金钗石斛类原球茎原生质体的分离

以具有较多生长点的金钗石斛PLBs为材料,研究通过酶解法获得原生质体的适宜条件。结果表明,酶液配比为φ=1%纤维素酶（Cellulase Onozuka R10）＋φ=0.4%果胶酶（Macerozyme Onozuka R10）＋0.65mol·L-1甘露醇,pH5.7;黑暗条件下,（25±2）℃,60r.min-1振荡酶解6h,800r.min-1离心分离4min,获得的原生质体质量较好。经计数和伊凡蓝染色法检测,用此法获得的金钗石斛PLBs原生质体鲜质量产量为（8.25±0.17）×105个.g-1,活性为（90.24±1.84）。（蓝光（470～75nm）激发活力检测发现较小的分生细胞具有较强的活力。

Isolation of the Protoplast from Dendrobium Nobile Lind1 PLBs

In order to obtain regeneration Dendrobium nobile Lind1 through protoplast isolation and cultivation,protoplasts were isolated from PLBs of Dendrobium nobile Lind1. The effects of various factors （composition of enzymes,digestion time,concentrations of osmotic pressure stabilize （mannitol）,centrifugation speed,purification methods,pretreatment of the material and the methods of vigor test） on the yield,viability and division of protoplasts were investigated. The results indicated that the optimum condition for protoplast isolation was established by using 1% cellulase R-10 and 0.4% macerozyme R-10 in 0.65 mol·L-1 mannitol （pH5.7） solution for cell wall digestion; the mixture of PLBs and enzyme solution was incubated at （25±2）℃ in dark for 6h on a rotary shaker with an agitation speed of 60 rpm,with adoption of 50—100μm filters to remove undigested tissues and debris,and after centrifugated at 800 rpm for 4min,the maximum yield （8.25±0.17）×105 per gram PLBs and the high viability （90.24±1.84）% were obtained. These data suggested that the small meristem cells of PLBs were good source of intact protoplast.