Agrobacterium tumefaciens gene transfer to Casuarina glauca, a tropical nitrogen-fixing tree

Transgenic calli of the tropical tree Casuarina glauca were produced using Agrobacterium tumefaciens-mediated gene transfer. Hypocotyls, cotyledons and epicotyls were excised from 30-60-day old Casuarina seedlings and cocultivated with Agrobacterium strain C58Cl(pGV2260) containing the binary vector BIN19GUSINT. Transformed calli were selected on nutrient medium supplemented with 0.5 mu M NAA, 2.5 mu M BA and 50 mg/l kanamycin. Some of the factors influencing T-DNA transfer to C. glauca explants were studied. Optimal transformation rates were obtained when explants from 45-day old seedlings were cocultivated for 3 days in the presence of 25 mu M acetosyringone, Transgenic buds differentiated on 10% of the calli grown on transformed epicotyls, Evidence for genetic transformation was obtained by beta-glucuronidase assay, PCR and Southern hybridization.