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<p>Cucumber (<i>Cucumis sativus</i> L.) is a globally essential vegetable crop but its limited genetic diversity poses significant challenges to breeding efforts aimed at improving environmental adaptability and stress tolerance. The present study optimizes protoplast fusion and in vitro regeneration to enhance genetic diversity of two Bangladeshi cucumber cultivars, Shosha and Khira. Protoplasts were isolated from leaf mesophyll using enzymatic digestion (2% Cellulase <i>R</i>-10, 0.6% Macerozyme <i>R</i>-10) and fused via 40% PEG-6000. Viability, assessed by Fluorescein Diacetate staining, was highest in Shosha (87.3%) and Khira (83.5%), while fused protoplasts showed 63.7% viability, with fusion success confirmed by dual fluorescence staining. Callus induction was optimized using Murashige and Skoog medium with varied plant growth regulators (NAA, BA, 2,4-D, KIN, BAP, IBA). Treatment BMT6 (MS salt +0.93 mg/l NAA +1.12 mg/l BA + 0.8% agar powder) yielded the largest mini-calli (4.50 mm) and macro-calli (76.60 mm), while HT8 (MS medium +1.0 mg/l 2,4-D +1.0 mg/l KIN) produced friable callus with high moisture content (92.5%). Somatic embryogenesis was most effective in SET13 (MS medium +2.0 mg/l BAP +1.0 mg/l NAA +1.0 mg/l KIN), achieving the largest embryos (22.43 mm) and highest formation rate (70.7%) in 59.3 days. Genetic diversity of regenerated hybrids was confirmed using RAPD markers (OPA-10, OPD-13, OPD-18), revealing 87.1% polymorphism. UPGMA clustering and Jaccard’s similarity analysis confirmed hybridity, with Shosha and hybrids clustering closely. These findings establish an efficient protocol for protoplast fusion and regeneration, supporting the development of genetically diverse, stress-resilient cucumber cultivars to address local cultivation challenges.</p> Graphical abstract <p></p>

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In vitro regeneration of microplants via protoplast fusion of two Cucumber cultivars (cucumis sativus L.)

  • 期刊论文

  • 发表日期：

    2025年10月8日

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  • Shakil Rahman Shoagh,
  • Abdur Rahim,
  • A. T. K. Mim,
  • Baishakhi Roy,
  • Jakia Sultana,
  • M. Nasiruddin

摘要

Cucumber (Cucumis sativus L.) is a globally essential vegetable crop but its limited genetic diversity poses significant challenges to breeding efforts aimed at improving environmental adaptability and stress tolerance. The present study optimizes protoplast fusion and in vitro regeneration to enhance genetic diversity of two Bangladeshi cucumber cultivars, Shosha and Khira. Protoplasts were isolated from leaf mesophyll using enzymatic digestion (2% Cellulase R-10, 0.6% Macerozyme R-10) and fused via 40% PEG-6000. Viability, assessed by Fluorescein Diacetate staining, was highest in Shosha (87.3%) and Khira (83.5%), while fused protoplasts showed 63.7% viability, with fusion success confirmed by dual fluorescence staining. Callus induction was optimized using Murashige and Skoog medium with varied plant growth regulators (NAA, BA, 2,4-D, KIN, BAP, IBA). Treatment BMT6 (MS salt +0.93 mg/l NAA +1.12 mg/l BA + 0.8% agar powder) yielded the largest mini-calli (4.50 mm) and macro-calli (76.60 mm), while HT8 (MS medium +1.0 mg/l 2,4-D +1.0 mg/l KIN) produced friable callus with high moisture content (92.5%). Somatic embryogenesis was most effective in SET13 (MS medium +2.0 mg/l BAP +1.0 mg/l NAA +1.0 mg/l KIN), achieving the largest embryos (22.43 mm) and highest formation rate (70.7%) in 59.3 days. Genetic diversity of regenerated hybrids was confirmed using RAPD markers (OPA-10, OPD-13, OPD-18), revealing 87.1% polymorphism. UPGMA clustering and Jaccard’s similarity analysis confirmed hybridity, with Shosha and hybrids clustering closely. These findings establish an efficient protocol for protoplast fusion and regeneration, supporting the development of genetically diverse, stress-resilient cucumber cultivars to address local cultivation challenges.

Graphical abstract