Development of an Efficient ‘One-Step Freezing’ Cryopreservation Protocol for a Georgian Provenance of Chestnut (Castanea sativa Mill.) Zygotic Embryos

  • Mariam GAIDAMASHVILI Iv. Javakhishvili Tbilisi State University, Faculty of Exact and Natural Sciences, Department of Biology, 1, Chavchavadze Ave. 0179, Tbilisi
  • Eka KHURTSIDZE Iv. Javakhishvili Tbilisi State University, Faculty of Exact and Natural Sciences, Department of Biology, 1, Chavchavadze Ave. 0179, Tbilisi
  • Carla BENELLI National Research Council (CNR), IBE/Institute of BioEconomy, via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze https://orcid.org/0000-0001-6068-8394
  • Maurizio LAMBARDI National Research Council (CNR), IBE/Institute of BioEconomy, via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze https://orcid.org/0000-0002-8898-7123
Keywords: chestnut; cryopreservation; dehydration; embryonic axes; vitrification; zygotic embryos

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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Published
2019-11-08
How to Cite
GAIDAMASHVILI, M., KHURTSIDZE, E., BENELLI, C., & LAMBARDI, M. (2019). Development of an Efficient ‘One-Step Freezing’ Cryopreservation Protocol for a Georgian Provenance of Chestnut (Castanea sativa Mill.) Zygotic Embryos. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 47(4). https://doi.org/10.15835/nbha47411653
Section
Research Articles