Agrobacterium-Mediated) Transformation of Cucumber (Cucumis Sativus L.) and Plant Regeneration

Abstract

ABSTRACTCucumber plant (Cucumis sativus L.) was transformed by EHA101 strain of A. tumefaciens harboring the binary vector pGA482GG plasmid carrying the marker genes the neomycin phosphotransferase II (npt II) determining resistance to kanamycin and p-ghicuronidase (GUS). Cotyledon and hipocotyl segments of in vitro grown seedlings were used as explants. These tissue segments were cultured on MS medium containing different hormone combinations and concentrations to identify optimum shoot regeneration condition. The highest number of shoot regeneration was observed on MS medium containing 2 mg/1 zeatin. In the tested explant tissues, the highest shoot formation was obtained from the proxiroal ends of cotyledon leaves which were cut transversely into two halves. Plant tissue segments were co-cultivated with A. tumefaciens for 2 days. Tissues, selected on the MS medium containing kanamycin (50 mg/1) were tested by histochemical GUS assay. Shoots regenerated from the transgenic tissue were cultured on the basal MS medium to induce root formation for four to six weeks. The rooted plants with 10 cm height were transferred to the soil for their adaptation to the natural environment. It was observed that transgenic plants had generally sterile flowers. To confirm presence of the transgenes, DNA from GUS+ transgenic cucumber plants was analysed by PCR. Thus an effective protocol for Agrobacterium-mediated genetic transformation of cucumber was optimised.