Studies On Protoplast Culture And Somatic Cell Fusion Techniques Of Diploid Potatos

The research studied ploidy on potato materials introduced from CIP and the effect of materials and pretreatment on coloration; protoplast culture basic condition and influencing factor and the effect of somatic fusion parameter on cell fusion frequency in diploid potato. The research results showed:(1) Obtaining materials was key component in potato ploidy identification. The cleavage cells in the root tip from tuber were more than root tip in vitro and chromosome were dispersive pretreated 5～8h with 0.1%colchicines, the coloration with carbolic acid rubin were more distinct than aceto-carmine. 60 potato materials introduced from CIP were identified, which include 10 diploid, 7 triploid and 43 tetraploid.(2) Osmotic concentration have important effect in protoplast isolation. The three genotype in the research have different osmotic concentration,â…¢-5 was 0.35～0.40, JK₃ was 0.35M,â…¡-10 was 0.30～0.45M; the enzyme combination greatly effected protoplast yield, the protoplast yield was highest in combination of 0.8%celluase and 0.4% pectinase and the isolation was more sufficiency; at the same time, the research compared the isolation result of the mesophyll cell and calli, it showed that viability have no difference, however, the yield of mesophyll cell was higher than calli; pretreatment before isolation improved the yield and viability of the protoplasts; the protoplast from potato mesophyll cell of different genotype have contrast in yield, viability and cell dimension; different homone combination greatly effected the differentiation of calli.(3) The fusion frequency was highest as the PEG6000 concentration was 30% in chemical fusion. In electrofusion aligment current and aligment time were key parameters to decide protoplast string and membrane contact. String frequency reached maximal under the aligment current 100v/cm, time 30s; when direct current was 1000v/cm, time was 100μs, one time , binuclear fusion frequency was maximal; cell fusion frequency of both chemical fusion and electrofusion have no difference, were above 35%, but in the time of the calli formation and differentiation, the cells from electrofusion were earlier than chemical fusion; fusion cells of different genotype combination have great contrast in cell culture. The fusion cells of the combination ofâ…¢-5+JK₃ cleavage fast, calli formed early and the plant regenerated, in the combination ofâ…¢-5+â…¡-10, only calli formed, only electrofusion was executed in the combination of JK₃+â…¡-10, but the cells doomed in early stage.