Efficient isolation and transient transformation of cowpea protoplasts

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. 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. 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.