In vitro propagation, transformation and biomass production and analysis in S. wagneriana Polak.

The species S. wagneriana Polak, sub-genus Calosphace, section Cardinales is a tropical ornamental species, the origin is Guatemala, Costa Rica; it is a large tropical sage with showy rosy – pink flowers. It is well adapted to the Mediterranean area and it is known for gardens and for pot plant production. In the frame of an international project on the improvement of the genus Salvia both for ornamental and medicinal purposes, S. wagneriana was studied for alternative uses. In 2004, Bisio et al. (Planta Medica 70: 452-457) described the identification of three new clerodane diterpenoids, hardwickiic acid and 1,10-dehydrosalviarin from the surface exudates of the aerial part of in vivo grown plants of S. wagneriana; and interesting antifeedant activity was observed by using the crude extract. Only several reports of in vitro culture on the genus Salvia were found in literature; some of them reported the possibility to identify and produce active compounds from hairy root, callus and cell biomass. No reports were found for S. wagneriana. The objective of this work was to study in S. wagneriana the micropropagation performances, the transformation with Agrobacterium rhizogenes and the biomass production and analysis. After several attempts, aseptic explants were obtained from apical and axillary buds of plant grown in the Hanbury Botanical Garden in Ventimiglia (I). The in vitro proliferation was evaluated in the presence of the cytokinins Benzyladenine and Kinetin; the BA (0.3 mgl-1) induced the highest multiplication rate. Root emergence was obtained in PGR-free medium and the plants were acclimatised promptly. Experiments on transformation with wild strains of Agrobacterium rhizogenes were carried out using leaf disks and stem fragments of micropropagated plants. Several HR lines were selected and isolated after transformation with the strain 15834 which showed the highest transformation percentage. The molecular evidence of the transformation was made by detection of the rolC gene in the putative transformed hairy roots. The HR liquid cultures, treated with elicitors (jasmonic acid and casein hydrolysate) were then analysed for the presence or absence of active compounds; a preliminary TLC control showed the presence of ursolic acid and β- sitosterol.