Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos

A transformation system was developed for the citrus variety tangelo (Citrus reticulata Blanco × C. paradisi Macf. ) cultivar "Page". Embryogenic cells from suspension cultures were bombarded with tungsten particles coated with plasmid DNA which contained the uidA gene encoding β-glucuronidase (GUS) and the nptⅡ gene conferring kanamycin resistance. More than 600 transient and 15 stable transformants were obtained per bombardment based on the number of GUS expression foci at 1 day and 8 weeks after bombardment. Pretreatment of cells with high osmotic potential (0.3 M sorbitol + 0.3 M mannitol ) was found to enhance both transient and stable transformation efficiendy. Between 2 and 3 months following bombardment, calli were formed on a medium containing kanamycin (100 mg/L). The majority of the kanamycin-resistant calli also expressed GUS activity. From these calli, embryos and plantlets were produced. Integration of the uidA and nptⅡ genes into the tangelo was confirmed by polymerase chain reaction (PCR) and Southern analysis.