为获得高质量的原生质体,给光皮树转化系统的建立奠定基础,以光皮树花药愈伤组织为材料,研究了愈伤组织结构、酶液组合、酶解时间及渗透压对原生质体分离效果及活力的影响,从而确定适合光皮树花药愈伤组织原生质体最佳分离条件.结果表明:淡黄绿色、疏松的愈伤组织在CPW+2％纤维素酶 R-10+0.3％蜗牛酶+0.25％果胶酶Y-23+13％甘露醇+0.6/L MES的酶解液中,黑暗条件下振荡酶解10 h,原生质体产量达到3.19×106个/g,具活力的原生质体为73.48％.%Anther callus of Swida wilsoniana were used as test materials to study the influencing factors on protoplast isolation, such as concentration and combination of enzyme, enzymolysis time and mannitol concentration, and ulteriorly, to determine the optimal separation condition that is appropriate for isolating protoplast of anther callus. The results show that the loose structure callus with light yellow and green color was mixed with enzyme liquid of CFW+ 2% CellulaseR-10 + 0.3% Snailase +0.25% Pectinase +13%mannitol +0.67L MES, and was oscillated slowly for 10 hours in dark, resulting the highest yield of protoplast go up to 3.19× 106 个 /g with viability of 73.48%. The research results would be a basis of establishing in vitro culture system of gene transform.
展开▼
