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<p>The present study demonstrates an efficient system for hairy root induction using two different strains (A4 and ATCC 15834) of <i>A. rhizogenes</i> with a non-invasive visual marker (RUBY) in two different explants (leaf and nodes) of <i>Coleus forskohlii</i>. <i>C. forskohlii</i> is a potent medicinal plant of the family Lamiaceae. It is the only natural source of forskolin, an essential bioactive molecule with several therapeutic applications in modern and traditional healthcare systems. The present study evaluated several factors influencing transformation efficiency, including explant type, pre-culture duration, acetosyringone concentration, bacterial strain, bacterial cell density, infection duration, and co-cultivation duration. The highest transformation efficiency (58.75 ± 0.14) was achieved using the A4 strain of <i>A. rhizogenes</i> with nodal explants that were pre-cultured for 2&#xa0;days, infected with bacterial cells at an optical density of 0.6 (OD<sub>600</sub>) for 20&#xa0;min, and co-cultivated for 60&#xa0;h on a basal MS medium supplemented with 100&#xa0;μM acetosyringone. The successful gene transformation was confirmed by the production of pink-coloured hairy roots expressing the RUBY reporter gene. The molecular analysis of these roots further validated the integration of the hptII gene through gel profiling of PCR amplification using hptII-specific primers. The presence of a band at 314&#xa0;bp confirmed the successful integration of the hptII gene. The method developed is helpful in the hairy root cultures of <i>C. forskohlii</i> for various applications, such as the transfer of novel candidate genes and genome editing studies to improve metabolic engineering pathways.</p>

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Optimization of Agrobacterium-mediated gene transformation using a non-invasive visual reporter gene (RUBY) in Coleus forskohlii (Willd) Briq.

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月17日

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  • Mahesh Kumar Badhepuri,
  • Phanikanth Jogam,
  • Vaishnavi Anumula,
  • Sathish Kumar Durgam,
  • Swathy Gottimukkula,
  • Venkataiah Peddaboina,
  • Nageswara Rao Singisala

摘要

The present study demonstrates an efficient system for hairy root induction using two different strains (A4 and ATCC 15834) of A. rhizogenes with a non-invasive visual marker (RUBY) in two different explants (leaf and nodes) of Coleus forskohlii. C. forskohlii is a potent medicinal plant of the family Lamiaceae. It is the only natural source of forskolin, an essential bioactive molecule with several therapeutic applications in modern and traditional healthcare systems. The present study evaluated several factors influencing transformation efficiency, including explant type, pre-culture duration, acetosyringone concentration, bacterial strain, bacterial cell density, infection duration, and co-cultivation duration. The highest transformation efficiency (58.75 ± 0.14) was achieved using the A4 strain of A. rhizogenes with nodal explants that were pre-cultured for 2 days, infected with bacterial cells at an optical density of 0.6 (OD600) for 20 min, and co-cultivated for 60 h on a basal MS medium supplemented with 100 μM acetosyringone. The successful gene transformation was confirmed by the production of pink-coloured hairy roots expressing the RUBY reporter gene. The molecular analysis of these roots further validated the integration of the hptII gene through gel profiling of PCR amplification using hptII-specific primers. The presence of a band at 314 bp confirmed the successful integration of the hptII gene. The method developed is helpful in the hairy root cultures of C. forskohlii for various applications, such as the transfer of novel candidate genes and genome editing studies to improve metabolic engineering pathways.