AN IMPROVED PROTOCOL FOR AGROBACTERIUM MEDIATED GENETIC TRANSFORMATION AND REGENERATION OF INDICA RICE (ORYZA SATIVA L. VAR. RAJENDRA KASTURI)
Agrobacterium mediated genetic transformation is widely adopted in rice as it is inexpensive and produces fewer transgene copies integration in genome compared to other methods of transformation. However, this method requires an efficient protocol for callus induction and regeneration after Agrobacterium infection. Recently, efforts have been made to develop efficient methods for Agrobacterium mediated transformation and regeneration of japonica, and very few indica rice varieties. Unfortunately, the local indica rice varieties have remained untouched as they are recalcitrant to regeneration, leading to low transformation efficiency. In present investigation, an improved Agrobacterium mediated genetic transformation protocol has been developed for local indica rice var. Rajendra Kasturi. The highest frequency of callus induction was observed in MS salt supplemented with 2,4 D (2.25 mg/L) Co cultivation of Agrobacterium with rice calli on medium containing 100 mM acetosyringone has yielded maximum transformation efficiency. The selection of transformed calli was done on medium containing 10 mg/l phosphinothricin (PPT) at which untransformed calli couldn't regenerate. Maximum regeneration frequency was observed on MS salt containing 2.5 mg/l BAP, 0.5 mg/l NAA and 1.25 mg/l kinetin along with 30 g/l sucrose and 1.5% sorbitol. Optimum rooting was observed in 2/5th strength of MS salts with 2 g of clarigel. The integration of transgene in T1 and T2 generation plants was confirmed by PCR. RT PCR analysis confirms the expression of BAR gene transcripts in T2 transgenic rice. Using this improved protocol indica rice variety var. Rajendra Kasturi was successfully transformed and raised to a full grown plant within 3 months of time.