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Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (03): 424-430.doi: 10.3724/SP.J.1006.2014.00424

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Isolation and Flow Purification of Endosperm Protoplast from Developing Seed of Maize

GUO Yan-Ping**,REN Cheng-Jie**,LI Zhi-Wei,WANG Wen-Bin,ZHANG Ren-He,LU Hai-Dong,LIU Jian-Chao,ZHANG Xing-Hua,XUE Ji-Quan*,GUO Dong-Wei*   

  1. College of Agronomy, Northwest A&F University / Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest Region, Ministry of Agriculture, Yangling 712100, China?
  • Received:2013-05-13 Revised:2013-10-20 Online:2014-03-12 Published:2014-01-16
  • Contact: 薛吉全,E-mail: xjq2934@163.com;郭东伟, E-mail: gdwei1973@126.com E-mail:158895300@qq.com

Abstract:

Maize endosperm is the main place for synthesizing starch and its development is very important for building grain. Isolation and purification of endosperm cell protoplasts can offer the homogeneous experimental materials for endosperm cultivation, transcriptome analysis and cell typing. On the basis of previous studies, this paper optimized a set of isolation and purification methods of maize endosperm protoplast through adjusting the combination and concentration of enzymes as well as using membrane stabilizer and osmotic stabilizer. A lot of crude protoplasts were obtained when endosperm tissue was digested in MS medium containing 0.5% macerozyme, 0.5% hemicellulase, 1.0% cellulose, 0.7–0.8 mol L-1 of osmotic stabilizer and 0.8 mol L-1 of membrane stabilizer for 4 h at 30ºC. Staining of Fluorescein diacetate (FDA) showed that purified protoplasts maintained more than 90% of viability. The viable protoplasts were accumulated and purified from the crude protoplast suspension by flow sorting technology. Some technical parameters and influencing factors about protoplast isolation and flow sorting were also discussed.

Key words: Maize, Endosperm, Protoplast, Endoreduplication, Isolation, Flow sorting

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