COMPARISON OF DIFFERENT PRECONDITIONING AND LOADING TREATMENTS WITH VANILLA (VANILLA PLANIFOLIA JACK.) APICES CRYOPRESERVED USING THE DROPLET-VITRIFICATION PROCEDURE

In order to improve post-cryopreservation survival of vanilla (Vanilla planifolia) apices, different preconditioning and loading treatments were studied comparing the effect of using sucrose or trehalose. In this work, vanilla apices were isolated from in vitro grown plants and subjected to a direct preculture on MS semisolid medium supplemented with 0.3 M sucrose or trehalose for one or seven days. Additionally, apices were also pre-conditioned after dissection by transferring them first to standard MS semisolid medium for seven days, followed by the same MS medium but containing 0.3 M sucrose or trehalose for one or seven days. After all pre-conditioning treatments, apices were loaded in solutions containing 0.4 M sucrose or trehalose mixed with 2 M glycerol, or in 0.8 M sucrose or trehalose mixed with 1 M glycerol, exposed to PVS2 or PVS3 vitrification solutios for 30 min, and then directly plunged into liquid nitrogen on droplets of PVS solution placed on aluminum foil strips.
Dehydration with PVS2 solution produced the best results after cryopreservation when apices were subjected to the pre-conditioning treatments with trehalose, and loading with sucrose-glycerol solutions. Nevertheless, the highest survival (57 and 62%) rates of cryopreserved apices were achieved using trehalose both in preconditioning and in loading solutions, followed by exposure to PVS3. The droplet-vitrification protocols which allowed improvements (from 30 to 60%) in survival of cryopreserved vanilla apices comprised: preconditioning of dissected apices on standard MS semisolid medium for seven days, followed by one or seven days on MS medium supplemented with 0.3 M trehalose, loading in 0.4 M trehalose + 2 M glycerol, and exposure to PVS3 for 30 min.