丹参悬浮培养细胞原生质体的制备和活力检测

Preparation and vitality detection of protoplast in Salvia miltiorrhiza Bunge

摘要:

对丹参悬浮培养细胞原生质体制备条件进行了研究，并利用FDA染色和钙离子荧光探针Fluo-3/AM装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为：悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%，离析酶0.5%；适宜的甘露醇浓度为0.4 mol/L；酶解时间为12 h；在600 r/min转速下离心5 min收集，纯化得到原生质体，其产量为1.1×106/g FW，FDA检测显示其活力为95%以上，荧光探针Fluo-3/AM可成功装载到原生质体中。

Abstract:

We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts’ vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600×g 5 min, and the protoplasts yield was 1.1×106 cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.