Genetic transformation and plant recovery from mature tissues of citrus sinensis L. Osbeck

Genetic transformation using mature tissue as explant is important for the precocious evaluation of the genetically modified characteristic. This research presents a protocol for adventitious organogenesis and genetic transformation of sweet orange varieties (Citrus sinensis L. Osbeck) using as explants internodal segments from adult plants maintained in the greenhouse. Organogenesis was induced in DBA3 medium supplemented with benzilaminopurine (1.0 mg l -1)+/naphthaleneacetic acid (0.5 mg l -1), in the dark at 279/2 8C until adventitious buds had formed and then transferred to a 16-h photoperiod. Histological analysis showed an indirect formation of the adventitious buds from callus that had proliferated at the cut ends of the explants. Transformation was done using the Agrobacterium tumefaciens strain EHA-105 containing the plasmid p35SGUSINT as a vector. The explants were cocultivated with Agrobacterium for 24 h, at 24 8C. Adventitious shoots were tested histochemically for GUS expression and micrografted into plantlets of the rootstock Carrizo citrange (C. sinensis L. Osbeck×/Poncirus trifoliata L. Raf.). Genetic transformation was confirmed by PCR and Southern blot analysis of the recovered plants.