Long-term Storage of Allium Germplasm by Cryopreservation

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1 Long-term Storage of Allium Germplasm by Cryopreservation Jiří Zámečník Crop Research Institute The Czech Republic

2 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

3 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

4 Crop structure of PGR in the Czech collections Fodder crops 7% Legumes (edible) 11% Aromatic 3% Ornamental 3% Fruit plants 6% Fibre crops 5% Oil crops 3% Potatoes and Root crops 6% Vegetables 15% Maize, alt. cereals Cereals 37% that is 76% of seed propagated collections

5 Conservation of Vegetatively Propagated Species 24% of all collections Field gene banks mostly fruit trees, vegetables garlic (Helena), medicinal and ornamental plants In vitro gene banks collection of potatoes, partially vegetables garlic with difficulties, hop, fruit trees and ornamental plants In situ conservation few endangered populations at particular localities, in cooperation with nature protection subjects On farm conservation e.g. fruit trees, garlic- landraces local growers Cryoconservation currently safe duplicates

6 What is Cryopreservation? Cryopreservation is the cooling of cells or tissues to ultra-low temperatures, typically to -196 C (boiling point of liquid nitrogen) Enable to interfere biological clock and to stop it for a while (decades) until it is thawed Basically, cryopreservation is a procedure to preserve the plant parts by arresting or slowing metabolic activity due to ultra low temparatures and amorphous state of plant matter Cryopreservation era arised with the accidentally understanding of the value of cryoprotectants in 1949 by Christopher Polge.

7 Liquid nitrogen is most widely used cooling medium for cryopreservation Why liquid nitrogen? Ultra-low temperature -196 C Vapour phase -135 C Chemically inert Relatively low cost Nontoxic Non flammable Independent on electricity Automatization of filling Dewar flask

8 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

9 Biological glass unknown behavior of water not fully understood yet garlic bulbil after deep drying (Allium sativum L. cv.djambul)

10 PVS3 Biological Glass glass

11 Intracellular ice crystallization Ice crystals vsf

12 Method - calorimetry Differential scanning calorimeter *Glass transition temperature *Crystalized water volume *Heat capacity DSC cela Referenční pánvička Vzorková pánvička Víčko Přívod promýv. plynu Chromelový disk Chromelový disk Blok topení Termočlánkový Alumelový drát svár Chromelový drát Termoelectrický disk (Konstantan)

How to Induce the Vitrification in Allium Physical pretreatment dehydration air drying freeze drying osmotic dehydration freezing rate Cryoprotectant pretreatment sugars, polyols, PVSx % w/v sucrose

13 How to Induce the Vitrification in Allium Physical pretreatment dehydration air drying freeze drying osmotic dehydration freezing rate Cryoprotectant pretreatment sugars, polyols, PVSx % w/v sucrose PVSL 5 PVS1 5 PVSL+ 15 PVS2 15 PVS3 50 Sucro se Amorphous phase with higher water content than MFCP or combination of both MF CP Ice Sikora Dupanov Kratochvíl Zámečník 2007 Macromol Sci B-Physics 46,71

14 QITMDSC Allium gh2o/ gdw

15 Glassy state Freezing Dangerous area Dehydration Safety area Tg Tg Tm Dehydration Thawing Dangerous area Temperature ( o C)

16 Natural Glass Biological Glass Glass Cryobiology vitrification Man-made Glass

17 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

18 Plant Cryopreservation Methods Based on Glass Formation Encapsulationdehydration fruit trees, potato, Allium Ultra-rapid freezing potato, hop Vitrification (PVS3) Allium, fruit trees Two- step freezing fruit trees dormant bud Zámečník Faltus Bilavčík 2007, Adv Hort Sci 21,247

19 garlic potato hop apple

20 garlic a potat b o hop c apple d Olomouc Allium 18

21 garlic potat o hop apple Zámečník,Faltus,Bilavčík & Kotková. Cryopreservation, Katkov (ed.), InTech

22 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

23 The Czech Republic Hop Research Inst. Žatec VS,VÚRV Karlštejn Genebank Cryobank Prague Potato Research Inst. Ltd. Havlíčkův Brod Research and Breeding Inst. of Fruit Trees, Holovousy RICP, GB Olomouc Lednice

24 Accesion Frequency Efficiency of the cryopreservation method Četnost regenerace garlic česnek zralé Ripe bulbils pacibulky nezralé Unripe bulbils pacibulky Regeneration (%) Regenerace (%) Zámečník et al.,2012, in Cryopreservation, Intech,

Cryobank Species in vitro CryoBank Solanum tuberosum L. 79 Humulus lupulus L 49 Allium sativum L. 137 Malus domestica BORKH. 17 Pyrus communis L. 24 Cerasus avium (L.) MOENCH 3 Cerasus vulgaris P.

