table3showedthatthemediumadding3%cane ;sugargainedthehighestcallusformationrateand ;significantlyhigherthanthemediaadding3%and5% ;maltose.Thecalluswasgreenandhadgrowthpower ;(Fig.5).Themediumadding3%maltosehadthe ;lowestcallusformationrate.

    ;Table3Theeffe~sofdifferentcarbonandit’Sconcentrationforcallusformation ;3.4Organinducementfrombalsumpear

    ;anthercallus

    ;TheresultshowedthatonlyPI(MS+ZT

    ;2.0+NAAO.05+0,5KT),P2(MS+KT1.0mg/L+2,4D

    ;0.5mgm)andP3(MS+HC100+KT1.0mgm+2,4D

    ;0.5mg/L)treatmentin16treatmentsinduced ;adventitiousroots.TherootinducingratesofP1,P2 ;andP3were70%,73%and75%(Table4).The

    ;averagerootnumberpercallusinPI,P2andP3were ;2.6,5.6and5.4(Fig.68).Therewasnoany

    ;adventitiousbudinducedinalltreatments. ;Table4TheeffectsofPGRSandHCfororganformationonanthercallus

    ;3.5ThechromosomeObservationOf

    ;adventitiousrootsfrombalsumpearanthercallus ;Thechromosomeobservationofadventitious ;rootsfromBestwishes,balsumpearanthercallus ;showedthatthechromosomenumberof69.67%root ;dividedpostureswas2n=3x=33(Fig.9),about20% ;was2n=3x+3=36(Fig.1O),about5%was2n=2x=22 ;(Fig.11)and4%rootdividedpostureswasn=x=l1 ;(Fig.12).Fewrootdividedposturesshowedlacking ;somechromosomes(Table51.

    ;15

    ;Primarystudyonanthercultureofbalsumpearforcallusandorganformation

    ;Note:Dateseparatedwithinaveragecountbyexcel ;4.Discussion

    ;4.1PGReffect

    ;PGRsarefactorswhichaffectcallusandembryoid ;formationfromantherculture.Manyresearchersfound ;thattherewasnocallusformationonnoauxinmedium.

    ;Eventhoughtherewascallusformationbychance,only ;lesscallushappenandthecalluscouldnotproliferate ;[8-9

    ;.IntheCuCllrbitacePplantantherculture,BA,IAA, ;NAA,KTand2,4.Dwereoftenaddedinthemedium. ;ThecombinationofthesePGRswasaddedaccordingto ;thecultureplantspeciesandculturetime[10J.Inthe ;Cucurbitaceaeplantantherculture,BAwasavery ;importantfactor.Whenadding6BAinthemedium.

    ;mostantherculturecouldinducecallus.IfaddingbOth ;BAand2,4-Dinthemedium,thecallusformationwas ;betterthanonlyaddingBA.Thistestalsoshowedthat ;thebestcallusformationmediumwasMS+BA0.5 ;mg/L+NAAO.2mg/L+2,4D0.5mg/L+KT2.0mg/L.

    ;OnthemediumwithoutKTand2.4-Dtherewaslower ;callusformationrate(10.14%).Thelower ;concentrationofKTand2,4Dalsocouldinducecallus.

    ;buttheratewaslowerthanhigherconcentrationofKT ;and2,4D.SoKT,BAand2,4-Dwerenecessaryfor ;callusformationfrombalsumpearanthercultureand ;theirconcentrationmustbehigher.DONGYanrong19]

    ;thoughtthatNAAconcentrationcouldnotbehigher ;than0.05me/I-intheCucurbitaceaeplantanther ;culture,butourtestshownthat0.2mg/LNAA ;l6

    ;combinedBAandKTSnMSbasemediumhadhigher ;callusformationrate.

    ;4.2Basemedium

    ;MSbasemediumwasusedinmostCucurbitaceae ;plantantherculture.Thistestalsoshowedthatitwasa ;suitablemediumforcallusformationofbalsumpear ;antherculture.N6medium(addingBA1.5

    ;mg,L+NAA0.05mg/L+2,4D0.5mg/L)obtained

    ;highercallusinducementrateinthetest.Punja,etal[11.

    ;thoughtN6wasasuitablebasemediumforinducing ;embryoidofcucumber(CucumissativusL.)andhigher ;2,4-Dconcentrationwasaimportantfactor.Lower ;NAAconcentrationcouldinduceembryoidcallus,but ;therewasnoembryoidofcucumber.Whetherornot ;balsumpearantherculturecouldbeinducedembryoid, ;itneedfartherstudy.

