how to cryoconserve a new plant species?€¦ · - dry silica gel in a closed container . 10 2....

How to Cryoconserve a New Plant Species? Bart Panis

2009

2014

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 ageing

(and cell death), is effectively stopped.

Practically: storage happens in big Dewar

flasks filled with liquid nitrogen

Freezing induced injury

1/ Effect of low (not always “freezing”

temperatures) (on membrane stability,

metabolism,……)

2/ Mechanical effects of extracellular ice crystals

at cell surfaces (shearing of tissues,

disconnection of cells)

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

6

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

7

Vitrification

8

Vitrification

Prevention of intracellular ice crystal formation. through ‘vitrification’

HOW???

1/ Concentration of

cellular solution

2/ Rapid cooling and

thawing rates

9

1. Concentration of cellular solution through air drying

- Sterile air from laminar air flow cabinet

- Dry silica gel in a closed container

10

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 (Mr Frosty)

11

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

12

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

13

5. Concentration of cellular solution through adaptive metabolism

14

• Problem: Most hydrated tissues do not withstand dehydration to

moisture contents needed for vitrification (20-30 %). Exceptions

are pollen, seed and somatic embryo of 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,…) / Loading

2/ Adaptive metabolism (hardening)

15

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.

16

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

Different cryopreservation protocols

18

Publications on cryopreservation (WoS)

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Cryopreservation Plant Cryopreservation

19

History of plant cryopreservation: key publications

• Sakai, first cryopreservation (1956 dormant buds of Mulberry), (1960

dormant buds of salix and populus)

• Nag and Street, 1973 ; Withers and King 1980; classical slow freezing

(carrot cell suspension)

• Stushnoff, 1987 dormant bud cryopreservation of apple

• Fabre and Dereuddre, 1990, encapsulation- dehydration of pear

• Sakai et al., 1990 PVS2 vitrification nucellar cells of navel orange

• Towill and Jarret, 1992 First “droplet vitrification” on sweet potato

• Schafer-Menuhr, 1996 potato droplet DMSO freezing.

• Sekizawa et al., 2011 (Dr. Niino) Cryoplate carnation

20

Cryopreservation of dormant buds (Akira Sakai (1956

dormant buds of Mulberry), (1960 dormant buds of Salix

and Populus))

Cold hardening (exposure to winter temperatures)

Freeze dehydration (-10, -20, -30°C)

Parameters to be optimised

• Hardening

• Cooling rate

• Holding temperature

21

Cryopreservation of dormant buds (Cecil Stushnoff, 1987

(dormant bud cryopreservation of apple))

Cold Hardening (Dormant field material)

Air dehydration at -5°C to 25-35% MC

Freeze dehydration at -1°C/hr to -35°C – hold 24hr


• Hardening

• Cooling rate


Only applicable to some species (need

dormant buds and grafting on rootstock);

example Apple, Mulberry, Cherry, Plum, Pear,

Grape

Grafting (no in vitro phases needed !)

22

Classical (slow) freezing protocol (Nag and Street, 1973 ;

Withers and King 1980; classical slow freezing (carrot cell

suspension)

Applied to shoot cultures, somatic embryos, cell suspension cultures,….

Cold hardening and/ or Osmotic dehydration and/or Sugar hardening

Penetrating cryoprotectants (often DMSO)+ non-penetrating

cryoprotectants

Freeze dehydration at 1°C /min to -35°C


• Hardening

• Cryoprotective mixture (Often including DMSO)

• Cooling rate


24

: Applicable to cell suspensions and callus (unorganised tissues)

: More limited application to organised tissues (meristems cultures)

Expensive cooling devices are sometimes needed

Problem Plasmolysis Extracellular ice

25

Encapsulation/dehydration

Applied to shoot tip cultures and somatic embryos

Typical protocol

1/ Encapsulation in alginate beads

2/ Treatment with high sucrose (1-2 M) for 1-5 days

3/ 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

26

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 protocol

Susceptibility towards high sucrose concentrations needed for

vitrification is very species dependent

27

4. Complete vitrification Applied to shoot cultures, somatic embryos, cell suspension cultures

Typical protocol

1/ Loading : LS : 2 M glycerol + 0.4 M sucrose

2/ Dehydration : PVS2 : 30 % glycerol + 15 % EG + 15 % DMSO

+ 0.4 M sucrose

3/ Rapid freezing

4/ Deloading : 1.2 M sucrose

Cold Hardening

Sugar hardening + osmotic dehydration + penetrating cryoprotectant


• Sugar hardening

• Loading

• Dehydration with vitrification solution (temp, time, composition,…)

