Abstract
Experiments were performed to determine the influence of various dehydration and vitrification treatment times on the ‘one-step freezing’ cryopreservation of embryonic axes (EAs), composed of zygotic embryos and cotyledon residuals, from mature seeds of a Georgian provenance of chestnut (Castanea sativa Mill.). Dehydration was carried out in laminar flow hood from 1 to 5 h, and vitrification experiments were carried out by immersion of EAs in PVS2 vitrification solution up to 120 min, both followed by direct immersion in liquid nitrogen. Both systems resulted in inducing specimen tolerance to ultra-rapid freezing, although to a different extent. Full germination of cryo-stored EAs after 5 h of dehydration (reducing moisture content from initial 66% to 21%) has been increased from 0% to 66.7%. A pre-treatment of EAs in PVS2 vitrification solution for 30 min produced fully developed plantlets at a rate of 55.6% in post-cryopreservation. Plantlet regrowth from cryopreservation was faster in EAs that underwent the dehydration/‘one-step freezing’ procedure. All the plantlet from cryopreserved EAs could be easily acclimatized, producing healthy potted plants. Finally, the TTC test showed to be useful for a fast evaluation of specimen survival after thawing and, as a consequence, to speed up the development of optimized cryo-protocols.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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Highlights
The forest ecosystems are of special significance for conservation of biodiversity of Georgia
In order to develop an efficient strategy for the longterm conservation of threatened chestnut Georgian provenances, the present study developed a step-wise ‘onestep freezing’ protocol for the cryopreservation of excised embryonic axes (EAs) from the Caucasus region, comparing two different cryo-techniques, i.e., specimen dehydration and PVS2-vitrification, both followed by the direct immersion of EAs in liquid nitrogen (LN) (‘one-step freezing’)
A decrease of moisture content (MC) was observed after each time of dehydration under the sterile air of laminar hood, significative starting from 2 h of dehydration and reaching a final of 21.4% of MC after 5 hours (Fig. 1A and B)
Summary
The forest ecosystems are of special significance for conservation of biodiversity of Georgia. The forest covers 39.9% of the territory of the country Their greater percentage (98%) is presented by mountainous forests and they have water regulating, soil protective, climate stabilizing functions. They are an important location of many relict, endemic and threatened species of plants and animals. European chestnut or sweet chestnut (Castanea sativa Mill.) is the dominant of mountainous forests of Western Georgia (150-1800 m). It occupies the most percentage of areas covered with forests Castanea sativa forests are developed in both West and East Georgia, but to the west of the country they occupy larger areas. Chestnut trees generally extend from 200 (West Georgia) up to 900–1000 meters above sea level, having the absolute upper limit at 1400 meters in sporadic locations of West and East Georgia (Nakhutsrishvili, 2013)
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