Efficient induction of embryogenic callus and biolistic-mediated transient transformation in the CP57-614 sugarcane cultivar (Saccharum officinarum L.)

Document Type : Research paper

Authors

1 Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

2 Faculty of Marine Science and Oceanography, Khorramshahr University of Marine Science and Technology, Khorramshahr, Iran.

Abstract

This study aimed to optimize embryogenic callus formation and transformation of the CP57-614 sugarcane variety using particle bombardment. Young leaf explants were cultured on modified Murashige and Skoog (MS) medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; auxin) (1, 2, and 4 mg L-1) and two Dicamba concentrations (4 and 6 mg L-1). The highest callus induction rate was achieved with 2 mg L-1 of 2,4-D. Subsequently, particle bombardment was used to introduce genetic constructs containing the uidA reporter gene driven by three promoters (CaMV35S, ubiquitin, and actin1) into the embryogenic calli. We assessed the impact of various biological, physical, and DNA parameters, including bombardment pressure, target distance, particle size, DNA amount, and osmotic pretreatment, through histochemical GUS assay to quantify blue spots. Results showed that the most effective combination involved bombardment at 1100 psi, 6 cm target distance, 1 μm gold particles coated with 1 μg DNA, under a vacuum of 25 inHg. Additionally, the best osmotic pretreatment utilized a solution containing 0.2 M sorbitol and 0.2 M mannitol. Notably, the construct driven by the ubiquitin promoter resulted in the highest transient expression of the uidA gene, suggesting its superiority for gene expression in this system.

Keywords

References

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History

  • Receive Date: 08 January 2024
  • Revise Date: 13 July 2024
  • Accept Date: 14 July 2024

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