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Key message <p>A composite hypocotyl–epicotyl–cotyledonary tri-complex (HECC) explant significantly improves soybean regeneration and <i>Agrobacterium</i>-mediated transformation efficiency (40.3%) while reducing genotype dependence, providing a rapid and robust platform for functional genomics.</p> Abstract <p>Soybean genetic transformation is hindered by low regeneration efficiency and considerable genotype dependency. Here, we present a hormone-optimized <i>Agrobacterium</i>-mediated transformation system employing a composite hypocotyl–epicotyl–cotyledonary tri-complex (HECC) explant that integrates multiple meristematic regions within a single explant and significantly improves regeneration and transformation efficiency. Compared to conventional single-meristem explant systems such as cotyledonary node and half-seed explants, HECC explants exhibited substantially higher regeneration frequencies and achieved an average transformation efficiency of 40.3%, calculated as the number of PCR-positive shoots obtained per infected explant in the JS-335 cultivar. Optimization of hormone combinations, particularly <i>trans</i>-zeatin riboside in shoot induction and elongation and indole-3-butyric acid (IBA) during rooting, promoted robust organogenic responses and reduced tissue damage. The system supported efficient transformation (26–41%) across multiple soybean cultivars, including JS-335, JS-2034, JS-2069 and PUSA-9712, indicating reduced genotype dependence compared with conventional explant systems. To demonstrate the applicability of this system for genome engineering, a CRISPR/<i>Cas9</i> construct targeting <i>GmSPL9c</i> (Glycine max SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9c), a transcription factor involved in regulating plant developmental transitions and shoot architecture, was introduced. Molecular analyses by PCR and qRT-PCR confirmed the integration and expression of <i>cas9</i> and <i>bar</i> genes, while segregation analysis in T<sub>1</sub> progeny demonstrated heritable transmission consistent with a 3:1 Mendelian segregation ratio. Functional selection by BASTA<sup>®</sup> spraying in the T<sub>1</sub> generation further validated glufosinate tolerance and inheritance of the <i>bar</i> transgene. Collectively, this HECC-based system provides a reliable and reproducible platform for efficient soybean transformation and delivery of genome-editing constructs for functional genomics applications.</p>

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Hormone-optimized hypocotyl–epicotyl–cotyledonary tri-complex explant system enables efficient soybean transformation with reduced genotype dependence

  • 期刊论文

  • 发表日期：

    2026年5月26日

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  • J. Sushmitha,
  • Durgeshwari Prabhakar Gadpayale,
  • Navita Bansal,
  • Ranjeet R. Kumar,
  • G. Rama Prashat,
  • Shivani Nagar,
  • Soham Ray,
  • Ashish Marathe,
  • R. Dinesh Kumar,
  • Akshay Talukdar,
  • Giriraj Kumawat,
  • Viswanathan Chinnusamy,
  • Suneha Goswami,
  • T. Vinutha

摘要

Key message

A composite hypocotyl–epicotyl–cotyledonary tri-complex (HECC) explant significantly improves soybean regeneration and Agrobacterium-mediated transformation efficiency (40.3%) while reducing genotype dependence, providing a rapid and robust platform for functional genomics.

Abstract

Soybean genetic transformation is hindered by low regeneration efficiency and considerable genotype dependency. Here, we present a hormone-optimized Agrobacterium-mediated transformation system employing a composite hypocotyl–epicotyl–cotyledonary tri-complex (HECC) explant that integrates multiple meristematic regions within a single explant and significantly improves regeneration and transformation efficiency. Compared to conventional single-meristem explant systems such as cotyledonary node and half-seed explants, HECC explants exhibited substantially higher regeneration frequencies and achieved an average transformation efficiency of 40.3%, calculated as the number of PCR-positive shoots obtained per infected explant in the JS-335 cultivar. Optimization of hormone combinations, particularly trans-zeatin riboside in shoot induction and elongation and indole-3-butyric acid (IBA) during rooting, promoted robust organogenic responses and reduced tissue damage. The system supported efficient transformation (26–41%) across multiple soybean cultivars, including JS-335, JS-2034, JS-2069 and PUSA-9712, indicating reduced genotype dependence compared with conventional explant systems. To demonstrate the applicability of this system for genome engineering, a CRISPR/Cas9 construct targeting GmSPL9c (Glycine max SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9c), a transcription factor involved in regulating plant developmental transitions and shoot architecture, was introduced. Molecular analyses by PCR and qRT-PCR confirmed the integration and expression of cas9 and bar genes, while segregation analysis in T1 progeny demonstrated heritable transmission consistent with a 3:1 Mendelian segregation ratio. Functional selection by BASTA® spraying in the T1 generation further validated glufosinate tolerance and inheritance of the bar transgene. Collectively, this HECC-based system provides a reliable and reproducible platform for efficient soybean transformation and delivery of genome-editing constructs for functional genomics applications.