體細胞雜交細胞融合課件

Chapter3

ProtoplastCulture&

SomaticHybridizationWenwuGuoChapter3

ProtoplastCulture&1ApplicationFusionandcultureDcess&hybridverificationDefinitionPart2

Somatichybridization（體細胞雜交）ApplicationFusionandcultureD21:DefinitionofSHSH,toplastfusion,aprocessofPPsfromtwodifferentparentswereinducedtofuseandthenregenerateintoplantlets,alsonamedasSH(A+B)Severaldifferentexpressions:體細胞雜交：Somatichybridization細胞融合：Cellfusion原生質(zhì)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:Cellengineering1:DefinitionofSHSH,i.e.pr3ComparisonofSHwithSexualcrossingComparisonofSHwithSexualc4PossibleFusionResultsofTwoGeneticallyDifferentProtoplasts=chloroplast=nucleusFusionheterokaryoncybridcybridhybridhybrid=mitochondriaPossibleFusionResultsofTwo5Terminology-1對稱雜種(symmetrichybrid)（雙親給雜種貢獻的遺傳物質(zhì)均為100%）非對稱雜種(asymmetrichybrid)（雙親給雜種貢獻的遺傳物質(zhì)不等（相對值）胞質(zhì)雜種（cytoplasmichybrid,Cybrid）：細胞質(zhì)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Terminology-1對稱雜種(symmetrich6Terminology-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(zhì)體-原生質(zhì)體融合：Subprotoplast-protoplastfusion

小原生質(zhì)體：Miniprotoplast微原生質(zhì)體：Microprotoplast胞質(zhì)體：Cytoplast胞質(zhì)體-原生質(zhì)體融合:Cytoplast-protoplastfusionTerminology-2供-受體融合：Donor-reci7Technicaldevelopmentalhistory1970s,establishandoptimizeofPPtechnique1972,tobaccointerspecificsomatichybrid1975,highCa-highpHPEGfusion;electro-fusionNumerousreports,somewerenotrealisticEarly1980s,modelplantstostaple/economiccrops,symmetrictoasymmetric,morerealisticLate1980s,manyreportsonperennialwoodyplants1990stodate,aconventional&safe‘biotech’forplantbreedingincitrus,rapeseed,wheatetc.Technicaldevelopmentalhistor8Potato+Tomato=

Pomato?Potato+Tomato=Pomato?92:protoplastfusionFusionmethodsFusionprocessFactorsaffectingfusion2:protoplastfusionFusionmet10FusionmethodsPEG-mediatedfusionElectro-fusionPolyethyleneglycol（PEG，聚乙二醇）Electrofusion,ElectricallyinducedfusionFusionmethodsPEG-mediatedfu111.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(zhì)和碳水化合物等形成H鍵，從而在原生質(zhì)體之間形成分子橋，從而使原生質(zhì)體發(fā)生粘連進而促進原生質(zhì)體融合；PEG能增加類脂膜的流動性，也使原生質(zhì)體的核、細胞器發(fā)生融合成為可能1.PEG-mediatedfusion

12Fusionsol:

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

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

CaCl22H2O0.79NaOH0.169SeveralstocksolutionstoprepareFusionsol:CaCl22H2O8-1013P1P2P1P2Mix,keepintact1minFusionFusionsolAddPEGDilutionWashAdddilutingsolMixsolsA&BAddmediumCultureScreeningPEGinducedfusionprocedureNote:MixsolutionsA&B(9:1)tomakeworkingsolutionP1P2P1P2Mix,keepintact1mi142．Electrofusion,ElectricallyinducedfusionThreeadvantagescomparedwithPEGfusionNotoxicitytoprotoplastsHigherfusionfrequencySimplermanipulationElectro-fusion:

AhighfrequencyACfieldisappliedbetween2electrodesimmersedinthesuspensionofprotoplasts-thisinduceschargesontheprotoplastsandcausesthemtoarrangethemselvesinlinesbetweentheelectrodestoformpearl-chain.Theyarethensubjecttoahighvoltagedischargewhichcausestheirmembranestofusewheretheyareincontact.2．Electrofusion,Electrically15Electro-fusioninstrumentinNKL,HZAUElectro-fusioninstrumentinN16TheprocessofelectricallyinducedfusionTheprocessofelectricallyin17FusionparametersAlternatecurrentvoltage(交流電壓)TimedurationofAC(交變電場處理時間)Directcurrentvoltage(直流高頻電壓)Pulsewidth(脈沖寬度)No.ofPulses(脈沖次數(shù))Fusionparameters18

