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1、單擊此處編輯母版標題樣式,單擊此處編輯母版文本樣式,第二級,第三級,第四級,第五級,*,Chapter 3,Protoplast Culture &,Somatic Hybridization,Wenwu Guo,Application,Fusion and culture,Devel. process & hybrid verification,Definition,Part 2,Somatic,hybridization,（體細胞雜交）,1: Definition of SH,SH, i.e. protoplast fusion, a process of PPs from two dif

2、ferent parents were induced to fuse and then regenerate into plantlets, also named as SH (A,+,B),Several different expressions:,體細胞雜交：,Somatic hybridization,細胞融合：,Cell fusion,原生質體融合：,Protoplast fusion,無性雜交：,asexual hybridization,超性雜交：,Parasexual,hybridization,超性融合：,Parasexual,fusion,細胞操作：,Cell manip

3、ulation,細胞工程,: Cell engineering,Comparison of SH with Sexual crossing,Items,Somatic fusion,Sexual cross,Time,時間,No season limit,Only bloom season,(,花期限制，母本花期,),Phylogeny,親緣關系,Can circumvent graft/sexual incompatibility,克服嫁接,/,有性不親和性,Can not,Results,結果,nuclear, cytoplasmic genomes recombined,Ploidy l

4、evel increaed,Double fertilization, no recombination and,ploidy,level unchanged,Possible Fusion Results of Two Genetically Different Protoplasts,=,chloroplast,=,nucleus,Fusion,heterokaryon,cybrid,cybrid,hybrid,hybrid,=,mitochondria,Terminology,-,1,對稱雜種,(symmetric hybrid),（雙親給雜種貢獻的遺傳物質均為100%）,非對稱雜種,(

5、asymmetric hybrid),（雙親給雜種貢獻的遺傳物質不等（相對值）,胞質雜種（,cytoplasmic,hybrid,Cybrid,）：,細胞質重組，細胞核未重組,對稱融合,（,symmetric fusion）,也稱標準融合（,Standard fusion）,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,-,2,供-受體融合：,Donor-recipient fusion,體配融合,（,gameto,-somatic fusion）（,性細胞與體細胞融合）,亞原生質體-原生質體融合：,Subproto

6、plast,-protoplast fusion,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-protoplast fusion,Technical developmental history,1970s, establish and optimize of PP technique,1972, tobacco,interspecific,somatic hybrid,1975, high Ca-high pH PEG fusion; electro-fusion,Numer

7、ous reports, some were not realistic,Early 1980s, model plants to staple/economic crops, symmetric to asymmetric, more realistic,Late 1980s, many reports on perennial woody plants,1990s to date, a conventional & safe biotech for plant breeding in citrus, rapeseed, wheat etc.,Potato + Tomato =,Pomato

8、 ?,2: protoplast fusion,Fusion methods,Fusion process,Factors affecting fusion,Fusion methods,PEG-mediated fusion,Electro-fusion,Polyethylene glycol （PEG,，聚乙二醇）,Electrofusion, Electrically induced fusion,1.,PEG-mediated fusion,Characteristics:,1) Merits include: low input, high frequency of hetero-k

9、aryons, no species limit; 2) Defects include: tedious fusion procedure, toxicity to cells,Working mechanism:,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(fā)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(fā)生融合成為可能,Fusion sol:,CaCl2 2H2O 8-10 mmol,KH2PO4 0.7 mmol,Mannitol,0.5,-1.0 mol,pH

10、 5.6,Inducing sol:,Fusion sol +,PEG 20-45,Diluting 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 0.169,Several stock solutions to prepare,P1,P2,P1 P2,Mix, keep intact,1,min,Fusion,Fusion sol,Add PEG,Dilution,Wash,Add diluting sol,Mix sols A & B

11、,Add medium,Culture,Screening,PEG induced fusion procedure,Note: Mix solutions A & B (9:1) to make working solution,2,Electrofusion, Electrically induced fusion,Three advantages compared with PEG fusion,No toxicity to protoplasts,Higher fusion frequency,Simpler manipulation,Electro-fusion,:,A high f

12、requency 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 to form pearl-chain. They are then subject to a high voltage discharge which causes their membran

13、es to fuse where they are in contact.,Electro-fusion instrument in NKL, HZAU,The process of electrically induced fusion,Fusion parameters,Alternate current voltage (,交流電壓,),Time duration of AC (,交變電場處理時間,),Direct current voltage (,直流高頻電壓,),Pulse width (,脈沖寬度,),No. of Pulses (,脈沖次數(shù),),Fusion process,Membrane fusion (,膜融合,),Cytoplasm fusion (,細胞質融合,),Nucleus fusion (,核融合,),Factors affecting PP fusion,PP quality (intact, viability etc),Fusion method (PEG or electro-fusion),Fusion parameters,(,電融合參數(shù)、溶液濃度等,),