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<p>A method of <i>Agrobacterium</i> mediated plant regeneration using petiole explants and transformation of pBI121 CaMV 35S:<i>GUS</i> construct and MDC123 CaMV 2 × 35S:Cas9:<i>MCAGL6</i>-sgRNA construct having <i>Bar</i> gene as plant selection marker was optimized for the first time in bitter melon. Different combination of cytokinins (BAP, Kinetin, TDZ) and auxins (2,4-D, IAA, NAA) were used in callus induction media (CIM) and shoot induction media (SIM) to regenerate plants from petiole (P) and leaf (L) explants taken from three varieties (BSS578, KSP1522, and KSP1277) and abbreviated as LCIM/PCIM and LSIM/PSIM. Leaf calli failed to induce shoot regeneration despite of 44.66 to 46.33% callus induction. However, significantly higher percentage (90.9%) of multiple shoot proliferation found in PSIM2 using petiole as explant with the callus induction of 57% in KSP1522. Based on the varietal response for callus and shoot induction, petiole explant mediated transformation protocol was selected and optimized for pre-culture period, acetosyringone concentration and phosphinothricin concentration (<i>Bar</i> gene as a selectable marker). <i>Agrobacterium</i> mediated transformation of <i>GUS</i> with plant selection marker kanamycin (80&#xa0;mg L<sup>−1</sup>) showed 10–11% transformation efficiency using 3&#xa0;days old pre-cultured explants and 250&#xa0;µM acetosyringone. Similar transformation efficiency percentage was observed for plant transformation of gene with phosphinothricin (0.5&#xa0;mg L<sup>−1</sup>) as plant selection. Thus, protocol efficiently works for both <i>GUS</i> <i>and</i> <i>Bar</i> insertion into bitter melon plants. The in vitro regeneration of bitter melon plant using petiole explant was more effective than leaf explant and variation in the percentage of plant regeneration capacity was highly varietal responsive.</p>

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Efficient Agrobacterium mediated plant regeneration using petiole explants and plant transformation of GUS and Bar gene in Momordica charantia L. plants

  • 期刊论文

  • 发表日期：

    2025年1月9日

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  • Sweta B. Kushwaha,
  • S. Vanchinathan,
  • Smita S. Lele,
  • G. Rama Prashat,
  • Suneha Goswami,
  • Ranjeet Ranjan Kumar,
  • Navita Bansal,
  • Arun Kumar,
  • Shelly Praveen,
  • Nandkumar Kunchge,
  • Jyoti S. Gokhale,
  • T. Vinutha

摘要

A method of Agrobacterium mediated plant regeneration using petiole explants and transformation of pBI121 CaMV 35S:GUS construct and MDC123 CaMV 2 × 35S:Cas9:MCAGL6-sgRNA construct having Bar gene as plant selection marker was optimized for the first time in bitter melon. Different combination of cytokinins (BAP, Kinetin, TDZ) and auxins (2,4-D, IAA, NAA) were used in callus induction media (CIM) and shoot induction media (SIM) to regenerate plants from petiole (P) and leaf (L) explants taken from three varieties (BSS578, KSP1522, and KSP1277) and abbreviated as LCIM/PCIM and LSIM/PSIM. Leaf calli failed to induce shoot regeneration despite of 44.66 to 46.33% callus induction. However, significantly higher percentage (90.9%) of multiple shoot proliferation found in PSIM2 using petiole as explant with the callus induction of 57% in KSP1522. Based on the varietal response for callus and shoot induction, petiole explant mediated transformation protocol was selected and optimized for pre-culture period, acetosyringone concentration and phosphinothricin concentration (Bar gene as a selectable marker). Agrobacterium mediated transformation of GUS with plant selection marker kanamycin (80 mg L−1) showed 10–11% transformation efficiency using 3 days old pre-cultured explants and 250 µM acetosyringone. Similar transformation efficiency percentage was observed for plant transformation of gene with phosphinothricin (0.5 mg L−1) as plant selection. Thus, protocol efficiently works for both GUS and Bar insertion into bitter melon plants. The in vitro regeneration of bitter melon plant using petiole explant was more effective than leaf explant and variation in the percentage of plant regeneration capacity was highly varietal responsive.