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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。
The effects of osmotic pressure (mannitol concentration), hydrolase concentration and enzymolysis time on the separation of mesophyll protoplasts from Petunia hybrida were studied. The results showed that the optimum conditions were as follows: 2% cellulase R-10 + 0.2% pectinase Y-23 + 0.4% isozyme R-10 + 20 mmol / L 2- (N- morphine) ethanesulfonic acid (MES) + 0.1% bovine serum albumin (BSA) and 0.11% anhydrous calcium chloride (CaCl 2) were used as enzymatic hydrolyzate, and then hydrolyzed by 0.5mol / L mannitol for 5h, and then protoplast was precipitated by centrifugation at 100r / The yield and activity of protoplasts were 2.90 × 106 / g and 88.1%, respectively, which could provide materials for subsequent protoplast culture and fusion.