Europe 64 endemic plants of Europe have become extinct in recent decades

1. COST Technical Committee "Agriculture, Biotechnology and Food Science"Proposal for a new COST ActionCryopreservation of crop species in EuropeACRONYM: CRYOPLANETProposing country: BelgiumAction Proposer : Bart Panis

2. Plant Genetic Resources: Current situation • Europe • 64 endemic plants of Europe have become extinct in recent decades • 24% of the species/subspecies of certain groups of European plants are in danger to be lost • Agricultural intensification has reduced the area under wetlands in Europe by some 60 % in the last decades with consequent threats on biodiversity • Worldwide • 100,000 plants representing 1/3rd of plant species are threatened • Since the 1970s large number of land races and wild relatives are sampled and stored ex situ • Now, about 6 million samples are held in national, regional, international and private genebank collections

3. Storage of Plant germplasm • In situ : Conservation in ‘normal’ habitat • rain forests, gardens, farms • Ex Situ : • Field collection, Botanical gardens • Seed collections • In vitro collection • Normal growth • Slow growth (temp, O2 ,H2O , medium ~) • Cryopreservation (-196°C) • (DNA Banks)

4. What is cryopreservation ? • What ? • Storage of living tissues at ultra-low temperatures (-196°C) • Use • Conservation of plant germplasm • Vegetatively propagated species (root and tubers, ornamental, fruit trees) • Recalcitrant seed species (Howea, coconut, coffee) • Conservation of tissue with specific characteristics • Medicinal and alcohol producing cell lines • Genetically transformed tissues • Transformation/Mutagenesis competent tissues (ECSs) • Eradication of viruses (Banana, Plum) • Conservation of plant pathogens (fungi, nematodes)

5. What is the ‘problem’ with plant cryopreservation ? • Cryopreservation procedures are now available for about 150-200 different plant species • But for each species and tissue type, the cryopreservation protocol needs to be empirically adapted in function of their • natural freezing resistance • explant size and type • water content • Most of the work on cryopreservation of plants has been performed in the framework of academic studies and involves only one or a few genotypes. Only few plant germplasm collections stored in liquid nitrogen currently exist (with a relatively limited amount of accessions). • No real standardised methods are available !!!

6. Examples of existing collections in liquid nitrogen? • National Seed Storage Laboratory (NSSL) (Fort Collins, Colorado, USA): 2,100 accessions of apple (dormant buds) • National Clonal Germplasm Repository (NCGR) of Corvallis (USA): 104 accessions of pear (shoot tips); • International Potato Centre (CIP) (Lima, Peru) : 345 potato accessions • Tissue Culture BC Research Inc.(Vancouver, BC, Canada) : 5000 accessions representing 14 conifer species • AFOCEL (Association Forêt Cellulose) of France, with over 100 accessions of elm (dormant buds); • National Institute of Agrobiological Resources (NIAR) of Japan, with about 50 accessions of mulberry. • IRD (Montpellier, France) : 80 accessions of oil palm • German Collection of Micro-organisms and Cell Cultures (DSMZ) (Braunschweig, Germany) : 519 old potato varieties • INIBAP, Laboratory of Tropical Crop Improvement, K.U.Leuven (Heverlee, Belgium) : 440 banana accessions

7. Why is cryopreservation not more widely applied? • the unavailability of efficient and robust cryopreservation protocols applicable to many plant species and diverse germplasm types • limited awareness of plant researchers unacquainted to recent developments in cryogenic storage methods • lack of coordinated research on plant cryopreservation. •  COST action like “CRYOPLANET” could make the difference

8. Why are the objectives of CRYOPLANET ? • Objective 1: To screen in detail the current utilization of plant cryopreservation in Europe. • Objective 2: To screen and compare the efficiency of existing plant cryopreservation protocols. • Objective 3: To improve fundamental knowledge about cryoprotection through the determination of physico-biochemical changes associated with tolerance towards cryopreservation. • Objective 4: To develop new plant cryopreservation protocols. • Objective 5: To assure the genetic stability and true-to-typeness of plants after cryopreservation. • Objective 6: To apply cryopreservation to European plant germplasm collections. • Objective 7: To proof the environmental, social and economic impact of plant cryopreservation.

9. WG1 Fundamental aspects of cryopreservation/cryoprotection and genetic stability Optimisation Feed back WG2 Technology, application and validation of plant cryopreservation Scientific programme of CRYOPLANET ?

10. Proteins Sugars Water thermal behavior Polyamines Membrane components Cytoskeletal protein Oxidative stress WG1: Fundamental aspects of cryopreservation/cryoprotection and genetic stability • 1.1. Fundamental aspect of cryopreservation and cryoprotection • Elucidation of the physico-biochemical background of cryoprotection and cryopreservation.

