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以湘豆3号大豆和Kabuli型鹰嘴豆幼嫩叶片为材料,研究了酶解种类及配比、酶解液中甘露醇浓度、酶解液p H和酶解时间对两种豆科植物幼嫩叶片原生质体产量和存活率的影响。结果表明,湘豆3号大豆叶片在含有0.5%纤维素酶Onozuka R-10、0.8%半纤维素酶Hemicellulase、0.8%离析酶Macerozyme R-10、0.4%果胶酶Pectolyase Y-23、0.1%2-(N-吗啡啉)乙磺酸(MES)和10%甘露醇的酶解液中(pH 6.0),27℃黑暗条件下恒温水浴振荡(45 r/min)酶解6 h,分离得到的原生质体产量和存活率最高;Kabuli型鹰嘴豆叶片在含有0.5%纤维素酶Onozuka R-10、0.8%半纤维素酶Hemicellulase、0.8%离析酶Macerozyme R-10、0.1%MES和10%甘露醇酶解液中(pH 4.8),27℃黑暗条件下恒温振荡水浴(45 r/min)7–8 h,分离得到的原生质体产量和存活率最高。通过上述件分离到的湘豆3号大豆和Kabuli型鹰嘴豆幼嫩叶片原生质体用于亚细胞定位的效果最好。
Taking the young leaves of Xiang-bean No.3 and Kabuli-type chickpea as materials, the effects of the types and proportions of the enzymes, the mannitol concentration in the hydrolyzate, the pH value of the hydrolyzate and the enzymolysis time on the growth of two leguminous plants Effect of Young Leaf Protoplast Yield and Survival. The results showed that the soybean leaves of Xiang Dou No.3 were cultured in the medium containing 0.5% cellulase Onozuka R-10, 0.8% hemicellulase, 0.8% Macerozyme R-10, 0.4% pectolyase Y- (PH 6.0) in 2- (N- morpholine) ethanesulfonic acid (MES) and 10% mannitol (pH 6.0), shaking in a constant temperature water bath at 45 ℃ for 6 h under dark conditions at 27 ℃, Of protoplast yield and survival rate were the highest; Kabuli-type chickpea leaves were cultured in a medium containing 0.5% cellulase Onozuka R-10, 0.8% hemicellulase Hemicellulase 0.8% Macerozyme R-10, 0.1% MES and 10% Mannitol hydrolyzate (pH 4.8), 27 ℃ dark conditions constant temperature shaking water bath (45 r / min) 7-8 h, the isolated protoplast yield and survival rate was the highest. Protoplasts of young leaves of Xiang bean No. 3 and Kabuli type chickpea isolated from the above pieces are most effective for subcellular localization.