March 1-2, 2011: Final Project Meeting at CRI, Prague, Czech Republic

• See for details the Final Meeting Website.
  

The meeting started with a welcome of the Head of the Department Molecular Biology, Jaroslava Ovesná. She presented the structure and the aims of the Crop Research Institute and welcomed the participants. The project coordinator Joachim Keller introduced then the guests to the meeting, Olivier Diana (GENRES office Brussels), Dave Astley (Chairman of the ECPGR Allium Working Group), Jan Engels (AEGIS Coordinator), Ana Maria Barata da Silva (Portuguese Genebank), Francisco Mansilla (Garlic Collection CIFA Cordoba, Spain).
Olivier Diana gave a survey on the activities of the projects within the GENRES call 870/2004 of the DG AGRI. He set this in the context of the European Strategy 2020 and the surrounding political project landscape such as the Natura 2000 network, the FP7 and LIFE project strategies. Jan Engels updated the participants about the present state of the AEGIS policy. The Memorandum of Understanding of AEGIS has been signed already by 26 countries. Dave Astley gave an actual picture about the distribution of the garlic and shallot accessions within the European Allium Database (EADB). Joachim Keller (P0) opened the presentation of the specific subjects in his overview about the project, its history, structure, parameters, achievements and problems in general.
Joachim Keller introduced the work package by coming back to the criteria for selecting the Core Collection, respectively the Most Appropriate Accessions MAA. The main subjects of his presentation were two activities belonging to WP1 (Documentation):

• The presentation of the first draft of the written documentation of the accessions envisaged for the Garlic and Shallot Core Collection. The EURALLIVEG Accession Catalogue (EAC) contains passport and characterization data as well as images of the respective accessions.This special part will be precluded by a general introduction about the collection and a short presentation of every partner.
• The websites of the Image database of IPK the "Garlic and Shallot Core Collection" (GSCC). Joachim Keller offered to host all the data within the IPK website, structured in analogy to IPK’s accessions and in the same arrangement as it is done in the EAC.

Finally some other documentation needs were stressed by Joachim Keller (P0): a document about the safety duplication including the legal aspects, a booklet about methods and technical guidelines and an action plan for the future management and further strategy.
Chris Kik (P4) reported conclusively about the problems in the molecular marker analysis, which were caused by the failure of the external assistance company Array-On. This was more or less a repetition of the subject as has been discussed in the third annual meeting. After this depressing summary of earlier facts, Christine Zanke (P0) came out with some new developments concerning some smaller analyses:

• confirming the unsuitability of most SNPs provided by Array-On for the intended analyses
• showing some results done by the laboratory of Frank Blattner within IPK concerning AFLP analyses of a limited selection of garlic and shallot accessions (more or less covering the core collection accessions) and
• by very encouraging new results of a SNP analysis test by the Company Trait Genetics, Gatersleben.

