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Callus

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Callus

    Callus

    For.Stud.China,2006,8f4):2529

    D0I10.1007/sl1632006.0032.5

    Callusinductionandplantregenerationfrommaturebluegrass

    (PoapratensisL.)seeds

    SunBi-poZhangWan-junDongJiangliJinYongshengWangTao

    StateKeyLaboratoryofAgro-biotechnology,ChinaAgriculturalUniversity,Beijing100094.P1LChina

    AbstractAprotocolwasdiscussedforhigl1e

    cientplantregenerationfromsevenbluegrassfPoapratensisL.)cuhivarsviaanin. directcalinsinductionandsomaticembryogenesismethod.Matureseedswereusedasexplantsforcallusinitiation.Callusinduction

    andproliferatione

    ciencieswereinvestigatedonNB.modifiedMS(MMS)andMSmedia.supplementedwith2.0mg-L-

    2,4dichlorophenoxyaceticacid(2,4

    D).TheMMSmediumperformedbest.BasedontheMMSmediurn,directandindirectcallus inductioneriectsofbluegrassfrommatureseedswerecomparedattherangeof1-5mg?L2,4.Dcontainedinthemediun1.

    Under

    thedirectcallusinductionmethod,themostsuitable2,4-Dconcentrationsvariedamongcultivars.Undertheindirectcallusinduction

    method,asignificantlyhighcallusinductionfrequency(93.33%-98.33%)wasobtainedandtherewerebarelyanystatisticallysig-

    nificantdifierencesamongthetestedgeneticallydiversecultivars.SomaticembryoswerepromotedontheMMSmediunlsupple

    mentedwith3mg-L-2,4

D,0.1mg?L-kinetinand0.8mg?L-CuSO4.Embryogeneticcallidevelopedintoplantletsont

    heMMS

    mediumcontainingdifierentconcentrationsofthidiazuronfTDZ),andthedifierentiationfre

    quenciesvariedintherangefrom

    20.15%to77.65%.The0.25mg?L-TDZwasgenerallythemostsuitableconcentrationforthe

    testedcultivars.

    Keywordsmatureseeds,bluegrass(Poapratens~L.),callus,tissuesculture,regeneration

    1Introduction

    Bluegrass(PoapratensisL.1isatypicalcoo1.season perennialgrass.Todevelophigh-qualitygrasses,im- portantaitssuchasyieldandtolerancetosalt.herbi cides,pests,stressandshadetoleranceshouldbeim. proved.However,conventionalbreedingtechniques aredifficultinbluegrassowingtothepredominance ofrhizomatousfSharman,1947;Etter,l951)and apomicticreproduction(BashawandFunk,1987)in thisspecies.Therefore,methodssuchasgenetic transformation,somatichybridizationandselectionof somaclonalvariantsappeartobemorepromising(Ha eta1.,2001).Toexploretheuseofthesemethods,an efficientinvitroculturesystemforbluegrassregen. erationisaprerequisite.Anumberofreportshave describedsystemsforcalluscultureandplantregen. erationinbluegrassusingdifferentexplantmaterials, suchasmatureandimmaturezygoticembryos

    fMcDormellandConger,1984;BoydandDale,1986), immatureinflorescences(vanderV_alketa1..1989), shoottipsfandJampates,l986),wholemature seeds(Krans,1981:vanderValketa1.,1989,1995; vanArketa1.,1991:GriffinandDibble,1995;Hodges

    eta1..19991andcoleoptile.1eaf,stemsectionsof seedlings(Keeta1..1996)andmatureseeds(Grin

    andDibble,1995).Inthesetissueculturesystems,the uthorforCo~espondenceE-mail:wangt@caueduCtl frequencyofplantregenerationsappearedtobethe highestfromimmatureinflorescencercvFylking) derivedcallus,at79%(vanderVa?【eta1..1989).Un.

    fortunately,inflorescencescouldbeusedasexplant onlyattheprecisestageofayear,which1imitsitsuse inthepractice.Matureseedsareeasytostoreand ada,ptedtoyear-roundresearch.So,inordertoim- provebluegrassquality,itisnecessarytoestablisha highfrequencyplantregenerationsystemandtoform matureseed.derivedcallus.

