Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers

More efficient conservation and use of vegetable genetic resources in Europe: ECPGR achievement and perspectives

The European Cooperative Programme for Plant Genetic Resources (ECPGR) is a regional network funded by the European countries and coordinated by Bioversity International. The Vegetables Network with representatives of 42 countries, is one of the crop specific ECPGR networks (http://www.ecpgr.cgiar.org/ networks/vegetables.html). It consists of six Working Groups (WGs), i.e., on Allium, Brassica, Cucurbits, Leafy Vegetables, Solanaceae and Umbellifer Crops. Sharing responsibilities for the ex situ conservation of European vegetable crops genetic resources is the highest priority of the Vegetables Network. It is foreseen that the rationalization of the collections will lead to higher cost-efficiency, and improvement of plant genetic resources documentation and quality. These efforts will be continued within the framework of the AEGIS initiative (A European Genebank Integrated System) (http://www.aegis.cgiar.org/). Challenges for the Vegetables Network include the identification of the so-called Most Appropriate Accessions (MAA) for each crop for their inclusion in the decentrally managed European Collection, and the development of agreed crop specific technical standards for conservation. Achievements of the Network in recent years include the development of European Central Crop Databases (ECCDBs), quality standards for collection man-agement of seed-propagated crops and cryopreserved material, safety duplication improvement and definition of minimum characterization descriptors. Several EU-funded projects have initiated and accelerated the activities of the WGs. Apart from further improvements within the framework of AEGIS, the Network is planning a number of other initiatives, such as improving collaboration at the global level (Allium), developing molecular characterization protocols (lettuce), filling the gaps in the conservation of wild relatives (Brassica and Umbellifer Crops), and improving the Network’s communication with the scientific community and the public at large

EURALLIVEG: Establishment of a European core collection by cryopreservation and virus elimination in garlic

A European project named EURALLIVEG (Vegetative Allium, Europe’s Core Collection, safe & sound) was performed under Council Regulation EC 870/2004 from 2007 until 2011. It was coordinated by IPK. The main focus of the project was the development of a European integrated Allium Core collection provided by national collections of Germany, Czech Republic, Poland, Italy, France, and Nordic countries. This collection was designed as a part of the European Genebank Integrated System (AEGIS) of the European Cooperative Program for Plant Genetic Resources (ECPGR). The project consists of five work packages: documentation, molecular screening, cryopreservation, virus elimination, and coordination. Main aim was identification of a core collection of the 200 most important garlic accessions. This collection was cryopreserved using vitrification, which is the most economic way for long term storage. A Cryobanks Network was organized by three project partners. This system ensures safe storage of material by adopting a safety duplicate strategy. The documentation of the collection used passport and characterization data, pictures and other information. In addition genetic fingerprinting with AFLP markers was conducted. In the project, the garlic accessions were introduced into meristem culture for virus elimination. Of the viruses present in garlic, Onion Yellow Dwarf (OYDV) and Leek Yellow Stripe (LYSV) Viruses are most harmful. Therefore, because of the impossibility to do all treatments and tests on all germplasm, it was decided to test the major part of the collection on OYDV and LYSV only. However, a specific part with higher priority (backbone subset) was examined for five viruses. Virus detection used ELISA testing

More efficient conservation and use of vegetable genetic resources in Europe: ECPGR achievements and perspectives

The European Cooperative Programme for Plant Genetic Resources (ECPGR) is a regional network funded by the European countries and coordinated by Bioversity International. The Vegetables Network with representatives of 42 countries, is one of the crop specific ECPGR networks (http://www.ecpgr.cgiar.org/ networks/vegetables.html). It consists of six Working Groups (WGs), i.e., on Allium, Brassica, Cucurbits, Leafy Vegetables, Solanaceae and Umbellifer Crops. Sharing responsibilities for the ex situ conservation of European vegetable crops genetic resources is the highest priority of the Vegetables Network. It is foreseen that the rationalization of the collections will lead to higher cost-efficiency, and improvement of plant genetic resources documentation and quality. These efforts will be continued within the framework of the AEGIS initiative (A European Genebank Integrated System) (http://www.aegis.cgiar.org/). Challenges for the Vegetables Network include the identification of the so-called Most Appropriate Accessions (MAA) for each crop for their inclusion in the decentrally managed European Collection, and the development of agreed crop specific technical standards for conservation. Achievements of the Network in recent years include the development of European Central Crop Databases (ECCDBs), quality standards for collection man-agement of seed-propagated crops and cryopreserved material, safety duplication improvement and definition of minimum characterization descriptors. Several EU-funded projects have initiated and accelerated the activities of the WGs. Apart from further improvements within the framework of AEGIS, the Network is planning a number of other initiatives, such as improving collaboration at the global level (Allium), developing molecular characterization protocols (lettuce), filling the gaps in the conservation of wild relatives (Brassica and Umbellifer Crops), and improving the Network’s communication with the scientific community and the public at large