AGROBACTERIUM - MEDIATED TRANSFORMATION OF CITRUS CELL SUSPENSION CULTURES

An efficient protocol for the genetic transformation of cell suspension cultures of several citrus cultivars using Agrobacterium is described. Cell suspension cultures of Citrus sinensis ‘Hamlin’, ‘Valencia’ and ‘OLL8’ (an early maturing ‘Valencia’ like somaclone), Citrus unshiu ‘Okitsu wase’ and Citrus reticulate ‘Ponkan’ and ‘W. Murcott’ were transformed using A. tumefaciens EHA105. It was determined that hygromycin was a better selection antibiotic than kanamycin and efficient transformation results were obtained only when an Agrobacterium strain containing hptII as a selectable marker was utilized. The EGFP protein was visualized in transgenic embryos within 6 weeks of transformation. Several transgenic plants were obtained using this protocol and hptII and egfp gene integration was confirmed. Amenability of cell suspension cultures to transformation using Agrobacterium would allow the transformation of any cultivar that can be induced in vitro as embryogenic cell masses, including special seedless sweet oranges or Satsuma mandarins and other difficult-to-transform cultivars of the mandarin/tangerine or lemon group.