    Thisworkwastargetedtoestablishahighfre. quencyplantregenerationsystemofbluegrassfrom matureseedsderivedcallus.includingtheselectionof asuitablemedium,optimizationofhormoneconcen- trationsforcallusinductionandplantletregeneration andtoshortentheperiodofplantletregeneration. 2Materialsandmethods

    2.1PIantmaterialsandcultureconditions Matureseedsofsevenbluegrasscultivars,Midnight, Bartitia,Naglade,Merit,Freedom,NassauandNublue wereusedasexplants.Fromthestart,theseedswere placedonthreelayersofwetfilterpaperin9cmdi

    ameterPetridishes,incubatedforsevendaysat7~C

26

    forvernalization.Thentheseedswerestir-

    face.steriliZedwith70%(v/v1ethanolforlmin.5% (w/v)sodiumhypochloritecontainingtwodropsof Tween.100perl00mLfor20minandrinsedsix timesinsteriledistilledwaterandplacedonmediafor callusformation.AUofthemediaweresolidifledwith 0.8%agarandadjustedtopH5.8beforeautoclaving at121.Cfor20min.AUtheplantmaterialswerecu1. turedat254-2.Cunderal4hour?day-'photoperiodof 35lamol?Il_z.S.unlessstatedotherwise.

    2.2Experimentl:mediaselection

    NB(Lieta1.,l993),modifledMS(MMS)(Zhangand Wlan2003)andMS(MurashigeandSk0o1962) mediasupplementedwith0.05mg?【『'kinetin(Kin)

    and2.0mg?L'2,4-dichlorophenoxyaceticacid(2,4.D) wereusedforcallusinductionfrommatureBartitia. Midnight.NagladeandMeritseeds.Observations weremadeeverytwodaysduringthecourseofcallus inductionandthestateofcalluswasrecorded.The callusinductionratioswerescoredfourweekslater Theweightsofthecallusweremeasuredaftereight weeks.

    2.3Experiment2:callusinduction

    Directcallusinduction.Maturebluegrassseedswere usedasexplants.Sterilizedseedswereplaceddirectly ontheMMSmedium.supplementedwithfivelevels of2,4.D(1.0,2.0,3.0,4.0and5.0mg?L-')anda combinationof0.05mg?【『'Kinforcallusinduction.

    Threeweekslate~thecallusinductionratioswere recorded.

    Indirectcallusinduction.Germinatedseedswere

    usedasexplants,whichweregerminatedonasolid MSmedium.Thenthegerminatedseedswereplaced ontheMMSmediunlsupplementedwith0.05mg?【『

    Kinandfivelevelsof2,4.D(1.0,2.0,3.0,4.0and5.0 mg?L-1toinducecallus.Threeweekslater,thecallus inductionfrequencywasrecorded.

    Subsequently,theMMSmediumsupplemented with2.0mg?L'2,4.D.0.1mg?【『Kin.and0.8

    mg?L-CuSO4(Haeta1.,2001)wasusedforembryo. geniccalluspromotion.Thecalliweresubcultured everytwoweeks.

    2.4Experiment3:conversionofembryogenic callustoplantlets

    Thefriable,compactandwhitetoyellowishembryo- geniecalliweresubculturedortransferredtoadiffer- entiationmediumforplantletdifferentiation.These mediaconsistedoftheMMSmediumsupplemented withfivelevelsofthidiazuron(TDZ)(O.062,5,0.125, ForestryStudiesinChina,Vo1.8,No.4'2006 0.25,0.5and1.0mg?【『),0.0lmg?【『2,4.Dand0.8

    mg?【『'CuSOd.Theembryogeniccalliwereincubated foroneweekindarkness.thenforthreeweeksina growthchamberwithal6hours/8hourslight/dark photoperiodOfl00lamol?m-Z.ssuppliedfromcool whitefluorescentbulbsandday/nighttemperaturesof 26/24.C.Smallvisiblegreenglobalembryosorgreen budsformedonthesurfaceoftheembryogeniccallus. Whentheshootsgrewto2-4cm,theyweretrans. ferredtopotscontainingahaIf-strengthMSmedium supplementedwith0.2mg?L-'NAAtopromoteroot

    development.Atierawel1.developedrootssystem formed,theregeneratedplantsweretransferredto12 cmflowerpotscontainingamixtureof1:1sphagnum peatmossandvermiculiteandmaintainedinagreen houseundersunnyconditionsof1733.C.

