Cryopreservation of woody species and cryotherapy
Maurizio Lambardi
Istituto per la Valorizzazione del Legno e delle Specie Arboree
CIMMYT
ICARDA
Svalbard Global Seed Vault
Form of conservation
Accessions Distribu-
tion
Seed banks
Cereals
Others
3.330.000 3.370.000
45%
45%
In field collections 700.000 10%
In vitro + cryopres. ???? ---
Total 7.400.000 100%
Source SoWPGR-2, FAO 2010
Ex situ conservation of genetic resources
About 1750 seed banks in
the world
Akira Sakai
Florence, July 2001
Milestone n° 1: 1960, Akira Sakai
CRYOPRESERVATION OF WOODY PLANT GERMPLASM
Populus sieboldii
Salix koriyanagi
Explants are treated in drops of DMSO (“dro- plet-method”) or PVS2 (“droplet-vitrification”) over strips of aluminium foils
The strips are inserted in cryovials filled with liquid nitrogen
The racks are immersed in liquid nitrogen
Thawing at room temperature and plating
Droplet-freezing techniques
Milestone n° 3: 1982, Luis Mroginski & K.K. Kartha
AXILLARY BUDS
Treatment with
PVS2 solution
Encapsulation-
-vitrification
Encapsulation-
-dehydration
Dehydration Dehydration + slow
cooling (-1 C/h)
Droplet
methods
“Two-step” freezing
(-1 C/min) SEEDS AND EM- BRYONIC AXES,
POLLEN
CELL AND CALLUS CULTURES
DORMANT BUDS
Conservation of clonal germplasm
high importance!
Fruit species, timber species (e.g., poplar), outdoor ornamentals
Shoot tips (axillary/apical microbuds) Dormant (winter) buds
Broad-leaf trees
Conservation of seed- propagated species Forest species
Seeds, embryonic axes Embryogenic callus
Conifers
Conservation of seed- propagated species high importance! Forest species
Seeds, embryonic axes Embryogenic callus
Conservation of clonal germplasm
Outdoor ornamentals, conifers with a well-developed micropropagation technique
Shoot tips (axillary/apical microbuds)
Shoot tips (=meristematic apexes from axillary and apical microbuds)
Largely utilized for the conservation in LN of fruit species Can be used naked or incapsulated in Ca-alginate Suitable for using with several cryo-techniques (PVS2-based vitrification, encapsulation-dehydration, encapsulation-vitrification, droplet-methods, cryo-V-plate)
Useful in cryotherapy
Advantages
Tens of explants from one single
jar of shoots
Thousands of explants
can be preserved even in small dewars
Genus Plant species
(n° Cultivar/rootstock)a
Cryopreservatio
n
techniqueb
Maximum
regrowth (%) Ref.
