Seed cryobiotechnology –

achievements and prospects

Hugh W. Pritchard & Dani Ballesteros

Royal Botanic Gardens Kew, Wakehurst Place, UK

h.pritchard@kew.org

Seed banks (-20°C, +LN) Fort Collins, USDA New Delhi,

NBPGR

Germplasm Bank of Wild Species, CAS

Millennium Seed Bank, Kew

Cro

ps

Wild

sp

ecie

s

Outline

ACHIEVEMENTS: 1. Cryobanking of orthodox seeds

i. Why, and what needs, cryo

2. Optimising orthodox seed storage i. The question of oil (seed, fern) ii. Hydration window (too dry) iii. Dormancy loss pre-treatment iv. Initial quality v. Measuring success - Salix

3. Partially drying-tolerant seeds and cryo

i. Ageing rate – citrus 4. Recalcitrant seeds

PROSPECTS: 1. Network for research and training 2. Strategy 3. Outreach

1. Cryobanking - diversity

Cryopreserved orchid seeds:

36 terrestrial species

24 epiphytic species

Popova et al (2016)

Embryo

Testa

1. Cryobanking - Medicago sativa cell line

Method: 8-day-old suspension culture cold 7% (w/v) DMSO in MS liquid medium cooled to -30°C at 0.5 °C min-1 20 min isothermal hold at -5°C cooling to -70°C at 9°C min-1 transfer to LN (-196°C) for 27 years rewarm at 40°С for 90 s. Volkova et al. (2015) CryoLetters

POX activity during culture (% of control)

Cell culture 4 days 20 days

Initial cell culture (no treatment)

90 ± 5 117 ± 7

Cell culture recovered after cryogenic storage

86 ± 5 118 ± 7

Cryobanking: plant cells, tissues, shoot-tips, buds, somatic embryos, zygotic embryos, pollen / fungal hyphae, spores.

1. Enhancing seed lifespan – lowering temperature

30 20 10 MC (FW)

Cryopreservation

Increasing rate of ageing

After Williams and

Leopold (1989) –

maize embryos Ice

6

1

10

100

1000

1 22 43 64 85 106 127 148 169 190 211 232 253 274

Species number

P5

0 (

ye

ars

)

22 % 53 %

1. Orthodox seed lifespan: 276 crop species; >10 years at 5° C, plus c. 25 years at -18°C (Li & Pritchard, 2009; modified from Walters et al 2005)

26 % of accessions (46 families) with significant viability loss in 20 years storage under seed bank conditions. (Probert et al., 2009, Ann Bot)

1. Cryobanking – lettuce and other seeds

• Ageing observable in accessions stored in cryo for > 10 years;

• Ageing rate below -15°C is faster that predicted by extrapolation from higher temperatures;

• Lettuce seeds have estimated half-lives of c. 500 and 3400 years in the vapour (-135°C) and liquid (-196°C) phases, respectively. NB. 100-200 years ata -18°C;

• Benefits of 18 C and -135 C storage lost is seeds pre-stored (aged) at 5°C first;

• Trend indicating viability loss in LN in 10-20 y in 12 species (no loss in 30 species).

1. Cryobanking – lettuce seeds

2i. Optimisation – oil seeds

0

5

10

15

20

25

30

35

1-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50

Nu

mb

er

of

sp

ecie

s

Lipid content (%)

10

Azadirachta indica seed (Leprince et al., 1998

Planta)

2i. Optimisation – oilseeds ultra-dry c

• Brassicas • Drying might

increases lifespan; • Silica gel, 2-3 %

moisture = ultra-dry

Ultra-dry, -5C, c. 40 years, UPM seed bank

Rapistrum rugosum

Elena Gonzalez-Benito

Sara Mira

But reduced lifespan on ultradrying is known in some Brassicas, legumes and orchids

Species Years storage

Germin-ation pre- storage,

%*

Germin-ation post

storage, % (n)

Germination post storage and after DSC, % (n)

Z-test ( P 0.05)#

Alyssoides utriculata 43 100 100 (6)§ 100 (6)§ NS

Alyssum saxatile 43 100 100 (21) 100 (31) NS

Arabis turrita 43 0 4 (26) ‡ 24 (45) ‡ S

Barbarea intermedia 43 95 100 (18) 93 (29) NS

Brassica napus 43 100 100 (24) 100 (13) NS

Coincya rupestris 43 92 86 (29) 100 (24) S

Conringia orientalis 44 0 100 (15) ‡ 100 (8) ‡ NS

Methods: Germination at 25 / 15C (generally)

