1

Introduction to CRYOBIOLOGY and PLANT CRYOPRESERVATION : Techniques and

Applications

Cryopreservation history

• Smith, 1952 (rabbit embryos survive -79°C)• Sakai, 1956 (Survival of Winter hardy tissues in LN after

dehydration)• Quatrano, 1968 (DMSO for cryoprotection of cultured flax cells)• Latta, 1971 (Cryopreservation of carrot cell cultures in LN)• 1970s-1980s : Application of Classical freezing techniques mostly

based on slow freezing• Engelmann et al, 1985 (pregrowth/desiccation on oil palm)• Uragami et al., 1989; Langis et al., 1989, Sakai et al., 1990;

Towell, 1990 (Application of vitrification solutions to plant tissues)• Fabre and Dereuddre, 1990; Dereuddre et al., 1990

(encapsulation/dehydration method)

Freezing induced injury

1/ Exposure of cellular components to low temperatures(In nature, during cryoprotection at 0°C and slow freezing)

2/ Mechanical effects of extracellular ice crystals at cell surfaces(Especially for organised tissues)

3/ Dehydration related effects (In nature, during cryopreservation when slow freezing rates are

applied) Results in solution and mechanical effects

4/ Injury due to intracellular ice formation⇒ Mechanical disruption of protoplasmatic structure, loss of semi-

permeability

(Freezing injury can also be expressed at the biochemical level : metabolic uncoupling leads to the production of free radicals.)

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 icecrystal 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

Vitrification

• Two requirements for vitrification 1/ Concentration of cellular solution

• Air drying• Freeze dehydration• Osmotic dehydration• Penetrating cryoprotective substances• Adaptive metabolism (hardening)

2/ Rapid cooling and thawing rates• Plunge cryovials in LN for cooling and thaw in warm water

bath (38°C)• If needed, use small cryovials (or freeze without vials)

1. Concentration of cellular solution through air drying

- Sterile air from laminar air flow cabinet

- Dry silica gel in a closed container

2

2. Concentration of cellular solution through freeze dehydration

Cooling rates : 0.3 to 10°C/min until -30 to - 50°C

- Computer driven cooling device

- stirred methanol bath

- propanol container

3. Concentration of cellular solution through osmotic dehydration

Non-penetrating cryoprotective substances

Sugars

Sugar alcohols

High molecular weight additives (PEG,….)

EG at low (0°C) temperature

4. Concentration of cellular solution through adaptive metabolism

Induced by temperature changes, changes in light regime, osmotic changes, ABA,…..

Result : increase in proteins, sugars, glycerol, proline, polyamines, glycine betaine,... which have (among others, see later) colligative effects

Induction is genetically defined

5. Concentration of cellular solution through the addition of penetrating cryoprotective substances

Colligative effect of penetrating cryoprotective substances

DMSO

Glycerol

amino acids

EG at high temperature (RT)

• Problem: Most hydrated tissues do not withstand dehydration to moisture contents needed for vitrification (20-30 %) due to solution and mechanical effects. Exceptions are pollen, seed and somatic embryo of most orthodox species.

• The key for successful cryopreservation thus lies in the induction of tolerance towards dehydration.

• How ?Non-colligative effects of

1/ Addition of cryoprotective substances (Sugars, glycerol, DMSO,…)2/ Adaptive metabolism (hardening)

1/ Addition of cryoprotective substances (non-colligative effects)

• Sugars– stabilisation of membranes– stabilisation of proteins

• Proline– protect cellular components during stress

• DMSO– free radical scavenger– increase of membrane permeability– protection of cytoskeleton during freezing

• Glycerol– Stabilisation of membranes

• ……..⇒ Mode of action is still far from understood; most cryoprotective

mixtures have been defined empirically.

