Projects

Specific Objectives

• Use European Allium Database to screen garlic and shallot germplasm.
• Screen 1600 garlic and 550 shallot accessions for redundant duplication by general molecular marker system.
• Confirm interrelationships by morphological character lists.
• Develop a structured Core Collection under elimination of redundant duplicates.
• Cryopreserve the 200 most important garlic accessions using vitrification.
• Exchange safety duplicates of cryopreserved garlic to establish the Tripartite Cryopreservation Genebank
• Disseminate CGP documents to facilitate joining other European partners.
• Virus elimination to free 125 most important garlic accessions from viruses, prove of virus-free state.
• Conclusions for future expanding preservation from garlic to shallot and other vegetative alliums.

Actions and means involved

Because of increasing awareness that vegetatively maintained germplasm is the most expensive part of plant genetic resources, a consortium of seven European partners comprising main collection holders of vegetatively propagated alliums, virus and molecular marker experts was established to use high-tech methods for rationalization and increase of storage safety and health conditions. Five work packages were developed: Documentation, Molecular screening, Cryopreservation, Virus elimination and Coordination.

Most important is the universal use of one molecular marker system for all accessions to ensure comparability in screening for duplicates in genetic patterns, which requires service of one laboratory to all countries. A half-centralized approach for germplasm storage in liquid nitrogen will be adopted joining laboratories of three countries, which allows connecting the advantages of economically favourable centralization and needed local safety splitting. Virus elimination increases germplasm quality facilitating its free exchange. This major investment in EU vegetative Allium genetic resources will result in upgrading the European Allium Database and considerable improvement of storage safety and health conditions of material.
 

WP 1 Documentation

The project is designated to be an integrated part of the European germplasm maintenance strategy. Therefore, the connections with the surrounding data management are maintained by close connection with the European Allium Database EADB managed by D.Astley, HRI Wellesbourne, UK. It is the first source for the duplicate screening, and it will then be the last target of the condensed information developed within the project.
 
All the other Work Packages function also with exact documentation only. Thus, the final safety duplication of cryo samples will be done on the base of the documentation developed in the project. The condensation of the documentation steps will be performed in the annual project reports.
 

The European Allium Database fundament for passport dokumentation

Coloured code used for the cryopreservation tubes

Etiquette printer

WP 2 Molecular duplicate screening

 
Undesired duplication (redundancy) is one of the main surplus cost factors in genebank management. As has been found in the past, morphological characters and passport data are not sufficient to find undesired duplication. Therefore, molecular markers which give considerable contribution to reach this goal, are used in this project. They are well developed in Allium, especially for garlic. External assistance is provided by the laboratory of the company Array-On at Gatersleben, which uses its services for all the garlic and shallot germplasm in the project. A novel technology is used which screens the material on the basis of SNP markers using polydimensional SNP analyses (micro-array technology). The collection curators send for this freeze-dried leaf samples, from which DNA is extracted in the laboratory of IPK (P0). Only P6 (NGB) performs the DNA extraction in the own institution. DNA is then forwarded to Array-On where the screening is done. CGN (P4) is then the final station, which does the integrative work, to analyse the material for duplicates using the cluster analysis UPGMA. Duplicates can finally be recognised and eliminated. Furthermore, new insights are expected into the infraspecific groupings of garlic and shallot.

 

Fresh leaf samples on ice

Preparing leaf material

Robotor for DNA isolation

WP 3 Cryopreservation

 
Cryopreservation is the main workpackage with respect to the manpower employed. This takes 65% of the project. This is caused by the high labour input required to introduce the material into cryopreservation. The economical effect of cryopreservation is, however, realized by the very low cost of the maintenance once the material is stored in liquid nitrogen.
 
The source organs for cryopreservation depend of the type of the material (bolting or non-bolting) and the season, because several organs are only available in some seasons. Bulbils are formed in summer. Then there is a dormant period, the main usability is from December until April. In non-bolting material, only the basal plate is usable, which is in the cloves from autumn to winter or in the compound bulb in spring. This source is also available in bolting garlic. Both types can be used in form of in vitro cultures, which is the normal donor material when it is in virus-free condition. In vitro cultures are best usable after cold preculture.
 
The basic method for cryopreservation envisaged here is vitrification. The principle consists in transferring small explants from shoot tips after dehydrating pretreatments into standardized cryoprotective solutions, which allow the tissue water to undergo glass transition during fast drop down of the temperature. The basic process characterized above is conducted in the three cryopreservation units of P0, P1, and P2. They will then exchange their safety duplicates under coordination of P1. All measures will be technically adjusted in agreements and will be subject to regular auditing procedures, which became standard in the present laboratory practice.
 

P1: Bulbil explants floating in PVS solution

P0: Transferring cryo tubes into liquid nitrogen

P0: Taking a rack with cryo material out of the tank

WP 4 Virus elimination

Virus diseases can cause dramatic yield losses in the crops. This danger is especially important in vegetatively propagated germplasm, because the major part of the viruses does not pass the seed stage of a normal generation plant cycle, which is, however, absent here. Of the five major viruses present in garlic, OYDV (onion yellow dwarf virus), SLV (shallot latent virus), LYSV (leek yellow stripe virus), GCLV (garlic common latent virus) and the Allexi-Virus group (previously know as mite-borne filamentous viruses), OYDV and LYSV are the most harmful virus species.
 
Virus tests are performed by ELISA technique and/or PCR. A final field performance test will be implemented by UNIBAS (p3) to document the impact of virus elimination on the health and yield components. Conclusions will be drawn about the further phytosanitary policy within garlic germplasm management and, beyond of the garlic germplasm also to shallot and other alliums.
 

Working with the ELISA technique

ELISA plate - yellow colour where still viruses

Various meristem explant sources

WP 5 Coordination

The coordinator (P0) is responsible for the overall management of the action. He will be assisted by three WP leaders who will manage the various WPs and will report to him. Decisions will be made during the annual project meetings with all partners present and based on majority vote. Major activities are supervision and writing of the annual progress reports, financial management of the project, organization of the annual project meetings by the local partners in close collaboration with the coordinator, contacts with the office at Brussels and the management of the project website.
 

Pre-project discussion in the ECPGR AWG meeting at Prague

WP Leaders’ Meeting at CGN Wageningen, March 06, 2008

Third Project Meeting at RIVC, Skierniewice, March 09 - 10, 2010

EURALLIVEG - Projects

project partners

cryo tank inside

in vitro multiplication