Standardization of Agrobactrium mediated transformation in strawberry

Genetic transformation studies were carried out to standardize a protocol for Agrobacterium-mediated gene transfer technique in strawberry (Fragaria x ananassa Duch. cv. Chandler). High frequency (41.66%) shoot regeneration was obtained on MS basal medium (macro and micro nutrients) supplemented with B₅ vitamins, 2% vitamins, 1 mg/l TDZ, 1 mg/l BA and 0.5 mgl⁻¹ IBA. High percentage root regeneration in in vitro developed shoots was obtained on MS medium supplemented with 1.0 mgl⁻¹ IBA. Kanamycin sensitivity (0–50 mgl⁻¹) was checked by the fresh weight of the explant/callus which was measured at an interval of 7 days till 35 days. Fresh weight decreased with increase in kanamycin concentration. The fresh weight of explant/callus and kanamycin concentration was found negatively correlated (r=-0.90) at different intervals of time. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter ?-glucuronidase (gus) gene in binary vector pBI121 along with kanamycin resistance gene (npt-II) was used for selection in both bacteria and plant. After co-cultivation, transformed cells (explant) were able to grow on the selective regeneration medium containing 50 mgl⁻¹ kanamycin and 500 mgl⁻¹ cefotaxime, while, control explant failed to grow or showed poor growth on the selective medium and turned brown/black after 35–40 days in culture. Pre-incubation of 48 hours and co-cultivation of 48 hours resulted in maximum putative transgenic shoot regeneration (2.70%) on the selective medium. The regenerated putative transgenic shoots were analyzed by GUS assay using biochemical method and only two shoots were found to be GUS positive.