    ;4.3Carbonsource

    ;MostCucurbitaceaeplantanthercultureused ;canesugarascarbOnsource.Someresearchersthought

    ;maltosewasmorepropitioustoCucurbitaceaeembryo ;inducementl.Canesugarwasbetterthanmaltose ;f0rcallusformationofbalsumpearantherculturein ;thetestandtherewasnoembryoinducement.Whether ;ornotmaltosewasgoodforembryoinducementfrom ;balsumpearantherculture.italsoneedfurtherstudy. ;4.4Organinducementfrombalsumpearcallus ;Theadventitiousrootswereinducedfrombalsum ;pearcallusonthemediaMS+KT1.0mg/L+2.4-D0.5 ;mg/L,MS+HCl00+KT1.0mg/L+2,4-D0.5mg,L ;Primarystudyonanthercultureofbalsumpearforcallusandorganformation

    ;andMS+ZT2.(NAA0.05+0.5KTinthetests.There ;wasnorootinducementonothermedia.Itshowedthat ;2.4Dwasaveryimportantfactorforrootinducement ;andthereisnoneedBAinthemedium.Therewasno ;adventitiousbudformationinalPGRcombinations.

    ;Someresearchersthoughtorganinducementfrom

    ;balsumpearcalluswasrelatedtothekindsofexplants. ;XUANPu.etalIsummedupthatthetendoforgan ;inducementratefrombalsumpearcalluswasepicotyl ;>stemtip>cotyledon>hypocotyls>leaf.Upto ;now,therewasnoanyreportaboutorganinducement ;fromhalsumpearantherculture.TAOJin.etal[15

    ;foundthattheadventitiOusbudinducementneeded ;lowerIAA/iPAs.Thereasonofdifficultorgan ;inducementfrombalsumpearmaybenotsuitable ;endogenesishormonelevel,suchashigherIAA ;contentandloweriPAs1161.Thecontinuineratimes

    ;alsoaffecttheendogenesishormonelevelandorgan ;inducement.ThefurtherstudyneedstoincreaseBA, ;KT,ZeatinconcentrationanddecreaseIAA,2,4D,

    ;NAAconcentrationintheorganinducementmedium. ;4.5Thechromosomenumbervariationinvitro ;culture

    ;Thevariationinvitroculturewascommonandthe ;variationcharacterswereabroad.NuanFeishi.fl[171

    ;foundthattherootchromosomeofgarlicregeneration ;plantshad100%variationrate.Novak118foundthat

    ;thepolyploidyrateincreasedandthediploidrate ;decreasedinthelongtimecalluscultureofgarlic. ;Therewasun--diploidandsub--diploidinducementin ;thegarlicculture.ZHANGSu-zhi[191a1sOfoundthat ;thevariationrateofgarlicroottipchromosomewas ;55.5%aftercontinueculturefor6times.Otherplant

    ;tissueculturealsoshowedthevariation.Therewere ;l4.7%haploid,57.5%diploid,5.1%triploid,22.9% ;tetraploid(doublediploid)and3.8%noneuploidin

    ;367regenerationplantsfromhotpepperantherculture ;l2o1

    ;.

    ;Antherof15genotypesofradish(Raph

    ;sativusL.)andrapemicrosporecultureshadbeen ;culturedinvitroandinducedtoundergo

    ;embryogenesisandplantformation.Thechromosome ;numbersofregeneratedradishandrapeplantswere ;complex.butmostofthemwerediploid15-61. ;Someresearchshowedthatthechromosome

    ;variationcamefromthetissueandorganculture.and ;frompreexistingvariationinexplants.The

    ;chromosomevariationincludedthenumber, ;frameworkandgenevariation,eta1.Inourtest,most ;chromosomenumbersofadventitiousrootsfrom ;balsumpearanthercalluswere2n=3x=33.Itbelonged ;chromosomenumbervariationandmayresultfrom ;nucleusfuse.Thereasonfornucleusfusemaybethat ;haploiddoubleintodiploid(2n=2x=22)firstly,then ;2n=2xandln=lxfusednucleusinto2n=3x=33.This ;alsoneedfurtherstudytoidentify.

    ;References:

    ;lWANGZuo,LIShuang-jie,YANGYongzong.

    ;PurificationofMCPandmeasurementofit’santi—virus

    ;effecttoCoxB3invitro.JournalofNanhuaUniversity: ;MedicalEdition,2002,30(1):13.(inChinese)

    ;2lWUGuo-xing,Z?

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