Presentation delivered by Dr Bart Panis at the International Agrobiodiversity Congress 2016, held in Delhi, India, 6-9 November.
Among other international endeavors, this presentation highlighted the efforts of the International Transit Centre in conserving plant genetic resources such as Musa (banana) for our consumption today and tomorrow.
Find out more about the India Agrobiodiversity Congress:
http://www.bioversityinternational.org/iac2016/
The dark energy paradox leads to a new structure of spacetime.pptx
Securing plant genetic resources for perpetuity through cryopreservation
1. Securing Plant Genetic Resources for Perpetuity
through Cryopreservation
Bart Panis, Ines Van den Houwe, Rony Swennen, Juhee Rhee and Nicolas Roux,
Bioversity International
6. 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:
6
7. 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
11. Cryopreservation
Cryopreservation 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
12. 12
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
13. 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....
16. 16
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).
17. 17
How to obtain more rapid freezing rates?
Cryotubes (about 6°C/sec )
Semen straws (about 60°C/sec)
Droplet vitrification (about 130°C/sec)
18. 18
Institute Country Crop Cryopreservation Method
Bioversity International, Leuven Belgium Banana Droplet vitrification
Crop 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
20. 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.
28. Use of bananas; fruit: desert banana, snack, cooking
banana (some plantains), matoke banana
29. Conservation of Musa spp. at the ITC
3 conservation methods:
In vitro active collection
1434 accessions
Cryopreserved base
collection 950 accessions
Lyophilized leaf tissue
collection 788 accessions
Off site black box safety back-up
(IRD, Montpellier, France)
34. 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).
38. Average regeneration rates per method and
per genomic group
0%
20%
40%
60%
80%
100%
AA
AAA
AAAA
AAAB
AAA-h
AAB
AAB-p
AB
ABB
Acuminata
balbisiana
blanco
varia
Totaal
42. • 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
43. 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”.
45. 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.
46. 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.
47. 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,