11. WG1: Fundamental aspects of cryopreservation/cryoprotection and genetic stability • 1.2. Genetic stability and authenticity • Assessment of the genetic integrity of plants to determine if they are ‘true to type’ after cryopreservation. • Assessments of phenotypic variation (morphological descriptors) • Cytological techniques to detect various types of chromosomal instability. (polyploidy, aneuploidy and other mitotic abnormalities) • Biochemical metabolite/protein (isozyme) profiles • Genomic DNA sequences that can be analysed using a range of hybridization and (PCR) techniques • Epi-genetic variation in chromatin and DNA methylation of gene sequences

12. WG2: Technology, application and validation of plant cryopreservation • 2.1. Technology aspects of cryopreservation • Applications of different cryopreservation protocols to different plant species and tissues.

13. WG2: Technology, application and validation of plant cryopreservation • 2.2. Impact and applications of cryopreservation in plants • genebanks, establishment of cryo-bank and dissemination of results

14. Organisation of CRYOPLANET • The Management Committee (MC) • Appointment of Action Chair, Vice-Chair(s) and WG Co-ordinators. • Planning of MC meetings and of Scientific Meetings and Workshops. • Assessment and report of the progress made by the different WGs • Promotion of co-operation and of data exchange between the WGs. • Promotion and approval of Short-Term Scientific Missions, • Preparation of the Annual Reports. • Establishment and update of a Web site • Organization of contacts and common workshops • Preparation of a EU 7th framework project

15. Organisation of CRYOPLANET • Two Working Groups • WG1 : Fundamental aspects of cryopreservation/cryoprotection and genetic stability • WG2: Technology, application and validation of plant cryopreservation • Working Group meetings will be organised on a yearly base • WG coordinators • Planning the appropriate Scientific Meetings. • Coordination of the activities within the WG. • Promoting joint research (f.e. STSMs) and common publications. • Report on the WG progress to the Action Chair and MC • Participation in the plenary and restricted MC meetings. • Inter-COST Workshops • Short-Term Scientific Missions (STSM),

16. Time table MC meeting: Management committee meeting; WG meeting: Working group meeting; STSMs: Short-term scientific missions; Workshop*: Timing of the Inter-COST Workshops will be defined in agreement with the Management committee of that specific Action.

17. Economic dimensions • 17 COST countries • 64 researchers • 50 research institutes

18. Dissemination • Articles in refereed scientific journals • Common reviews, books • A public website (information about the project, the achievements, services/consultancies offered and announcements of training workshops. • Information on the official webpages of the collaborating institutions. • The consortium will organize workshops for scientists, germplasm curators regulatory bodies and policy makers • At the end of the Action, the consortium will offer its expertise as a service to the EU. • Presentations at International Conferences, for promoting the European know-how and increasing the international collaboration. • Teaching activities in Universities at undergraduate and post-graduate level. Young scientists and engineers will thus be trained and informed on the latest developments in cryopreservation.

19. Acknowledgements • Carpentier Sebastien, Druart Philippe, Geelen Danny, Swennen Rony, Tsvetkov Ivaylo, Bilavcik Alois, Faltus Milos, Zamecnik Jiri, Antonius-Klemola Kristiina, Häggman Hely, Nukari Anna, Rokka Veli-Matti, Uosukainen Marjatta, Dussert Stéphane, Engelmann Florent, Grapin Agnès, Harvengt Luc, Malaurie Bernard, Péros Jean-Pierre, Trontin Jean-François, Höfer Monika, Keller Joachim, Meier-Dinkel Andreas, Pinker Ina, Schumacher Heinz Martin,Benelli Carla, Caboni Emilia, Damiano Carmine, Dulloo Ehsan, Lambardi Maurizio, Hausman Jean-François, Criel Bram, Harrouni Cherif, de Klerk Geert-Jan, Raemakers Krit, Bach Anna, Lisek Anna, Mikuła Anna, Niedzielski Maciej, Pukacki Pawel M., Rybczyński Jan J., Sochacki Dariusz, Zimny Janusz, Miguel Célia, Reis Moura Isabel, Oliveira¨Margarida,Halmagyi Adela, Palada Magdalena, Rakosy-Tican Elena, Gavrilenko Tatjana, Salaj Terezia, González-Benito Elena, Revilla M. Ángeles, Vieitez Ana, Pâques Marc, Feki Lotfi, Aylin Ozudogru Elif, Ozden-Tokatli Yelda, Benson Erica, Cripps Ryan, Grout Brian, Harding Keith, Lynch Paul T., Pritchard Hugh W.,Wetten Andy