2i. Optimisation - seed quality after 43 years

12

Approaches

He

at flo

w (m

W/g

FW

)

5,5

6,0

6,5

7,0

7,5

Temperature (Cº)

-80 -60 -40 -20 0 20

5,0

5,5

6,0

6,5

7,0

7,5

a

b

c

d

e

f

g

h

ij

k lmn

o

pq

2i. Optimisation - thermal fingerprinting

More

saturated fats

More

unsaturated

fats

He

at f

low

(m

W )

Temperature (°C )

17 wild brassicas

Differential scanning calorimeter

Crystallisation / melting

Cooling / warming 10 oC min-1.

13

Species Banked, month / year

Initial germin-ation, % (years

storage)

Germin-ation, % (years

storage)

Germin-ation, % (years

storage)

Germin-ation, % (years

storage)

Alliaria petiolata

9 / 1979 92 (4) 55 (23) 24 (32) NT

Brassica napus

8 / 1973 100 (0) 100 (13.6) 100 (23.6) 100 (33.6)

Lesquerella gordonii

11 / 1995 96 (3) 0 (8) NT NT

Rorippa palustris

6 / 2000 100 (0.25) 100 (10.75) NT NT

2i. Optimisation - Kew seed quality at 34 years

Methods: drying at 15% RH, 15C; store at -18C; various germination

14

2i. Optimisation - thermal fingerprinting Kew seeds

-100 -80 -60 -40 -20 0 20 40 80

100

120

140

160

180

200

220

240 H

eat F

low

(m

W g

-1 F

W)

Temperature (oC)

A.petiolata

L.gordonii

R.palustris

B.napus

Crystallisation / melting

15

2i. Optimisation – lipid composition

VLFA Saturates

Po

or

G

oo

d

Ballesteros et al. (2019) Plant & Cell Physiology

2i. Optimisation - fern spores as a model system

Temperature (oC)

Pow

er p

er

dry

mas

s (m

W g

-1)

-150 -100 -50 0 50

Ageing rate at -20°C > 5°C with lipids conformational changes

Hard shield fern; Dryopteridaceae

Dani Ballesteros

Chris Walters

2i. Optimisation - dry fern spores in cryo

Ballesteros et al (2019) Plant and Cell Physiology

no lipid crystallization in cryo

2ii. Optimisation – hydration window

Salix caprea seed have a hydration window that balances

desiccation stress and ice formation (too wet)

Seed ageing / reduced vigour

2iii. Optimisation – initial quality (Populus deltoides and Salix sp; 8-15 % moisture)

Normal germination (%) after LN storage / LN exposure

Salix spp

2iv. Optimisation – dormancy breaking (ginseng)

Fresh fruit Depulped fruit (‘seed’) Opened (after warm)

embryo

embryo

2iv. Optimisation – dormancy breaking

Ginseng needs cold wet treatment to enable embryo to grow. This reduces tolerance to drying (and cryo)

Hydration window

Cold treated E:E = 0.9

Dehisced E:E = 0.5

2v. Optimisation – measuring success

Radicle emergence overestimates success when

measured at normal germination

3. Optimisation – fast ageing after drying!

Embryos at 10% MC

4. Recalcitrant seeds – temperate species seed axes > 20 y

Aesculus somatic embryos on

embryonic axes

Ballesteros & Pence (2019) Cryobiology

Comparative study in embryo axes of 12 Quercus sp. from UK, Spain and Lebanon.

4. Drying and cryo of (temperate) seed axes

Pablo Bernal

Natalia Fanega

4. Mechanisms of cryo-tolerance

Investigate the causes for structural collapse during partial desiccation and LN exposure in embryonic axes of recalcitrant seeds.

X-ray micro-Computer Tomography (CT)

image: Maria Conejero

4. Evolution of freezing tolerance / avoidance

New structural and functional traits gained. 1. Herbs: overwinter underground

(perennation) 2. Trees: deciduousness 3. Trees: small conducting vessels

Zanne et al. (2014) Nature 506.7486: 89

4. Recalcitrant seeds – reducing heterogeneity

2 cm

Aesculus hippocastanum

– horse chestnut.

