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1、棉花高效體細(xì)胞胚發(fā)生及同步控制培養(yǎng)體系研究*曹景林張獻(xiàn)龍金雙俠楊細(xì)燕朱華國(guó)付莉莉(華中農(nóng)業(yè)大學(xué)作物遺傳改良國(guó)家重點(diǎn)實(shí)驗(yàn)室，湖北武漢430070)摘要：棉花體細(xì)胞胚胎發(fā)生的頻率低，且體細(xì)胞胚的發(fā)育存在著不同步性，給體細(xì)胞胚胎發(fā)生發(fā)育過(guò)程的生理學(xué)、生物化學(xué)和分子生物學(xué)的研究帶來(lái)許多困難。本研究以陸地棉品種Coker201為材料，建立了一種簡(jiǎn)單有效的棉花體細(xì)胞胚高頻率發(fā)生和同步發(fā)育的培養(yǎng)體系。經(jīng)過(guò)多次繼代獲得的胚性愈傷首先在液體培養(yǎng)基中振蕩培養(yǎng)2d使其分散，然后過(guò)30目篩去除大的顆粒，重新懸浮于同樣的液體培養(yǎng)基中。懸浮培養(yǎng)14d后，過(guò)50目篩，將篩上的胚性愈傷重懸浮于新鮮的培養(yǎng)基中，-1-1并
2、用吸管吸取懸浮液，將其均勻地接種在表面墊有濾紙的同成分固體培養(yǎng)基（附加2.46μmolLIBA和0.70μmolLkinetin）上。培養(yǎng)21d后，用墊有濾紙的相同的固體培養(yǎng)基繼代培養(yǎng)。利用這種培養(yǎng)體系，獲得的體細(xì)胞胚數(shù)量分別是單純懸浮培養(yǎng)和固體培養(yǎng)（不墊濾紙）的16.5倍和4倍。其中，球形胚、魚(yú)雷形胚和子葉胚的同步發(fā)生率分別為70.2%、52.3%和73.0%。關(guān)鍵詞：棉花；體細(xì)胞胚胎發(fā)生；同步控制；培養(yǎng)體系A(chǔ)nEfficientCultureSystemforSynchronizationControlofSomaticEmbryogenesisinCotton(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