Actinidia
A. chinensis (1) Vitrif 52 Xu et al. (2006)
A. deliciosa (1) En-dehy 45 Zhai et al. (2003)
Actinidia spp. (9) En-dehy 95 Bachiri et al. (2001)
Actinidia spp. (3) En-dehy 56 Wu et al. (2001a)
Diospyros Diospyros spp. (2) Vitrif 30 Ai and Luo (2003)
Diospyros spp. (3) Droplet-vitrif 80 Niu et al. (2009)
Malus
M. domestica (5) En-dehy 88 Paul et al. (2000)
M. domestica (4) Droplet 80 Wu et al. (2000)
M. domestica (2) En-dehy 78 Kushnarenko et al. (2006)
M. domestica (1) Vitrif 80 Liu et al. (2008)
M. domestica (4) Vitrif / En-dehy 80 Kushnarenko et al. (2009)
M. domestica (4) Droplet-vitrif 70 Halmagyi et al. (2010a)
M. domestica (2) Droplet-vitrif 68 Halmagyi et al. (2010b)
M. domestica (1) En-dehy 72 Forni et al. (2010)
M. domestica (4) Droplet 70 Condello et al. (2011)
M. pumila (3) En-dehy 87 Hao et al. (2001)
M. pumila (1) Vitrif (PVSL)c 81 Liu et al. (2004)
Olea
O. europea Vitrif 15 Benelli et al. (2001)
O. europea Vitrif 30 Nisi et al. (2006)
O. europea Vitrif 38 Lynch et al. (2007)
Prunus
P. cerasifera Droplet-vitrif 20 Vujovic et al.(2011)
P. domestica Vitrif 57 De Carlo et al. (2000)
P. dulcis Vitrif 88 Channuntapipat et al. (2000)
P. dulcis En-dehy 62 Shatnawi et al. (2000)
P. dulcis Droplet 58 De Boucaud et al. (2002)
P. dulcis Vitrif 67 Al-Ababneh et al. (2003)
P. dulcis En-dehy 60 Al-Ababneh et al. (2003)
P. dulcis Vitrif 80 Wirthensohn et al. (2006)
Prunus hybrids TSF 74 De Boucaud et al. (2002)
P. persica Droplet 32 De Boucaud et al. (2002)
Prunus rootstock Droplet-vitrif 52 De Boucaud et al. (2002)
P. salicina Vitrif 60 Zhao et al. (2008b)
High post-thaw recovery with optimized procedures
Shoot tips (=meristematic apexes from axillary and apical microbuds)
Problematics
Cryo-techniques are poorly efficient
in species with low proliferation rates (e.g., walnut, chestnut, olive, …)
Results are often cultivar-dependent
Slow recovery of shoot culture lines in post-cryopreservation
The “from field-to field”
is highly time-consuming
Over 2,200 accessions of APPLE cryopreserved at the USDA/NCGPR of Fort Collins, U.S.A.
Cryopreservation of dormant buds Towill L.E. e Ellis D.D., 2008. Cryopreservation of dormant buds. In: Reed B.M. (ed) Plant Cryopreservation. A Practical Guide. Springer, New York, pp. 421-442.
-196°C
Winter collection of scions and cold harde ning at -5°C (8 wks)
Slow cooling up to -30°C (-1°C/hour)
Thawing and rehydration in damp peat at 2°C (2 wks)
Chip budding
After 6 wks
After 1-2 wks
Uni-nodal segments, cold hardened at 4°C and dehydrated up to a MC of 35-26% (6-8 wks)
-5°C
Rootstock: Malus floribunda, cv Radian
-196°C
Tissue culture-based technique: “vitrification” of shoot tips
Dormant bud-based cryopreservation technique
Advantage: tissue culture not required !!
Lambardi M., Previati A., Benelli C., Da Re F., De
Carlo A., Ellis D., 2010. Acta Hort., in press (1st Int.
Symp. on Cryopreservation in Horticultural Species.
Leuven, 5-8 April 2009).
from the field to LN in almost 1/2 time
from LN to the field in 1/4 of the time
about 50% of the handlabour required
Dormant bud cryotechnique vs.
Tissue culture-based cryotechnique
Problematics - 1
The technique requires to be optimized for each cv, especially as for moisture content of uni-nodal sections before LN
…but…..
