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Background <p>Cowpea (<i>Vigna unguiculata</i>) 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.</p> Results <p>Sequential optimization identified a working enzyme solution containing 1.3% cellulase, 0.25% pectinase, 0.3% macerozyme, and 0.45&#xa0;M mannitol, with a 13&#xa0;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&#xa0;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.</p> Conclusions <p>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.</p>

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Efficient isolation and transient transformation of cowpea protoplasts

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月27日

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  • Xuewen Qiu,
  • Chuntao Zeng,
  • Shuyue Zhang,
  • Yutong Zhang,
  • Liuyang Cheng,
  • Yudi Gan,
  • Youxin Yang,
  • Caijun Wu

摘要

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.