虎仗原生质体分离纯化及电融合初步研究

以野生虎杖根状茎芽为外植体诱导虎杖愈伤组织,选取生长状况良好的愈伤组织作为原生质体分离材料,通过L16(44)正交试验对虎杖原生质体分离条件进行优化.结果表明:1.4%纤维素酶R-10,0.1%果胶酶,17%甘露醇的酶液,酶解时间7h,虎杖原生质体分离效果最好.在80r/min振荡分离后虎杖原生质体得率为46.2%.以800r/min的离心速度纯化,纯化的原生质体得率为43.8%,其中原生质体的成活率为75%.通过对虎杖原生质体进行融合,原生质体的融合率为20.2%.

Centrifugation Purification and Protoplasts Electrofusion of Reynoutria japonica

Take the well growth callus of Reynoutria japonica as the material,adopting the method of orthogonal test and using the orthogonal table of L16(44) to research the four factors influencing the protoplast separation,which contain the cellulase density,the pectase density,the mannitol density,the enzymolysis time.The optimum condition was obtained when using 1.4% cellulase Onozuka R-10,0.1% pectinase and 17% mannitol enzyme fluid to enzymolysis the call of Reynoutria japonica by the 80 r/min cot vibration raise for 7 h,the maximum yield of protoplasts is 46.2%.After 800 r/min centrifugal purification,the yield of the protoplast after purified is 43.8% and the survival rate of the protoplast is 75%.Electrofusion technique was applied to fusing the protoplasts.The fusion rate is 20.2%.