only one trial per year can be carried out, due to grafting time
a large number of good-quality rootstocks is necessary every year
Good healing Failure
Problematics - 2
the “species-specificity” of the technique
apple
Not idoneous for species that cannot be reproduced by budding
Olive
Olive grafting
Pear
Cherry
Dormant buds vs. PVS2 vitrification
8 racks x 10 cryoboxes x 25 cryovials x 10 shoot tips
= 20,000 units of conservation
Dewar of 120 lt of liquid nitrogen
8 racks x 10 cryoboxes x 7 bags x 5 segments
= 2,800 units of conservation
7 : 1
Problematics - 3
Abies alba
Pinus cembra
Fagus sylvatica
Abies viable non viable empty total +LN viable
ctrl ctrl 4 12 4 20 25 (-LN)
15 A40CV 3 7 10 20 15
16 ARTCV 4 7 9 20 20
17 A40AL 3 10 7 20 15
18 ARTAL 5 10 5 20 25
Fagus viable non viable empty total +LN viable
ctrl ctrl 20 0 0 20 100 (-LN)
9 FN40CV 8 0 0 8 100
10 FNRTCV 8 0 0 8 100
11 FN40AL 11 1 0 12 92
12 FNRTAL 12 0 0 12 100
13 FV40AL 9 3 0 12 75
14 FVRTAL 11 1 0 12 92
Pinus viable non viable empty total +LN viable
ctrl ctrl 20 0 0 20 100 (-LN)
1 CN40CV 8 0 0 8 100
2 CNRTCV 8 0 0 8 100
3 CV40CV 8 0 0 8 100
4 CVRTCV 8 0 0 8 100
5 CN40AL 7 3 0 10 70
6 CNRTAL 7 3 0 10 70
7 CV40AL 7 3 0 10 70
8 CVRTAL 7 3 0 10 70
%
When seed cryopreservation is very easy……
TTC test
Pistacia vera L.
Desiccation: silica gel, 8 h ► Cryopreservation: direct immersion in LN
► Thawing: 37°C, 5 min ► Germination: hormone-free MS
0
20
40
60
80
100
20 16,9 14,5 12,5 11,7 11,4 9,8MC (%)
Ger
minat
ion
(%)
CONTROL LN+
Time (h) 0 1 2 4 8 12 24
Natural germinability: 100%
90% germinability after seed cryopreservation
Seedling from a cryopreserved seed
Ozden-Tokatli Y, Ozudogru EA, Gumusel F, Lambardi M (2007) CryoLetters, 28: 83-94.
‘Foetifera’
C. aurantium ‘Virgatum’
0
20
40
60
80
100
C. v
olka
mer
iana
C. a
uran
tium
x C
. par
adisi
C. s
inen
sis
C. s
inen
sis
'Obl
ongu
s'
C. a
uran
tium
'Virg
atum
'
C. lim
on 'F
oliis
var
iega
tis'
C. a
uran
tium
'Foe
tifer
a'
C. lum
ia 'P
yrifo
rmis'
C. m
edica
C. lim
on
C. lim
onm
edica
'Per
etto
ne'
C. lim
onm
edica
'Fio
rent
ina'
C. lim
etta
di R
angp
ur
C. m
eyer
Ge
rmin
ab
ilit
y (
%)
75%
-LN +LN
Lambardi M, Halmagyi A, Benelli C, De Carlo A, Vettori C (2007). Adv. Hort. Sci. 21:198-202.
De Carlo A, Lambardi M, Ozudogru EA (2011). Meth. in Molec. Biol., 710. Springer, pp. 185-200.
