Establishment of Callus Induction, Cell Suspension Culture, and Agrobacterium-mediated Transformation system for Iranian Rice Cultivars

Up to now, a large number of optimized plant systems have been established for various purposes in Japonica and Indica rice. Based on genetic diversity in rice cultivars, this study established highly efficient protocols for in vitro callus induction, cell suspension cultures, and genetic transformation for some Iranian rice cultivars using mature embryos. In this study, the effect of different concentrations of 2,4-D (0.0, 1.0, 1.5, and of 2.0 mg L-1) on callus induction were investigated in 10 cultivars. Regarding higher callus induction frequency and mean weight of fresh calli, Hashemi, Binam, and Kazemi cultivars were selected for the experiments. The effects of kinetin (0.0, 1.0, and 2.0 mg L-1) and sucrose (30.0 and 60.0 g L-1) concentrations were tested to improve the biomass yield of a cell suspension culture. The MS medium supplemented with 0.5 mg L-1 2,4-D, 2.0 mg L-1 kinetin, and 60.0 g L-1 sucrose exhibited the maximum cell growth in the selected cultivars. The transformation efficiency for different bacterial strains (EHA105, LBA4404, and AGL(-1)), OD600 (0.1, 0.3, 0.6), the concentration of acetosyringone (50, 100, 200 mu M), and co-cultivation period (1, 2, 3 days) were evaluated. The presence and expression of gusA gene in transgenic cultivars were determined by GUS histochemical assay, PCR, and RT-PCR analysis. The result showed that Hashemi cultivar had the highest cell biomass and efficiency of genetic transformation (58%) with EHA105 at bacterial OD600= 0.3, in 100 (??M) acetosyringone and two days of co-culture time. The findings offer insights for genetic transformation studies in Iranian rice cultivars.