{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "College of Agronomy, Jiangxi Agricultural University",
    "Dongtai Agricultural Technology Extension Center, Dongtai City Bureau of Agriculture and Rural Affairs"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 8,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Xuewen Qiu"
    },
    {
      "affs": [
        0
      ],
      "name": "Chuntao Zeng"
    },
    {
      "affs": [
        0
      ],
      "name": "Shuyue Zhang"
    },
    {
      "affs": [
        0
      ],
      "name": "Yutong Zhang"
    },
    {
      "affs": [
        1
      ],
      "name": "Liuyang Cheng"
    },
    {
      "affs": [
        0
      ],
      "name": "Yudi Gan"
    },
    {
      "affs": [
        0
      ],
      "name": "Youxin Yang"
    },
    {
      "affs": [
        0
      ],
      "name": "Caijun Wu"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/7h1ef9h9cj2m5.bib",
  "cited_by": 0,
  "doi": "10.1186/s12870-026-09619-y",
  "fulltext_url": "https://doi.org/10.1186/s12870-026-09619-y",
  "id": "7h1ef9h9cj2m5",
  "issns": [
    "1471-2229"
  ],
  "landing_url": "https://doi.org/10.1186/s12870-026-09619-y",
  "language": "en",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "QK1-989"
    ],
    [
      "openalex",
      "1312|Plant tissue culture and regeneration"
    ]
  ],
  "openalex": "W7171366680",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-07-01",
  "publisher": "BMC",
  "ris": "https://research.draconitic.com/paper/7h1ef9h9cj2m5.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:45:47Z",
      "native_id": "b4293bbd0cd24a33b5c04ff93e776a53",
      "source": "doaj",
      "url": "https://doi.org/10.1186/s12870-026-09619-y"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Plant tissue culture and regeneration",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Efficient isolation and transient transformation of cowpea protoplasts",
  "title_text": "Efficient isolation and transient transformation of cowpea protoplasts",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:45:47Z",
  "url": "https://research.draconitic.com/paper/7h1ef9h9cj2m5",
  "venue": "BMC Plant Biology",
  "versions": []
}