ArticleBMC plant biology2026
Efficient isolation and transient transformation of cowpea protoplasts.
Article in BMC plant biology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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8 authors.
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Abstract
backgroundCowpea (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.
resultsSequential 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.
conclusionsThis 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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