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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
Parameters to be optimised
• Hardening
• Cooling rate
• Holding temperature
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
Parameters to be optimised
• Hardening
• Cryoprotective mixture (Often including DMSO)
• Cooling rate
• Holding temperature
23
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
Parameters to be optimised
• 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
30
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
Temperature (°C)Exo Up Universal V3.3B TA Instruments
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
Temperature (°C)Exo Up Universal V3.3B TA Instruments
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
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
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 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
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
Droplet vitrification
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
0
10
20
30
40
50
60
70
80
90
100
2 weken 4 weken 8 weken
tijd van observatie
herg
roei
(%)
20min
3u
6u
hergroei
0
10
20
30
40
50
60
70
80
90
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
20
40
60
80
100
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
0
10
20
30
40
50
60
70
80
90
100
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 after 21 days since cryopreservation procedure
Explant state 21 days after thawing
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
Solanum commersonii after 28 days since cryopreservation procedure
65
Solanum commersonii
%
Explant state 28 days after thawing
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
%
Explant state 8 days after thawing
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
Solanum tuberosum cv Désirée
%
Explant state 14 days after thawing
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
Solanum tuberosum cv Désirée
%
Explant state 21 days after thawing
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
Solanum tuberosum cv Désirée
%
Explant state 28 days after thawing
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
20
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
(%
)
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)
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
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