    2.5Datacollectionandstatisticalanalysis AIltheexperimentswerearrangedinrandomizedde. signs.Theexplantsofdifferentcultivarswereplated andculturedon9cmPetridishes.eachdishcontain. iI1g30explants.Eachexperimentwasreplicatedfour times.Thecallus.inductionratiowasdefinedasthe numberofcalliformedperseeddividedbythenuln- berofviableseeds.Aviableseedwasdefinedasa seedthatgerminatedorproducedcalliontheca1. 1us.inductionmediunl(Wangeta1..2002).Thefi'e. quencyofcallusdifferentiationwasdefinedasthe percentageofcallusclumpsthatproducedplantlets dividedbythetotalnumberofembryogeniccallifor plantconversion.

    Thedata,expressedaspercentages,weresubjected toanarcsinetransformationpriortostatisticalanalysis Differencesamongtreatmentmeanswereanalyzedby LSDtestwithSPSSsoftware(versionl0.0.SPSS Inc.)

    3Resultsanddiscussion

    Genotypedifferenceisabottle.neckprobleminmany gramineousplanttissuecultures.Largely,MSandNB mediaareusedingramineousplanttissueculturesto overcomegeneticdifferences(HoqueandMansfield, 2004).Toselectthepropermedium,NB,MMSand

    MSmediasupplementedwith0.05mg?L-'Kinand2 mg?L'2,4.Dwereusedforcallusinductionfrom

    bluegrassgeneticdifferentcultivars(Merit,Bartitia, MidnightandNuglode).Theresultsshowedthat,on MMSmediathetestedfourcultivarsconsistentlyob. tainedrelativelyhighercallus.inductionfrequencies thanonMSandNBmedia(Tablel1.Accordingtothe averagecallusweightandcallusstate,theMMSme. diUlYlwasmoresuitableforcallusproliferationofPre. neticallydiversebluegrasscultivars.Amongthefour testedcultivars,threeofthemobtainedthehighest

SunBi

    poeta1.:Callusinductionandplantregenerationfrommaturebluegrass(Poapratens~L.)see

    ds

    Table1EFectofdiFerentmediaoncallusinduction(unit:% Table2Comparisonofcallusproliferationculturedonthree differentmedia(unit:mg)

    Themeanswithineachcolumnfollowedbydifferentletters aresignificantlydifferentaccordingtotheLSDtest.05). Thesameasinfollowingtables.

    callusweightonMMSmediumaftereightweeks.The callusproliferationratesvariedamongdifferentculti

    vars.Meritperformedthebest.Itscallusgrewmore quicklYthantheothercultivars(Table2,.

    BasedontheMMSmedium,thecallusinduction

    responsesofsevenbluegrasscultivarswereinvesti

    gatedunderdifferentconcentrationsof2,4.D.Seeds wereplacedontheMMSmediumsupplementedwith

0.05nag?L-Kinatdifierentconcentrationsr15

    mg?L-1of2,4.D,directlyforcallusinduction,orafter theseedsgerminatedontheMSmedium,wereputon thesameMMSmediumcontainingdifferent2.4D

    andKincombinationsforcallusformation(indirect callusinductionfromseeds).Theresultsindicatedthat, underdirectcallusinductionexperiments,givendif- ferentcultivars.themostsuitable2.4Dconcentrations

    forcallusinductionvariedandthecallusinduction 27

    ratiosvariedaswel1.Naglodeshowedthehighestca1. 1usinductionfrequency(86.96%1with2mg?L-2,4.D. followedbyMerit(80.06%1th3mg?L-2,4D,

    Nassue(79.23%1with1nag?L-2.4DandBartitia

    (78.84%1wim3mg?L-2,4D.Thelowestca1.

