Applied plant cryobanking at CGIAR plant

collection: from protocol development to genebank

management Bart Panis, Ines Van den houwe, Bart Piette and Nicolas Roux

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• More then 800 million people are undernourished and 200 million children under five years of age are underweight.

• The world’s population is expected to reach 10,500 million by 2050.

• Reliable and sustainable improvements in yield will be needed to meet the demands of this growing population.

The conservation and sustainable utilization of plant genetic resources are the keys to improving agricultural productivity and sustainability

Some facts

HOW???

• In crease land?

• Improve cultural practices

(fertilizer, crop rotation,…)

• Increase biotic (diseases)

and abiotic stress resistance

•Increase yield

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Plant Diversity that is used is limited

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Food security is a strong, sustainable, local or regional food system that ensures access to affordable, nutritious and culturally appropriate food to all people at all times.

1. Availability (own production + markets)

2. Access (Resources)

3. Utilization (metabolism of food)

4. Stability (chronic or temporary)

5

What are is the CGIAR ?

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CGIAR (Consultative Group on International Agricultural

Research) is a global partnership that unites organizations

engaged in research for a food secure future.

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Research at the CGIAR is centred around CRPs

How is crop germplasm stored at the CGIAR

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)

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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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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BUT 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 exemple

through cryopreservation of seed or embryos

Store vegetative tissues

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?

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

Zone I : no vitrification

Zone II : Unstable glass

some vitrification

devitrification

Zone III : vitrification

devitrification ?

Zone IV : vitrification

no devitrification

TOXIC

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

Air drying

Penetrating cryoprotective substances

Osmotic dehydration

Freeze dehydration

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

Prevention of intracellular ice crystal formation. through ‘vitrification’

HOW???

1/ Concentration of

cellular solution

2/ Rapid cooling and

thawing rates

• Slow (classical) freezing

• Encapsulation-dehydration

• Droplet freezing

• Fast Preculture (+ dehydration) freezing

• Vitrification (PVS2 , PVS3,…)

• Encapsulation-vitrification

• Droplet vitrification

• V-cryo-plate procedure

Methods for cryopreservation

Cryobanking at IITA

Dr. Badara Gueye

Where

Crop collections at IITA

• IITA holds international clonal crop collections of Yam, Cassava and Banana.

• Currently conservation mainly happens in field bank and in vitro genebanks (8 species of yam (Dioscorea spp.) and 1 species of cassava (Manihot esculenta Crantz))

• Future: Integrated conservation strategy using Cryobanking for yam and cassava

Cryobanking at IITA (conclusion)

• Cassava cryobanking protocol ready to be used (institutional

support, facilities, staff, equipment, plant material, cryo strategy

and protocol in place)

• Waiting for minimum QMS to be fully in place

• Cassava cryobank planned to start in 2015

• Research still ongoing to optimise protocols for yam cryobanking

Cryopreservation at CIP

Reiner Vollmer/Ana Panta/Dave Ellis

Where

Cryopreservation at CIP

Droplet vitrification

CIP’s cryo-team

Cryopreservation at CIAT

Roosevelt Escobar

Where

The in vitro cassava collection at CIAT

• 6643 accessions (data of 2014).

– 5341 landraces, 408 breeding lines

– Manihot spp: 25 wild spp (883 genotypes)

• Distribution of germplasm following phytosanitary requirements

– In 1979-2012, 36036 samples (6492 accessions) have been

distributed to 71 countries

• Cryobanking: safety

duplication

www.ciat.cgiar.org

CIAT's cryopreservation research team

and technical support people

Liliana Muñoz.

Research Assistant

Auradela Rios.

Lab Technician

Adrian Nunez.

Lab Technician

Carlos Dorado.

Field Technician

Roosevelt Escobar

Associated. Team coordinator

Daniel Cruz

Limsys development

Ericson Aranzales

In vitro gene bank Coordinator

Gerardo Gallego

Lab Coordinator

Joe Tohme

Agro biodiversity Team Leader

Cryobanking at Bioversity International

Where

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 129 million tonnes (2009,

FAO)(Apple: 71 million tonnes)

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

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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Slow growth • T 16°C

• PPF 25µmol.m-2.s-1 (24/24h)

Type of material • proliferating shoot tips

Culture medium • MS salt and vitamins, 30g.l-1

sucrose, 2.25 mg .l-1 BA, 0.175 mg .l-1 IAA, 2g.l-1 Gelrite

Number of replicates • 20

Storage capacity • 12 months (4-22

months)genotype effect !

