Biolistic transformation of sugarcane (Saccharum officinarum L.) with the Oryza sativa L. H+-PPase gene for improved salt stress tolerance
Keywords:biolistic transformation, OVP1, sugarcane, salinity stress, tissue culture
Several biotic and abiotic stresses adversely affect sugarcane crop. Amongst the abiotic stresses, salinity causes substantial losses to sugarcane yield. Lack of conducive environment for sugarcane flowering and unstable genome are the main constraints in sugarcane crop improvement via conventional breeding. Therefore, genetic engineering of sugarcane using Oryza sativa L. H+-PPase gene (OVP1) to confer salt tolerance is the preferred alternative strategy. In the present research, tissue culture and biolistic transformation protocols for sugarcane were optimized. For callus induction, Dichlorophenoxyacetic acid (2,4-D) was most effective. Maximum callus induction (50.22%) was achieved on CM-3 augmented with 2.5 mgL-1 2,4-D. The induced calli was subjected to biolistic transformation. For biolistic transformation, the gold particles (0.6 and 1.0 µm) and plasmid DNA (100 and 50 ng) were used for 1X and 2X bombardment. Significant differences (P≤0.05) were observed amongst the gold particles and plasmid DNA concentrations. Maximum transformation frequencies were observed when calli were bombarded 2X with 0.6 µm gold particles coated with 100 ng of DNA (23.60%). Selection and regeneration of transgenic calli were performed on MS media augmented with BAP (2.5 mgL-), NAA (1 mgL-1) hygromycin (25 mgL-1) and 2% coconut water. Regenerated plants after root induction on MS media containing NAA (2 mgL-1) and 2% coconut water were transferred to loam and clay (1:1) and acclimatized to greenhouse condition. The transgenic plants were confirmed through PCR and RT-PCR.
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