A press conference about EURALLIVEG was held in order to increase the awareness of the project in the public of the Czech Republic. It was presented by Olivier Diana, Jan Engels, Joachim Keller (P0), Jaroslava Ovesná, Helena Stavělíková (P1) and Jiří Zámečník (P1).
In the WP 3 (Cryopreservation) the partners presented their results in their specific contributions. Renata Kotková (P1) referred additionally on results of her PhD work. So far Jiří Zámečník (P1) reported that 14 accessions are missing in the general summarization. Data of them will be provided as soon as they will have been recorded, at latest by the completion of the final technical report. In detail the bolting accession are more than due and in non-bolting and on the back bone collection remain lacks. This is mainly caused by problems Jiří Zámečník (P1) referred on. Finally he presented the strategy for the Safety Duplicate action which is intended to be performed in the last week of March. For WP 4 (virus elimination) Vito Miccolis (P3) and Luciana Altieri (P3) reported about the activities concerning virus elimination and the backbone collection. The method is suitable in principle. It needs, however, more time than expected. This concerns the problems of difficult elimination of the allexi virus complex, in too slow growth of the regenerants from meristem culture and in the tendency to fall into dormancy of many of the accessions. Christine Zanke (P0) reported in the presentation belonging to the WP5 (Coordination) on the project meetings, training couses, the website of EURALLIVEG and underlined the deadlines of the reporting for the final technical and financial reports.
The second day was organised as a workshop on aspects of germplasm preservation in Allium. It started with a presentation of Jaroslava Ovesná (CRI).She worked on AFLP analyses of the Czech garlic using 286 markers. Furthermore, analyses were presented on S-alk(en)yl cystein, which is pharmaceutically important (concerning the ratio alliin/methiin). Leona Leišová-Svobodová tested five viruses by means of real-time PCR. Joachim Keller (P0) referred on a project to analyse costs of cryopreservation in comparison with the cost of field culture. This was performed in the frame of a Master's Thesis in IPK together with the Leibniz University of Hannover. A Small Competitive Project financed by AEGIS and coordinated by Christine Zanke (P0) was presented in which unripe inflorescence bases were used for cryopreservation.
The next part of the workshop consisted in a demonstration of the preparation actions technically organized by the staff of CRI. The preparation of explant isolation from in vitro plants and bulbils for cryopreservation as well as of meristem explants for virus elimination were demonstrated. A workshop discussion followed. The next presentation focussed on political aspects of cryopreservation within Czech Republic. Ladislav Dotlačíl, the National Coordinator, presented the general situation of germplasm preservation in the various institutions and gave information about the vegetatively propagated material in detail, especially concerning the role of cryopreservation. Helena Stavělíková (P1) described the situation in the Czech garlic and shallot genebank at Olomouc and mentioned four companies using garlic from the collection.
Towards the end of the meeting future aspects were stressed. Ana Maria Barata da Silva (Portugal) presented the Portuguese collection which started in 1994 and pointed out that they had performed genetic analyses by AFLP (28 markers), morphological and biochemical (of allicin) analyses of 367 garlic accessions. Francisco Mansilla (Spain) also presented the collection and several accessions in detail. Both guests emphasized that they are very interested to collaborate with the project consortium and be included in any future discussion about the Allium germplasm preservation strategy.
The meeting was closed by a visit to the baroque monastery St. Margaret and its Romanesque undercroft from 11th century as well as a good farewell dinner in the adjacent restaurant. In conclusion, we can state that the whole meeting was performed in a very open and collegial atmosphere, encouraging all participants to go on with their efforts to collaborate also in future beyond the termination of the project. We thank the staff of the host institution CRI for the good organization and nice arrangements to present our main objects. We gratefully thank some companies for sponsoring the meeting and, thus, supporting the very good performance. Visitors representing these companies were Radovan Tuma (Agriculture Union), Martina Beránková (TARGO) and Martin Dindos (ALLIVICTUS).

Single bulbs of shallot and bulbs of garlic accessions

In vitro plantlets in the growth chamber

Participants of EURALLIVEG and visitors of the final meeting

March 9-10, 2010: Third Annual Project Meeting at RIVC (now InHort), Skierniewice, Poland

The meeting started with the welcome, given by the Director of RIVC, Franciszek Adamicki, and the main organizer Teresa Kotlinska (P2) and was opened by the coordinator, Joachim Keller (P1), followed by a short introduction of all participants. The aims of the meeting were the annual reporting of the results, discussions on achievements and problems of the last year as well as to point out which requirements are needed to fulfil the project parameters. The first presentation was a general review of the project philosophy and the international situation of the genetic resources preservation, which is the background for the project. It was given by Joachim Keller. The final composition of the garlic core collection was discussed by Christine Zanke (P0). IPK (P0) will contribute with 36 bolting and 20 non-bolting accessions, CRI (P1) with 36 and 26 and RIVC (P2) with 37 and 20, respectively. The Italian (non-bolting) material will be split into 9 accessions for P0 and 6 for P1. The French material (6 accessions) will be stored by P1. Since the structuring by means of molecular markers is not possible yet, the final documentation of the 200 accessions of the cryopreserved core collection needs to be focussed onto morphological description and passport data. For this examples were given.
The state of the art of the backbone subset (WP3, virus elimination) was given as follows by Vito Miccolis (P3):

