In Planta transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice ( Oryza sativa L.) cultivar

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T1 seeds. T0 plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T1 was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T2 generation. Transformation efficiency was found to be 30–32% at the T0 generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na+/K+, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T2 generation.