Optimization of Minimal Inhibitory Dose of Selective Agent (Basta) for Selection of Transgenics in Sugarcane

Abstract

Success rate in the selection of putative transformants is mainly dependent upon the dose rate of selective agent. Selection pressure determines the survival frequency of transformant and non transformant cells. Dose of selective agent, which is lethal for the non transformants, would be the optimum for the transgenic selection. We investigated the optimum dose of basta (selective agent), in two sugarcane genotypes viz., S-2003-us-359 and S-2003-us-127, in which we have already established a proficient in vitro regeneration system (Ijaz et al., 2012; Anjum et al., 2012). Different dosages of basta viz., 1 mg/L, 3 mg/L, 5 mg/L, 7 mg/L and 10 mg/L were studied for regeneration media (RM) of both genotypes. For determining the optimal inhibitory dose of basta on regeneration, 21 days old calli of S-2003-us-359 were induced on CIM1, 28 days old calli of S-2003-us-127 were induced on CIM2 and were shifted to RSM1 and RSM2 respectively. In genotype S-2003-us-359, early callus death was observed on RSM having 10 mg/L basta, and RSM having 5 mg/L, 7 mg/L, 10 mg/L basta proved to be lethal for S-2003-us-127 with early death of calli. For genotype S-2003-us-127, among 5 mg/L, 7 mg/L and 10 mg/L basta levels, 5 mg/L basta was selected for regeneration media as optimal dose for the selection of transgenics of this genotype. For transgenic selection in both sugarcane genotypes, the affectivity of selected optimal doses of basta was checked. Twenty one (21) days old calli of S-2003-us-359 and 28 days old calli of S-2003-us-127 were bombarded with bar gene and plant were selected at 10 mg/L and 5 mg/L of basta respectively. Putative transgenic plants with integrated copy of bar gene were confirmed with PCR analysis.