• one Italian and three German accessions were sent to P0 completely virus-free, 2 have still some viruses.
• five Czech and two French accessions had virus-free plants and were sent to P1, three of them were lost on the transport and need to be sent again. Of two accessions several plants, which still contain some but not all viruses, were sent separately.
• five Polish, one Italian and two French accessions were sent virus-free to P2.

Since the total of the backbone set is still not reached, UNIBAS needs to test the material further for all 5 viruses in order to have the option to rearrange the plants designated for the main virus elimination set into the backbone set in case fully virus-free material could be obtained. Concerning the main virus elimination set, the re-sending to the partners started with 2 accessions to P0, 4 acc. to P1, and 2 acc. to P2. Others are ready to be tested by ELISA. Discussion arose concerning the field performance tests planned by UNIBAS. Due to the delay in the fulfilment of the backbone set, it will not be possible to do a full performance test within the project period. Helena Stavělíková (P1) presented the situation of the field management and virus elimination activities at the Olomouc collection place. The special situation of the Polish garlic collection, which includes 373 accessions, was presented more in detail by Teresa Kotlinska (P2) at the beginning of WP3 (Cryopreservation). The partners presented their accessions already being in cryopreservation. In total 59 bolting, 11 non-bolting and one accession of the backbone subset were cryopreserved, which means, that only 35.5 % of the required 200 cryopreserved accessions are finished. Planning and logistics of safety duplicate exchanges was discussed by Jiří Zámečník (P1). He presented the planned transport route coming from Prague via Gatersleben to Skierniewice and retour. This will be done in an open transport car driven by a staff member of CRI. The distance is 2200 km in total. The day was ended by a visit to the laboratory of P2 guided by Marta Olas-Sochacka and Teresa Kotlinska. The plantlets in the preculture phase were presented. Their quality is well-suited for cryopreservation. One of the main progress steps is that the laboratory got a new clean bench, which improves the work power for in vitro culture and cryopreservation. At the second day Chris Kik (P4) reported on the problems appearing during the molecular screening, which resulted in a general failure of the SNP analysis. No concluding duplicate screening could be done. Additional activities at IPK were presented by Christine Zanke (P0). At the end, only four primers were found to be really usable, which is by far too insufficient. Concluding discussions were followed how to proceed with the molecular analysis in order to find finally some results. Svein Solberg (P6) is a new representative of NordGen replacing Agnese Kolodinska Brantestam, who is in maternity leave. He presented the new structure of NordGen. Florence Esnault (P5) gave a summary about the contribution of INRA to the project consisting of providing standard material of shallot according to the presentation of Le Thierry D’Ennequin et al. (1997). Christine Zanke (P0) summarized the obtained results and gave an overview about the meetings so far organized. At the end, the delay of fulfilling deliveries and milestones in comparison to the project document was characterized. The deadline for the delivery of the contributions of the next financial and technical annual reports was fixed. Since the core collections of garlic and shallot need to be documented until summer 2010, it is now necessary to fix the accessions finally. A list of the final accessions needs to be sent to Christine Zanke by March 19, 2010, and will be included in the next annual technical report. Joachim Keller (P0) concluded the meeting with the expression of hope that, in the last project year, all efforts will be concentrated on fulfilment of the parameters as good as possible to give the activities to continue the European Garlic and Shallot Core Collection a good perspective beyond the project period. The day was ended by a piano performance in the concert hall of the institute’s main building giving a very good concluding atmosphere and encouragement for further activities together in the project. The participants thank the well-organized and dedicated activities of Teresa Kotlinska, Marta Olas-Sochacka and the other staff of the laboratory to give the meeting a good background.

