Transformation of rice (Oryza sativa L.) using hygromycin (hpt) selector and -glucuronidase (gus) reporter genes by in planta method

Transgenic plants are often produced by tissue culture-dependent methods that require careful preparation and selection of explants, selection of transformed cells and regeneration of transgenic plants. In this in planta transformation method, the mature embryos of soaked seed were pierced by an Agrobacterium tumefaciens -coated needle or scalpel. The inoculated seeds were germinated and grew under no sterile conditions. In order to evaluate the effects of cultivars (Hashemi and Gerdeh), A. tumefaciens strains (EHA105 and LBA4404) and Three methods of inoculation (0. 5 mm Ø needle, 0. 5 mm Ø needle together with vacuum infiltration, Wounding with a scalpel) on transformation efficiency. A CRD based factorial experiment with three replications was applied. Integration of the transgene into the genome of transformed plants (T0 and T1) was confirmed using PCR method, resistance of leaf tissues to hygromycin and histiochemical GUS assay. Accordingly, the highest significant transformation efficiency (11. 72 %) was obtained in Hashemi cultivar wounded with a scalpel. Transformation efficiency of the same cultivar inoculated by a needle under vacuum infiltration was significantly higher than treatment of inoculation in the absence of vacuum. Seventy T0 seedlings were selected randomly and analysed further at T1 generation, which ~19 confirmed to be positive.