seed cryobiotechnology achievements and prospects€¦ · tkc • tkc is a research (7 posts) and...
Post on 17-Oct-2020
1 Views
Preview:
TRANSCRIPT
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
top related