Developing efficient cryopreservation procedures (conservation in liquid nitrogen, at -196°C) and establishment of a cryobank for the long-term storage of endangered plant genetic resources of Georgia

Project leaders

Maurizio Lambardi, Mariam Gaidamashvili

Agreement

GEORGIA - SRNSF - Shota Rustaveli National Science Foundation

Call

CNR/SRNSF biennio 2018-2019 2018-2019

Department

Biology, agriculture and food sciences

Thematic area

Biology, agriculture and food sciences

Status of the project

New

Research proposal

Conservation plays a fundamental role in order to prevent the loss of plant species with ecological and economic importance. The anthropogenic pressure, the introduction of alien species, as well as domesticated species, and chronic weed infestation have dramatic effects on plant diversity, which is reflected in an increase in the number of threatened species. Therefore maintenance of plant genetic resources in situ and ex situ has two main objectives, the conservation of genetic diversity at large, and of selected varieties with economic and agronomic value.
In situ conservation methods alone are insufficient for saving endangered species. Additional approaches, like storage in seed banks, field gene collections, in vitro collections and botanical gardens complement the preservation programs for plant biodiversity. They are classified as ex situ strategies, which mean to maintain the biological material outside their natural habitats. Storage of desiccated seeds at low temperature, the most convenient method to preserve plant germplasm is not applicable to plant species that do not produce seeds, or plants with non-orthodox seeds, as well as to vegetatively propagated plants. Preservation only in field collections is risky and can lead to genetic erosion because of pests, diseases and adverse weather conditions. The maintenance of in vitro collections is labour-intensive and there is always the risk of losing accessions due to contamination, human error or somaclonal variation.
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. Under these conditions, biochemical and most physical processes are completely arrested. The plant material can thus be stored without alteration or modification for a theoretically unlimited period of time. In addition, cultures are stored in a small volume, protected from contamination and require very limited maintenance. In this context, cryopreservation is a sound alternative to seed- and in-field banks for the long-term storage of plant germplasm. However, it should be regarded as complementary to the traditional in situ and ex situ approaches to germplasm preservation, so becoming a strategically important support to traditioanal in-field banks to provide a real guarantee against accidental loss of plant genetic resources.
So far, cryopreservation procedures have been developed for about 200 plant species. With the development more innovative cryopreservation protocols, based on the prevention of intra- and extra-cellular ice crystals by means of cell dehydration followed by direct immersion of explants in liquid nitrogen, the cryostorage of genetic resources has become a realistic target for many plant species. Nevertheless, despite the fact that cryogenic procedures are now being developed for an increasing number of recalcitrant seeds and in vitro tissues/organs, the routine utilization of cryopreservation for the preservation of plant biodiversity is still limited. A large amount of the work is still performed in the framework of academic studies and involves only one or a few accessions per plant species. This is mainly due to the (i) complex and time-consuming optimization of procedures, which is always required before an efficient cryopreservation protocol is established for a new species or accession, (ii) limited awareness of plant researchers unacquainted to recent developments in cryogenic storage methods, (iii) lack of coordinated research on plant cryopreservation.
To address these shortfalls, the project aims to establish efficient cryopreservation procedures for long-term conservation of threatened woody plant species of Georgia (Caucasus Biodiversity Hotspot region), with the final goal to generate a cryobank of endangered national woody germplasm. Emphasis will be placed on using this approach to priority woody species that are included in the Red List of Georgia according to the criteria defined by the International Union for Conservation of Nature (IUCN). The project also aims to extend cooperation with European researches by means of collaboration with the research group of the CNR-IVALSA, Trees and Timber Institute, Laboratory of "In Vitro Technologies and Cryopreservation", and by scientific contribution to International Conferences and Journals.
As for the expected outcomes, cryopreservation of plant germplasm has obvious advantages over in vitro storage in terms of space saving and improved phytosanitation. Other benefits of cryopreservation are: (i) the possibility to put in conservation a wide range of plant organs and tissues, (ii) the low conservation cost, (iii) the maintenance of specimens in absolute genetic and sanitary security. However, the storage of specimens at -196°C does not aim to replace the traditional in situ and ex situ approaches to tree germplasm preservation. Rather, it should be regarded as complementary, in order to develop a multi-option approach for the conservation and use of genebanks, to provide a real guarantee against accidental loss of plant genetic resources. Hence, by taking advantage of the internationally recognized expertise of the CNR-IVALSA in the field of plant cryopreservation, the creation of a plant cryobank in Georgia (the first in the Country) would be a milestone towards the safeguard of threatened woody plant genetic resources of the Caucasus Biodiversity Hotspot region.
The proposed plant species (see "Obiettivi") will be selected on the basis of their economic and ecological importance for the region.

Research goals

The emphasis will be put on forest woody Angiosperms and Gymnosperms with non-orthodox seeds and a very limited storage period, as well as vegetatively-propagated trees. The list of target plants will include: Castanea sativa Mill. (IUCN category VU, A2), Juglans regia L. (IUCN category VU, A2), Taxus baccata L. (IUCN category VU, B1b (i,ii). Selected broad-leave and coniferous species are the dominants of forest phytocoenosis of Georgia and remain on the verge of extinction due to the low self-renewal and diseases.
Objective 1: To screen in detail the current utilization of plant conservation measures in region. Current research on plant preservation in region is limited and not coordinated. Hence, a listing of all initiatives that are currently ongoing is essential.
Objective 2: To screen and compare the efficiency of existing plant cryopreservation protocols, and to test them with the selected Georgian endangered species.
Objective 3: To develop and optimize plant cryopreservation protocols. The resulting cryopreservation protocols will be: efficient (at least 50 % post-thaw recovery), user-friendly (technically not too demanding and with a limited number of steps), fast (thus applicable to large amounts of germplasm), broad (applicable to a wide variety of plant species and tissues).
Objective 4: To apply cryopreservation to existing ex situ germplasm collections, as well as to in vitro collections, and create a cryobank of endangered Georgian woody plant germplasm.

Last update: 18/08/2025