Cryopreservation of olive embryogenic cultures

The aim of this work was to optimize a protocol for the cryopreservation of embryogenic cultures of olive (Olea europaea L.). Exposure time to loading solution and PVS2 significantly influenced the regrowth rate of both organized and non-organized tissues. Organized tissues were more sensitive to prolonged treatments with vitrification solutions compared to non-organized tissues. Three cryopreservation protocols were compared using non-organized tissues: the "classical" vitrification protocol, an ultra-fast freezing method using droplet vitrification on aluminium foil strips and a "classical" slow freezing method (1 degree C per min). The best results were obtained using the droplet vitrification method after a 60 min dehydration period with PVS2. Under these conditions, all cryopreserved cultures showed renewed embryogenesis six weeks after thawing. A long-term (7-8 weeks) sucrose preculture had a significant effect on the initial response of the cultures, allowing particularly to protect cells against the toxic effects of the vitrification solution.