Cryopreservation of woody species and cryotherapy

Maurizio Lambardi

Istituto per la Valorizzazione del Legno e delle Specie Arboree

….and today!!! Plant cryo-meetings 20 years ago…

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

Milestone n° 2: 1990, Akira Sakai – Jean Dereuddre

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

Pre-grafting tests

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

Cryopreservation of seeds and embryonic axes

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

Cryopreservation of embryogenic callus lines

Olive

Ash

Cypress

Horsechestnut

Norwey spruce

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)

Introducing cryotherapy…………….

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

Relations among virus

eradication, meristem

size and shoot

development

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