‘陇东’紫花苜蓿原生质体最佳分离条件

Study on Protoplasts Isolation Conditions of Medicago sativa L.cv.'Longdong'

以‘陇东’紫花苜蓿(Medicago sativa L.cv.‘Longdong’)下胚轴愈伤组织为材料,研究酶液组合、酶解时间、酶液渗透压、愈伤组织继代培养时间及预处理措施对原生质体分离效果的影响。结果表明:获得产量最高(8.8×105个·g-1)、活力最高(88.55%)的原生质体最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶、酶解时间为10h、酶液渗透压即甘露醇浓度为0.55mol·L-1,愈伤继代培养天数为12d、预处理措施为4℃、黑暗条件下放置24h。

Hypocotyl callus of wild Medicago sativa L.cv.＇Longdong＇ was used to study the effect of enzyme combination,enzymolysis time,osmotic pressure,subculture time and pretreatment measures on protoplast isolation.The result showed that the optimum enzyme solution combination for the highest yield（8.8×105·g-1） and viability（88.55%） of protoplast were as follow：2% cellulase ＋ 0.5% pectinase ＋ 0.3% driselase,the optimum enzymolysis time was 10 h,the optimum enzyme osmostic pressure（viz.mannitol concentration） was 0.55 mol·L-1,the optimum subculture time was 12 d and the optimum pretreatment measure was 24 h in dark at 4 ℃.