Vol. 84 No: 3

Title:
Genetic transformation of Calibrachoa excellens via Agrobacterium rhizogenes: Changing morphological traits

Authors:
M.C. GENNARELLI, J.C. HAGIWARA, D. TOSTO, M.A. ÁLVAREZ, M. BORJA and A.S. ESCANDÓN

pp: 305-311

Abstract:
Leaves and stem segments of in vitro-cultured Calibrachoa excellens were used as explants to obtain transgenic hairy roots after inoculation with the following 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 root and callus proliferation, while only 10% of the explants showed root and/or callus development using 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, rol B, rol C, and rol D genes, and the absence of the vir D1 gene, confirming the absence of Agrobacterium. Southern blotting indicated the presence of one inserted copy in one transformation event, and two copies 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 rol A and rol B genes was confirmed in four out of 26 plants recovered that showed some hairy root characteristics. For the first time, transgenic hairy root plants of the genus Calibrachoa have been regenerated, which would allow transgene incorporation into a breeding programme.

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