25 Cryobank Species in vitro CryoBank Solanum tuberosum L. 79 Humulus lupulus L 49 Allium sativum L. 137 Malus domestica BORKH. 17 Pyrus communis L. 24 Cerasus avium (L.) MOENCH 3 Cerasus vulgaris P.MILLER 10 Cerasus x effusa HOST 3 Fragaria x ananassa DUCH 34 Armeniaca vulgaris LAM 5 Vitis vinifera L. } 3 Lonicera caerulea L. 17 var. edulis Turcz. ex Herder Celkem 381 current state (Oct 2017) In Cryobank 13 species 381 Acc in cryovials from 6 collection keepers

26 Cryobank Storage Technology in Dewar flask in liquid nitrogen cryovials barcode CRYO. CZ

hormones in medium up to 1 week, 1 year % of regeneration safe

27 Plant Genetic Resources Documentation in the Czech Republic passport data characterisation and evaluation data genebank storage data hormones in medium up to 1 week, 1 year % of regeneration safe storage temperature (onset Tg + alfa) suggested thawing method regeneration method

28 Probabilistic Tools for Cryopreservation Management (Dussert et al. 2003) S = n 1 + n 2 P> 0.95 n 2 n 1 P obs (%) methods

CryoLetters 24, 149-160 (2003) DEVELOPMENT OF

MANAGEMENT OF CRYOPRESERVED PLANT GERMPLASM COLLECTIONS

29 CryoLetters 24, (2003) DEVELOPMENT OF PROBABILISTIC TOOLS TO ASSIST IN THE ESTABLISHMENT AND MANAGEMENT OF CRYOPRESERVED PLANT GERMPLASM COLLECTIONS Stéphane Dussert, 1 *, Florent Engelmann 1,2 and Michel Noirot 1

30 Allium Tripartite Cryobanking CRI Czech INRA Cryo banks German Polish IPK UNIBAS RIVC Zanke, Zamecnik, Kotlińska, Olas, Keller, Acta Hort. (ISHS) 908:431

31 Cryotherapy Cryo-knife Allium 4 viruses 20 acc avg. 60% Potato 3 viruses 12 acc % Hop 2 viruses 12 acc 10-25% Samples of garlic where OYDV was detected (Ct < 45) Samples of garlic no positive on OYDV (Ct > 50)

32 Collaboration Norway project Dag-Ragnar Blystard NIBIO Zhibo Zhang Min-Rui Wang Finland - Saja Rantala Luke Sweden - Svein Oivind Solberg Slovakia, Germany, GB, Poland, Argentina, Thailand

33 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

34 FEASIBILITY STUDY FOR A SAFETY BACK- UP CRYOPRESERVATION FACILITY INDEPENDENT EXPERT REPORT: JULY 2017 Authors: Acker, J.P.; Adkins, S.; Alves, A.; Horna, D.; Toll, J. Pages: 100 p. Publisher: Bioversity International Publication Year: 2017 Publication Format: PDF Language: EN

2017 Publication Format: PDF ISBN 13: 978-92-9255-073-8 Language: EN FEASIBILITY STUDY CRYO BACK-UP Percentage of the

35 2017 Publication Format: PDF ISBN 13: Language: EN FEASIBILITY STUDY CRYO BACK-UP Percentage of the total holdings of 15 institutes for 30 crops that are maintained in cryopreservation, in in vitro culture and in the field.

Potato 47% Potat47 % Banana and plantain 13% Mulberry 15% Strawberry 3% Allium 7% Coffee 5%

36 FEASIBILITY STUDY CRYO BACK-UP Sweet potato 0,4% Taro 0,04% Apple 2% Artichoke 0,05% Representation of different crops in the cryopreserved collections of the 15 institutes. Potato 47% Potat47 % Banana and plantain 13% Mulberry 15% Strawberry 3% Allium 7% Coffee 5% Citrus 1% Cassava 3% Other fruit trees 1% Other crops 0,4% Other berries Hop 1% 1% Grape 0,04% Mint 2%

37 FEASIBILITY STUDY CRYO BACK-UP Proportion of accessions in cryopreservation out of the total number of accessions held collectively by the 15 institutes. Note: Berries include Morus spp. (mulberry), Fragaria spp. (strawberry), Rubus spp., Ribes spp., and Lonicera caerule

38 FEASIBILITY STUDY CRYO BACK-UP

39 Outline Introduction Theory Biological Glass Cryopreservation Methods Cryopreservation of Czech GR Global Cryopreservation Conclusion

40 Conclusion Svalbard Global Seed Vault Global Cryo Vault, where?

41 Thank you for your attention

FEASIBILITY STUDY FOR A SAFETY BACK-UP CRYOPRESERVATION FACILITY INDEPENDENT EXPERT REPORT: JULY 2017

FEASIBILITY STUDY FOR A SAFETY BACK-UP CRYOPRESERVATION FACILITY INDEPENDENT EXPERT REPORT: JULY 2017 Bioversity International is a global research-for-development organization. We have a vision that agricultural