Standardize various parameters for Agrobacterium-mediated genetic transformation for tomato (Solanum lycopersicum L.)

The study was conducted to standardize various parameters for Agrobacterium-mediated genetic transformation of tomato (Solanum lycopersicum L). Agrobacterium-mediated transformation factors for tomato explants viz. cotyledon were optimized using β-glucuronidase (GUS) as a reporter. The Agrobacterium tumefaciens strain GV3101 contained npt II (plant selectable marker gene providing resistance to kanamycin) under the control of nopaline marker gene providing promoter (pNOS) and the Cry 1 Ac coding region containing a plant intron linked to the cauliflower mosaic 35 S (CaMV 35 S) promoter, was used for co-cultivation with explants from genotype Punjab Upma. The various parameters for transformation were optimized including bacterial concentration, co-cultivation period, acetosyringone concentration and pre- selection of explants on different concentration of kanamycin antibiotic. Results for bacterial concentration and co-cultivation were obtained based on the percentage of GUS expression and explants mortality percentage, while for acetosyringone concentration result were based on GUS expression percentage. Agrobacterium tumefaciens strain GV 3101 at concentration (OD600=1.0) diluted culture (1:20) for 20 minutes, followed by co-cultivation and 2 days co-cultivation period showed the maximum Gus expression and minimum explants mortality in explants of tomato. For acetosyringone concentration 25 (µM) showing higher transformation percentage. The transgenic plants were selected on the medium containing 30 mg/l kanamycin. The protocol developed showed very high efficiency of transformation for tomato genotype Punjab Upma.