Hyoscyamine-6-B-Hydroxylase Gene: Obtention Of Recombinant Hairy Roots Of Brugmansia

L. SETTEN, M. CAPELLO, S. PITTA-ALVAREZ, M. A. ALVAREZ AND P. MARCONI

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión Anual SAIB; 2006

Current experimental work is being performed in order to obtain hairy roots of Brugmansia candida that over-express h6h cDNA and the enzyme hyoscyamine-6-â-hydroxylase (H6H). The cDNA encoding H6H was isolated and cloned from anthers of B. candida flowering plants. The gene was cloned in the vector pJIT with the double promoter CAMV35S and nos terminator. The size of the insert was 1.1 Kb, encoding a sequence of 344 amino acids that was highly homologous to the same enzyme derived from other Solanaceae. Agrobacterium rhizogenes LBA 9402 was transformed with this construct by triparental mating. The colonies were screened by subculture in YMB medium with rifampycin (5 mg/L), obtaining the strain LBA-H6H. Plants of B. candida maintained in vitro were used to infect them with A. rhizogenes strains LBA 9402 and LBA-H6H. Subsequently, A. rhizogenes strains LBA9402-PR and 15834-PR (gift of Dr. Rocha) and LBA 9402-Hn (gift of Dr. Oksman-Caldentey) were used in order to obtain HR lines over-expressing cDNA for H6H from Hyoscyamus muticus and H. niger, respectively. The induction of hairy roots from B. candida plants was successful (80%). The hairy roots that appeared at the infection sites were excised and cultured individually on B5/2 liquid medium supplemented with sucrose 15 g/l, ampicillin 2 g/l, rifampycine 5 mg/L, kanamycin 30 mg/L and agar 8 g/l. We are now in the process of choosing the clones based on their growth index and alkaloid production.