石斛兰叶片原生质体分离条件研究

[目的]探讨了石斛兰原生质体分离过程中的影响因素,建立最佳的原生质体制备体系。[方法]以石斛兰叶片为材料,采用正交设计研究纤维素酶浓度、酶解时间、温度、pH值以及甘露醇浓度对石斛兰原生质体分离的影响。[结果]以3%纤维素酶R-10＋0.6%果胶酶Y-23为混合酶液,0.5mol/L甘露醇为渗透压稳定剂,在26℃的黑暗条件下,pH值为5.4的酶液组合中,黑暗条件下振荡,酶解15h,获得的原生质体产量最高。[结论]该制备体系为后续原生质体培养和体细胞杂交奠定良好的基础。

Isolation Conditions of Protoplast in Dendrobium Leaves

[Objective] To investigate the isolation conditions of protoplast in Dendrobium and establish a best protoplast preparation system.[Method] Dendrobium leaves were used as research materials.The effects of cellulase concentration,hydrolysis time,temperature,pH value,and mannitol concentration on the separation of the protoplasts in Dendrobium were studied using orthogonal design.[Result] The highest yield of protoplast was obtained in the conditions including the mixed enzyme solution of 3.0% Onozuka R-10+0.6% pectinase Y-23,the osmotic stabilizer of 0.50 mol/L mannitol,in dark and pH 5.4 for 15 h at 26 ℃.[Conclusion] The preparation system lays a good foundation for subsequent protoplast culture and somatic cell hybridization.