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<p><i>In vitro</i> regeneration was successfully achieved through both somatic embryogenesis and direct organogenesis using the shoot apical meristem as an explant of pearl millet. AgNO<sub>3</sub>, an anti-necrotic compound, increased the frequency of induction of embryogenic callus from 30 to 75%. Embryogenic callus development progressed from the globular to the scutellar stage, as visualized by scanning electron microscopy (SEM). Auxin transport inhibitor was evaluated for the first time to improve <i>in vitro</i> regeneration from rhizogenous pearl millet callus. Notably, 2,3,5-triiodobenzoic acid (TIBA) at 0.75&#xa0;mg L<sup>−1</sup> significantly improved the regeneration frequency from 21.6% to 33%. In addition to improving <i>in vitro</i> regeneration systems, we also developed an efficient <i>Agrobacterium</i>-mediated transformation system for pearl millet. The <i>Agrobacterium</i> strain EHA105, harbouring the modified binary vector pBI121 (NBRI1.2), was used to optimize the transformation conditions. It contains <i>npt</i>II and <i>uid</i>A as selectable and visual reporters, respectively. Explant age, infection, and vacuum infiltration duration, concentrations of acetosyringone, kanamycin, and cefotaxime, and the inclusion of anti-necrotic compounds were all examined. Inclusion of cotton leaf extract during <i>Agrobacterium-</i>mediated transformation improved the transformation efficiency. In addition, supplementing the recovery medium with L-Cysteine increased both transient and stable GUS expression frequency. The presence of the <i>uid</i>A and <i>npt</i>II genes in T<sub>1</sub> progeny was further confirmed by PCR, which produced the expected 510&#xa0;and 531&#xa0;bp amplicons, respectively. Real-time PCR revealed a 3.5-fold increase in the <i>uid</i>A gene expression in the T<sub>1</sub> progeny.</p>

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Role of anti-necrotic compounds and auxin inhibitor on regeneration and Agrobacterium-mediated transformation of pearl millet (Pennisetum glaucum L.)

  • 期刊论文

  • 发表日期：

    2026年4月1日

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  • Anshu Sahu,
  • Uma Gupta,
  • Shashi Kashyap,
  • Liptikanta Bhoi,
  • Indraneel Sanyal

摘要

In vitro regeneration was successfully achieved through both somatic embryogenesis and direct organogenesis using the shoot apical meristem as an explant of pearl millet. AgNO3, an anti-necrotic compound, increased the frequency of induction of embryogenic callus from 30 to 75%. Embryogenic callus development progressed from the globular to the scutellar stage, as visualized by scanning electron microscopy (SEM). Auxin transport inhibitor was evaluated for the first time to improve in vitro regeneration from rhizogenous pearl millet callus. Notably, 2,3,5-triiodobenzoic acid (TIBA) at 0.75 mg L−1 significantly improved the regeneration frequency from 21.6% to 33%. In addition to improving in vitro regeneration systems, we also developed an efficient Agrobacterium-mediated transformation system for pearl millet. The Agrobacterium strain EHA105, harbouring the modified binary vector pBI121 (NBRI1.2), was used to optimize the transformation conditions. It contains nptII and uidA as selectable and visual reporters, respectively. Explant age, infection, and vacuum infiltration duration, concentrations of acetosyringone, kanamycin, and cefotaxime, and the inclusion of anti-necrotic compounds were all examined. Inclusion of cotton leaf extract during Agrobacterium-mediated transformation improved the transformation efficiency. In addition, supplementing the recovery medium with L-Cysteine increased both transient and stable GUS expression frequency. The presence of the uidA and nptII genes in T1 progeny was further confirmed by PCR, which produced the expected 510 and 531 bp amplicons, respectively. Real-time PCR revealed a 3.5-fold increase in the uidA gene expression in the T1 progeny.