Seed cryobiotechnology fundamental aspects - NBPGR€¦ · 3). Fundamental - Measuring seed moisture •Moisture content by weighing after forced air oven drying (103°C, 17 h; 130°C,

Seed cryobiotechnology –

fundamental aspects

Hugh W. Pritchard

Royal Botanic Gardens Kew, Wakehurst Place, UK

[email protected]

Introduction – seed types

Ger

min

atio

n (%

)

0

20

40

60

80

100

V. paradoxa

S. cuminii

D. caffra

S. cocculoides

Khaya senegalensis

Moisture content - fresh weight basis (%)

0 10 20 30 40 50

0

20

40

60

80

100

T. emetica

L. microcarpa

S. birrea

X. americana

K. africana

A

B

3-7 % MC, preferably -18°C for seed

banking

Recalcitrant

Ort

ho

do

x

Outline

1) History 19th Century – latent life of seeds 20th Century – cryopreservation 21st Century - cryobiotechnology

2) Policy arena Aichi Biodiversity Targets

GSPC; PSESP; Exceptional Species GPA FGR ITPGRFA

2) Fundamental aspects

1) History – 19th Century at Kew (1899)

© 2015 Royal Botanic Gardens, Kew

William Turner Thiselton-Dyer (3rd Director of Kew)

4

1) History – 19th century at Kew (1899)


Mustard seed 88% germination; all others 100%

Thiselton-Dyer (1899) Proc. Royal Soc., Lond. 5

Germination “unimpaired”, but some seeds can be brittle

Poppy

Cryogen, temperature / time

Species Reference

‘froze mercury’ (-39°C) / 15 min

wheat, barley, rye, broad bean

Edwards & Colin (1834)

-57 or –110°C / 30 or 20 min

9 species, including California poppy

Wartman (1860)

liquid air / 110 h

12 sorts from wide range of families

Brown & Escombe (1897)

liquid hydrogen (-250°C) / 1 – 6 h

several kinds Thiselton-Dyer (1899)

liquid air / 176 days

sweet clover Busse (1930)

1) Late 19th and early 20th century advances

6

3 32 406

3230

7830

13400

0

5000

10000

15000

Num

ber

of

refe

rences

to p

lant

cry

opre

se

rvation

Decade of publication

1) History - 20th century plant cryopreservation


Google Scholar

7

Decade No. of

papers Material

1955-1964 0 -

1965-1974 0 -

1975-1984 8 cultured plant cells = protoplasts = meristems

1985-1994 95 cultured plant cells > meristems > zygotic embryos > somatic embryos > winter buds = pollen

1995-2004 269 cultured plant cells > meristems > zygotic embryos > somatic embryos > winter buds

2005-2014 603 meristems > zygotic embryos (including seeds and axes) > somatic embryos > cultured plant cells = protocorms > winter buds = pollen;


1) History - 20th century expansion of plant cryopreservation: Web of Science

8

1) 21st Century - cryobiotechnology

July 2017 (Acta Horticulturae)

January 2016 (Biotech Adv)

Introduction of the term

WANG Qiaochun

March 2018 (Biodiv & Cons)

May 2018 (Plant Cell Rep)

Gayle Volk


Use of the term

Statistics. Definition: Biotechnology ……as the application of science and technology to living organisms as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services.

Biotechnology……. as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock.

Biotechnology… .means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.


Developing the definition of the term

Definition Cryobiotechnology……. is the use of modern technologies to understand the response of biological systems to low temperature (cryo) environments, whether natural or imposed, and leading to the production of knowledge, goods and services, including the preservation of cells and tissues for use by industry, agriculture, medical science and conservation agencies

Cryobiotechnology

Natural processes

Adaptation, resilience, FPD,

supercooling, etc.

Fundamental science

Omics, structural biology, in vitro,

model systems, etc.

Applied cryopreservation &

technology

Biobanking for agriculture, medicine,

conservation; cryotherapy; design (D-

plate, etc.)


