Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

Plant Methods

Methodology

Wenjing Li¹  Jun Li¹  Shanhe Liu¹  Caihua Zhang¹  Ke Zhang²  Zixuan Wang³  Dongmei Zhang³  Yunze Fu⁴  Xiubo Yang⁴ 

[1] State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, 071001, Baoding, China;Hebei Key Laboratory of Plant Physiology and Molecular Pathology, College of Life Sciences, Hebei Agricultural University, 071001, Baoding, China;State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, 071001, Baoding, China;Hebei Key Laboratory of Plant Physiology and Molecular Pathology, College of Life Sciences, Hebei Agricultural University, 071001, Baoding, China;Key Laboratory of Crop Growth Regulation of Hebei Province, Hebei Agricultural University, 071001, Baoding, China;State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, 071001, Baoding, China;Key Laboratory for Crop Germplasm Resources of Hebei, Hebei Agricultural University, 071001, Baoding, China;State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, 071001, Baoding, China;Key Laboratory of Crop Growth Regulation of Hebei Province, Hebei Agricultural University, 071001, Baoding, China;

关键词: Protoplast isolation;    PEG-mediated transfection;    scRNA-seq;    Genome editing;    Cotton;   

DOI  :  10.1186/s13007-023-00983-6

 received in 2022-11-15, accepted in 2023-01-15,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundCotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton.ResultsWe established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies.ConclusionsThe protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

【 授权许可】

CC BY   
© The Author(s) 2023

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