To cryopreserve sugar beet shoot tips using an encapsulation-dehydration technique, cold hardening of in vitro plants was needed to obtain high survival rates after freezing. Cold acclimation not only enhanced dehydration and freezing tolerance, but also induced several changes in sugar beet shoots. Plants contained greater amounts of sucrose, D-glucose and D-fructose and the fatty acid composition of lipids changed. Furthermore, the unsaturation level of membrane lipids, estimated by the (C-18:2 +C-18:1)/C-16:0 ratio, increased after cold hardening. These changes were correlated with better survival rates after cryopreservation.