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<p>A reliable and efficient <i>Agrobacterium</i>-mediated genetic transformation protocol was developed for <i>Chamomilla matricaria</i>, a medicinally significant plant with growing biotechnological relevance. A direct regeneration system was optimized using leaf explants, identified as the sole responsive tissue for shoot organogenesis. Among various cytokinins evaluated, meta-topolin (mT) exhibited the highest morphogenetic potential. The optimal regeneration response (96.0 ± 6.92%) and maximum shoot proliferation (14.6 ± 0.08 shoots per explant) were observed on Murashige and Skoog (MS) medium supplemented with 2.0&#xa0;mg/L mT and 0.1&#xa0;mg/L NAA, significantly outperforming conventional BAP-based combinations (84.3 ± 6.35% regeneration; 5.4 ± 1.53 shoots per explant). Supplementation with adenine sulphate (1.5&#xa0;mg/L) improved shoot quality in BAP-treated cultures (9.3 ± 1.52 shoots per explant) but remained inferior to mT-mediated responses. Rooting was successfully achieved on hormone-free half-strength MS medium, indicating adequate endogenous auxin levels. For selection, 8&#xa0;mg/L kanamycin was optimal, enabling the recovery of transformed shoots with minimal toxicity. Transformation efficiency was further enhanced by optimizing bacterial density (OD<sub>600</sub>&#xa0;= 0.6), acetosyringone concentration (100 µM), and infection duration (15&#xa0;min). Transgene integration and expression were confirmed through PCR amplification of the <i>nptII</i> gene and GUS histochemical assays. This optimized protocol represents the first reported system for direct regeneration and stable genetic transformation in <i>Matricaria chamomilla</i>, offering a reliable and efficient platform for genetic enhancement and functional genomics investigations in this species.</p>

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Direct regeneration-based protocol for efficient Agrobacterium-mediated genetic transformation of Chamomilla matricaria

  • 期刊论文

  • 发表日期：

    2025年9月11日

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  • Sabika Akram,
  • Zakir Husain,
  • Gazala Parween,
  • Tanya singh,
  • Kahkashan Khatoon,
  • Zafar Iqbal,
  • Parmeshwar Singh,
  • Pooja Singh,
  • Laiq ur Rahman

摘要

A reliable and efficient Agrobacterium-mediated genetic transformation protocol was developed for Chamomilla matricaria, a medicinally significant plant with growing biotechnological relevance. A direct regeneration system was optimized using leaf explants, identified as the sole responsive tissue for shoot organogenesis. Among various cytokinins evaluated, meta-topolin (mT) exhibited the highest morphogenetic potential. The optimal regeneration response (96.0 ± 6.92%) and maximum shoot proliferation (14.6 ± 0.08 shoots per explant) were observed on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L mT and 0.1 mg/L NAA, significantly outperforming conventional BAP-based combinations (84.3 ± 6.35% regeneration; 5.4 ± 1.53 shoots per explant). Supplementation with adenine sulphate (1.5 mg/L) improved shoot quality in BAP-treated cultures (9.3 ± 1.52 shoots per explant) but remained inferior to mT-mediated responses. Rooting was successfully achieved on hormone-free half-strength MS medium, indicating adequate endogenous auxin levels. For selection, 8 mg/L kanamycin was optimal, enabling the recovery of transformed shoots with minimal toxicity. Transformation efficiency was further enhanced by optimizing bacterial density (OD600 = 0.6), acetosyringone concentration (100 µM), and infection duration (15 min). Transgene integration and expression were confirmed through PCR amplification of the nptII gene and GUS histochemical assays. This optimized protocol represents the first reported system for direct regeneration and stable genetic transformation in Matricaria chamomilla, offering a reliable and efficient platform for genetic enhancement and functional genomics investigations in this species.