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<p>The efficient regeneration and transformation protocols are crucial for accelerating functional genomics studies and crop improvement in <i>Cucurbita pepo</i>. This study aimed to establish an in vitro regeneration and transient transformation system in <i>C. pepo</i> var. <i>clypeata</i> (Scallop squash) cv. Punjab Chappan Kaddu-1 (PCK-1). Cotyledon, cotyledonary node and hypocotyl explants excised from in vitro germinated seedlings were cultured on shoot induction media supplemented with varying concentrations of growth regulators. Only cotyledonary node explants showed higher regeneration potential (90.3%) on the Murashige and Skoog (MS) medium supplemented with 1.0&#xa0;mg/l 6-benzylaminopurine (BAP) for effective shoot induction and elongation. The elongated shoots were successfully rooted on half-strength MS medium containing 0.5&#xa0;mg/l indole-3-butyric acid (IBA) plus 0.5&#xa0;mg/l naphthaleneacetic acid (NAA). Regenerated plants were then transferred to glasshouse conditions, with a survival rate of 60%, producing healthy and phenotypically normal plants. Using this optimized system, <i>Agrobacterium</i>-mediated transient transformation was successfully achieved with the <i>RUBY</i> reporter gene. Two strains of <i>Agrobacterium</i>,<i> EHA105</i> and <i>LBA4404</i>, were evaluated on cotyledonary nodes, and <i>EHA105</i> showed higher transformation efficiency than <i>LBA4404</i>. Furthermore, using an <i>Agrobacterium</i> suspension (OD₆₀₀ = 0.2) supplemented with 150 µM acetosyringone and co-cultivation for 3 days at 28&#xa0;°C was found to be effective for optimal transformation. Red-colored patches appeared within three days, indicating successful <i>RUBY</i> gene expression, which was further confirmed by PCR analysis. The transient transformation protocol will be effective in accelerating functional genomics in <i>C. pepo</i> and the utilization of genome editing for crop improvement.</p> Graphical Abstract <p></p>

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Development of an efficient in vitro regeneration and non-invasive transient transformation system in Cucurbita pepo var. clypeata (Scallop squash)

  • 期刊论文

  • 发表日期：

    2026年6月30日

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  • Riya Rani,
  • Ajmer Singh Dhatt,
  • Madhu Sharma,
  • Jiffinvir Khosa

摘要

The efficient regeneration and transformation protocols are crucial for accelerating functional genomics studies and crop improvement in Cucurbita pepo. This study aimed to establish an in vitro regeneration and transient transformation system in C. pepo var. clypeata (Scallop squash) cv. Punjab Chappan Kaddu-1 (PCK-1). Cotyledon, cotyledonary node and hypocotyl explants excised from in vitro germinated seedlings were cultured on shoot induction media supplemented with varying concentrations of growth regulators. Only cotyledonary node explants showed higher regeneration potential (90.3%) on the Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 6-benzylaminopurine (BAP) for effective shoot induction and elongation. The elongated shoots were successfully rooted on half-strength MS medium containing 0.5 mg/l indole-3-butyric acid (IBA) plus 0.5 mg/l naphthaleneacetic acid (NAA). Regenerated plants were then transferred to glasshouse conditions, with a survival rate of 60%, producing healthy and phenotypically normal plants. Using this optimized system, Agrobacterium-mediated transient transformation was successfully achieved with the RUBY reporter gene. Two strains of Agrobacterium, EHA105 and LBA4404, were evaluated on cotyledonary nodes, and EHA105 showed higher transformation efficiency than LBA4404. Furthermore, using an Agrobacterium suspension (OD₆₀₀ = 0.2) supplemented with 150 µM acetosyringone and co-cultivation for 3 days at 28 °C was found to be effective for optimal transformation. Red-colored patches appeared within three days, indicating successful RUBY gene expression, which was further confirmed by PCR analysis. The transient transformation protocol will be effective in accelerating functional genomics in C. pepo and the utilization of genome editing for crop improvement.

Graphical Abstract