Bioversity International in vitro banana collection (1445 accessions)

Regenerable Musa tissues, suitable for cryopreservation

• Seed

• Zygotic embryos

• Embryogenic cell suspensions

• Somatic embryos

• Meristem cultures

2 methods for cryopreservation of banana

meristems

•Droplet vitrification of ‘cauliflower

like’ meristem clumps

•Droplet vitrification of tiny

meristems (1 mm ) excised from

in vitro plants

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Droplet - vitrification

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Average post-thaw regeneration rates of banana accessions

grouped according their genomic constitution after the application

of three different cryopreservation protocols.

0

10

20

30

40

50

60

70

80

90

100

AB

B

AA

Bpl

AA

B

AA

A

AA

Ah

AA

M. acc

Ensete

Genomic group

Po

st-

thaw

reg

en

era

tio

n (

%)

Simple freezing

(meristem clumps)

Vitrification (meristem

clumps)

Vitrification (apical

meristems)

Comparison of the 3 cryopreservation protocols

Simple freezing

Vitrification of proliferating

cultures

Vitrification of apical meristemsa

Time needed before cryopreservation can take placeb

5-16 months 5-16 months 5-7 months

Preparation of starting material difficult difficult easy

Preparation of meristem for freezing easy easy very difficult

N° of explants which can be cryopreserved per person per day

600 400 60

User friendliness of protocol +++ ++ +

a Results obtained at K.U.leuven

b Time between an accession is received from the ITC and it can be used for cryopreservation

Laboratory of Tropical Crop Improvement

Protocol •ABB cultivars: Scalps

•AAB cultivars: Scalps

•AAB plantains: Scalps / Meristems

•AAA cultivars: Scalps / Meristems

•AA cultivars: Scalps / Meristems

•AAA Highland bananas: Meristems

•AA wild: Meristems

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)

Storage container

-4x locator 6 plus Thermolyne (4860 tubes)

-1x locator 4 plus Thermolyne (3240 tubes)

Black Box (at IRD, Montpellier)

-809 accessions ( dry shipper )

Some data of 10 years cryobanking

Number of accessions cryopreserved

0

100

200

300

400

500

600

700

800

900

1000

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

Cryopreservation is applicable to all Musa

accessions

0

50

100

150

200

250

300

in vitro

in cryo

Average regeneration rates per method and per

genomic group

0%

20%

40%

60%

80%

100%

reg scalps

reg meristems

Future of plant cryopreservation at the CGIAR?

Situation of Cryobanking at the CGIAR

• “Efficient” cryopreservation protocols for many important

CGIAR crops are already available and are being applied.

More experimental work on sweet potato, yams, ARTC is needed

• Choice between different methods (slow freezing, vitrification

droplet vitrification, encapsulation dehydration).

• QMS will soon be applied to the cryobanking activities at the

CGIAR

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; 50-100 accessions can be

cryopreserved /person/year)

•Presence of endogenous microorganisms

•Tissues survive cryopreservation but do not grow out

“normally”

Acknowledgments

Global Diversity Trust :

INIBAP/IPGRI/ Bioversity International

DGIS (Directorate General of International Collaboration, Belgium)

KU Leuven/Bioversity International Hannelore Strosse, Hans Krohn, Karen Reyniers, Bart Piette, Edwige André, Yves Lambeens, Zenaida Managuelod, Madelyn Ibana, Guoyu Zhu (In vitro group, Leuven), Ines Van den houwe, Els Kempenaers (ITC, Leuven), Rony Swennen, Nicolas Roux (BI)

CIAT: Roosevelt Escobar, Daniel Debouck, Joe Tohme & co

CIP: Ana Panta, Reiner Vollmer, Dave Ellis, Willy Roca & co

IITA: Guye Badara, Dominique Dumet, Abigael Adeyemi, Michael Abberton & co

QMS: Erica Benson, Keith Harding

www.bioversityinternational.org

Thank you