棉花高效体细胞胚发生及同步控制培养体系研究

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1、棉花高效体细胞胚发生及同步控制培养体系研究*曹景林张献龙金双侠杨细燕朱华国付莉莉(华中农业大学作物遗传改良国家重点实验室，湖北武汉430070)摘要：棉花体细胞胚胎发生的频率低，且体细胞胚的发育存在着不同步性，给体细胞胚胎发生发育过程的生理学、生物化学和分子生物学的研究带来许多困难。本研究以陆地棉品种Coker201为材料，建立了一种简单有效的棉花体细胞胚高频率发生和同步发育的培养体系。经过多次继代获得的胚性愈伤首先在液体培养基中振荡培养2d使其分散，然后过30目筛去除大的颗粒，重新悬浮于同样的液体培养基中。悬浮培养14d后，过50目筛，将筛上的胚性愈伤重悬浮于新鲜的培养基中，-1-1并

2、用吸管吸取悬浮液，将其均匀地接种在表面垫有滤纸的同成分固体培养基（附加2.46µmolLIBA和0.70µmolLkinetin）上。培养21d后，用垫有滤纸的相同的固体培养基继代培养。利用这种培养体系，获得的体细胞胚数量分别是单纯悬浮培养和固体培养（不垫滤纸）的16.5倍和4倍。其中，球形胚、鱼雷形胚和子叶胚的同步发生率分别为70.2%、52.3%和73.0%。关键词：棉花；体细胞胚胎发生；同步控制；培养体系AnEfficientCultureSystemforSynchronizationControlofSomaticEmbryogenesisinCotton(Gossypiumh

3、irsutumL.)*CAOJing-Lin,ZHANGXian-Long,JINShuang-Xia,YANGXi-Yan,ZHUHua-Guo,FULi-Li(NationalKeyLaboratoryofCropGeneticImprovement,HuazhongAgriculturalUniversity,Wuhan430070,Hubei,China)Abstract:Lowefficiencyofsomaticembryogenesisandasynchronousembryodevelopmentbringsalotofdifficultiestothephysiolog

4、ical,biochemicalandmolecularbiologicalstudiesofembryogenesisprocessesincotton.Inthepresentstudy,asimpleandefficientmethodwasdevelopedtoimprovesomaticembryogenesisfrequencyandsynchronousdevelopmentofmasssomaticembryosfromculturedcellsofGossypiumhirsutumcv.Coker201.Theembryoniccalliobtainedaftersev

5、eralroundsofsubculturewerescatteredinliquidmediumbyshakingfor2dandthenresuspendedinthesameliquidmediumafterdiscardingthelargercallusaggregatesover30mesh-size-sieve.Thesuspensionsculturedfor14dwerefilteredthrough50mesh-size-sieveandthentheaggregatesoverthesievewereinoculatedontothesurfaceofWhatman

6、filterpaperplacedonthesolidmediumcontaining2.46µmol-1-1LIBAand0.70µmolLkinetintoculturefor21dfollowedbysubculturing.Thesomaticembryoamountobtainedbyusingnewsystemwas15.5-foldhigherthanthatbysuspensioncultureand3-foldhigherthanthatbysolidculture(withoutfilterpapers)atthesamecultureperiod,andinthed

7、ifferentstagesofsomaticembryodevelopment,70.2%forglobular,52.3%fortorpedo-shapedand73.0%forcotyledonaryembryoswereobtainedrespectivelyduringtheculture.Suspensionculturecombinedwithsolidculturewithfilterpaperwereconside