3

2/ Adaptive metabolism (hardening)

• Temperature changes, changes in light regime, osmotic changes, sugar treatment, ABA treatment,…..induces the production of certain proteins, sugars, glycerol, proline, polyamines, glycine betaine, ….. which have also non-colligative effects

– prevention of precipitation of molecules– prevention of intracellular ice formation (Anti freeze proteins,...)– stabilisation of membranes

• changed lipid composition (saturation (FAD8), length, amount...)• accumulation of sugars• production of membrane protecting by hydrophillic polypeptides

(LEA proteins, …)– Induction of anti-oxidative mechanisms– Induction of genes coding molecular chaperones preventing protein

denaturation (hsp90, ……)• Sugar hardening and cold hardening are most frequently applied

⇒ Mode of action of hardening is still far from understood

All cryopreservation protocols are combinations of 2 or more of cryogenic strategies (except for seed and somatic embryos)

• Cold Hardening + Penetrating cryoprotective substances (Carnation shoots)

• Cold Hardening + Freeze dehydration (mulberry shoots)• Cold Hardening + Air drying (mulberry shoots)• Cold Hardening + Air drying + Freeze dehydration (apple shoots)• Cold Hardening + 0smotic dehydration + Freeze dehydration (pear

shoots)• Cold Hardening + 0smotic dehydration + penetrating cryoprotective

substances +Freeze dehydration (Rubus shoots)• Cold Hardening + 0smotic dehydration + penetrating cryoprotective

substances (apple shoots)• Cold Hardening + Sugar Hardening + 0smotic dehydration +

penetrating cryoprotective substances (mulberry shoots)• Sugar Hardening + 0smotic dehydration + penetrating cryoprotective

substances (mint shoots)• Sugar Hardening + Air drying (oil palm somatic embryos) • Sugar Hardening + osmotic dehydration + penetrating cryoprotective

substances (Banana shoots)• Penetrating cryoprotective substances + Freeze dehydration

(Classical freezing method (banana cell suspensions))• ……….

1. Air drying Applied to orthodox seed, zygotic embryos and pollen

Zygotic embryos of wild Musaspp. (Villalobos et al., 1992)

☺ :Simple No sophisticated equipment needed

:Limited application (pollen, seed, zygotic embryos of orthodox seed species)

2. Classical freezing protocolApplied to shoot cultures, somatic embryos, cell suspension cultures,….

Cold hardening Osmotic dehydration + Penetrating cryopr. substances + Freeze dehydrationSugar hardening

Parameters to be optimised• Hardening• Cryoprotective mixture (Often including DMSO)• Cooling rate• Holding temperature

Nucellar cells of navel orange (Kobayashi, 1990)

Cryoprotection : 5%DMSO, 1.2 M sucrose

Effect of cooling rate Effect of prefreezing temperature

Pea apices (Uemura, 1981)

- 40°C, LN

Cold hardening : 0°C, 10 days

Preculture : 2 days 5 % DMSO

Dehydration : 0.5°C/min

Carnation shoots (Uemura & Sakai, 1980)

Effect of preculture/hardening Effect of cryoprotectant

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Cassava Shoot tips (Escobar et al., 1997)

☺ :Applicable to cell suspensions and callus of a wide range of plant species (unorganised tissues)

:More limited application to organised tissues (meristems cultures)Expensive cooling devices are sometimes needed

C4 medium : 4E medium + 1 M Sorbitol + 0.1 M DMSO + 0.11 M Sucrose

Cryoprotection : 1 M Sorbitol + 1.28 M DMSO + 0.11 M Sucrose

Freeze Dehydration : 0.5 °C/min from 5 to-15°C, 25 °C/min to -20°C,

1°C/min to -40°C

3. Encapsulation/dehydrationApplied to shoot tip cultures and somatic embryos

Typical protocol1/ Encapsulation in alginate beads2/ Treatment with high sucrose (1-2 M) for 1-5 days3/ Air Drying (to 20-30 % moisture content)4/ Rapid freezing (LN plunge)

Cold hardening + Sugar Hardening + 0smotic dehydration + Air drying

Parameters to be optimised : • Hardening• Sugar treatment (concentration, length, regime)• Air drying

Shoot tips of pear (Scottez et al., 1992)

Effect of cold hardening Effect of sucrose concentration

Sugar preculture : essential to survive

dehydration

Cold hardening : essential to survive

dehydration

Microspore embryos of oilseed rape (Uragami, 1993) Shoot tips of pear (Scottez et al., 1992)

☺ : Easy handling of alginate beads No cryoprotective agents besides sugars are needed No slow cooling devices are needed

:Labour intensive cryopreservation protocolSusceptibility towards high sucrose concentrations needed for

vitrification is very species dependent

4. Complete vitrificationApplied to shoot cultures, somatic embryos, cell suspension cultures

Typical protocol1/ Loading : LS : 2 M glycerol + 0.4 M sucrose2/ Dehydration : PVS2 : 30 % glycerol + 15 % EG + 15 % DMSO + 0.4 M sucrose3/ Rapid freezing 4/ Deloading : 1.2 M sucrose

