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

Ke Zhang; Shanhe Liu; Yunze Fu; Zixuan Wang; Xiubo Yang; Wenjing Li; Caihua Zhang; Dongmei Zhang; Jun Li

doi:10.1186/s13007-023-00983-6

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

Plant Methods. 2023 Jan 18;19(1):5. doi: 10.1186/s13007-023-00983-6.

Authors

Ke Zhang^#^{1

2

3}, Shanhe Liu^#^{1

2}, Yunze Fu^#^{1

3}, Zixuan Wang^{1

4}, Xiubo Yang^{1

3}, Wenjing Li^{1

2}, Caihua Zhang^{1

2}, Dongmei Zhang^{5

6}, Jun Li^{7

8}

Affiliations

¹ State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, Baoding, 071001, China.
² Hebei Key Laboratory of Plant Physiology and Molecular Pathology, College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China.
³ Key Laboratory of Crop Growth Regulation of Hebei Province, Hebei Agricultural University, Baoding, 071001, China.
⁴ Key Laboratory for Crop Germplasm Resources of Hebei, Hebei Agricultural University, Baoding, 071001, China.
⁵ State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, Baoding, 071001, China. zhangdongmei1108@hebau.edu.cn.
⁶ Key Laboratory for Crop Germplasm Resources of Hebei, Hebei Agricultural University, Baoding, 071001, China. zhangdongmei1108@hebau.edu.cn.
⁷ State Key Laboratory of North China Crop Improvement and Regulation, College of Agronomy, Hebei Agricultural University, Baoding, 071001, China. junli@hebau.edu.cn.
⁸ Hebei Key Laboratory of Plant Physiology and Molecular Pathology, College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China. junli@hebau.edu.cn.

^# Contributed equally.

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.

Keywords: Cotton; Genome editing; PEG-mediated transfection; Protoplast isolation; scRNA-seq.

Abstract

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