Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer

The method for plant transformation and antibiotic selection has been well established for several citrus varieties. Transgenic citrus suspension cells, however, have never been produced and maintained, thus, a procedure for transformation, antibiotic selection, and maintenance of transgenic suspension cells is still required. Transgenic citrus suspension cells containing the npt II gene forresistance to an aminoglycoside antibiotic would be very valuable for chromosome transfer experiments. To determine the lethal antibiotic concentration where growth inhibition of suspension cells would occur, the antibiotics kanamycin sulfate, geneticin, and paromomycin sulfate were used. Paromomycin sulfate at 50 µg/ml was found to be the best antibiotic forselection. Geneticin and kanamycin were not appropriate forselection of transgenic Valencia sweet orange suspension cells. For protoplast transformation, the electroporation parameters and incubation procedures were established. Pulses of 400, 500, and 700 V/cm were evaluated. The best electroporation settings were pulse of 500 V/cm and capacitance of 1000 µF. To secure high survival rates of the protoplasts, it was essential to add BH3 medium immediately after electroporation. Transgenic suspension cells of Valencia sweet orange containing the npt II and the ß-glucuronidase (Gus) gene were produced and maintained in 14 day subculture cycles in medium containing 50 µg/ml of paromomycin sulfate. This concentration was also very efficient for selection of transgenic Valencia embryos, since non-transgenic embryos were bleached