Opening remarks of Joachim Keller

The new laminar flow box used at InHort

Explants on plates or in tubes after cryopreservation

October 20, 2009: Small Technical Meeting Cryopreservation and In vitro Culture" at RIVC (now InHort) Skierniewice, Poland

The local organizer was Teresa Kotlinska, Research Institute of Vegetable Crops (RIVC, now renamed to InHort), together with her colleagues. The main aim of the meeting was the updating of work results and discussion of technical and technological details of in vitro culture and cryopreservation in WP 3, mainly to facilitate technology transfer between the participating laboratories. The meeting was opened by a warm welcome of Franciszek Adamicki, the director of RIVC, and a small visit to the historical main building of the institute, where a part of the meeting was conducted.
After the start by Joachim Keller, Christine Zanke gave information about the situation in WP 2, where the molecular duplicate analysis in the laboratory of the external assistance failed; thus, giving no usable results for the project. The implications of this problem were discussed vehemently. The selection of 200 accessions of the cryo-core collection needs to be done by the traditional methods only, i.e. using the passport data and morphological characters to avoid duplication as much as possible. In the following part, the partner presented their state of the art. At present 42 accessions of bolting and 6 accessions of non-bolting garlic are in cryopreservation. A number of 52 accessions of non-bolting garlic are in the in vitro phase. Luciana Altieri reported on results of virus elimination. Some factors were discussed during and after the presentations. Afterwards the group visited the laboratory of P2. The final discussion was about the date of the next annual project meeting. It was decided to postpone the next meeting to March 9-10, 2010. The meeting ended with a very well organized dinner in a good and collegial atmosphere. Then participants thanked the local organizer and all their colleagues for their hospitality and the good organization of the meeting.

Working place for multiplication and cryopreservation

Marta Olas presented the her growth chamber (Heraphyt, Heraeus)

In vitro plantlets in this growth chamber

January 27, 2009: Special technical Meeting for WP 2: Molecular Screening at IPK, Gatersleben

The meeting was organized to discuss the further progress of the molecular marker analysis for duplicates and to overcome some difficulties observed in the last period. The programme was structured into four presentations which were provided at the internal website, a problem discussion and an agreement what will be done in the next three weeks.
The title of the four presentations were:

• "Collection and preparation of the leaf material for screening" presented by Christine Zanke (IPK, P0)
• "Development of SNP-markers and SNP-screening" presented by Dirk Fischer (Array-On)
• "Analyses of SNP data of European garlic accessions" presented by Robbert van Treuren (CGN)
• "Interpretation of the cluster analysis, garlic duplicate set" presented by Chris Kik (CGN, P4)

Finally it was emphasized by all participants, that the problems so far occurring are problems of the whole project community and not only of one of the participants. Insofar, it is in the interest of all of us to overcome that as much as possible. Correspondingly, the meeting was performed in a very constructive and collegial atmosphere.

December 4, 2008: Small technical meeting for WP 3: Quality Management of the In Vitro Culture and Cryopreservation Processes" at CRI, Prague

The local organizer was Jiří Zámečník, Crop Research Institute (CRI, P1), together with his colleagues. The meeting was organised to improve the knowledge and abilities of the participants with respect to the various steps of in vitro culture and cryopreservation of garlic. Another aim was to discuss the problems of Cryo-Knife which had been marked by the reviewers of the Brussels office after reviewing the Technical Report. Christine Zanke (P0) presented a Powerpoint presentation starting with the general structure of the accessions to be conserved by each partner. She gave detailed results of P0 and showed typical pictures of in vitro cultures prior to and after cryopreservation. Marta Olas (P2) gave a overview about the results of P2 including pictures of in vitro cultures. Jiří Zámečník (P1) showed a summary table about the whole WP. Afterwards online demonstrations were performed of the standard preparation of explants for cryopreservation from in vitro plantlets or bulbils and meristem explants for virus elimination. The size of explants from bulbils and from in vitro cultures used for cryopreservation is clearly larger than that of meristem explants used for virus elimination. A presentation of the culture rooms and cold rooms for alternating temperature preculture of P1 followed. A decision about the further strategy concerning cryo-knife was made. The meeting showed that such kind of discussions are, indeed, very necessary for the further improvement of collaboration. The meeting was conducted in an open and critical atmosphere, which was supported by the informal character of the meeting. The organiser Jiří Zámečník (P1) offered a very good surrounding by all the social background activities like lunch and dinner and an easy-to-reach accommodation.

