陇东野生紫花苜蓿愈伤组织原生质体游离条件的筛选

Selection for Protoplasts Dissociate Conditions of Callus of Native Medicago sativa L. cv. Longdong

以陇东野生紫花苜蓿子叶诱导的愈伤组织为材料,研究了酶解时间、酶液组合、酶液中甘露醇浓度、预处理措施及愈伤组织继代培养时间对其原生质体游离效果的影响。结果表明：当酶解时间为10h、酶液组合为2%纤维素酶＋0.5%果胶酶＋0.3%离析酶＋0.3%半纤维素酶＋0.3%崩溃酶、甘露醇浓度为0.60mol/L、预处理措施为CPW-10溶液中浸泡1h、愈伤继代培养天数为12~14d时,游离出的原生质体产量最高,可达19.5×105个/g,存活率高达89.7%。

The tested material was cotyledons callus of Native Medicago sativa L.cv.Longdong,and the effects of enzymolysis time,enzyme combination,mannitol concentrations in the mixed enzyme solution,subculture time and pretreatment measures on protoplast dissociation were studied.The results showed that the optimal conditions for the highest yield（19.5×105 g-1）and viability（89.7%） of protoplast were as follows： the optimal enzymolysis time was 10 hours,the optimal enzyme combination was 2% Cellulase Onozuka R-10 ＋ 0.5% Pectinase Y-23 ＋ 0.3% Macerozyme R-10 ＋ 0.3% Driselase ＋ 0.3% Hemicellulase Onozuka,the optimal mannitol concentration in enzyme solution was 0.60 mol/ L,the optimal callus subculture time was 12 to 14 days and the optimal pretreatment measure was 1h in CPW-10 solution.