Cotyledon with hypocotyl segment as an explant for the production of transgenic citrullus vulgaris schrad (watermelon) mediated by agrobacterium tumefaciens

Abstract

In this study, we describe the successful production of transgenic watermelon using Agrobacterium tumefaciens after optimizing a couple of transformation parameters using cotyledon with hypocotyl segment from 5 day old in vitro seedlings as target tissues. It was found that the best condition for transformation of watermelon was using bacteria at a concentration of OD600 0.6, inoculation for 30 min and 3 day of co-cultivation. Addition of 200 µM acetosyringone in the medium helped to further increase the transformation efficiency. Agrobacterium tumefaciens strain EHA101 was found to be more effective to infect this watermelon variety as compared to LBA 4404 strain. Wounding of the explant meristems to induce multiple shoot was best carried out after 3 day. Transformed explants were selected on medium containing 5 mg L-1 hygromycin. A total of 31 putative transformed plants were recovered with 25 shoots were found to be GUS positive after transformation of 110 cotyledons. A total of 15 GUS positive shoots amplified the hpt gene using PCR. However, only 7 shoots were successfully regenerated into whole plants.