Effect of preparatory treatments for the cryopreservation of olive (Olea europaea L., cv Moraiolo) microbuds from micropropagation.

Various procedures are today available for the cryopreservation of microbuds from micropropagation, among which encapsulation-vitrification, droplet-vitrification, and V-cryo-plate and are the most used and successful. However, a necessary condition for the effectiveness of these techniques is to appropriately prepare the microbuds to tolerate the very rapid lowering of temperature, due to their direct immersion in liquid nitrogen (-196°C). The tested cryopreservation methods included the preparation and use of synthetic seeds, the droplet-vitrification on aluminium foil strips, and the V cryo-plates with microbuds placed on solid bars and covered with calcium alginate. The preparatory treatments were applied to olive synthetic seeds, as well as in droplet-vitrification and V-cryo-plates methods, all prepared with microbuds (Olea europaea L., cv Moraiolo) from micropropagation, in an effort to induce them to tolerate the direct immersion in liquid nitrogen. The microbuds were pre treated with different concentrations of sucrose (0.5, 0.75, and 1 M) before exposure to the loading solution (LS) and the PVS2 solution. In addition, multiple exposure durations to PVS2 (30, 45, 60, 75, 90, and 105 min) were tested to identify the optimal time for the subsequent use in cryopreservation. Recovery rates from synthetic seeds containing 0.5 M sucrose after 105 minutes of exposure to PVS2 showed the highest germination rate (100%), demonstrating the best tolerance of microbuds to the toxic effects of concentrated cryoprotective solutions. This result was followed by exposure times of 90 minutes (71.6%) and 75 minutes (62.2%). In a subsequent trial, the effects of mannitol and sucrose on the ‘germination’ (=conversion to plantlets) of synthetic seeds were also evaluated. Results demonstrated that mannitol was more beneficial than sucrose in terms of both the speed and quality of ‘germination’, with mannitol-treated synthetic seeds exhibiting higher germination rates and faster regrowth of encapsulated microbuds. These findings suggest that mannitol may be more effective than sucrose in improving the post-cryopreservation response of olive synthetic seeds. Trials to test the efficacy of such protocols in inducing tolerance to cryopreservation are currently underway.