PJB-2019-664
OPTIMIZATION OF TISSUE CULTURE AND TRANSFORMATION OF THE SUGARCANE VARIETY (CP 77/400) WITH THE PROMOTER OF OSC3H52 GENE
Mohammad Sayyar Khan
Abstract
Sugarcane growth and productivity is negatively impacted by abiotic stresses. Transgenic approach using abiotic stress-responsive genes has been the method of choice to produce stress tolerance. However, tissue culture optimization and transformation are challenging in sugarcane depending on the type of variety, gene construct and transformation method. In the present work, we optimized callus induction and transformation of the sugarcane variety CP-77-400 with the promoter region of OsC3H52 gene to analyze its regulatory function. Calli were induced using different callus induction media (CIM). The promoter region of C3H52 gene that belongs to zinc finger protein family was cloned in two types of expression vectors i.e. pBI221 and pGreenII0129 and subsequently transformed to sugarcane calli through agrobacterium and biolistic transformation methods. The putative transgenic calli were confirmed with PCR and GUS analysis. Amongst the various media used for callus induction, CIM3 (5 mg L-1 2,4-D + 10% coconut water) showed maximum callus induction (93%). Biolistic transformation using recombinant pGreenII0129 at 8.5µg concentration resulted in higher efficiency of transformation (28%). While the agrobacterium mediated transformation using recombinant pBI221 plasmid gave 13% transformation efficiency. Amongst the various concentrations of acetosyringone used in agrobacterium mediated transformation, 100 µM showed higher efficiencies (13%) of transformation. Selection of the transformed calli was performed at 25 mg L-1 Hygromycin and 25 mg L-1 Hygromycin + 300 mg L-1 cefetoxime for 15 days, both for biolistic and agrobacterium mediated transformation, respectively. The transformed calli were then successfully confirmed through PCR and GUS expression.
To Cite this article:
Download
