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Preparation and Purification of Protoplast from Nervilia fordii (Hance) Schltr. Leaves

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【Author】 ZHANG Xiao-li1, HE Rui1*, TONG Jia-yun1,2, ZHAN Ruo-ting1, LIN Chao1 1. Key Laboratory of Chinese Medicinal Resources from Lingnan of Ministry of Education, Research Centre of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, China; 2. College of Chinese Traditional Medicine,Guangzhou University of Chinese Medicine, GuangZhou, Guangdong 510006, China

【摘要】 [Objective] To set up protoplast preparation and purification method from Nervilia fordii (Hance) Schltr. leaves. [Method] Taking the yield and survival rate of protoplast as indices, the effects of different factors, including pretreatment, enzymolysis method, enzymolysis temperature, centrifugation speed, illumination, and sieve mesh number were studied for the separation and purification of protoplast from N. fordii leaves. [Result] The optimum process for protoplast isolation was obtained as follows: N. fordii leaves were first pretreated for 1 h in CPW solution containing 13% mannitol, and then incubated at (25±1)℃ in enzyme solution under dark conditions; after the enzymolysis, the enzyme solution was filtrated through 200 mesh screen and centrifuged at 800 r/min for 8 min. The filtration and centrifugation were repeated once before microscopic examination. Large amount of intact and clean protoplast with very few cell debris was obtained. [Conclusion] The preparation system has been successfully established, which lays foundation for establishing protoplast regeneration system from N. fordii leaves.

【基金】 Supported by National Natural Science Foundation of China (30701090);the Scientific Research Foundation for the Returned Overseas Chinese Scholars,Ministry of Education, P. R. China (20091001)
  • 【分类号】R284.1
  • 【下载频次】39
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