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Olive somatic embryos have been successfully cryopreserved using the droplet-vitrification method on aluminum foil strips. Although acceptable recovery rates have been obtained after rewarming, the influence of this cryopreservation protocol on the somatic embryogenesis process is unknown. To evaluate the effect of cryopreservation on olive somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process. In order to analyze the influence of the genotype, this investigation was carried out in two independent lines. During the proliferation step, only the line T1 was affected by cryopreservation, with higher fresh weight increases. Although similar total embryos were produced per culture, freezing in liquid nitrogen significantly improved the maturation pattern in the line P5. Better germination results were also found in this embryogenic line. The genotype plays a key role, largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Embryogenic cultures established after rewarming retained their regeneration capacity, with no evident negative effects affecting their regeneration capacity.

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Cryopreservation is considered the best technique for the safe, long-term conservation of embryogenic cultures. However, before integrating it into a somatic embryogenesis system, the influence of cryopreservation on the final production of plants should be investigated. The objective of this investigation was to evaluate the effect of cryopreservation on the regeneration performance of olive embryogenic cultures as well as on the quality of the plants obtained and their response to ex vitro establishment. In order to analyze the influence of the genotype, all the investigations were carried out in two genetically distinct embryogenic lines. The results obtained revealed no variation in the regeneration potential or the quality of the regenerated plants due to cryopreservation. The subsequent multiplication, rooting, and acclimatization steps were not influenced by cryopreservation either, although a significant genotype × cryopreservation interaction was found for shoot length during the multiplication step. The genotype played an important role, determining the quality of the regenerated plants and some aspects of the multiplication and rooting phases. This investigation revealed that the droplet-vitrification procedure optimized for the cryopreservation of olive somatic embryos can be efficiently used for the long-term conservation of olive embryogenic lines.