Discussion about the multiplication of in vitro plantlets

Culture rooms at the CRI, Prague presented by Jiří Zámečník

Cold rooms for alternating temperature preculture

July 08-09, 2008: Second annual project meeting at UNIBAS, Potenza

The local organizer was Vito Miccolis (P3), University Potenza, together with his colleagues. They provided a perfect organization of all the events and the transportation and were very perfect in organising local services. The aim of the meeting was the annual reporting of the results, discussions on achievements and problems of the first year as well as on the further programme and to see the special situation of the garlic laboratory and field work of the Italian Partner P3. Also methodological and logistic questions had been discussed. Joachim Keller (P0) revised the general strategy of the project referring on the situation of genetic resources in garlic and shallot and gave a short report of the AEGIS activities. Then Kristiina Antonius presented the Nordic Genebank System and its recent changes. The main feature is a closer connection between plant, animal and forest genetic resources. Christine Zanke (P0) discussed the numbers, the selection criteria and classification of accessions provided to the project together with the official duplicates known by comparing the data sets of the European Allium Database. The different WPs were reminded and the overlapping nature of the tasks cryopreservation and virus elimination was, again, justified. In order to clear up the responsibilities of the partners, the specific numbers of accessions for each group was presented as it became also a part of the annexes to the project report. For WP2, molecular markers, an overview about the preparation steps to produce the DNA and the justification of the work, which consists in a cost reduction when eliminating undesired redundancy, was given by the WP leader. Chris Kik (P4) pointed out that the subcontractor Array-On (and his collaboration partner GenXPro) had difficulties which results in a delay of 2.5 months. Then the main cryopreservation results were reported by the different project partners. Vito Miccolis (P3) gave an overview about the accessions provided by the partners for virus elimination for the backbone subset and further details were given about the material transfers and movement. Luciana Altieri (P3) added more details on the accessions used for virus elimination and pointed out that there were some unforeseen problems. Later the practical details were discussed at the place where the work is running, which concerns the laboratories of the University of Basilicata. A visit to the fields of the Metaponto farm completed the programme. The participants finally visited the vegetable processing company "Centrone" at Conversano, which is mainly active in producing conserves, amongst them also such of bulbous crops.

Looking at harvested Allium plants

Guided tours through the labs

Looking at the Allium fields at Metaponto farm

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

The local organizer, Chris Kik, CGN Wageningen (WP leader P4), ensured perfect organization of the meeting including hotel reservation, local transportation and meeting dinner. The aim of the meeting was to prepare the first year’s annual interim reports and to further improve the internal communication between all the work package leaders. After a short welcome by Chris Kik (WP leader, P4) the actual activities and results of the work packages were discussed. For WP1, documentation, Joachim Keller (P0) presented the list of the garlic bolting and non-bolting accessions, which are envisaged to come into cryopreservation in course of the entire project. For WP2, molecular markers, organization and timing were excellent for collecting leaf materials for DNA extraction and their postage as well as the DNA extraction. Problems arose due to the delay in EST marker analyses in garlic. Christine Zanke (P0) and Jiří Zámečník (WP leader, P1) reported about the activities within WP3, Cryopreservation, including amount of accessions in vitro, in cryopreservation and cryo-knife results. Vito Miccolis (WP leader, P3) presented the actual results of the virus elimination. The EURALLIVEG website was presented to all participants. At the end the tables for the declaration of expenses were presented and explained in detail. Finally Chris Kik gave a good introduction into the seed storage and greenhouse reproduction and comparative trials of this well-organized and very active genebank.

