TRANSFORMATION OF CITRUS EMBRYOGENIC CELLS USING PARTICLE BOMBARDMENTAND PRODUCTION OF TRANSGENIC EMBRYOS

A transformation system was developed for the citrus variety tangelo ( Citrus reticulata Blanco x C. paradisi Macf.) cultivar 'Page'. Embryog enic cells from suspension cultures were bombarded with tungsten parti cles coated with plasmid DNA which contained the uidA gene encoding be ta-glucuronidase (GUS) and the nptII gene conferring kanamycin resista nce. More than 600 transient and 15 stable transformants were obtained per bombardment based on the number of GUS expression foci at 1 day a nd 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 efficiency. Between 2 and 3 months following bombardment, calli were formed on a medium containing kanam ycin (100 mg . l(-1)). The majority of the kanamycin-resistant calli a lso expressed GUS activity. From these calli, embryos and plantlets we re produced. Integration of the uidA and nptII genes into the tangelo genome was confirmed by polymerase chain reaction (PCR) and Southern a nalyses.