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<p>Efficient genetic transformation continues to be a major bottleneck in soybean (<i>Glycine max</i> L.) genome editing. We used a stable and reproducible <i>Agrobacterium</i>-mediated transformation system to compare cotyledonary node (CN) and half-seed (HS) explants for CRISPR/Cas9-based editing. A binary vector harboring a CaMV 35&#xa0;S-driven bar gene for glufosinate selection and a Cas9-sgRNA cassette targeting the <i>Apetala1</i> (<i>AP1</i>) homolog was introduced into <i>Agrobacterium tumefaciens</i>. Transformation efficiencies were 28.15% (CN) and 22.0% (HS). The CN method showed higher transformation efficiency, faster shoot elongation, and a shorter regeneration cycle, whereas HS explants had fewer escapes. Quantitative PCR confirmed Cas9 integration and expression, with HS-derived lines showing higher transcript accumulation (4.17 ± 0.13 to 6.06 ± 0.10-fold) than CN-derived lines (2.17 ± 0.02 to 5.70 ± 0.02-fold). The optimized hormone regime (zeatin, GA₃, and IBA) significantly enhanced shoot elongation and overall regeneration. Stable integration was validated through T₁ inheritance and glufosinate resistance assays. This comparative framework establishes a robust and reproducible platform for efficient, high-throughput genome editing in soybean.</p>

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Optimized Agrobacterium-mediated CRISPR/Cas9 transformation in soybean through comparative evaluation of cotyledonary node and half-seed explants

  • 期刊论文

  • 发表日期：

    2025年12月15日

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  • Durgeshwari Prabhakar Gadpayale,
  • J. Sushmita,
  • Navita Bansal,
  • Shalini Singh,
  • Ch Prasanna Kumari,
  • C. R. Nagesh,
  • Ranjeet Ranjan Kumar,
  • G. Rama Prashat,
  • Shivani Nagar,
  • Soham Ray,
  • R. Dinesh Kumar,
  • Arun Kumar,
  • Akshya Talukdar,
  • Aashish Marathe,
  • Giriraj Kumawat,
  • Viswanathan Chinnusamy,
  • T. Vinutha,
  • Suneha Goswami

摘要

Efficient genetic transformation continues to be a major bottleneck in soybean (Glycine max L.) genome editing. We used a stable and reproducible Agrobacterium-mediated transformation system to compare cotyledonary node (CN) and half-seed (HS) explants for CRISPR/Cas9-based editing. A binary vector harboring a CaMV 35 S-driven bar gene for glufosinate selection and a Cas9-sgRNA cassette targeting the Apetala1 (AP1) homolog was introduced into Agrobacterium tumefaciens. Transformation efficiencies were 28.15% (CN) and 22.0% (HS). The CN method showed higher transformation efficiency, faster shoot elongation, and a shorter regeneration cycle, whereas HS explants had fewer escapes. Quantitative PCR confirmed Cas9 integration and expression, with HS-derived lines showing higher transcript accumulation (4.17 ± 0.13 to 6.06 ± 0.10-fold) than CN-derived lines (2.17 ± 0.02 to 5.70 ± 0.02-fold). The optimized hormone regime (zeatin, GA₃, and IBA) significantly enhanced shoot elongation and overall regeneration. Stable integration was validated through T₁ inheritance and glufosinate resistance assays. This comparative framework establishes a robust and reproducible platform for efficient, high-throughput genome editing in soybean.