Importance of in vitro culture for developing cryopreservation strategies of woody plants

Plant cryopreservation has greatly evolved in recent years, being today a safe cost-effective complementary approach to the traditional ex situ conservation of plant biodiversity. A milestone in the cryopreservation of woody plant material dates back to 1990, when Akira Sakai and co-workers developed the Plant Vitrification Solution n°2 (PVS2) which showed to be very effective for the induction of cell vitrification during ultra-rapid freezing in liquid nitrogen. Since then, the number of PVS2-based protocols, mainly developed for the cryopreservation of shoot tips, increased yearly while, at the same time, new and effective encapsulation- and droplet-based methods were proposed. A range of different cryo-techniques is now available for the cryostorage of woody plant germplasm, allowing the safe long-term conservation in liquid nitrogen of different organs and tissues coming from tissue culture, such as (i) shoot tips, obtained in vitro by axillary or apical buds and used naked or incapsulated in Ca-alginate beads. They are the most used explants with broad-leaf trees, provided that optimized protocols of micropropagation have been achieved; (ii) somatic embryogenic callus, largely used with conifer species for which efficient micropropagation procedures from mature stock plants are rarely available; (iii) seeds and embryonic axes, useful material for the long-term preservation of both seed-propagated, and vegetatively-propagated species, provided that the latter have polyembrionic seeds, such in citrus. Seeds and embryonic axes take advantage from in vitro culture for their development after the recover from liquid nitrogen. The optimization of effective cryo-protocols passes through a correct interpretation of results in post-cryopreservation in terms of "survival" and "regrowth" of explants.