2014 PLOS ONE: 90 reads (c. 20 y-1)

Reads on ResearchGate with ‘cryopreservation’ in title or keywords

2016 Plant Diversity: 289 reads (c. 100 y-1)

2016 CryoLetters: 171 reads (c. 60 y-1)


2017 Acta Horticulturae:

428 reads (> 200 y-1)

2016 Biotech Adv: 1255 reads (> 350 y-1)

Cryobiotechnology in title

Reads on ResearchGate with ‘cryobiotechnology’ in title


2) Policy – Conventional on Biological Diversity

2) Policy - Aichi Biodiversity Targets: CBD Aichi Targets: Strategic Goal C:

Improve the status of biodiversity by safeguarding

ecosystems, species and genetic diversity

16

2). Policy – PSESP (China)

120 sp = 70 shrubs / trees; 1 fern; 38 orchids; 11 cycads

2). Policy - Exceptional Species

2) Policy - GSPC and many threatened plants

Wyse et al. (2018) Nature Plants

Recalcitrant

Orthodox (-20°C)

Pro

po

rtio

n o

f sp

ecie

s

The 75% target is unattainable without

urgent investment into alternative techniques.

2). Global Plan of Action for FGR

27 priorities

2). International Treaty for PGR for Food and Agriculture

Annex I • 64 crops or crop complexes

• 76 genera (Agropyron, Lathyrus, Solanum)

Pritchard (2016) Ind J of PGR 29, 292-7

2). Policy – ITPGRFA (Pritchard, 2016)

Information on c. 200 species. Notable exceptions: Canavalia ensiformis, Coronilla varia

3). Fundamental Aspects – drying and cooling

Camellia

sinensis Litchi

chinensis

Castanea

mollissima Pisum

sativum

Eriobotrya

japonica

3). Fundamental - Seeds are hygroscopic: lose (and take up) water

Air

Wet seed in air with low RH

Seed Water potential

gradient

Air

Air and seed at equilibrium

Seed

• Seed oil content – more oil = lower MC

• RH – lower RH = lower eMC (equilibrium MC)

• Temperature – higher temp = lower the MC

• Adsorption OR desorption (hysteresis)

What affects the extent of drying?

What affects the drying rate?

• Proximity to eMC – closer = slower rate

• Seed size and structure – great mass = slower rate

• Depth of seeds – thicker depth = slower airflow

• Air speed/ventilation – slower airflow = slower rate

• Temperature – cooler = slower rate

3). Fundamental – drying

Seeds need to be dry enough to avoid ice formation on cooling

3). Fundamental – Isotherms (MC vs RH)

RH (%)

Mo

istu

re C

on

ten

t (

% D

W)

M

oistu

re Co

nten

t (% FW

) barley (2 % oil)

lettuce (35% oil) Isotherms are oil

content and temperature-dependent Roberts and Ellis (1989)

Ann Bot

Colder

temperature

desorption

adsorption

Zone I II III Freezable

water

Bound water

Tigh

tly-

bo

un

d w

ater

27

3). Unfrozen water content

Landolphia kirkii axes (Pammenter et al., 1991, Plant Phys.)

0.37 g H2O /g DW

0.28 g /g

3). Fundamental - drying methods

Options: Ambient drying (sun/shade) / Dry room / Drying cabinet / Incubator drier / Silica gel, dried rice, charcoal

Cooled incubator:

18ºC, giving an

RH of ~15%.

Protecting seeds from direct sun to reduce risk

of heat damage

Hermetically sealed room at 10-15% RH and 10 - 25C (FAO / IPGRI Genebank Standards). Handle large volume.

3). Fundamental - Drying with desiccants

© The Hardy Orchid Society

3:1 volume desiccant: seed, i.e. dried rice, charcoal, silica gel

3). Fundamental - Drying with controlled RH (seeds, leaves, pollen)

http://prometheuswiki

.publish.csiro.au/

RH: 5 % = silica 13% = LiCl.H2O 23 % = KAc 33 % = MgCl2 or CaCl2

40 % = Zn(NO3)2

53 % = Mg(NO3)2

66 % = NH4NO3 75 % = NaCl 85 % = KCl 94 % = KNO3

3). Fundamental - Measuring seed moisture

• Moisture content by weighing after forced air oven drying (103°C, 17 h; 130°C, 1-4 h) by ISTA method

• Equilibrium relative humidity (RH) using Rotronic Hygrometer.

Humidity dial

3). Fundamental – seed ageing (deaths over time)

Time units

% V

iab

ility

Sigma,

Decreasing MC (or temperature)

3). Fundamental - Drying and longevity (days) at 60°C

Astronium urundeuva,

Medeiros et al., 1998 SS&T

KE Cw

Species 11 % MC = 1

day lifespan

(sigma, σ)

3). Fundamental - Pre-storage RH and lifespan

10 %

x 2

Dactylorhiza fuchsii

Dendrobium anosmum

Broken stick

3 Orchids at 40°C

Pritchard et al.