28

: Protocol applied to a wide range of culture types and plant species

No slow cooling devices are needed

: Susceptibility to ‘toxic’ Vitrification solution is species dependent

rather time consuming and labour intensive protocol

29

Differential scanning calorimetry

31

0.91°C

-1.36°C208.6J/g

-20

-15

-10

-5

0

5

10

He

at

Flo

w (

W/g

)

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

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

Banana meristems not treated with PVS2

(control meristems)

-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

He

at

Flo

w (

W/g

)

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


Banana meristems treated with PVS2 for a

too short (1 hour) period

-110.64°C(I)1.236J/g/°C

-2

0

2

4

He

at

Flo

w (

W/g

)

-150 -100 -50 0


PVS2

32

-113.92°C(I)0.9561J/g/°C

-42.73°C(I)1.989J/g/°C

-2

-1

0

1

2

3

He

at

Flo

w (

W/g

)

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


-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

He

at

Flo

w (

W/g

)

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


Banana meristems treated with PVS2 for 2

hours (no crystallization)

Banana meristems treated with PVS2 for 2

hours (cold crystallization)

33

• Slow (classical) freezing

• Dormant bud freezing

• Encapsulation-dehydration

• Droplet freezing

• Fast Preculture (+ dehydration) freezing

• Vitrification (PVS2 , PVS3,…)

• Encapsulation-vitrification

• Droplet vitrification

• V-cryo-plate procedure

• D-cryo plate procedure

Most used methods for cryopreservation

34

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


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


Slow freezing Dormant bud freezing

USDA-ARS, Fort Collins and Corvallis USA Citrus species,

grape, garlic, mint,

fruit trees.

Vitrification


Slow freezing Dormant bud freezing

36

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).

37

How to obtain more rapid freezing rates

• Freezing in partially solidified nitrogen (sludge) which has

a temperature of about –208°C (instead of –196°C in

case of liquid nitrogen)

• A closer contact between the tissue and the cooling

agent. (Cryotubes (about 6°C/sec )).

– Semen straws (about 60°C/sec)(potato)

– droplet vitrification (about 130°C/sec)

38

Droplet - vitrification of apical meristems

39

Different steps in developing a droplet vitrification protocol

1. Define accessions representative for the collection

Include drought/cold resistant as well as sensitive plants.

2. Check accessions for the absence of contamination

Especially, slow growing endogenous bacteria that often do not

interfere with normal in vitro multiplication but only appear (and

interfere) with cryopreservation

3. Define shoot multiplication medium

Many plants are needed

4. Define meristem outgrowth medium

meristem tip culture media are often different from those used for

normal shoot multiplication

5. Define toxicity level for LS solution

40

Different steps in developing a droplet vitrification protocol

6. Define toxicity level (against PVS2 at 0°C) for LS

treated tissues

7. Define optimal lenght of PVS2 treatment after

cryopreservation

Banana (Panis et al.,

2005)

8. (Define optimal size of explant for cryopreservation)

9. Redefine regrowth conditions

Different steps in developing a droplet vitrification protocol for Sweet potato

1. Define accessions

representative for the

collection (10)

2. Check accessions for the

absence of contamination

OK

CIP- number

Collecting number Accession name

Country of Origin

440166 UGA Tanzania Tanzania UGA

420530 ARB 620 Camote Mata Serrano PER

440669 I00207 Cinitavo PHL

421028 DLP 5314 Espelma PER

420665 DLP 905 Trujillano PER

440031 NCSU 240 Jewel USA

442764 IITA-CL 663 TIS 87/0029 NGA

400989 DLP 1424 Ibarreno ECU

400040 DLP 3212 Manchester Hawk JAM

440145 CMR IRA 1112 CMR 1112 CMR

3. Define shoot multiplication

medium

Number of nodes developed after 6 weeks of

culture

4. Define meristem outgrowth

medium (0.5 mg/l GA3)

