Comm. Appl. Biol. Sci, Ghent University, 71/1, 2006 3 PROTEOME AND SUGAR ANALYSIS OF ABIOTIC STRESS UNDERLYING CRYOPRESERVATION IN POTATO B. CRIEL 1,2 , J.-F. HAUSMAN 2 , M. OUFIR 2 , R. SWENNEN 1 , B. PANIS 1 & J. RENAUT 2 1 Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, K.U.Leuven, Kasteelpark Arenberg 13, BE-3001 Heverlee, Belgium 2 EVA, Centre de Recherche Public – Gabriel Lippmann 41, rue du Brill, LU-4422 Belvaux, GD Luxembourg INTRODUCTION Cryopreservation complements classical conservation methods, which are carried out in the field or in vitro. It involves the storage of biological material in liquid nitrogen (-196°C). At this temperature, all chemical and physical processes are stopped, allowing a safe storage over an unlimited period of time. However, at this moment the development of improved cryopreserva- tion protocols for potato is mainly based on a trial-and-error approach. Drought acclimation is known to improve recovery after cryopreservation in potato and other species. In this study, drought acclimation is used to ana- lyze the mechanism underlying tolerance towards cryopreservation. MATERIAL AND METHODS Plant material Plants (Solanum tuberosum, cv. Désirée by Gaspard Bauhin (1591)) were precultured for 21 days on regular MS and MS supplemented with 0.055M, 0.11M and 0.22M sorbitol. Immediately, after pre-treatment, leaf and shoot tip samples were collected and stored at –80°C for subsequent proteomic and carbohydrate analyses. Cryopreservation Cryopreservation was carried out after 21 days of pre-treatment on the me- dia discribed above. Potato shoot tips were cut from pre-treated plants and incubated for 15 minutes in Loading Solution, containing 9% glycerol (v/v), and 0.4M sucrose. Afterwards, shoot tips were incubated for 50 minutes on ice in Plant Vitrification Solution 2 (PVS2), containing 30% glycerol, 15% ethylene glycol, 15% DMSO (all v/v) and 0.4M sucrose. Subsequently, shoot tips were placed on an aluminum foil strip in droplets of PVS2 and plunged into liquid nitrogen (Panis et al., 2005). Thawing was done at room tempera- ture in a highly osmotic Recovery Solution, containing 1.2M sucrose, to pre- vent osmotic shock. After cryopreservation, shoot tips were transferred into the dark for 1 week (Panis et al., 2005). During the first day of post-culture, shoot tips were maintained on MS media, containing 0.3M sucrose. After- wards, regular MS media were used. After 30 days, recovery was calculated as the percentage of newly formed shoots.