Cryopreservation of olive somatic embryos using the droplet-vitrification method: The importance of explant culture conditions

- Growth conditions of somatic embryos significantly affected their subsequent response to cryopreservation.
- Culture of explants in liquid medium significantly improved cryopreservation.
- Culture growth phase at which explants were sampled significantly affected post-rewarming recovery.
- Optimal growth phase depended on culture method.
- Improved recovery achieved in liquid medium may be explained by stimulation of cell proliferation and secondary embryo formation.

This study reports on successful cryopreservation of olive somatic embryos by using the droplet-vitrification method on aluminium foil strips. All explants tested showed high sensitivity to both cryoprotective solutions and freezing. Nevertheless, slightly higher recovery values were achieved when using the most heterogeneous explants, i.e., somatic embryos 1-6 mm in size. Growth conditions of somatic embryos significantly affected their subsequent response to cryopreservation. Post-rewarming culture recovery percentage was significantly affected by the culture method, in solid or liquid medium, the culture growth phase at which explants were sampled and the interaction between both factors. Significantly better results were obtained when somatic embryos were previously cultured in liquid medium for 28 days. At this conditions, 60% of samples resumed embryogenesis, thus ensuring the safe long-term conservation of this type of embryogenic structures. As revealed by the histological analysis, increased recovery observed in this treatment could be explained by stimulation of cell proliferation and secondary embryos formation, thus increasing the presence of meristematic and embryogenic cells, more prone to withstand cryopreservation treatments.

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