Securing Plant Genetic Resources for Perpetuity through CryopreservationBart Panis, Ines Van den Houwe, Rony Swennen, Juhee Rhee and Nicolas Roux, Bioversity InternationalInternational Agrobiodiversity Congress, New Delhi, India

How is plant germplasm stored

Methods of conservation

• 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)

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Seed conservation at GCIAR

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More than 700 000 accessions are preserved as seed

Svalbard Global Seed Vault

• Ensuring that the genetic diversity of the world’s food crops is preserved for future generations

• The Svalbard Global Seed Vault opend in Svalbard in 2008• 820,000 seed samples are already catalogued, coded and

moved into the Vault:

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Seed storage at -20°C not applicable to all crops!

• Crops that produce “recalcitrant” seed (avocado, mango, mangosteen, lychee, cocoa, rubber tree,…)

• Crops from which we like to preserve a specific gene combination (fruit trees, potatoes, cassava,…)

• Sterile crops (bananas), no seed vailable

• Find other ways of storing the seed for example through cryopreservation of seed or embryos (in case of recalcitrant seed)

• Store vegetative tissues

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40000 vegetatively propagated accessions need to be preserved

Methods of conservation

• 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)

What is cryopreservation?

CryopreservationCryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) −196 °C (the boiling point of liquid nitrogen).

At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped.

Practically: storage happens in big Dewar flasks

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Almost all cryogenic strategies rely on the prevention of intracellular ice crystal formation. The only way to prevent ice crystal formation at ultra-low temperatures without an extreme reduction of water content is through ‘vitrification’ (solidification of a solution without ice-crystals).

Freezing induced injury

Vitrified Crystallized

Prevention of intracellular ice crystal formation. through ‘vitrification’

HOW?

1/ Concentration of cellular solution

2/ Rapid cooling and thawing rates.

Air drying

Penetrating cryoprotective substances

Osmotic dehydration

Freeze dehydration

Adaptive metabolism : (temperature, light, osmotic changes, ABA....)

Different cryopreservation protocols

• Air drying• Classical (slow) freezing• Encapsulation-dehydration• Vitrification• Droplet freezing• Fast Preculture (+ dehydration) freezing• Encapsulation-vitrification• Droplet vitrification • V-cryo-plate procedure.

Cryopreservation methods for plant tissues

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What is droplet-vitrification?

COMBINATION OF • Classical vitrification (with PVS2 or PVS3 or….)

AND• The application of ultra fast freezing and ultra fast

warming (to avoid respectively crystallization and cold crystallization).

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How to obtain more rapid freezing rates?

Cryotubes (about 6°C/sec )

Semen straws (about 60°C/sec)

Droplet vitrification (about 130°C/sec)

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Institute Country Crop Cryopreservation Method Bioversity International, Leuven Belgium Banana Droplet vitrificationCrop Research Institute, Prague Czech

Republic Potato, garlic, hops

Droplet vitrification

International Center for Tropical Agriculture (CIAT), Cali

Colombia cassava Droplet vitrification Encapsulation/dehydration

International Institute of Tropical Agriculture (IITA), Ibadan

Nigeria Yam, banana, cassava

Droplet vitrification

International Potato Center (CIP), Lima Peru Potato Straw vitrification Droplet vitrification

Julius Kühn-Institut (JKI), Institut für Züchtungsforschung an Obst, Dresden

Germany Strawberry/ Fruit trees

Vitrification Dormant bud freezing

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Genebank Department, Gatersleben

Germany Potato, garlic, mint Droplet freezing Droplet vitrification

National Agrobiodiversity Center (NAAS), RDA, Suwon South Korea Garlic Droplet vitrification

Tissue Culture and Cryopreservation Unit, NBPGR, Delhi

India Banana, chives, medicinal plants, berries, fruit trees.

Vitrification Droplet vitrification Slow freezing Dormant bud freezing

USDA-ARS, Fort Collins and Corvallis USA Citrus species, grape, garlic, mint, fruit trees.

Vitrification Droplet vitrification Slow freezing Dormant bud freezing

Cryobanking at Bioversity International

Importance of banana, plantains, cooking bananas

• Staple food for 400-1,000 million people

• Produced in >120 countries

• Banana and plantain (Musa spp.): Largest fruit crop in the world with an annual production of 145 million tonnes (2013, FAO)(Apple: 81 million tonnes)

• International banana trade: yearly turnover of ~6 billion USD.

