Development of Agrobacterium-mediated in planta transformation protocol through coleoptile in rice

Genetic modification of rice is mainly carried out by Agrobacterium-mediated transformation of callus accompanied by tissue culture. It is time consuming, laborious and unapplicable for cultivars unable to induce callus. In this study, we have reported a novel gene transfer protocol that involves pulling out primary leaf from coleoptile and injection of Agrobacterium culture into the empty channel. Out of 25 plants survived after injection of Agrobacterium tumefaciens EHA105 culture harboring pCAMBIA1301-RD29A-AtDREB1A, 8 T₀ plants revealed the expected size of around 811 bp corresponding to AtDREB1A gene and Southern blotting analysis on 18 T₁ plants suggested introgression of AtDREB1A. 3 T₂ lines (7-9, 12-3, 18-6) exhibited accumulation of free proline and soluble sugars, yet increase of chlorophyll content, but decrease of electrolyte leakage and methane dicarboxylic aldehyde under cold stress condition at the vegetative growth stage. Yield components investigation on T₂ lines showed earlier heading date and no yield loss compared to wild type plants grown under normal condition. GUS expression analysis and integrated transgene detection in T₀ and T₁ plants followed by evaluation of cold stress tolerance in T₂ lines suggest the advantage of this in planta transformation protocol to obtain transgenic rice.