Studies On The Techniques Of Improving The Frequency Of Two Cell Fusion In Potato Somatic Electrofusion

Somatic electrofusion techniques are of great importance in the potato biotechnology breeding theoretically and practically. Electric-field induced protoplasts fusion as an newly developed method. has recently showed bright practical prospect ,but the low frequency of two cell fusion is the main problem in the practice of the potato somatic electrofusion because the two haploid cell fusion can keep the same chromosome number of a species. In order to obtain the high two cell fusion frequency in mass somatic fusion experiments, the optimum electric paramete and other effective factors in potato electrofusion were studied .The mesophyll cells from an anther derived dihaploid strain 4-3 and the hypocotyls from true potato seeds of S.tuberosum L. dihaploid strain 1 -.15were used as the donor tissues .The results showed that the high vigor ,stable membrane protoplasts could be obtained when the materials cultured under sunlight and 25C combined with accelerating shalker rotation speed from 40r/min to 50-55r/min ahead of protoplasts purification 30 mm. Homologous electrofusion experiments reveled that at the intermediate protoplasts densities(5 X I 04/ml),using low AC field( 1-15. 200Vpp/cm.4-3 23OVpp/cm)and moderate duration(40s),the desirable ell chain with two protoplasts of the two materials could reach about 36%.lt was found that adjustment of Vpp intensity during cell alignment could increase desirable ell chain frequency from about 36% up to 45%,after treatment with 0.8kv/cm DC square pulse for 150 ,42% optimal frequency of two cell fusion were obtained. The results of experiments on the heterofusion proved that ,at low densities(5>( I 04/ml),by adjusting the ratio of two materials protoplasts which exists inherent difference in fusability could get higher heterofusion frequency . In the present experiments, at the ratio of 1:2( 1-15 hypocotyl protoplasts: 4-3 mesophyll protoplasts), the binary heterofusion frequency reached 26%. It has been noted that with adjusting AC intensity could obviously increase binary heterofusion frequecies during the course of cell alignment in both homologous and heterologous electrofusion experiments. At the present studies, under the optimum conditions, the frequecies of binary heterofusion reached 32% which increased 7% at the average nevel compared with unadjusted conditions. In addition the studies also indicated that it is very important to add fresh , unchanged osmolarity medium timely during culturing fusion products, it is unadvisable to reduce medium osmolarity from protoplasts division stage to callus differentiation stage.