体细胞杂交细胞融合课件.ppt

1、Chapter 3Protoplast Culture&Somatic HybridizationWenwu GuoApplicationFusion and cultureDevel.process&hybrid verificationDefinitionPart 2 Somatic hybridization（体细胞杂交）（体细胞杂交）1:Definition of SH SH,i.e.protoplast fusion,a process of PPs from two different parents were induced to fuse and then regenerate

2、 into plantlets,also named as SH(A+B)Several different expressions:体细胞杂交：体细胞杂交：Somatic hybridization 细胞融合：细胞融合：Cell fusion 原生质体融合：原生质体融合：Protoplast fusion 无性杂交：无性杂交：asexual hybridization 超性杂交：超性杂交：Parasexual hybridization 超性融合：超性融合：Parasexual fusion 细胞操作：细胞操作：Cell manipulation 细胞工程细胞工程:Cell engineer

3、ingComparison of SH with Sexual crossingItemsSomatic fusionSexual crossTime时间时间No season limitOnly bloom season(花期限制，母本花期花期限制，母本花期)Phylogeny亲缘关系亲缘关系Can circumvent graft/sexual incompatibility克服嫁接克服嫁接/有性不亲和性有性不亲和性Can notResults结果结果nuclear,cytoplasmic genomes recombinedPloidy level increaedDouble fert

4、ilization,no recombination and ploidy level unchangedPossible Fusion Results of Two Genetically Different Protoplasts=chloroplast=nucleusFusionheterokaryoncybridcybridhybridhybrid=mitochondriaTerminology-1l对称杂种对称杂种(symmetric hybrid)（双亲给杂种贡献的遗传物（双亲给杂种贡献的遗传物质均为质均为100%）l非对称杂种非对称杂种(asymmetric hybrid)（双亲

5、给杂种贡献的遗（双亲给杂种贡献的遗传物质不等（相对值）传物质不等（相对值）胞质杂种（胞质杂种（cytoplasmic hybrid,Cybrid）：）：细胞质重组，细胞核未重组细胞质重组，细胞核未重组l对称融合对称融合（symmetric fusion）也称标准融合（也称标准融合（Standard fusion）l非对称融合非对称融合（asymmetric fusion）：）：融合前对一方进行融合前对一方进行处理，使其染色体丢失一部分处理，使其染色体丢失一部分Terminology-2l供供-受体融合：受体融合：Donor-recipient fusionl体配融合体配融合（gameto-so

6、matic fusion）（）（性细胞与体细胞性细胞与体细胞融合）融合）l亚原生质体亚原生质体-原生质体融合：原生质体融合：Subprotoplast-protoplast fusion 小原生质体：小原生质体：Miniprotoplast 微原生质体：微原生质体：Microprotoplast 胞质体：胞质体：Cytoplastl胞质体胞质体-原生质体融合原生质体融合:Cytoplast-protoplast fusionTechnical developmental historyl1970s,establish and optimize of PP technique1972,tobac

7、co interspecific somatic hybrid1975,high Ca-high pH PEG fusion;electro-fusionNumerous reports,some were not realisticlEarly 1980s,model plants to staple/economic crops,symmetric to asymmetric,more realisticlLate 1980s,many reports on perennial woody plantsl1990s to date,a conventional&safe biotech f

8、or plant breeding in citrus,rapeseed,wheat etc.Potato+Tomato=Pomato?2:protoplast fusionFusion methodsFusion processFactors affecting fusion Fusion methodsPEG-mediated fusionElectro-fusionPolyethylene glycol（PEG，聚乙二醇）Electrofusion,Electrically induced fusion1.PEG-mediated fusion Characteristics:1)Mer

9、its include:low input,high frequency of hetero-karyons,no species limit;2)Defects include:tedious fusion procedure,toxicity to cells Working mechanism:PEG分子具有轻微的负极分子具有轻微的负极性，可与具有正极性基团的水、蛋白质和碳水化合物等性，可与具有正极性基团的水、蛋白质和碳水化合物等形成形成H键，从而在原生质体之间形成分子桥，从而使原键，从而在原生质体之间形成分子桥，从而使原生质体发生粘连进而促进原生质体融合；生质体发生粘连进而促进原生质体

10、融合；PEG能增加能增加类脂膜的流动性，也使原生质体的核、细胞器发生融合类脂膜的流动性，也使原生质体的核、细胞器发生融合成为可能成为可能Fusion sol:CaCl2 2H2O 8-10 mmol KH2PO4 0.7 mmol Mannitol 0.5-1.0 mol pH 5.6Inducing sol:Fusion sol+PEG 20-45Diluting sol:sol A(g/100 ml)pH 6.0 sol B(g/100 ml)pH 10.5 Glucose 7.21 Glycin 0.375 CaCl2 2H2O 0.79 NaOH 0.169Several stock

11、solutions to prepareP1P2P1 P2Mix,keep intact 1 minFusionFusion solAdd PEGDilutionWashAdd diluting solMix sols A&BAdd mediumCultureScreeningPEG induced fusion procedureNote:Mix solutions A&B(9:1)to make working solution2Electrofusion,Electrically induced fusion Three advantages compared with PEG fusi

12、onlNo toxicity to protoplastslHigher fusion frequencylSimpler manipulationElectro-fusion:A high frequency AC field is applied between 2 electrodes immersed in the suspension of protoplasts-this induces charges on the protoplasts and causes them to arrange themselves in lines between the electrodes t

13、o form pearl-chain.They are then subject to a high voltage discharge which causes their membranes to fuse where they are in contact.Electro-fusion instrument in NKL,HZAUThe process of electrically induced fusionFusion parametersAlternate current voltage(交流电压交流电压)Time duration of AC(交变电场处理时间交变电场处理时间)

14、Direct current voltage(直流高频电压直流高频电压)Pulse width(脉冲宽度脉冲宽度)No.of Pulses(脉冲次数脉冲次数)Fusion processMembrane fusion(Membrane fusion(膜融合膜融合)Cytoplasm fusion(Cytoplasm fusion(细胞质融合细胞质融合)Nucleus fusion(Nucleus fusion(核融合核融合)Factors affecting PP fusion lPP quality(intact,viability etc)lFusion method(PEG or electro-fusion)lFusion parameters(电融合参数、溶液浓度等)