Establishment of an Agrobacterium-mediated transformation system for Fortunella crassifolia

Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 degrees C on kanamycin free shoot regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm(-3) kanamycin and 300 mg dm(-3) cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm(-3) kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved.