甘蔗愈伤组织原生质体酶法分离研究

METHOD OF PROTOPLAST ISOLATION FROM SUGARCANE CALLUS BY ENZYMOLYSIS

本研究以甘蔗愈伤组织为主要材料,进行了解离酶类、酶浓度、酶解条件及前处理研究;参试甘蔗栽培品系和野生资源比较研究;悬浮培养细胞解离研究等.当纤维素酶占酶类比值0.727～0.744时,可以游离出较多原生质体.2%纤维素酶+0.7%果胶酶组合既经济适中,效果又好.处于指数生长期的愈伤组织在上述配方中离解,经27±1℃温和振荡、筛网过滤,漂浮纯化、离心洗涤,可获得10~4个/ml以上有活力的甘蔗愈伤组织原生体.

The sugarcane calluses as primary maiierials in isolation of protoplasts, the type and concentration of enzymes, the conditions of isolation and pretreatment, a comparison between cultivated species and wild resource, isolation of the cell suspension cultures etc. were investigated. It was found that constitution 2% cellulase (Onozuka R-10) , 0.7% pectinase was the best. It could isolate more pro-oplasts when the specific value of cellulase was 0.727-0.747.The result obtained 104/ml calluses protoplasts of sugarcane, with the calluses at exponential growth phase in the constitution, and shak-et for 6hr at 27±1°C, filter by 37μm sieve, by float purification, by centrifugation and washing.