Use of bananas - Starch corn (Ensete)

Use of bananas - fibres (Musa texitilis)

Use of bananas - beer bananas (East-African Highland bananas)

Use of bananas - male flower = vegetable

Use of bananas - feed

Use of bananas - flowers

Use of bananas; fruit: desert banana, snack, cooking banana (some plantains), matoke banana

Conservation of Musa spp. at the ITC

3 conservation methods:

In vitro active collection1434 accessions

Cryopreserved base collection 950 accessions

Lyophilized leaf tissue collection 788 accessions

Off site black box safety back-up(IRD, Montpellier, France)

Cryopreservation of banana

Regenerable Musa tissues, suitable for cryopreservation

• Seed• Zygotic embryos• Embryogenic cell suspensions• Somatic embryos• Meristem cultures

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10-15 meristems per hour!

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Droplet - vitrification

When is an accessions considered as safely stored?

• 3 independent successful repetitions

• 95% certainty that at least 1 plant can be regenerated per repetition (Dussert et al., 2003).

Black Box (at IRD, Montpellier, France)

• 905 accessions (dry shipper).

Some data of 10 years cryobanking

Number of accessions cryopreserved

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 20150

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Cryopreservation is applicable to all Musa accessions

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Average regeneration rates per method and per genomic group

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Download a copy: bit.ly/2fwv3zN

Cryopreservation protocols developed in collaboration Potato (CIP, Peru; CRPGL, Luxemburg; VIR,

Russia) Ulluco (CIP, Peru) Sweet potato (CIP, Peru) Chicory (CRA, Gembloux, Belgium) Strawberry (CRA, Gembloux, Belgium) Taro (SPC, Fiji) Pelargonium (INH, Angers, France) Date Palm (Université de Sfax, Tunesia) Banana (CIRAD, France; NBPGR, India) Thyme (Univ Alicante, Spain) Olive (IFAPA, Malaga, Spain) Hop (Univ Oviedo, Spain) Photinia (CNR, Firenze, Italy; Gebze, Turkey) Vitis (CNR, Palermo, Italy, PFR, Palmerston, NZ) Apple (Fruit Tree Research Institute, Italy) Cassava (IITA, Nigeria, CIAT, Colombia) Tomato (Univ. Politecnica de Valencia , Spain) Bituminaria (Univ. Politecnica de Valencia , Spain) Narcissus (Daffodil) (University of Krakow, Poland) Galanthus (Snowdrop) (University of Krakow,

Poland) Lily (PRI, the netherlands) Rose (University of Krakow, Poland)

Future of plant cryopreservation

• National Seed Storage Laboratory (NSSL) (Fort Collins, Colorado, USA): 2,100 accessions of apple (dormant buds)

• International Potato Centre (CIP) (Lima, Peru) : about 1000 potato accessions

• Tissue Culture BC Research Inc.(Vancouver, BC, Canada) : 5000 accessions representing 14 conifer species

• IPK (Gatersleben, Germany) German Collection of Micro-organisms and Cell Cultures (DSMZ) (Braunschweig, Germany) : 1500 old potato varieties, garlic, mint

• RDA (South Korea) 1000 garlic accessions• Laboratory of Tropical Crop Improvement, K.U.Leuven

(Heverlee, Belgium) : 950 banana accessions• International Center for Tropical Agriculture (CIAT, Cali,

Colombia), 540 cassava accessions• NBPGR (National Bureau for Plant Genetic Resources), Delhi,

wide variety of species.

Largest cryopreserved collections of vegetatively propagated crops

Are all problem solved?

• Chase for funds for routine application of cryopreservation of a collection of vegetatively propagated crops (main problem remains cost of labour; 40-100 accessions can be cryopreserved /person/year)

•Presence of endogenous microorganisms

•Tissues survive cryopreservation but do not grow out “normally”.

A global safety back up for all crops?

A global safety back up for all crops?

Svalbard Global Seed Vault - safety back up for seed propagated crops

Future idea: the Global Cryo Vault – Safety back up for vegetatively propagated crops through Cryopreservation. Where? ITC Leuven.

Future idea – Global Cryo Vault

a. Serve as cryopreservation black box for other centres

b. Support the cryopreservation work of other centers.

c. Development of an International “knowledge centre” for plant cryopreservation research.

Acknowledgements• Commission of the European Communities, specific Cooperative Research

programme Quality of Life and Management of Living Resources, • Global Diversity Trust• World bank• Gatsby foundation• BMGF• INIBAP/IPGRI/ Bioversity International • DGIS (Directorate General of International Collaboration, Belgium)• RTB CRP• In vitro and cryopreservation: Hannelore Strosse, Karen Reyniers, Bart

Piette, Edwige André, Yves Lambeens, Zenaida Managuelod, Madelyn Ibana, Guoyu Zhu, Hans Krohn, Kevin Longin, Ines Van den houwe, Els Kempenaers, Pablo Caceres, Evert Bruyninckx, Miranda Van Meensel, Hamid Moshafi, Alex Henneau,

www.bioversityinternational.org

Thank you