1. Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis.
- Author
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Zhang, Ke, Liu, Shanhe, Fu, Yunze, Wang, Zixuan, Yang, Xiubo, Li, Wenjing, Zhang, Caihua, Zhang, Dongmei, and Li, Jun
- Subjects
PLANT protoplasts ,GENE expression ,RNA sequencing ,GENE transfection ,FUNCTIONAL genomics ,GENOME editing ,COTTON growing - Abstract
Background: Cotton 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. Results: We 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 × 10
5 /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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]- Published
- 2023
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