Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.28

HAIRY ROOT INDUCTION IN HELICHRYSUM ITALICUM AND H. STOECHAS FOR THE PRODUCTION OF SECONDARY METABOLITES

M. AMORETTI, A. DI GUARDO, P. FILOTTI, B. RUFFONI, A. GIOVANNINI

Istituto Sperimentale per la Floricoltura, Corso degli Inglesi 508, 18038 Sanremo (Imperia)

Agrobacterium rhizogenes, flavonoids, everlasting, H. angustifolium, no food plants

Helichrysum italicum (Roth) G. Don and H. stoechas (L.) Moench are two medicinal species of the Mediterranean region, belonging to the genus Helichrysum (family Asteraceae). The plants are evergreen shrubs growing in arid soils, from the cliffs above the sea to the hills, where they flower from May to August. The bright yellow flower heads contain principally essential oils, flavonoids (helichrysin A and B) and tannins. H. italicum and H. stoechas have been used in folk medicine because of their antibacterial, antitoxic, diuretic and antiallergic properties; moreover the flowering stems are dried and used as "everlasting flowers". The neoplastic roots (hairy roots), induced by the soil Gram negative bacterium Agrobacterium rhizogenes, are a biosynthetically active tissue that can express root-specific metabolic pathways as efficiently as intact plant. The secondary metabolite synthesis is not strictly limited to those that are normally produced in roots of differentiated plants (Flores et al., Plant Physiol., 1993, 101: 363-371).

We report a protocol for hairy root induction in H. italicum and H. stoechas. Leaf and root tissues of micropropagated plant clones (Giovannini et al., Acta Horticolturae, in press) were co-cultivated with a solution of A. rhizogenes wild type strain 15834 (ATCC). After 3 days the explants were transferred to a medium without growth regulators, containing basal salts and vitamines MS (Murashige and Skoog, Physiol. Plant., 1962, 15: 473-497), 30 g/l sucrose and 8 g/l agar (Basal Medium), supplemented with Cefotaxime (100 mg/l). The number of explants developing roots was counted after 30, 60 and 90 days. One hundred percentages of root explants developed new roots after 30 days of culture, in the two species. After 90 days, new roots were observed in 92,3% of H. italicum and in 18,7% H. stoechas leaf explants. Several putative hairy roots were isolated and further propagated on Basal Medium. PCR analysis was performed on root total genomic DNA, using specific primers for the amplification of A. rhizogenes rolC and virC1 genes. A rolC gene amplification band of 600bp was detected in 3 putative H. italicum hairy root lines; virC1 amplification band of 326 bp was visualised only in the bacterium sample. H. italicum hairy root cultures were established in liquid Basal Medium and were cultivated in light and dark conditions, in continuous shaking. The pH of the medium was monitored for five-week culture.

H. italicum hairy root culture is a fast growing active tissue that can be exploited for the production of secondary metabolites and for the scaling up in bioreactor.