FusionprocessMembranefusion(膜融合)Cytoplasmfusion(細胞質(zhì)融合)

Nucleusfusion(核融合)

FusionprocessMembranefusio19FactorsaffectingPPfusion

PPquality(intact,viabilityetc)Fusionmethod(PEGorelectro-fusion)Fusionparameters(電融合參數(shù)、溶液濃度等)FactorsaffectingPPfusion

20Chapter3

ProtoplastCulture&

SomaticHybridizationWenwuGuoChapter3

ProtoplastCulture&21ApplicationFusionandcultureDcess&hybridverificationDefinitionPart2

Somatichybridization（體細胞雜交）ApplicationFusionandcultureD221:DefinitionofSHSH,toplastfusion,aprocessofPPsfromtwodifferentparentswereinducedtofuseandthenregenerateintoplantlets,alsonamedasSH(A+B)Severaldifferentexpressions:體細胞雜交：Somatichybridization細胞融合：Cellfusion原生質(zhì)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:Cellengineering1:DefinitionofSHSH,i.e.pr23ComparisonofSHwithSexualcrossingComparisonofSHwithSexualc24PossibleFusionResultsofTwoGeneticallyDifferentProtoplasts=chloroplast=nucleusFusionheterokaryoncybridcybridhybridhybrid=mitochondriaPossibleFusionResultsofTwo25Terminology-1對稱雜種(symmetrichybrid)（雙親給雜種貢獻的遺傳物質(zhì)均為100%）非對稱雜種(asymmetrichybrid)（雙親給雜種貢獻的遺傳物質(zhì)不等（相對值）胞質(zhì)雜種（cytoplasmichybrid,Cybrid）：細胞質(zhì)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Terminology-1對稱雜種(symmetrich26Terminology-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(zhì)體-原生質(zhì)體融合：Subprotoplast-protoplastfusion

小原生質(zhì)體：Miniprotoplast微原生質(zhì)體：Microprotoplast胞質(zhì)體：Cytoplast胞質(zhì)體-原生質(zhì)體融合:Cytoplast-protoplastfusionTerminology-2供-受體融合：Donor-reci27Technicaldevelopmentalhistory1970s,establishandoptimizeofPPtechnique1972,tobaccointerspecificsomatichybrid1975,highCa-highpHPEGfusion;electro-fusionNumerousreports,somewerenotrealisticEarly1980s,modelplantstostaple/economiccrops,symmetrictoasymmetric,morerealisticLate1980s,manyreportsonperennialwoodyplants1990stodate,aconventional&safe‘biotech’forplantbreedingincitrus,rapeseed,wheatetc.Technicaldevelopmentalhistor28Potato+Tomato=

Pomato?Potato+Tomato=Pomato?292:protoplastfusionFusionmethodsFusionprocessFactorsaffectingfusion2:protoplastfusionFusionmet30FusionmethodsPEG-mediatedfusionElectro-fusionPolyethyleneglycol（PEG，聚乙二醇）Electrofusion,ElectricallyinducedfusionFusionmethodsPEG-mediatedfu311.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(zhì)和碳水化合物等形成H鍵，從而在原生質(zhì)體之間形成分子橋，從而使原生質(zhì)體發(fā)生粘連進而促進原生質(zhì)體融合；PEG能增加類脂膜的流動性，也使原生質(zhì)體的核、細胞器發(fā)生融合成為可能1.PEG-mediatedfusion

32Fusionsol:

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

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

CaCl22H2O0.79NaOH0.169SeveralstocksolutionstoprepareFusionsol:CaCl22H2O8-1033P1P2P1P2Mix,keepintact1minFusionFusionsolAddPEGDilutionWashAdddilutingsolMixsolsA&BAddmediumCultureScreeningPEGinducedfusionprocedureNote:MixsolutionsA&B(9:1)tomakeworkingsolutionP1P2P1P2Mix,keepintact1mi342．Electrofusion,ElectricallyinducedfusionThreeadvantagescomparedwithPEGfusionNotoxicitytoprotoplastsHigherfusionfrequencySimplermanipulationElectro-fusion:

AhighfrequencyACfieldisappliedbetween2electrodesimmersedin