In Vitro Callogenesis and Agrobacterium-Mediated Transformation of Globe Artichoke

Micropropagation techniques have been widely applied in globe artichoke (C. cardunculus L. var. scolymus), however, efficient protocols for the establishment of in vitro callogenesis and organogenesis, a pre-requisite for Agrobacteriummediated genetic transformation, have not been set up so far. We developed an efficient protocol for callus induction from leaf explants of three globe artichoke genotypes of the varietal type ‘Romanesco’; after just one week culture callus formed with an efficiency close to 100%. Leaf explants were transformed with Agrobacterium tumefaciens Agl0 01-124 strain, harboring the binary vector pCAMBIA 2301 with the gene marker GUS, under the control of CaMV 35S promoter. After two weeks, about 30% of the calli obtained from infected leaf explants were positive to GUS assay. INTRODUCTION Globe artichoke (Cynara cardunculus var. scolymus L.) is an herbaceous perennial plant native to the Mediterranean and whose cultivation is widely distributed all over the world, even though it is mainly concentrated in Mediterranean countries. In recent years the increasing demand for functional foods has led to a renewed interest in this crop, which is one of the richest natural sources of bioactive phenolic compounds, like caffeoylquinic acids (CQAs). Micropropagation techniques have been well established for clonal propagation of globe artichoke (Ancora, 1986; Barba et al., 2004; Cadinu et al., 2004), on the other hand the regeneration of adventitious shoots from somatic tissues has been only occasionally reported (Ordas et al., 1990, 1991; Kchouk et al., 1997; El-Bahr et al., 2001). The difficulty in obtaining the induction of in vitro callogenesis and the following regeneration of a whole plant from the callus, hampers the possibility to generate stable genetically transformed globe artichoke plants. (Ordas et al., 1991; Kchouk et al., 1997). Here we report on the setting up of an efficient protocol for the induction of in vitro callogenesis from globe artichoke leaf explants as well as on the obtainment of stable genetically modified calli from leaf explants infected with Agrobacterium tumefaciens. MATERIALS AND METHODS In Vitro Culture and Callogenesis The starting materials were virus-free plantlets, obtained via meristem tip culture (Barba et al., 2004; Cadinu et al., 2004) of three globe artichoke clones (C3-RPO, C3-RR and SAROM) of the varietal type ‘Romanesco’. The plantlets were grown on MS (Murashige and Skoog, 1962) medium including vitamins and BAP (2,22 M), pH 5.7, 23°C. On the whole 108 combinations of light/dark exposures (photoperiod of 16 h light/8 h darkness, 48 h darkness after 16 h light/8 h darkness and full darkness), hormone Proc. 7 IS on In Vitro Culture and Horticultural Breeding