白及原生质体的分离与纯化

doi:10.7525/j.issn.1673-5102.2016.05.023

植物研究 ›› 2016, Vol. 36 ›› Issue (5): 795-800.doi: 10.7525/j.issn.1673-5102.2016.05.023

• 研究简报 • 上一篇

白及原生质体的分离与纯化

徐德林¹, 张林¹, 储士润¹, 韦欣², 钱刚¹, 郑明辉³

1. 遵义医学院细胞生物学教研室, 遵义 563000;
2. 遵义医学院医学与科技学院, 遵义 563000;
3. 遵义医学院寄生虫教研室, 遵义 563000

收稿日期:2016-04-15 出版日期:2016-09-15 发布日期:2016-09-27
通讯作者: 郑明辉,E-mail:ivying0209@hotmail.com E-mail:ivying0209@hotmail.com
作者简介:徐德林(1981-),男,博士,主要从事中药材遗传育种研究。
基金资助:
国家自然科学基金（31560079）；贵州省中药现代化科技产业研究开发专项（黔科合ZY字[2013]3002号）；贵州省科技厅2014年联合基金（黔科合LH字[2014]7549号）；2015年国家大学生创新训练计划项目（201513653003）；遵义市2014年度“15851”人才工程（201424）

A Protocol for the Isolation and Purification of Protoplast from Bletilla striata Leaves

XU De-Lin¹, ZHANG Lin¹, CHU Shi-Run¹, WEI Xin², QIAN Gang¹, ZHENG Ming-Hui³

1. Department of Cell Biology, Zunyi Medical University, Zunyi 563000;
2. Medicine and Technology School, Zunyi Medical University, Zunyi 563000;
3. Department of Parasite, Zunyi Medical University, Zunyi 563000

Received:2016-04-15 Online:2016-09-15 Published:2016-09-27
Supported by:
Natural Science Foundation of China(31560079);The Modernization and Industrialization Project of TCM in Guizhou Province(QKH-ZY[2013]3002);The Cooperation Program of Guizhou Province(QKH-LH[2014]7549);College Students’ Innovative Training Project of China in 2015(201513653003);The 15851# Talent Projects of Zunyi City in 2014(201424)

摘要/Abstract

摘要： 为建立起白及原生质体分离纯化的技术体系，以白及组培苗叶片为材料，经不同方式预处理后，用不同浓度的纤维素酶、果胶酶、离析酶和甘露醇组合进行酶解，将释放出来的原生质体用过滤和离心的方法进行纯化，最后用荧光素双醋酸酯（FDA）法对纯化的原生质体进行活力率测定，以期系统建立起酶解法从叶片中分离纯化出白及原生质体的技术流程。结果发现在低温黑暗+0.75 mol·L^-1甘露醇条件下对叶片进行预处理、酶液组合为1.5%纤维素酶Cellulase R-10+0.4%果胶酶Pectolyase Y-23+0.5%离析酶Macerozyme R-10+0.75 mol·L^-1甘露醇、酶解液pH值保持在5.8、摇床转速120 r·min^-1、酶解4 h时，得到的原生质体产量最大，达4.72×10⁶个·g^-1，活力率也达90.4%。本研究构建了用白及叶片分离和纯化原生质体的技术流程，为白及遗传资源的拓展和遗传改良奠定了一定的物质和技术基础。

关键词: 白及, 原生质体, 分离, 纯化, 技术体系

Abstract: To construct a protocol of isolation and purification of protoplasts from the seedling leaves of Bletilla striata. After a series of pretreating assay, the leaves were enzymatic hydrolyzed with mixed solution of mannitol, Cellulase R-10, Pectolyase Y-23 and Macerozyme R-10 for certain hours. Then the newly released protoplasts were purified through filtration and centrifugalization. Finally, the harvest protoplastswere detected in the survival rate by means of fluorescein diacetate (FDA). The preprocessing of seedlings living in dark field for 12 h and the leaves were immersed in 0.75 mol·L^-1 mannitol for 1 h was the most effective way for protoplasts isolation. The enzymes mixture of 0.75 mol·L^-1 mannitol+1.5% Cellulase R-10+0.4% Pectolyase Y-23+0.5% Macerozyme R-10 was the optimism composition of enzyme solution. The pH of 5.8 and 4 h enzyme dissolve with the revolving speed of 120 r·min^-1 at room temperature was the best environmental condition. Under this optimal condition, abundant of 4.72×10⁶ protoplasts with survival rate of 90.4% could be harvested from each gram leaf. This study gave a necessary help for protoplasts culture, cell fusion and genetic transformation of B.striata.

Key words: Bletilla striata, protoplast, isolation, purification, protocol

中图分类号:

Q945.4

徐德林, 张林, 储士润, 韦欣, 钱刚, 郑明辉. 白及原生质体的分离与纯化[J]. 植物研究, 2016, 36(5): 795-800.

XU De-Lin, ZHANG Lin, CHU Shi-Run, WEI Xin, QIAN Gang, ZHENG Ming-Hui. A Protocol for the Isolation and Purification of Protoplast from Bletilla striata Leaves[J]. Bulletin of Botanical Research, 2016, 36(5): 795-800.

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白及原生质体的分离与纯化

A Protocol for the Isolation and Purification of Protoplast from Bletilla striata Leaves

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