Cryopreservation of polyembrionic seeds
MC
% C. aurantium
0
10
20
30
40
50
60
0 4 8 12 16 20 24 28
Disidratazione (ore)
Conte
nuto
in a
cqua (
%) C. aurantium ‘Foetifera’
Dehydration (h)
MC
% C. limon (Volkameriana)
0
10
20
30
40
50
60
0 4 8 12 16
Disidratazione (ore)
Conte
nuto
in a
cqua (
%) C. volkameriana
Dehydration (h)
MC
%
Dehydration (h)
C. lumia
0
10
20
30
40
50
60
0 4 8 20 24 28 32 36 40
Disidratazione (ore)
Conte
nuto
in a
cqua (
%) C. lumia ‘Pyriformis’
C. sinensis
0
10
20
30
40
50
60
0 4 8 12 16 20 24
Disidratazione (ore)
Conte
nuto
in a
cqua (
%)
C. sinenis
Dehydration (h)
MC
% C. aurantium x C. paradisi
0
10
20
30
40
50
60
0 4 8 12 16 20 24 28 32 36
Disidratazione (ore)
Conte
nuto
in a
cqua(%
) C. aurantium x C. paradisi
Dehydration (h)
MC
%
Dehydration trend
Problematic
Seeds with large cotyledons cannot be cryopreserved entire
Cryopreservation of embryonic axes
Slow cooling
Direct immersion in LN
Conifers
Effective protocols developed for 14 species from 3 genera
Explant Embryogenic callus or
suspension cells
Preculture 0.2-0.4 M sorbitol
Pre-freezing 5% DMSO (30 min) sorbitol/sucrose/PEG
Cooling rate
0.3-0.5°C/min or -70/-80°C
Thawing 37°C-45°C
Ozudogru E.A., Lambardi M., 2015. In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)
Explant Embryogenic callus or somatic embryos
Pre-freezing 10% DMSO (30 min)
(+ 1M sucrose)
Cooling rate
0.2-1.0°C/min
Thawing RT-40°C
Broadleaf trees: slow cooling
Ozudogru E.A., Lambardi M., 2015. In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)
Broadleaf trees: “one-step freezing”
Effective protocols developed for 11 species from 10 genera
Explant Embryogenic callus or
somatic embryos (G/H/T/Cot)
Preculture 0.3-0.7 M sucrose
(4°C)
Pre-freezing PVS2 (30-180 min) ENC+DEHY/DEHY
Thawing RT-45°C
Ozudogru E.A., Lambardi M., 2015. In Vitro Embryogenesis in Higher Plants. SPRINGER (in press)
CRYOPROTECTION: WPM + 120-210 gr/l sucrose + 7.5% DMSO, 30-90 min, at 0ºC
SLOW COOLING to -40 C (-1 C/min)
Cryopreservation by SLOW COOLING
WPM + 150 g/l sucrose
WPM + 120 g/l sucrose
60 MINUTE-TREATMENT (Sucrose + DMSO)
WPM + 180 g/l sucrose
WPM + 210 g/l sucrose
day 0
day 42
TTC (=TEZ) TEST (2,3,5- triphenyltetrazolium chloride)
1.0% TTC, overnight at 30°C and darkness
day 0
day 14 2 months
day 42
Recovery from LN: >90%
Horsechestnut (Aesculus hippocastanum)
CRYOPRESERVATION
Explants: Portion of embryogenic callus
Crio-technique: PVS2-based (90’ at 0°C)
Thermotherapy Shoo-tip excision (0.5-1.0 mm)
Shoot-tip
colture
Shoot development Virus-free plant
37°C
Traditional approach to recover virus-free plants
Virus eradication by cryotherapy
REVIEW PAPER
Wang Q., Panis B., Engelmann F., Lambardi M. & Valkonen J.P.T., 2009. Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation. Annals of Applied Biology, Vol. 154: 351-363
Courtesy of Qiaochun Wang
Virus Species Eradication in post- cryopreservation (%)
Reference
Plum Pox Virus (PPV, Sharka) Prunus 75 Brison et al., 1997
Banana Streak Virus
(BSV)
Banana 90 Helliot et al., 2002
Tomato Mosaic Virus
(TMV)
Banana 30 Helliot et al., 2002
Grapevine Virus A
(GVA)
Grape 97 Wang et al., 2003
Potato Virus Y (PVY) Patato 95 Wang et al., 2006
Potato LeafRoll Virus
(PLRV)
Patato 86 Wang et al., 2006
Sweet Potato Feathery Mottle Virus (SPFMV)
Sweet potato 100 Wang et al., unpublished
Sweet potato chlorotic stunt virus (SPCSV, Crinivirus)
Sweet potato 100 Wang et al., unpublished
Raspberry Bushy Dwarf Virus (RBDV)
Raspberry
35
Wang et al., unpublished
Sweet Potato Little Leaf Phytoplasma
Sweet potato 100 Wang et al., 2007