Genetic transformation of sour orange using Agrobacterium rhizogenes

Sour orange (Citrus aurantium L.) plants were regenerated from Agrobacterium rihzogenes-transformed roots. To obtain the transformed roots, internodal stem segment from in vitro germinated seedings were cocultured for 96 h with Agrobacterium rhizogenes strain A4 with the ESC4 plasmid that contains the nptⅡ and gus genes. The highest efficiency was obtained using a suspension of 1×108 cells mL-1 and 96 h of coculture. Under these conditions, 91 % of the explants produced transformed roots with an average of 3.6 roots per explant. For shoot regeneration, segments of transformed roots were cultured on regeneration medium with 5.0 mg L-1 benzyladenine (BA) and 1.0 mg L-1 napthaleneacetic acid (NAA). In this medium,18% of the transformed root segments produced adventitious shoots, with an average of 1.25 shoots per explant. Gus activity was evident in the trandformed rootsm shoots and regenerated plans. The presence of the foreign genes was confirmed by PCR analysis and Southern blot. Afterwards, this system was used to generate sour orange roots and shoots transformed with the gene that codes for the capsid protein of the Citrus Tristeza Closterovirus.