engleski

CRYOPRESERVATION AND CRYOTHERAPY OF GRAPEVINE (Vitis vinifera L.)

INTRODUCTION – Cryopreservation is a method for long-term conservation in which explants are stored at the ultra-low temperature of liquid nitrogen. In the case of grapevine, cryopreservation protocols have not been defined enough for wide application to numerous cultivars. Cryotherapy is a novel technique, which eliminates plant pathogens during exposure to ultra low temperatures. Plant cryopreservation is a complex procedure during which explants are exposed to potentially stressing events. Genetic stability of regenenerated plants is a part of cryopreservation protocol establishment. AIMS AND SCOPES – The main goals of this research are: to determine the efficient cryopreservation protocol for Croatian native grapevine cultivars and international cultivars, to test efficiency of cryotherapy in virus eradication and to check genetic stability of regenerated plants. This research was performed aiming to introduce this technique in general viticulture. MATERIALS AND METHODS – In vitro explants of Croatian (Plavac mali, Maraština-(plus their infected genotypes), Pošip and Škrlet) and international cultivars (Chardonnay, Cabernet Sauvignon, Merlot and Pinot Noir, within their infected genotypes) were taken for cryopreservation experiment, which had been established previously on cultivar Portan. Droplet- vitrification protocol was performed. Regenerated two-months in vitro plants from infected genotypes (control and cryopreserved apices) were subjected to ELISA testing. AFLP markers were employed to test genetic stability on cultivar Portan. Eight AFLP primer combinations were used on cryopreserved and non-cryopreserved shoot tips (sampled after sucrose preculture, loading and treatment with half-strength PVS2 solution) for a total of 43 plants tested. RESULTS AND DISCUSSIONS –The basal cryopreservation protocol, performed on international cultivars revealed: intermediate regrowth was achieved with Chardonnay (30%) and high with Merlot (70%), before and after cryopreservation, which was comparable to previously published results on 10 Vitis cultivars, which produced an average recovery of 64%. Similar results were obtained with healthy and virus infected cultivars. The same protocol, performed on Croatian cultivars, showed a low survival and recovery with only one (Maraština) of the four cultivars tested. This low success might be due to the fact that cryopreservation experiments were performed using cultures which had been introduced in vitro very recently and that the in vitro culture conditions employed were not adapted to the multiplication of these Croatian cultivars. The results of the cryotherapy experiments revealed that a high proportion of Chardonnay (100%) and Cabernet Sauvignon (78%) plants were virus-free after being subjected to cryopreservation. Control plants showed a high proportion of healthy plants, almost the same as with LN exposed ones for both viruses. AFLP markers revealed no significant differences in AFLP profiles after sucrose preculture, treatment with the loading solution and with half-strength PVS2, whereas an increase in polymorphic fragments was observed in non- cryopreserved and cryopreserved samples treated with PVS2 solution, the number of polymorphic fragments increasing with longer durations of exposure to PVS2 solution. CONCLUSIONS AND POSSIBLE APPLICATIONS – Cryotherapy as a method can be efficient for virus eradication even in the preconditioning steps of a cryopreservation protocol. A genetic stability study should be included in post- cryopreservation experiments.

grapevine; cryopreservation; cryotherapy; genetic stability

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