Optimization of isolation and transfection conditions of maize endosperm protoplasts

Yufeng Hu; Dalin Song; Lei Gao; Babatope Samuel Ajayo; Yongbin Wang; Huanhuan Huang; Junjie Zhang; Hanmei Liu; Yinghong Liu; Guowu Yu; Yongjian Liu; Yangping Li; Yubi Huang

doi:10.1186/s13007-020-00636-y

Optimization of isolation and transfection conditions of maize endosperm protoplasts

Plant Methods. 2020 Jul 9:16:96. doi: 10.1186/s13007-020-00636-y. eCollection 2020.

Authors

Yufeng Hu^{1

2}, Dalin Song^{1

2}, Lei Gao^{1

2}, Babatope Samuel Ajayo^{1

2}, Yongbin Wang², Huanhuan Huang², Junjie Zhang³, Hanmei Liu³, Yinghong Liu², Guowu Yu², Yongjian Liu², Yangping Li^{1

2}, Yubi Huang^{1

2}

Affiliations

¹ State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Chengdu, China.
² College of Agronomy, Sichuan Agricultural University, Chengdu, 611130 China.
³ College of Life Science, Sichuan Agricultural University, Ya'an, 625014 China.

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-suspension in 1 ml MMG. The EPS was used to express GFP protein, analyze the subcellular location of ZmBT1, characterize the interaction of O2 and PBF1 by bimolecular fluorescent complementation (BiFC), and simultaneously analyze the regulation of ZmBt1 expression by ZmMYB14.

Conclusions: The described optimized conditions proved efficient for reasonable yield of viable protoplasts from maize endosperm, and utility of the protoplast in rapid analysis of endosperm-trait related genes. The development of the optimized protoplast isolation and transfection conditions, allow the exploitation of the functional advantages of protoplast system over biolistic system in conducting endosperm-based studies (particularly, in transient analysis of genes and gene regulation networks, associated with the accumulation of endosperm storage products). Such analyses will be invaluable in characterizing endosperm-trait related genes whose functions have not been identified. Thus, the EPS will benefit the research of cereal grain yield and quality improvement.

Keywords: Endosperm; Genes’ functions; Maize; Protoplast; Response surface; Transient system.