GENETIC-TRANSFORMATION AND PLANT-REGENERATION OF WATERMELON USING AGROBACTERIUM-TUMEFACIENS

Abstract

Adventitious shoots formed on the Proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of 'Sweet Gem' were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter-beta-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl-1 BA and 200 muM beta-hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl-1 BA, 250 mgl-1 carbenicillin, and 100 mgl-1 kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl-1 BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.