Agrobacterium-mediated genetic transformation of ‘Hamlin’ sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic "Hamlin" sweet orange (Citrus sinensisa(L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regeneeating plants by direct orgranogenesis. Higher percentage of tansformation was obtained with explants collected from seedling germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in cilture medium supplemented with 100 μM of acetosyringone. Genetic transformation was confirmed by performing β-glucoronidase (GUS) assays and, subseqently, by PCR amplification for the nptⅡ and GUS genes.