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<p>This study investigated the functional activity of two developmentally regulated promoters—CcKIP1 from <i>Cenchrus ciliaris</i> and <i>HvNUC</i> from <i>Hordeum vulgare</i>—by testing them in <i>C. ciliaris</i>, a drought-tolerant forage grass, using the <i>uidA</i> reporter gene system. A constitutive <i>CaMV35S</i> promoter was used as a positive control. The constructs were subsequently introduced into 56- and 72-wk-old embryogenic calluses <i>via</i> biolistic transformation at four rupture disc pressures (600, 900, 1100, and 1350 psi). Of these pressures, 1100 psi was identified as optimal for transient <i>uidA</i> expression. Transient expression in 56-wk-old callus was highest with <i>CaMV35S::uidA</i> (~ 14%), followed by <i>CcKIP1::uidA</i> (approximately 10%) and <i>HvNUC::uidA</i> (approximately 7%). The corresponding stable expression frequencies were 6 to 7% for <i>CaMV35S</i>, 4% for <i>CcKIP1</i>, and 3% for <i>HvNUC</i>. In contrast, the transformation efficiency decreased in 72-wk-old callus across all the constructs. Hygromycin selection (used for the <i>CaMV35S</i> and <i>CcKIP</i>1 constructs) was more stringent than kanamycin selection (used for <i>HvNUC</i>), as evidenced by a sharper reduction in resistant callus during selection. Regeneration was observed in callus transformed with <i>CcKIP1::uidA</i> (one plant) and <i>HvNUC::uidA</i> (two plants), whereas <i>CaMV35S::uidA</i>-transformed callus failed to regenerate despite higher expression levels. However, transgenes could not be detected in the regenerated plants. Overall, both the <i>CcKIP1</i> and <i>HvNUC</i> promoters were active in <i>C. ciliaris</i> embryogenic tissues. These findings demonstrate the potential of developmentally regulated promoters in improving transformation and regeneration strategies for monocot species with limited amenability to genetic engineering.</p>

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CcKIP1 and HvNUC promoters activity in Cenchrus ciliaris L.

  • 期刊论文

  • 发表日期：

    2026年3月13日

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  • Shipra Goyal,
  • Pankaj Kumar Agnihotri,
  • Vishvas M. Kulkarni,
  • Vishnu Bhat

摘要

This study investigated the functional activity of two developmentally regulated promoters—CcKIP1 from Cenchrus ciliaris and HvNUC from Hordeum vulgare—by testing them in C. ciliaris, a drought-tolerant forage grass, using the uidA reporter gene system. A constitutive CaMV35S promoter was used as a positive control. The constructs were subsequently introduced into 56- and 72-wk-old embryogenic calluses via biolistic transformation at four rupture disc pressures (600, 900, 1100, and 1350 psi). Of these pressures, 1100 psi was identified as optimal for transient uidA expression. Transient expression in 56-wk-old callus was highest with CaMV35S::uidA (~ 14%), followed by CcKIP1::uidA (approximately 10%) and HvNUC::uidA (approximately 7%). The corresponding stable expression frequencies were 6 to 7% for CaMV35S, 4% for CcKIP1, and 3% for HvNUC. In contrast, the transformation efficiency decreased in 72-wk-old callus across all the constructs. Hygromycin selection (used for the CaMV35S and CcKIP1 constructs) was more stringent than kanamycin selection (used for HvNUC), as evidenced by a sharper reduction in resistant callus during selection. Regeneration was observed in callus transformed with CcKIP1::uidA (one plant) and HvNUC::uidA (two plants), whereas CaMV35S::uidA-transformed callus failed to regenerate despite higher expression levels. However, transgenes could not be detected in the regenerated plants. Overall, both the CcKIP1 and HvNUC promoters were active in C. ciliaris embryogenic tissues. These findings demonstrate the potential of developmentally regulated promoters in improving transformation and regeneration strategies for monocot species with limited amenability to genetic engineering.