Optimization of isolation and transfection conditions of maize endosperm protoplasts

作者：Yufeng Hu, Dalin Song, Lei Gao, Babatope Samuel Ajayo, Yong‐Bin Wang, Huanhuan Huang, Junjie Zhang, Hanmei Liu, Yinghong Liu, Guowu Yu, Yongjian Liu, Yangping Li, Yubi Huang · 发表于：Plant Methods · 年份：2020 · DOI：10.1186/s13007-020-00636-y · 被引用次数：35 · 研究领域：Plant tissue culture and regeneration、Transgenic Plants and Applications、Plant Molecular Biology Research

Abstract Background Endosperm-trait related genes are associated with grain yield or quality in maize. There are vast numbers of these genes whose functions and regulations are still unknown. The biolistic system, which is often used for transient gene expression, is expensive and involves complex protocol. Besides, it cannot be used for simultaneous analysis of multiple genes. Moreover, the biolistic system has little physiological relevance when compared to cell-specific based system. Plant protoplasts are efficient cell-based systems which allow quick and simultaneous transient analysis of multiple genes. Typically, PEG-calcium mediated transfection of protoplast is simple and cost-effective. Notably, starch granules in cereal endosperm may diminish protoplast yield and integrity, if the isolation and transfection conditions are not accurately measured. Prior to this study, no PEG-calcium mediated endosperm protoplast system has been reported for cereal crop, perhaps, because endosperm cells accumulate starch grains. Results Here, we showed the uniqueness of maize endosperm-protoplast system (EPS) in conducting endosperm cell-based experiments. By using response surface designs, we established optimized conditions for the isolation and PEG-calcium mediated transfection of maize endosperm protoplasts. The optimized conditions of 1% cellulase, 0.75% macerozyme and 0.4 M mannitol enzymolysis solution for 6 h showed that more than 80% protoplasts remained viable after re-suspe...