5. Define toxicity level for LS

solution

hergroei

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100

2 weken 4 weken 8 weken

tijd van observatie

herg

roei

(%)

20min

3u

6u

hergroei

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100

Jewel TIS 87/0029 Ibarreno Tanzania

cultivar

herg

roei

(%)

1/2 MS

Hirai

MS

6. Define toxicity level (against PVS2 at 0°C) for LS treated tissues

60 min PVS2 is toxic

7. Define optimal lenght of PVS2 treatment after cryopreservation

0

20

40

60

80

100

120

0 15 30 45 60

PVS 2 (min)

Su

rviv

al

(%)

2 primordia

4 primordia

8. Define optimal size of explant for cryopreservation

0

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120

2 2P 4 4P

Plant (%)

Regeneration (%)

Survival (%)

Protocol

Survival, even shoot growth is not a problem but plant regeneration is

CONCLUSIONS

Post-thaw plant regeneration rate : 2-49 % depending on the cv

•Redefine regrowth conditions ??????

•Age of plants for meristem exision

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plant

reg

surv

Different steps in developing a droplet vitrification protocol for Cassava

1. Define accessions

representative for the

collection

6

2. Check accessions for the

absence of contamination

OK

3. Define shoot multiplication medium

MS medium, no Plant growth regulators

4. Define meristem outgrowth medium

• MS with 2,22µM Ba 1 week, afterwards MS

• MS with Kin (2.32 µM) and GA3 (0.144 µM)

5. Define toxicity level for LS solution

20 min

6. Define toxicity level (against PVS2 at 0°C) for LS

treated tissues

120 min PVS2 is toxic

7. Define optimal lenght of PVS2 treatment after

cryopreservation

45 min

Influence of cryoprotection and freezing

1. Cryoprotection = lethal

2. M Bra 856 highest plant regeneration

Influence of regeneration medium

– Medium of reference

– Dumet: callus formation

– Charoensub: Root formation

1. Absolute control; slight difference

2. Cryoprotection no difference

3. Cryopreserved, no difference

Influence of regeneration medium

Histology

Conclusions

Regeneration of cryoprotected and cryopreserved meristems of

cassava is problematic

Reason?

- Partial survival of meristem? Adopt preculture/

cryoprotection/ cryopreservation protocol

- Survival is OK but regeneration is problematic ? Adopt

regeneration conditions

Further microscopical study needed.

Tissues survive but fail to regenerate into a plant

ABIOTIC STRESS IN POTATO TO DEVELOP IMPROVED CRYOPRESERVATION TECHNIQUES OF POTATO

PhD of Rakel Folgado

Cryopreservation through droplet vitrification

Experimental setup

• Here you can add bullets

• This is a level 1 bullet

• This is again a level 1 bullet

– This is a level 2 bullet

• This is a level 3 bullet

S. commersonii S. tuberosum cv

Désirée

Survival

Tip growth

Recovery

Differentiate Survival of Tip, Tip Growth and Plant Recovery

ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

9896

96 100

42

54

38

28

00

00

0

10

20

30

40

50

60

70

80

90

100

Solanum commersonii after 8 days since cryopreservation procedure

62

Explant state 8 days after thawing

Solanum commersonii

%

ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

9896 96 100

44

77

54

64

614

33

0

10

20

30

40

50

60

70

80

90

100

Solanum commersonii after 14 days since cryopreservation procedureExplant state 14 days after thawing