    1usinductionfrequencywas70.66%fromFreedom with1mg-L-2,4.D(Table31.Clearly,allthetested cultivarsachievedrelativelyhighcallusinductionra

    tiosunder2.4Dconcentrationsof1-3mg?L-.Incon- trast,underindirectcallusinductionexperiments,all thetestedgeneticallydiversecultivarsachievedhigh callusinductionratios(93.33%98.33%1.Therewere

    nostatisticallysignificantdifferences(Table4,Fig. 1A1.Consideringthegoodcallusstate.3mg-L- 2.4Dwasselectedastheoptimalcallusinduction medium.

    Anextremelylongregenerationperiodisanother troublesomeproblemingramineousplanttissuecul

    ture.Animportantreasonisthatcallusgrowsslowly onsubculture.Inthisexperimentthecalluscultured

    onMMSmediumproliferatedquicklV_anditssize increasednearlyfourtimesinonemonth.Thecallus tumedbrightyellowish.Thedifferencesbetweenthe MMSandMSmediareflecttheirorganiccomponents: theMMSmediumusesvitaminBf9.9nag?L-thia

    mine,4.5mg?L-nicotinicacid,9.5mg?L-pyridoxine) and2.0g?Lcaseinenzymatichydrolysate(Sigma)to substitutetheorganiccomponentsofMS.Obviously, VitaminB.whichcombinescaseinenzymatichydro. 1ysateasorganicsupplements,issuitableforbluegrass callusinduction,proliferationandembryogenesis. Furthermore,thedecreaseddifferentiationabilityof callus,withprolongedsubculturetime,canalsobe avoided.Aftertwoorthreesubculturesontheem- bryogenicpromotionmedium(MMSmediumsup.

    plementedwith2mg?L-2,4D.0.1mg?L-Kin.and

    0.8mg?L-CuSO41,thecalligrewto0.4-_I).6cmin diameter,mostofthemappearingwithembryogenic Table3Effectofdifferent2.4-Dconcentrationsondirectcallusinduction(unit:%

    Table4Effectofdifferent2.4Dconcentrationsonindirectcallusinduction(unit:%

28

    Table5EffectofdifferentTDZconcentrationsoncallusregeneration(unit:%1

    ForestryStudiesinChina,Vo1.8,No.4,2006

    characteristics:friable.compact.whitetopaleyellow (Fig.1B,.Thentheembryogeniccalliweretransferred t0theMMSmediumcontainingdifferentconcentra

    tions0fTDZf0rplantletsconversion.0nthedifferen. tiationmedium,thebrightyellowish,compactem

    bryogeniccalluscontinuedincreasinginsizeand

    manyvisibleembryostructuresemergedafterabout threeweeks.Subsequently,afteranintervalofseveral daysthegreenglobularstructuresgerminatedand shootsformed(FiR.1C,.ThemostsuitableTDZcon

    centrationsfordifie:rentcalluscultivardif.ferentiation variedonlyalittle.Meritachievedthegreatestcallus direntiationratio(77.65%,ofthetestedcultivars with0.5mg?LTDZfollowedbyBartitiaf71.43%) with0.125mR?LTDZ.Freedomf5o.I2%1with0.5 mR?LTDZ.Midnight(46.23%1with0.25mg?L

    TDZandNaglode(20.15%,with0.25mg?LTDZ

    (Table51.UnderthetestedTDZconcentrationsall testedcultivarsobtainedarelativelyhighdifferentia

    tionfrequencyattheTDZlevel0.25mR?L.Hence. weselected0.25mR?LTDZastheoptimalconcen

    trationforcallusdifferentiation.

    Whentheshootswereabout2_4cmlongwithor

    withoutroots,theywereplacedinahalf-strengthMS mediumtotriggerrootproduction(Fig.1D).Two weekslater.plantletswithextensiverootsystemswere transferredtoflatsandmaintainedinagreenhouse undersunlightat173.CrFig.1E).Alloftheplant

    letswithextensiverootsystemssurvivedinflatsone week1ater

    Callusinductionanddifferentiationaretwocritical stagesinbluegrassplantletsregeneration.Theresults indicatedthatahighcallusinductionfrequencydid

    notimplyahighdifferentiationratio.Thedirectcallus inductionfrequencyandthecallus.differentiationfre. quencyvariedsignificantlyamongthecultivarsand

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