Cold Hardening Sugar hardening + osmotic dehydration + penetrating cryoprotective substances

Parameters to be optimisedSugar hardeningloadingdehydration with vitrification solution (temp, time, composition,…)

Differential scanning calorimetry (WP1)

The application of vitrification solutions leads to vitrification of plant tissues

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DSC= A technique for measuring heat flows during the cooling / warming process

-110.64°C(I)1.236J/g/°C

-2

0

2

4

Hea

t Flo

w (W

/g)

-150 -100 -50 0

Temperature (°C)Exo Up Universal V3.3B TA Instruments

PVS2 solution: only vitrification

0.91°C

-1.36°C208.6J/g

-20

-15

-10

-5

0

5

10

Hea

t Flo

w (W

/g)

-140 -120 -100 -80 -60 -40 -20 0 20

Temperature (°C)Exo Up Universal V3.3B TA Instruments

-113.40°C(I)0.6299J/g/°C

-42.37°C(I)1.190J/g/°C

-11.88°C

-27.95°C12.33J/g

-36.38°C

-34.18°C16.62J/g

-2

0

2

4

Hea

t Flo

w (W

/g)

-140 -120 -100 -80 -60 -40 -20 0 20

Temperature (°C)Exo Up Universal V3.3B TA Instruments

-111.98°C(I)0.4365J/g/°C

-41.72°C(I)0.2190J/g/°C

-1.42°C

-4.20°C13.76J/g

4.52°C

1.14°C19.74J/g

-3

-2

-1

0

1

Hea

t Flo

w (W

/g)

-140 -120 -100 -80 -60 -40 -20 0 20

Temperature (°C)Exo Up Universal V3.3B TA Instruments

-111.53°C(I)1.010J/g/°C

-41.78°C(I)0.3380J/g/°C

-3

-2

-1

0

1

Hea

t Flo

w (W

/g)

-140 -120 -100 -80 -60 -40 -20 0 20

Temperature (°C)Exo Up Universal V3.3B TA Instruments

Crystallization

Melting

Crystallization

Vitrification

Vitrification

DevitrificationVitrification

Toxicity of vitrification solutions

Shoot tips of tropical crops (Takagi et al., 1998)

How to survive PVS2 toxicity ?

Cold Hardening

Sugar Hardening

Loading

Application of PVS at 0°C

Typical vitrification protocol

Rooting

Meristemselection

Loading Dehydration 0.7mlcryotube

LN2 storageThawingUnloadingBlottingRecovery

O

PC

FC

Wrapping

0oC25oC

Ice

Tropical monocots (Thinh et al., 2000)

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Mulberry shoot tips (Niino et al., 1992)

Effect length of PVS2 treatment

Day 0 Day 10 Day 30

Banana shoot tips

☺ : Protocol can be applied to a wide range of culture types and plant species No slow cooling devices are needed

:Susceptibility towards ‘toxic’ Vitrification solution is very species dependent

5. Preculture / dehydrationApplied to shoot cultures, somatic embryos

Typical protocol1/ Sugar preculture2/ Air drying3/ Rapid freezing

Sugar hardening + Air drying

Parameters to be optimisedSugar hardening (length, sugar concentration)Air drying (Moisture content)

Asparagus stem segments (Uragami et al., 1990)

Preculture on 0.7 M sucrose for 2 days

Desiccation with silicagel

Effect of dehydration

Oil palm somatic embryos (Dumet et al., 2000)

Sucrose preculture (7 days)

Dessication with silicagel (16 h)( water content 19-36 %)

Starting material

Drying in silicagel

Regeneration 3 weeks after cryopreservation

Sucrose preculture (7 days), desiccation with silicagel (16 h)( water content 19-36 %)

Oil palm somatic embryos (Dumet et al., 1993)

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Preculture/dehydration protocol : conclusion

• ☺ : Very simple No cryoprotective agents besides sugars are needed No slow cooling devices are neededVery high sample throughput

• : Only applicable to a limited amount of plant species/tissues

–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)

Uses of cryopreservation

Cryopreservation of Embryogenic cells

• Their initiation is difficult and time consuming (up to 2 years !)

