矮牵牛叶肉原生质体分离条件的优化

Optimization for Protoplast Isolation from Leaves of Petunia hybrida

以幼嫩叶片为试材,探讨了渗透压(甘露醇浓度)、水解酶浓度、酶解时间等关键因素对矮牵牛叶肉原生质体分离效果的影响。研究结果表明,以2%纤维素酶R-10+0.2%果胶酶Y-23+0.4%离析酶R-10+20 mmol/L 2-(N-吗啡啉)乙磺酸(MES)+0.1%牛血清白蛋白(BSA)+0.11%无水氯化钙(CaCl_2)为酶解液,在0.5mol/L甘露醇浓度下静置酶解5h,1 100r/min离心2min沉淀原生质体,原生质体产量及活性分别为2.90×106个/g和88.1%,可为后续原生质体培养及融合提供材料。

Young leaves of Petunia hybrida were used to investigate the key factors affecting the efficiency of mesophyll protoplast isolation.The results indicated that the optimal protocol is to digest the leaves in enzyme solution consisted of 2% Cellulase R-10+0.2% Pectolyase Y-23+0.4% Macerozyme R-10+20mmol/L MES+0.1% BSA+0.11% CaCl_2 at 0.5 mol/L mannitol with no shaking for 5hand then centrifuge at 1 100r/min for 2min and brake to precipitate protoplasts.Protoplast yields were up to 2.9×106 protoplasts per gram and the viability was 88.1%.These protoplasts can meet the technical demands of protoplast culture and fusion.