Abstract

Studies related to the genome engineering in plants require an efficient protocol with high frequency callus induction and regeneration. This present study was conducted at PRR Biotech laboratory, Hyderabad in the year 2020 and aimed to develop an efficient protocol to achieve a high frequency callus induction and genetic transformation of RPBio-226, a Samba Mahsuri rice variety. In-vitro callus induction was carried out using MS media supplemented with different concentrations of 2,4-D viz., 1.0, 1.5, 2.0, 2.5 mg/L singly and in combinations with BAP viz., 0.1, 0.2, 0.4, 0.6 mg/L. Shoot induction was carried out using MS media supplemented with BAP 1.0, 1.5, 2.0, 2.5 mg/L and Kinetin 0.5, 1.0, 1.5, 2.0 mg/L and the Best response observed with 2,4-D and BAP at 2.0 and 0.1 mg/L, respectively. Agrobacterium tumefaciens strain EHA105 harboring PBI 121 plasmid (binary vector) was used in the process of rice transformation. An experiment was planned to use various parameters viz. 0.1, 0.2, 0.3, 0.4 and 0.5 ODs of Agrobacterium cells at 600nm. Embryogenic calli were co-cultivated with Agrobacterium supplemented with different concentrations of acetosyringone (AS) (0, 50, 100 and 150 μM) and 100 μM was found to give the best response for transformation. After Agrobacterium infection, infected calli were selected on the selection media with kanamycin as selection agent. The best hormone for shoot induction was BAP at 1.5 mg/L and kinetin at 0.5 mg/L and highest number of roots was achieved with 0.1 mg/L NAA in combination with 0.2 mg/L IBA. Regenerated plantlets were successfully acclimatized on soilrite mix showing the highest survival rate after 4 weeks of acclimatization. The npt II and Gus specific primers were used for PCR to identify stably transformed progeny that survived kanamycin treatment. GUS Histochemical Analysis of Callus was also carried out for the confirmation of transformation which helps us to generate transgenic plants in future.