Projects


Overall objective

Establishment of European Core Collection of vegetative alliums, covering garlic including molecular characterization, cryopreservation and virus elimination, and molecular characterization of shallot.


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.
 


 
The Work Packages


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 Dave 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 documentation

Colour code used for the
cryopreservation tubes

Label 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, it was intended to use molecular markers, which should give a considerable contribution. Molecular markers are well-developed in Allium, especially for garlic. The attempt was made to use external assistance by a laboratory which, unfortunately, did not reach this goal. A novel technology was applied on the basis of SNP markers using micro-array technology. Since the failure was not caused by the technique itself, SNP analyses are still seen as the main way to find undesired duplicates, In course of the action, the collection curators sent freeze-dried leaf samples, from which DNA was extracted in the laboratory of IPK (P0). Only P6 (NordGen) performed the DNA extraction in the own institution. DNA was then forwarded to the analysis laboratory. CGN (P4) was the final station, which analysed the work. Since we did not come to a finalization of this task, a limited AFLP survey was done on the EURALLIVEG garlic and shallot core collections in IPK (P0). As a result of this, first impressions on the infraspecific structure of the preserved genepools were obtained.

 

Fresh leaf samples on ice

Preparing leaf material

Robot 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 was conducted in the three cryopreservation units of P0, P1, and P2. They exchanged then their safety duplicates under coordination of P1. All measures were technically adjusted in agreements and were subject to regular auditing procedures, which became standard in the present laboratory practice. The safety-duplicated cryo-collection hosted in the genebanks of three countries is legally fixed by a Consignment Agreement between the three partner institutions.
 

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 were performed by ELISA technique. A further field performance test will be implemented later by UNIBAS (P3) to document the impact of virus elimination on the health and yield components. Conclusions will be drawn later 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
viruses still present

Various meristem
explant sources


WP 5 Coordination


The coordinator (P0) was responsible for the overall management of the action. He was assisted by three WP leaders who managed the various WPs and reported to him. Decisions were made during the annual project meetings with all partners present and based on majority vote. Major activities were 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. The further maintenance of the EURALLIVEG collection and the activities based on this project will be supported by the chairmanship of the EURALLIVEG coordinator in the ECPGR Allium Working Group.
 

Pre-project discussion
in the ECPGR AWG meeting
Prague, January 20, 2006

WP leaders’ meeting at CGN
Wageningen, March 06, 2008

Third project meeting at RIVC (now InHort),
Skierniewice, March 09 - 10, 2010


EURALLIVEG - Projects

 

cryo tank inside
cryo tank inside

 

preparing leaf material
preparing leaf material

 

in vitro multiplication
in vitro multiplication