Rationale

Many of the world’s major food plants produce seeds that undergo maturation drying, and are thus tolerant to extensive desiccation and can be stored dry at low temperatures. Seeds of this type are termed orthodox. Storage of such orthodox seeds is the most widely practiced method of ex- situ  conservation of plant genetic resources (90% of the 6.1 million accessions stored in genebanks are maintained as seeds).

In contrast to orthodox seeds, a considerable number of species, predominantly tropical or subtropical in origin, such as coconut, cacao and many forest and fruit tree species, produce seeds which do not undergo maturation drying and are shed at relatively high moisture content.  Such seeds are unable to withstand desiccation and are often sensitive to chilling. They therefore cannot be stored under the conventional seed storage conditions described above, i.e., low moisture content and low temperature. Seeds of this type are called recalcitrant and have to be stored in moist, relatively warm conditions to maintain viability. Of more than 7,000 species for which information on seed storage behaviour has been published, approximately 3% are recorded as recalcitrant and an additional 4% as possibly recalcitrant.

There are other species for which conservation as seed is problematic. Firstly, there are those that do not produce seeds at all and are consequently propagated vegetatively, for example banana and plantain (Musa  spp.). Secondly, there are crops such as potato (Solanum tuberosum ), other root and tuber crops such as yams (Dioscorea  spp.), cassava (Manihot esculenta)  and sweet potato (Ipomoea batatas) , and sugarcane (Saccharum  spp.) that have either some sterile genotypes and/or some that produce orthodox seed. However, as is the case for grape (Vitis vinifera ), these seeds are highly heterozygous and are consequently of limited use for the conservation of certain genotypes. These crops are usually propagated vegetatively to maintain genotypes as clones.

For long-term storage, cryopreservation, i.e., storage at ultra low temperature (liquid nitrogen, -196 OC), is employed. At this temperature, all cellular divisions and metabolic processes are stopped. The plant material can thus be stored without alteration or modification for a theoretically unlimited period of time. Moreover, small volumes of culture can be stored protected from contamination, and require very limited maintenance. Cryopreservation is currently the only safe and cost-effective option for the long-term conservation of genetic resources of problem species. With plants, cryopreservation is used for long-term storage of recalcitrant and intermediate seed species, vegetatively propagated plants, rare and endangered species and biotechnology products such as metabolite-producing cell lines and genetically engineered material. In the last 15 years, new vitrification-based techniques have been developed including vitrification, encapsulation-dehydration, encapsulation-vitrification and droplet-vitrification.

Cryopreservation protocols have been established for a large range of plant species from temperate and tropical countries. The development and application of cryopreservation is much more advanced for vegetatively propagated species than for recalcitrant seed species. A growing number of examples of large scale, routine applications of cryopreservation can be found for orthodox seeds of short lived and endangered species, pollen of horticultural crops, non-orthodox seed species, dormant buds of fruit and forestry species, in vitro  shoot tips of vegetatively propagated plants and biotechnology products. Large scale, international projects focusing on the development and application of cryopreservation for plant species are underway, with funding coming from various sources, including the EU (COST Action 871 “Cryopreservation of crop species in Europe”) or the Global Crop Diversity Trust (Development and refinement of cryopreservation protocols for the long-term conservation of vegetatively propagated crops). Dramatic progress has been made during the last 15 years in the development of cryopreservation techniques and there are a steadily increasing number of situations where cryopreservation is being used routinely for long-term storage of plant genetic resources.

Based on this assessment of the current state of the art of cryopreservation, the following priority areas for research have been identified:

• Development of new (vitrification-based) techniques & application to additional species, including vegetatively propagated and recalcitrant species;
• Fundamental studies aiming at understanding mechanisms related to resistance to dehydration and freezing;
• New utilizations of cryopreservation (cryotherapy).
• Management of cryopreserved collections and integration of cryopreservation in global conservation strategies.

Objectives

The project has two main objectives, to:

1. Develop cryopreservation techniques for a vegetatively-propagated crop, yam and transfer;
2. Apply these techniques to other selected crop species.

Actions planned

WP1 Develop cryopreservation protocols for in-vitro plants from apex of yam

Task 1. Selection and multiplication of accessions (2-3 accessions per species).
Task 2. Comparison of different techniques.
Task 3. Histological analysis of explants during cryoconservation.

WP2 Pilot tests of selected cryopreservation protocols

Task 1. Selection and multiplication of accessions (about 10 accessions per species).
Task 2. Transfer protocols to partners.

WP3 Apply the protocols that are developed to other species

Task 1. Select target species.
Task 2. Test selected protocols on a limited number of accessions.

Project team