TRANSFORMATION OF THE OSC3H52 GENE PROMOTER IN TISSUE CULTURE OF SUGARCANE

M. S. Khan1*, W. Ahmad1, M. Z. Ahmad2, M. Ullah1 and A. Jan1

1Genomics and Bioinformatics Division, Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan.

2Dr. M. Ajmal Khan Institute of Sustainable Halophyte Utilization (ISHU),

University of Karachi, Karachi, Pakistan

*Corresponding author’s email: sayyarkhankazi@aup.edu.pk

ABSTRACT

Sugarcane growth is negatively affected by abiotic stresses. Genetic engineering of sugarcane with abiotic stress-responsive genes has been the method of choice to confer abiotic stress tolerance. In the present work, we optimized callus induction and transformation of the sugarcane variety CP-77-400 with the promoter region of the OsC3H52 gene to analyze its regulatory function under drought and salt stress. Calli were induced using different callus induction media (CIM). The promoter region of the OsC3H52 gene that belongs to the zinc finger protein family was cloned into two different expression vectors i.e. pBI221 and pGreenII0129, which were subsequently transformed to sugarcane calli through agrobacterium and biolistic transformation methods. Among various callus induction media, CIM3 (5 mg L-1 2,4-D + 10% coconut water) showed high callus induction (93%). Biolistic transformation using recombinant pGreenII0129 at 8.5µg/µl 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 cefotaxime for 15 days, both for biolistic and agrobacterium mediated transformation, respectively. Transformed calli were then successfully confirmed through PCR and GUS expression. The regulatory role of the OsC3H52 promoter in transgenic calli was further evaluated under drought and salt stress. Quantitative real-time PCR analysis of the GUS reporter gene showed high expression in transgenic calli under drought and salt stress conditions.

Key words: Abiotic stress, Callus induction, Gene Transformation, GUS Analysis, Saccharum officinarum.