Determination of minimal inhibitory concentration of kanamycin as selective agents and marker genes for use in Agrobacterium mediated transformation in sugarcane

Abstract

Efficient genetic transformation of sugarcane requires an effective dose of selective antibiotics/agents for differentiating actively transformed cells from non-transformed ones. The objective of the present study was to optimize and establish lethal minimal inhibitory concentration of kanamycin that could be used to select true transformed shoots of sugarcane. A step-wise increase doses of kanamycin antibiotic from 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 and then to 120 mg L-1 as a selection agent were evaluated for their effectiveness in transformation of sugarcane elite variety CoS 96268. In vitro regenerated shoots were inoculated on MS medium containing 0.8 mg L-1 BAP + 0.1 mg L-1 NAA with various concentrations of kanamycin. At lower concentrations of kanamycin (10-40 mg L-1), the growth of shoots were unaffected and the survival rates were substantially high (100%). Chlorosis starts at a kanamycin concentration of 50 mg L-1. Survival percentage of the non-transformed shoots was decreasing gradually with the step-wise increase of kanamycin concentration. The most effective concentration of kanamycin was found to be 100 mg L-1 at which all the non-transformed (susceptible) shoots were died after four weeks of inoculation. Maximum transformed shoots were obtained at 100 mg L-1 kanamycin for sugarcane with a death rate of more than 95% of non-transformed shoots on selection medium. Among the different antibiotics doses tested for selection of transformants, kanamycin at 100 mg L-1 was found to be optimum. So worked out for the selection of transformed shoots of CoS 96268 could be used as selective npt II marker in other sugarcane transformation practices.