Welcome to the Meeting at CGN

Visiting of the green house

Participants in front of the CGN office building

April 12-13, 2007: Start-up Meeting at IPK, Gatersleben

Soon after the start of the project on April 1, 2007, all participants came to IPK Gatersleben for the first meeting. After the welcome address of the Vice-Director of the genebank, Andreas Börner, and a review of the project documents, two guests gave a presentation. This was first Jan Engels from Bioversity International, Rome, who is responsible there for the European AEGIS policy. He explained the main topics of this strategy. After that, Dave Astley, HRI Wellesbourne, who runs the European Allium Database, presented it as being the fundamental basis for any documentation of accessions related to EURALLIVEG. The main part were the presentation and discussions about the work packages of the project together with a walk through the laboratory of the cryopreservation group and some practical demonstrations, e.g. about freeze-drying for leaf samples used for DNA analyses. The meeting was conducted in a very open atmosphere, characterized by vivid discussion around the nine main talks covering all aspects of the project. At the end, a visit to the Allium fields and to the contracted analysis laboratory of Array-On completed the programme. In the evening, the participants met in an atmosphere of friendship in the Potato House in Quedlinburg.

Looking at the Allium fields

Special discussion about an accession

Equipment demonstration in Array-On's lab

March 31- April 4, 2008: Training course on techniques of cryopreservation in IPK (P0) for RIVC (now InHort - P2)

An additional training course on techniques of micropropagation and cryopreservation of in vitro plants was organised in IPK (PO) for Renata Kotková (CRI, P1). In this one week Renata learned which methods are used for the sterilisation of the bulbils and how to isolate explants under the microscope. She isolated explants which are used for in vitro cultivation or for cryopreservation. In the very short time Renata successfully studied all instructions and helpful information, how to multiply in vitro garlic plants, which media need to be used for cryopreservation and the method for cryopreservation, carried out at IPK. The course was successful for all and a nice pleasure.

Sterilisation of bulbils for isolation of explants

Preparation of shoot tips for in vitro multiplication

Rewarming tubes in water bath after cryopreservation

May 21 - June 1, 2007: Training course on techniques of virus elimination in IPK (P0) for UNIBAS (P3)

The first meeting was very successful. Within these two short weeks we have passed the basic techniques of preparing meristems from ripe bulbs and bulbils from the field and ELISA testing of different plant material to Luciana Altieri (P3). Luciana brought her own material with her, garlic plants from five different accessions. These materials were used for virus screening and establishment of meristem culture. We prepared meristems from sterilized bulbs under the microscope and transferred them to solid growth media, which Luciana took then back to Italy. We made two different ELISA tests with material from bulbs, bulbils, in vitro plants and field plants for testing all important viruses. In the very short time we successfully showed Luciana all instructions and helpful information, which media need to be used for virus elimination and how to multiply in vitro garlic plants.

Filling up ELISA plates

Preparation of meristems

Multiplication of in vitro plants

April 16-27, 2007: Training course on techniques of cryopreservation in IPK (P0) for RIVC (now InHort - P2)

Within these two weeks we have passed the basic techniques on cryopreservation to Marta Olas (P2). Marta brought her own material with her, ripe bulbils from last year. We used this material for our cryopreservation training. After the sterilisation of the bulbils, we isolated the explants under the microscope and transferred them to solid growth media. After one day of incubation, the explants were cryopreserved. Marta also learned how to test the survival rates of cryopreserved explants, which media are used for cryopreservation and multiplication of in vitro plants. The course was successful for all and a nice pleasure.

Preparation of shoot tips for cryopreservation

Transferring explants into liquid nitrogen

Multiplication of in vitro plants