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<p>A direct organogenesis-mediated <i>Agrobacterium tumefaciens</i>-based transformation system was developed in pea (<i>Pisum sativum</i> L. cv. Ageta 6) using green-synthesized bioengineered nanoparticles (BENPs) to improve transformation efficiency. Titanium dioxide (TiO₂), silver (Ag), and acetosyringone (AS)-doped AS–Ag–TiO₂ BENPs were synthesized through a green hydrothermal approach and evaluated under optimized co-cultivation, sonication, and vacuum infiltration conditions. The synthesized BENPs were previously characterized through XRD, SEM, EDAX, HR-TEM, SAED, XPS, and cyclic voltammetry analyses, confirming ultrafine crystalline nanostructures and stable nanocomposite formation. Regenerated putative transformants were assessed through RAPD-based genetic fidelity analysis and further validated by histochemical GUS assays, <i>bar</i> gene-specific PCR amplification, and RT-PCR expression analysis. Among all treatments evaluated, AS–Ag–TiO₂ BENPs at 4 ppm produced the highest transformation efficiency (30.66%), followed by AS–TiO₂ (23.33%), Ag–TiO₂ (18.33%), and TiO₂ (13.33%), whereas the conventional AS-mediated transformation system produced only 8.66% transformation efficiency under identical optimized conditions and BASTA<sup>®</sup> selection pressure. RT-PCR analysis further demonstrated relatively enhanced <i>bar</i> transcript accumulation in AS–Ag–TiO₂-treated transformants compared with other BENPs-assisted treatments. The enhanced transformation response may be associated with improved <i>Agrobacterium</i> attachment, <i>vir</i> gene activation, intracellular T-DNA transfer, and enhanced explant survival during co-cultivation. In addition, the AS–Ag–TiO₂ nanocomposite may facilitate proton-coupled electron transfer (PCET)-associated alternative protonation mechanisms supporting enhanced <i>VirA/VirG</i> signalling and transformation competency. Collectively, the present study establishes an efficient and cost-effective BENPs-assisted transformation platform for recalcitrant legumes and highlights the potential application of multifunctional nanocomposites in plant genetic transformation and nano-enabled biomolecule delivery systems.</p>

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Synergetic role of As-Ag-TiO2 nanoparticles on organogenesis dependent Agrobacterium mediated pea (Pisum sativum L.) genetic transformation

  • 期刊论文

  • 发表日期：

    2026年7月9日

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  • Ajithan Chandrasekaran,
  • Magdalin Sylvia Singarayar,
  • Thangamuniyandi Pilavadi,
  • Prabhjot Kaur,
  • Geung-Joo Lee,
  • Manickavasagam Markandan

摘要

A direct organogenesis-mediated Agrobacterium tumefaciens-based transformation system was developed in pea (Pisum sativum L. cv. Ageta 6) using green-synthesized bioengineered nanoparticles (BENPs) to improve transformation efficiency. Titanium dioxide (TiO₂), silver (Ag), and acetosyringone (AS)-doped AS–Ag–TiO₂ BENPs were synthesized through a green hydrothermal approach and evaluated under optimized co-cultivation, sonication, and vacuum infiltration conditions. The synthesized BENPs were previously characterized through XRD, SEM, EDAX, HR-TEM, SAED, XPS, and cyclic voltammetry analyses, confirming ultrafine crystalline nanostructures and stable nanocomposite formation. Regenerated putative transformants were assessed through RAPD-based genetic fidelity analysis and further validated by histochemical GUS assays, bar gene-specific PCR amplification, and RT-PCR expression analysis. Among all treatments evaluated, AS–Ag–TiO₂ BENPs at 4 ppm produced the highest transformation efficiency (30.66%), followed by AS–TiO₂ (23.33%), Ag–TiO₂ (18.33%), and TiO₂ (13.33%), whereas the conventional AS-mediated transformation system produced only 8.66% transformation efficiency under identical optimized conditions and BASTA® selection pressure. RT-PCR analysis further demonstrated relatively enhanced bar transcript accumulation in AS–Ag–TiO₂-treated transformants compared with other BENPs-assisted treatments. The enhanced transformation response may be associated with improved Agrobacterium attachment, vir gene activation, intracellular T-DNA transfer, and enhanced explant survival during co-cultivation. In addition, the AS–Ag–TiO₂ nanocomposite may facilitate proton-coupled electron transfer (PCET)-associated alternative protonation mechanisms supporting enhanced VirA/VirG signalling and transformation competency. Collectively, the present study establishes an efficient and cost-effective BENPs-assisted transformation platform for recalcitrant legumes and highlights the potential application of multifunctional nanocomposites in plant genetic transformation and nano-enabled biomolecule delivery systems.