體細胞雜交細胞融合第1頁/共21頁Chapter3

ProtoplastCulture&

SomaticHybridization第2頁/共21頁ApplicationFusionandcultureDcess&hybridverificationDefinitionPart2

Somatichybridization（體細胞雜交）第3頁/共21頁1:DefinitionofSHSH,toplastfusion,aprocessofPPsfromtwodifferentparentswereinducedtofuseandthenregenerateintoplantlets,alsonamedasSH(A+B)Severaldifferentexpressions:體細胞雜交：Somatichybridization細胞融合：Cellfusion原生質體融合：Protoplastfusion無性雜交：asexualhybridization超性雜交：Parasexualhybridization超性融合：Parasexualfusion

細胞操作：Cellmanipulation細胞工程:Cellengineering第4頁/共21頁ComparisonofSHwithSexualcrossingItemsSomaticfusionSexualcrossTime時間NoseasonlimitOnlybloomseason(花期限制，母本花期)Phylogeny親緣關系Cancircumventgraft/sexualincompatibility克服嫁接/有性不親和性CannotResults結果nuclear,cytoplasmicgenomesrecombinedPloidylevelincreaedDoublefertilization,norecombinationandploidylevelunchanged第5頁/共21頁PossibleFusionResultsofTwoGeneticallyDifferentProtoplasts=chloroplast=nucleusFusionheterokaryoncybridcybridhybridhybrid=mitochondria第6頁/共21頁Terminology-1對稱雜種(symmetrichybrid)（雙親給雜種貢獻的遺傳物質均為100%）非對稱雜種(asymmetrichybrid)（雙親給雜種貢獻的遺傳物質不等（相對值）胞質雜種（cytoplasmichybrid,Cybrid）：細胞質重組，細胞核未重組對稱融合（symmetricfusion）也稱標準融合（Standardfusion）非對稱融合（asymmetric

fusion）：融合前對一方進行處理，使其染色體丟失一部分第7頁/共21頁Terminology-2供-受體融合：Donor-recipientfusion體配融合（gameto-somaticfusion）（性細胞與體細胞融合）亞原生質體-原生質體融合：Subprotoplast-protoplastfusion

小原生質體：Miniprotoplast微原生質體：Microprotoplast胞質體：Cytoplast胞質體-原生質體融合:Cytoplast-protoplastfusion第8頁/共21頁Technicaldevelopmentalhistory1970s,establishandoptimizeofPPtechnique1972,tobaccointerspecificsomatichybrid1975,highCa-highpHPEGfusion;electro-fusionNumerousreports,somewerenotrealisticEarly1980s,modelplantstostaple/economiccrops,symmetrictoasymmetric,morerealisticLate1980s,manyreportsonperennialwoodyplants1990stodate,aconventional&safe‘biotech’forplantbreedingincitrus,rapeseed,wheatetc.第9頁/共21頁Potato+Tomato=

Pomato?第10頁/共21頁2:protoplastfusionFusionmethodsFusionprocessFactorsaffectingfusion第11頁/共21頁

FusionmethodsPEG-mediatedfusionElectro-fusionPolyethyleneglycol（PEG，聚乙二醇）Electrofusion,Electricallyinducedfusion第12頁/共21頁1.PEG-mediatedfusion

Characteristics:1)Meritsinclude:lowinput,highfrequencyofhetero-karyons,nospecieslimit;2)Defectsinclude:tediousfusionprocedure,toxicitytocells

Workingmechanism:

PEG分子具有輕微的負極性，可與具有正極性基團的水、蛋白質和碳水化合物等形成H鍵，從而在原生質體之間形成分子橋，從而使原生質體發生粘連進而促進原生質體融合；PEG能增加類脂膜的流動性，也使原生質體的核、細胞器發生融合成為可能第13頁/共21頁Fusionsol:

CaCl22H2O8-10mmolKH2PO40.7mmolMannitol0.5-1.0molpH5.6Inducingsol:Fusionsol+

PEG20-45％Dilutingsol:solA(g/100ml)pH6.0solB(g/100ml)pH10.5Glucose7.21Glycin0.375

CaCl22H2O0.79NaOH0.169Severalstocksolutionstoprepare第14頁/共21頁P1P2P1P2Mix,keepintact1minFusionFusionsolAddPEGDilutionWashAdddilutingsolMixsolsA&BAddmediumCultureScreeningPEGinducedfusionprocedureNote:MixsolutionsA&B(9:1)tomakeworkingsolution第15頁/共21頁2．Electrofusion,Electricallyinducedfusion

ThreeadvantagescomparedwithPEGfusionNotoxicitytoprotoplastsHigherfusionfrequencySimplermanipulationElectro-fusion:

AhighfrequencyACfieldisappliedbetween2electrodesimmersedinthesuspensionofprotoplasts-thisinduceschargesontheprotoplastsandcausesthemtoarrangethemselvesinlinesbetweentheelectrodestoformpearl-chain.Theyarethensubjecttoahighvoltagedischargewhichcausestheirmembranestofusewheretheyareincontact.第16頁/共21頁Electro-fusioninstrumentinNKL,HZAU第17頁/共21頁Theprocessofelectricallyinducedfusion第18頁/共21頁FusionparametersAlternatecurrentvoltage(交流電壓)TimedurationofAC(交變電場處理時間)D