• Once initiated, they can be subject to :– somaclonal variation– microbial contamination

• Prolonged culture periods result in loss of morphogenic capacity

Regenerable suspensions should be safely stored in liquid nitrogen, since they are the material of choice for genetic engineering of banana

Example : Problems related to the use of embryogenic cell suspensions in banana

Applications of embryogenic cell suspensions in banana• Mass clonal propagation (1 g of cells gives

rise to 16000 to 70000 embryos)

• Material of choice for genetic engineering– Particle bombardment– Agrobacterium mediated transformation

• Only source of regenerable protoplasts– Somatic hybridisation– transformation through electroporation

• Use for mutagenesis

• After cryopreservation suspension cells need to be :

– Viable

– able to give rise to an embryogeniccell suspension

– true-to-type (Côte et al., 2000)

– retain their characteristics (transformation competence)

Agrobacterium mediated transformation of cryopreserved cell suspensions of banana

RB LBgusAt p neo tp

pFAJ3000

intron

Agrobacterium Strain : EHA101

Plasmid

Suspensions

THP (Three Hand Planty)

THP control

THP c4/96 , thaw12/99

THP c9/96 , thaw12/99

THP c5/97 , thaw12/99

gusA : reporter gene : β-glucuronidase

neo : selectable marker : neomycin phosphotransferase

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Transient GUS expression

400 µm

suspensions nb spots/66µl SCV (n=4)

THP no cryo 988a

THP cryo 10/4/96 1196a

THP cryo 27/9/96 931a

THP cryo 22/5/97 1361a

Stable transformation after selection on 50 mg/l geneticinsuspensions Clumps /66µl SCV

(n=4)

Shoots/66µl SCV

(n=4)

Regeneration

frequency (%)

THP no cryo 42a 20 47

THP cryo 10/4/96 51a 19 37

THP cryo 27/9/96 46a 12 25

THP cryo 22/5/97 25b 14 57

Cryopreservation of banana nematodes

Why ? For many experiments, nematode populations with different pathogenicity from different regions are needed. Out of practical reasons only 9 Radopholus similis populations are now stored).

Material and Methods : Radopholus similis populations from Ghana, Cuba, Indonesia and Uganda cultured on carrot disks or alfalfa callus. Rapid freezing in liquid nitrogen !

Results• All populations under investigation gave riseto surviving (moving) nematodes (1-17 %)

• Surviving nematodes are able to reproduce • Pathogenicity of cryopreserved nematodes is now under investigation

Viral diseases (CMV, BSV,BBTV, …..) are a constrain to banana production and to cross-border germplasm movement

A dramatic delay in the distribution of high yielding and newly bred varieties to small farmersEradication through

•in vitro culture

•meristem culture

•chemotherapy

•thermotherapy

•electrotherapy

•cryotherapy.

Cryotherapy (viruseradication)

Virus detection by ELISA - 1st test on in vitro plants- 2d test on in vivo plants

CMV and BSV infected Williams plants

In vitro cultures CRYOPRESERVATION

3 0 230

0 230

020406080

100

bud c

ulture

(3/96

)

meriste

m cultu

re (0

/8)

cryop

rotec

tion (

1/45)

cryoth

erap

y (24

/79)

CMV ERADICATION BY CRYOPRESERVATION

% o

f hea

l thy

p lan

ts

1st test2d test

HYPOTHESIS

X 10

X 60

•Cryopreservation act as a micro-scalpel

•Virus eradication is probably based on the uneven distribution of viruses in plants

IMMUNOLOCALISATION OF CMV PARTICLES (Gold-Silver Enhancement)

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Acknowledgments

Commission of the European Communities, specific Cooperative Research programme Quality of Life and Management of LivingResources, INIBAP/IPGRI (International Network for the Improvement of Banana and Plantain, Montpellier, France)DGIS (Directorate General of International Collaboration, Belgium) Angelo Locicero, Bertrand Helliot (FUSAGx, Belgium)Hannelore Strosse, Karen Reyniers, Bart Piette, Edwige André, Yves Lambeens, Zenaida Managuelod, Madelyn Ibana, Guoyu Zhu (In vitro group, Leuven)Annemie Elsen, Tom Van Wauwe, Salvador Ferrandis Valtera(Nematology group , Leuven)Serge Remy, Laszlo Sagi (Molecular group, Leuven)Ines Van den houwe, Els Kempenaers (ITC, Leuven)Rony Swennen (Leuven)Stéphane Dussert (IRD, France)

Laboratory of Tropical Crop Improvement