The effect of antibiotics on the shoot regeneration in apple cultivar “Golden Delicious”

Abstract

The effect of antibiotics hygromycin, cefotaxime and meropenem on shoot regeneration in apple cultivar “Golden Delicious” was evaluated to optimize the protocol for Agrobacterium tumefaciens-mediated genetic transformation. The careful determination of antibiotics concentration is important step to ensure efficient elimination of Agrobacterium and appropriate selection process without escapes and detrimental effect on plant regeneration. Healthy and fully expanded young leaves of in vitro grown apple shoots were cut transversely into three pieces that were used as explants for shoot regeneration. Regeneration frequency, the mean number of shoots per explant and the index of shoot-forming capacity were calculated after 8 weeks of culturing on Murashige and Skoog medium containing 5 mg L-1 thidiazuron (TDZ) and 0.3 mg L-1 indole- 3-butyric acid (IBA), supplemented with hygromycin (0, 2, 4, 5, 6, 8 and 10 mg L-1), cefotaxime (0, 100, 200, 300, 400, 500 and 600 mg L-1) or meropenem (0, 25, 50, 75, 100, 125 and 150 mg L-1). Results indicated that the highest index of shoot-forming capacity was obtained at 300 mg L-1 cefotaxime (3.67) comparing to control (2.93) and other antibiotic treatments. The increase of hygromycin concentration significantly decreased regeneration efficiency, while at concentrations of 5 mg L-1 and higher it completely inhibited shoot regeneration. Therefore, the present study outlines the use of cefotaxime at 300 mg L-1 and a stepwise selection procedure with the final hygromycin concentration of 5 mg L-1 as a suitable system for A. tumefaciens-mediated genetic transformation of apple cultivar “Golden Delicious”.