Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange

Transgenic Washington navel orange [Citrus sinensis (L.) Osbeck] plants were obtained using Agrobacterium-mediated transformation of seedling epicotyl tissue. An average of 45% (58 out of 128 segments) of the epicotyl segments produced shoots expressing the β-glucuronidase (GUS)-intron reporter gene when using Agrobacterium strain C58 C1, compared to 29% (38 out of 128 segments) for EHA101-5 and 0% for LBA4404. Co-culture of 21-day-old Washington navel epicotyl stem segments gave greater transformation efficiency than co-culture of 35- or 56-day-old stem segments. After 6 weeks, regenerated shoots were micro-grafted in vivo onto seedling rootstocks of Carrizo citrange. Stable integration of the transgene sequence was confirmed by expression of the plant intron-containing GUS gene, PCR and Southern hybridization. The apomictic (non-zygotic) state of the transgenic plants was confirmed by isoenzyme and random amplified polymorphic DNA analyses. More than 50 transgenic plants have been obtained and are growing in the greenhouse.