《植物生理学报》 2011, 47(5): 495-500

‘陇东’紫花苜蓿原生质体最佳分离条件

陶茸, 师尚礼*, 李玉珠, 李剑锋

甘肃农业大学草业学院, 草业生态系统教育部重点实验室, 中-美草地畜牧业可持续发展研究中心, 兰州730070

通信作者：师尚礼；E-mail: shishl@gsau.edu.cn；Tel: 甘肃农业大学草业学院, 草业生态系统教育部重点实验室, 中-美草地畜牧业可持续发展研究中心, 兰州730070

摘　要：

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10 h、酶液渗透压即甘露醇浓 度为0.55 mol•L-1, 愈伤继代培养天数为12 d、预处理措施为4 ℃、黑暗条件下放置24 h。

关键词：‘陇东’紫花苜蓿; 下胚轴; 愈伤组织; 酶解条件; 原生质体

收稿：2011-03-01   修定：2011-04-12

资助：农业部公益性行业(农业)科研专项(nyhyzx07-022)和现代 农业产业技术体系建设专项资金

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

TAO Rong, SHI Shang-Li*, LI Yu-Zhu, LI Jian-Feng

Sino-U.S. Centers for Grazingland Ecosystem Sustainability, Key Laboratory of Grassland Ecosystem of Ministry of Education, College of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China

Corresponding author: SHI Shang-Li; E-mail: shishl@gsau.edu.cn; Tel: 甘肃农业大学草业学院, 草业生态系统教育部重点实验室, 中-美草地畜牧业可持续发展研究中心, 兰州730070

Abstract:

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 ℃.

Key words: Medicago sativa L. cv. ‘Longdong’ ; hypocotyls; callus; enzymolysis conditions; protoplast