Sapindaceae

29

50 kPa (381 mm = 15 in Hg)

PVS2: 30% glycerol, 15% DMSO, 15% ethylene glycol in M&S medium + 0.4 m sucrose

4. Laurus nobilis embryonic axis: 0°C

Viability Regrowth

Nadarajan & Pritchard (2014) PLoS ONE

30

4. ‘Personalised’ vitrification solutions (Kim and Lee, 2012)

LS: 4 concentrations (Glyc + Sucr) VS: PVS2 = A at 6 concentations (Glyc + DMSO + EG + Sucr)

or VS: PVS3 = B at 4 concentrations (Glyc + Sucr)

Cytotoxicity Size and permeability of materials

Osm-otic

Chem-ical

Tiny (callus) Small (1 mm, meristem)

Medium (2 mm, shoot tip)

Large / semi-permeable (bulb, rhizome)

T T B5-80%, A3-70-80%; RT

B3-90%, A3-70-80% (RT)

B1-100%, A3-90% (RT)

B1-100%

T S B5-80% B3-90% B1-100% B1-100%

S T A3-70-80% (RT) A3-80-90% (RT) A3-90% (RT) A3-90% (RT)

S S A3-70-80% (ICE), B5-80%, B3-90%

A3-80-90% (ICE), B3-90%

A3-90% (ICE), B1-100%

-

31

Hedeoma todsenii (Todsen’s pennyroyal; Lamiaceae)

cryobanked 7 – 13 y.

Dr Valerie Pence (CREW)

4. Exceptional species – shoot tips

Pence et al. (2017) In vitro Cell Dev Biol - Plant

DV

Cry

o s

urv

ival

(%

)

4. Australian species – shoot tips

Method Material: species Reference

DV: Droplet vitrification

Shoot-tips: Androcalva adenothalia (79%) Androcalva perlaria (82%) Drummondita ericoides (85%) Eremophila virens (15%) Hemiandra gardneri (25%) Lomandra sonderi (30%) Philotheca basistyla (100%) Synaphea stenoloba (88%) Synaphea quartzitica (76%)

Funnekotter et al. (2017)

Cryo mesh Anigozanthus viridus (85%) Conospermum galeatum (75%)

Funnekotter et al. (2017)

VIV: Vacuum infiltration vitrification

Embryos: Laurus nobilis (70%) Shoot-tips: Loxocarya sonderi (10%)

Nadarajan & Pritchard (2014) Funnekotter et al (2015)

VIV 50 kPa (381 mm = 15 in Hg)

PVS2: 30% glycerol, 15% DMSO, 15% ethylene glycol in M&S medium + 0.4 m sucrose

Dispersed relationships, few in the tropics where most plant diversity

5. Prospects – the right place?

5. Prospects - networks and knowledge sharing

COST Action 871: Cryopreservation of Crop Species in Europe (2006-10; Bart Panis):

• 21 countries • 5 training workshops + 24 STSM early career scientists

• c. 40 PR publications per annum

2011 2014 NBPGR, New Delhi

In vitro conservation and cryopreservation training

35

5. Global Agenda for Plant Cryobiotechnology

Global Agenda for Plant Cryobiotechnology

Images: MCRH, USDA, Britannica, Trees4Future, Kew

2019-2029

5. GAPC

Industrial sectors:

1. Agriculture

2. Forestry

3. Horticulture

4. Pharma, medicine

5. Conservation

1. Clonal, shoot tips, global crops, fruit trees;

2. Shoot-tips, somatic embryos, micropropagation;

3. Shoot-tips, micropropagation, pollen;

4. Suspension cultures, callus;

5. Seed, embryos, shoot-tips, pollen etc;

Sponsors / supporters?

Research material & needs:

5. Prospects - outreach

Kew Science Festival: LN ice cream

MSB TKC

• TKC is a research (7 posts) and training centre for comparative cryobiotechnology that aims to reveal mechanisms of low temperature adaptation and tolerance.

• TKC complements the MSB at Wakehurst in widening the banking options beyond the seeds of higher plants.

Wellcome Trust Millennium Building

5. The Kew Cryosphere

Summary -

1. Wide range of techniques and protocols are now available for use;

2. Still require empirical study (oil composition / crystallisation, hydration window, initial quality, prestorage dormancy release; measuring success);

3. Continue to modernise the perception of cryo studies (if NGS and $);

4. Need to train scientists from biodiversity rich countries;

5. Need more networking and co-funding.

Acknowledgements