CONICET | Buscador de Institutos y Recursos Humanos

Leaves and stem segments of in vitro cultured Calibrachoa excellens, were used as explants to obtain induced transgenic hairy roots after inoculation with the following four strains of Agrobacterium rhizogenes: LBA 15834, LBA 8490, LMG 155, and A13. Only leaves showed sensitivity towards Agrobacterium infection. A. rhizogenes strain LBA 15834 was the most virulent among the four strains tested with 33 % of leaves showing roots and callus proliferation, while only 10 % of the explants showed root and/or callus development with strain A13 and no responses were observed in explants infected with the other two strains used. Two different transformation events were recovered as transgenic plants regenerated from hairy roots via spontaneous organogenesis. These plants showed, to different degrees, the classic symptoms of hairy root disease. PCR analysis of the recovered plants showed the presence of the rol A, B, C and D genes, and the absence of the vir D gene, confirming the absence of Agrobacterium. Southern blotting indicated the presence of two inserted copies in one transformation event, and three in the other. Morphological and physiological differences were observed between the independent transformation events as well as several differences, such as flower colour and leaf shape, between plants of the same event. Pollen from one of the transformed plants was used to cross with commercial hybrids of Calibrachoa, and the presence of the rolA and rolB genes was confirmed in four of 26 plants recovered that showed some hairy root characteristics. In conclusion, transgenic hairy roots-plants have been regenerated for the first time in the Calibrachoa genus and this material could be incorporated into a breeding programme.