| Summary: | 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 × 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. 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.
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