刺槐愈伤组织原生质体的分离和 PEG 融合1）

摘要

With the immature zygotic embryos callus material of Roib nia pseudoacacia L., we studied the protoplast isolation and protoplast fusion with PEG .The main three factors were the enzyme concentration , dissociation time and osmotic pressure ( the enzyme concentration includes the concentrations of cellulase R-10, pectinase Y-23 and macerozyme R-10). By su-crose isodensity centrifugation , the best conditions to get amount protoplasts from the immature zygotic embryos callus ma -terial of Robinia pseudoacacia L.were 2.5%of cellulase R-10, 0.6%enzyme of pectinase Y-23, 1%of macerozyme R-10, 11%of mannitol, and 10 h of hydrolysis time.Under these conditions, the yield of protoplasts was 2.24×106/mL FW, and the activity was 92.13%.Thus, the actual yield of protoplasts was 2.06×106/mL FW.In terms of fusion research, with the method of PEG6000-induced, the integration time and the concentration of PEG 6000-related factors were discussed. The fusion rate was 10.97%with 25%PEG6000-induced and the time lasting of 30 min.%以刺槐未成熟合子胚的愈伤组织为材料，试验通过正交试验设计，对酶液质量分数（纤维素酶R－10、果胶酶Y－23、离析酶R－10）、分离时间和分离液渗透压（即甘露醇质量分数）3个影响因素分析，并采用蔗糖等密度离心法中的上浮法纯化后，得到分离最佳条件：纤维素酶R－10质量分数为2．5％，果胶酶Y－23质量分数0．6％和离析酶R－10质量分数1％，甘露醇质量分数11％，酶解时间10 h。在此条件下，原生质体产量达22．4×106个/mL，活力为92．13％，原生质体净得率为2．06×106个/mL。在此基础上，采用PEG诱导法，对原生质体的融合时间和融合剂PEG6000质量分数等相关影响因素进行了相关探讨，结果表明最佳融合条件为25％的PEG6000诱导下，融合30 min，融合率达到10．97％。