Efficient isolation and transient transformation of cowpea protoplasts
- 1College of Agronomy, Jiangxi Agricultural University
- 2Dongtai Agricultural Technology Extension Center, Dongtai City Bureau of Agriculture and Rural Affairs
Abstract
Background Cowpea (Vigna unguiculata) is an important legume crop, but reproducible protoplast isolation and transient expression systems remain limited. We developed a leaf mesophyll protoplast isolation procedure and a polyethylene glycol (PEG)-mediated transient transformation system for cowpea. We also characterized physiological and transcriptional changes associated with enzymatic digestion. Results Sequential optimization identified a working enzyme solution containing 1.3% cellulase, 0.25% pectinase, 0.3% macerozyme, and 0.45 M mannitol, with a 13 h digestion period. Across the sequential optimization experiments, protoplast yield reached up to 4.34 × 10⁷ protoplasts g⁻¹ fresh weight and viability exceeded 95%. In the digestion-time experiment, the 13 h treatment yielded 4.19 × 10⁷ protoplasts g⁻¹ fresh weight with 97.84% viability. Enzymatic digestion altered antioxidant enzyme activities, oxidative-status indicators, and the expression of stress- and cell-state-associated genes. These molecular measurements describe the physiological state of freshly isolated protoplasts but do not demonstrate regeneration competence. The optimized PEG-mediated assay achieved a final transformation efficiency of 22.54%. Transient expression of VuPPR95-EGFP and its chloroplast-localized fluorescence demonstrated the suitability of the system for rapid subcellular localization analysis. Conclusions This study provides a reproducible cowpea protoplast isolation and transient expression platform for gene-expression and subcellular-localization assays. The protocol improves protoplast recovery and viability relative to an earlier unsuccessful cowpea PEG-transfection attempt, while its moderate transformation efficiency and untested regeneration capacity define clear priorities for further development.
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BibTeX
@article{qiu2026efficient,
title = {{Efficient isolation and transient transformation of cowpea protoplasts}},
author = {Xuewen Qiu and Chuntao Zeng and Shuyue Zhang and Yutong Zhang and Liuyang Cheng and Yudi Gan and Youxin Yang and Caijun Wu},
journal = {BMC Plant Biology},
year = {2026},
month = jul,
doi = {10.1186/s12870-026-09619-y},
url = {https://doi.org/10.1186/s12870-026-09619-y},
note = {CC BY},
}
RIS
TY - JOUR
TI - Efficient isolation and transient transformation of cowpea protoplasts
AU - Xuewen Qiu
AU - Chuntao Zeng
AU - Shuyue Zhang
AU - Yutong Zhang
AU - Liuyang Cheng
AU - Yudi Gan
AU - Youxin Yang
AU - Caijun Wu
PY - 2026
DA - 2026/07/01
JO - BMC Plant Biology
DO - 10.1186/s12870-026-09619-y
UR - https://doi.org/10.1186/s12870-026-09619-y
AB - Background Cowpea (Vigna unguiculata) is an important legume crop, but reproducible protoplast isolation and transient expression systems remain limited. We developed a leaf mesophyll protoplast isolation procedure and a polyethylene glycol (PEG)-mediated transient transformation system for cowpea. We also characterized physiological and transcriptional changes associated with enzymatic digestion. Results Sequential optimization identified a working enzyme solution containing 1.3% cellulase, 0.25% pectinase, 0.3% macerozyme, and 0.45 M mannitol, with a 13 h digestion period. Across the sequential optimization experiments, protoplast yield reached up to 4.34 × 10⁷ protoplasts g⁻¹ fresh weight and viability exceeded 95%. In the digestion-time experiment, the 13 h treatment yielded 4.19 × 10⁷ protoplasts g⁻¹ fresh weight with 97.84% viability. Enzymatic digestion altered antioxidant enzyme activities, oxidative-status indicators, and the expression of stress- and cell-state-associated genes. These molecular measurements describe the physiological state of freshly isolated protoplasts but do not demonstrate regeneration competence. The optimized PEG-mediated assay achieved a final transformation efficiency of 22.54%. Transient expression of VuPPR95-EGFP and its chloroplast-localized fluorescence demonstrated the suitability of the system for rapid subcellular localization analysis. Conclusions This study provides a reproducible cowpea protoplast isolation and transient expression platform for gene-expression and subcellular-localization assays. The protocol improves protoplast recovery and viability relative to an earlier unsuccessful cowpea PEG-transfection attempt, while its moderate transformation efficiency and untested regeneration capacity define clear priorities for further development.
SN - 1471-2229
ER -