Development of ‘one-step freezing’ cryopreservation procedures for the long-term conservation of timber species

Author: Mariam mariamgaidamashvili
Co-authors: E. Khurtsidze, T. Kutchava, E. Makharashvili, N. Lortkipanidze
Keywords: cryopreservation; dehydration; vitrification; zygotic embryos, encapsulation.
Annotation:

Conservation plays a fundamental role in order to prevent the loss of plant species with ecological and economic importance. Cryopreservation or freeze-preservation at ultra-low temperature (-196°C) in liquid nitrogen is the only technique currently available to ensure the safe and cost-efficient long-term conservation of the germplasm of problem species, including non-orthodox seed species and vegetatively propagated plants. Optimization of cryogenic storage methodology has been achieved for individual plant specimens, which limits the routine use of above method. Our work aims to develop effective ‘one-step freezing’ cryopreservation procedures for the long-term conservation of some timber species. 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%) have 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.