以大花蕙兰的叶片、根为试材,分析不同酶液组合、摇床转速、酶解温度、材料酶液比、酶解时间等因素对其原生质体分离的影响。大花蕙兰叶片、根经过90 min质壁分离预处理后,在温度28 ℃,于摇床上50 r·min-1振荡酶解6 h,叶、根分别在酶解液为纤维素酶2.0%+果胶酶0.1%+离析酶0.2%+崩溃酶0.2%和纤维素酶2.0%+果胶酶0.3%+离析酶0.1%+崩溃酶0.1%中得到最高产量的原生质体。
Taking Cymbidium hybridum leaves and roots as material, the effects of enzyme combination, shaking speed, enzymolysis time, enzymolysis temperature, and the ratio of materials enzyme on protoplast isolation were studied. The results showed that:after 90 min pretreatment for plasmolysis, protoplast yield from leaves was the highest using mixed enzyme (2.0% cellulose + 0.1% pectinase + 0.2% macerozyme + 0.2% driselase) with shaking speed 50 r·min-1 at 28 ℃ for 6 h. And protoplast yield from roots was the highest using mixed enzyme (2.0% cellulose + 0.3% pectinase + 0.1% macerozyme + 0.1% driselase) under the same conditions.
[1] 朱根发. 大花蕙兰市场发展新动向[J]. 中国花卉盆景, 2004(7): 32—33.
[2] Pindel A. Optimization of isolation conditions of Cymbidium protoplasts[J]. Folia Horticulturae, 2007,19(2): 79—88.
[3] 柳玉晶. 百合原生质体分离及培养的研究[D].哈尔滨: 东北农业大学硕士学位论文, 2006.
[4] 张学英. 草莓(Fragaria ananassa Duch.)原生质体培养及植株再生研究[D]. 保定: 河北农业大学博士学位论文, 2004.
[5] 沈前华,杨拍云,郭金平,杨宁生,钟青萍. 大花蕙兰原生质体分离的影响因素研究[J]. 江西农业学报, 1996,8(1): 36—40.
[6] 唐克轩. 中草药生物技术[M]. 上海: 复旦大学出版社, 2005: 62—67.
[7] 乔永旭,张永平,王桂兰,陈超,王燕. 蝴蝶兰原生质体提取方法的优化[J]. 植物生理学通讯, 2008,44(6) : 1177—1180.
[8] 周蓉,陈小娟. 花生叶片原生质体游离和培养因子的研究[J]. 中国油料作物学报, 1999,21(1): 7—9.
[9] 高丽霞,蒋冬梅. 桑树原生质体分离条件的优化[J]. 贵州农业科学, 2012,40(2): 20—22.