Solanum commersonii

%

ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

9896

96 100

75

96

8988

23 28

2118

0

10

20

30

40

50

60

70

80

90

100



Solanum commersonii

%

ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

9896 96 100

75

9694

90

35

77

6572

0

10

20

30

40

50

60

70

80

90

100


65

Solanum commersonii

%


ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

94 98 98

90

21

38

17

300

00

0

10

20

30

40

50

60

70

80

90

100

Solanum tuberosum cv Désirée after 8 days since cryopreservation procedure

Solanum tuberosum cv Désirée

%


ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

94 98 98

90

25

52

42 36

09

6

20

10

20

30

40

50

60

70

80

90

100

Solanum tuberosum cv Désirée after 14 days since cryopreservation procedure


%


ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

94 98 98

90

68

77

84

56

4

1714

60

10

20

30

40

50

60

70

80

90

100

Solanum tuberosum cv Désirée after 21days since cryopreservation procedure


%


ControlSucrose

SorbitolCold

Recovery Rate

Tip Growth Rate

Survival Rate

94 98 98

90

7479

84

56

6

29

21

30

10

20

30

40

50

60

70

80

90

100

Solanum tuberosum cv Désirée after 28days since cryopreservation procedure


%


0

10

20

30

40

50

60

70

80

90

100

Control Sucrose Sorbitol Cold

Re

co

ve

ry R

ate

(%

)

S.commersonii

Désirée

Recovery rate of two varieties 28 days after thawing

Potato cryopreservation : conclusions

It is essential to follow growth for at least 4 weeks after cryopreservation in order

to determine the success or failure in cryopreservation

Re-growth needs to be tested, not just survival

The three stress pre-treatments improve the recovery after cryopreservation in

S. commersonii

General conclusion

Make sure that a plant can be regenerated after cryopreservation!

Coconut with RDA

Pelargonium (In collaboration with Institut National d'Horticulture, Angers)

0

10

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30

40

50

60

70

80

90

100

A Happy

Thought

Cahors Elsie Panaché Renard

Bleu

Stellar

Artic

Balcon

Lilas

Balcon

Rouge

Su

rviv

al

(%)

0

10

20

30

40

50

60

70

80

90

100

A Happy

Thought

Cahors Elsie Panaché Renard

Bleu

Stellar

artic

Balcon

Lilas

Balcon

Rouge

Re

gro

wth

(%

)

http://upload.wikimedia.org/wikipedia/commons/a/a2/Geraniaceae.jpg

POTATO (in collaboration with CIP)

2 mm

4 mm

A B

C

D

2 mm

4 mm

2 mm

4 mm

A B

C

D

Date Palm (in collaboration with University of Sfax)

http://upload.wikimedia.org/wikipedia/commons/c/c0/Phoenix_dactylifera2.jpg

http://upload.wikimedia.org/wikipedia/commons/1/17/Dates_on_date_palm.jpg

Thyme (in collaboration with the University of Oviedo)

Exposure time (min)0 30 60 90 120 180

Surv

ival (%

)0

20

40

60

80

100

a

ab

abab

bc

c

Olive (in collaboration with the University of Malaga)

0

2

4

6

8

10

12

14

Control 30´ -LN 30´+LN Vial

30´ +LN Drop

60´ -LN 60´ +LN Vial

60´ +LN Drop

SF -LN SF +LN

Re

gro

wth

ra

te

Treatment

a

a

b

cd

bcbc

bc

dd

B

Presentation title Name Surname, Title Date

Cassava

0

20

40

60

80

100

120

2 2P 4 4P

Plant (%)

Regeneration (%)

Survival (%)

Sweet potato

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 (University of Valencia, Spain)

Bituminaria (University of Valencia, Spain)

Narcissus (Daffodil) (University of Krakow, Poland)

Galanthus (Snowdrop) (University of Krakow, Poland)

Lily (PRI, the netherlands)

Rose (University of Krakow, Poland)

Yacon (University of Prague, Czech republic )

Coconut (RDA, South Korea)

Spuce (Slovac academy of science)

Pine (Slovac academy of science)

Ash (CNR, Firenze, Italy)

Byrsonima (UFLA, Brasil)

Callerya (INTAS , China)

Stevia (KU Leuven)

Raspberry (FTRI, Italy)

Many efficient cryopreservation protocols for many species are available; are all problem solved?

• Some species remain cryopreservation “difficult” (sweet

potato, cassava)

• Tissues survive cryopreservation but do not grow out

“normally”

• 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

• Back up (cfr Svalbard)

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, Kevin Longin, Natalia Sleziak, 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)

QMS: Erica Benson, Keith Harding

www.bioversityinternational.org

Thank you

how to cryoconserve a new plant species?€¦ · - dry silica gel in a closed container . 10 2....

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