PHD COURSE IN VEGETAL CROPS AND ENVIRONMENT XXI CICLE (VII CICLE N.S.) " Micropropagation like opportunity to development in Marche nursery " PhD candidate: Dott. Luca Vita Tutor: Prof. Bruno Mezzetti POLITECNIC UNIVERSITY OF MARCHE – AGRONOMIC FACULTY DIPARTMENT OF ENVIRONMENTAL SCIENCE AND OF VEGETAL CROPS Key words: micropropagation, artichoke, cynara, raspberry, Rubus idaeus, sage, Salvia officinalis, oregano, Origanum vulgare, vivaism, in vitro protocols Abstract This study want to supply the techniques of in vitro cultivation of four species of interest to the Marche local nursery. These species were: a local ecotype artichoke (Cynara cardunculus var. Scolymus L.) the 'Artichoke of Montelupone', a raspberry cultivar (Rubus idaeus L.) var. Rossana, and two aromatic plants such as oregano (Origanum vulgare L.) and sage (Salvia officinalis L.). What emerged from the study were summarized in four micropropagation protocols, one for each species studied, with whom we tried to get the most efficient, simple and secure method for a rapid propagation of local interest species in order to obtain material that respond to required characteristic, like genetic stability, health and uniformity for the production and wholesale nurseries for recultivation. Related to artichoke the developed protocol to provide carducci 2-4 cm like explant from the field and then the levy from the top of meristems after a treatment with 2% of active chlorine for 20 minutes sterilization, the explant thus obtained are started to in vitro culture by using the Murashige and Skoog, 1962 (MS) substrate, supplemented with 0.25 mg/l of 6-BAP. Healthy and viable explants were forced to the proliferation phase with the same substrate but modified with different cytokinine concentrations. The most efficient plant growth combination for inducing proliferation of this species resulted BAP 0.5 mg/l combined with 0.2 mg/l of indole butyric acid (IBA). The concentration of gelling agent, agar, was also increased to 8 mg/l so to reduce vitrification problems. The best rooting results were obtained by eliminating the cytokinine concentration in the substrate of the last subculture and just keeping 0.2 mg/l IBA. The subculture time of 20 days resulted able to soustain a proliferation rate higher then 2. The sensitive stage for this specie was the choice of the mother plant and removal of the meristem. This protocol resulted efficient for the production of healthy certified mother plants for the carducci production. For raspberry, the protocol developed from this study is based on the removal of the meristem from buds collected in open field, and sterilization with a solution of sodium hypochlorite and water at 2% of active chlorine, for 20 minutes, then the meristems were transferred in vitro on MS substrate added 0.25 mg/l of 6-BAP. This medium resulted appropriate to produce a proliferation rate of about 2.4, at each subculture of 20 days. Rooting of raspberry plants was obtained by using the same substrate but without PGR. This protocol can be applied for the production of complete plants of commercial interest. Regarding sage, the protocol provides a 1 cm in length microtalee sterilization by 2% of active chlorine for 20 minutes, then, better proliferation rate was obtained by using MS medium added with 0.25 mg/l of 6-BAP and with agar concentration increased to 8 mg/l, so to reduce explant vitrification. At these conditions resulted a proliferation rate of about 1:3 explants. To achieve sage in vitro plant rooting was not sufficient the the simple removal of cytokinin, This confirm the necessity to use for this specie different rooting media including auxins or even activated carbon, so to better stimulate plant elongation and rooting before of an acclimatation period of about 20 - 30 days. Related to origano’s trials, results confirm what is available in literature, the sterilizations were positive when performed on seeds treated with 0.5% of active chlorine, for 5 minutes, and then transferred to the proliferation substrate of MS containing 0,25 mg/l of 6-BAP, to obtain a proliferation rate of 5. The rooting protocol resulted relatively simple because it is sufficient to lengthen the time of subculture with 7 days more obtain a 100% rooting rate. At the end we can confirm the versatility of the micropropagation technology and components primarily regarding the substrate composition as described by MS (1962). This experience was useful for the purpose designated in theory, techniques and detailed protocols developed by this work can be taken in account for local nursery production of high quality commercial plants. This experience can be considered useful to be transferred for the propagation of other specie of interested for the regional production system.