(1999) Lindleyana

2nd ‘rule’ = 10% decrease

in RH doubles longevity:

seeds, spores, pollen

-10% =

Doubling

Broken stick?

Steiner & Ruckenbauer (1995) SSR

Hordeum vulgare and Avena sativa

81-90% germination after 110 years

Storage at 10-15°C and 3% MC

Perez-Garcia et al (2007) SS&T

37 Brassicaceae species

High germination after 38-40 years

Storage at c. -5°C and < 3% MC (over silica gel)

3). Fundamental - ultra-dry

c. 5% eRH

Ulmus carpinifolia

At higher temperatures,

the Q10 is about 2.

Tompsett (1986) Ann Bot

-75°C

-13°C

21°C

31°C

36°C

42°C

52°C

3). Fundamental - Longevity increases with reducing temperature

11 % MC,

31°C > 10

day lifespan

(sigma, σ)

-75°C

-13°C

21°C

31°C

36°C

42°C

52°C

3). Fundamental - Relative effect of temperature becomes less when cooler

10°C

60°C

Ulmus carpinifolia

Beneficial effect of cooling is

relatively less as temperature

falls

Tompsett (1986) Ann Bot

8 species from 6 families; 350 survival curves

Dickie et al. (1990) Ann Bot

3) Fundamental - Relative effect of temperature is same across species

- -

Universal

temperature

constants

Available for 19 species (17 / 76 genera = 22%) on ITPGRFA: Beta, Brassica, Cicer, Eleusine, Helianthus, Hordeum, Malus, Oryza, Pennisetum, Phaseolus, Phleum, Pisum, Sorghum, Trifolium, Triticum, Vigna, Zea.

log σ = KE - CW logm - CHt - CQt 2

Distribution of seed deaths

(lifespan)

Sensitivity to moisture level

Sensitivity to temperature

(Ellis & Roberts, 1980; Ann Bot)

3). Fundamental - The viability equation

http://www.kew.org/data/sid

Viability equation module:

55 species

Data: storage behaviour, weight / mass,

dispersal, germination, oil and protein

content, morphology and salt tolerance

3). Fundamental - Seed Information Database

(SID)


3). Fundamental – temperature Quadratic not predict survival at ultra-cold temperature

- -

Alfonse de Candolle. 19th

Century

But ultracold

not dead in

minutes! © 2015 Royal Botanic Gardens, Kew

3). Fundamental – temperature Exponential no better an approximation!

Dickie et al., (1990)

Annals of Botany

Over estimate longevity at low temperatures?


3). Fundamental - biophysics

30 20 10 MC (FW)

Cryopreservation

Increasing rate of ageing

After Williams and Leopold (1989) – maize embryos

Drying

stress

Ice

1

2

3

43

3. Structural biology (seeds): summary

Cooling

(recalcitrant seeds)

Cooling/time

(oily seeds)

Fluid

(hydrated

systems)

Glass

Lipid

crystal

Ice

Glass

3). Fundamental - Coffea Arabica (water and lipid)

Dussert et al 2001, Physiol. Plant.

3). Fundamental - sensitive Cuphea seeds with c. 25 °C melt and cold crystallisation

Crane et al (2003); Volk et al (2006) Planta

Pow

er /

dry

mas

s

(mW

atts

/ m

g)

Temperature (°C)

Control

Cold treated*

46

*germination fell by 32-74%

3). Fundamental - Unusual behaviour associated with specific lipids: 35 Cuphea sp.

Crane et al (2003) Planta

Cuphea

carthagenensis

Species C12+C14

(%)

(C12+C14) /

(C8+C10) Tolerant of

–18°C

C. procumbens 4 0.05 Yes

C. gaumeri 11 1.5 Yes

C. wrightii 59 2 No*

C.

carthagenensis

76 8 No*

C. parsonia 78 10 No*

48

3). Longer-term cold sensitivity of dry oilseeds

Carica papaya Elaeis guineensis

Coffea arabica

Azadirachta indica

Partial desiccation tolerance but rapid viability loss at -20C, due to lipid crystallisation?

3). Fundamental - Recalcitrant seeds

2 cm

Aesculus hippocastanum – horse chestnut.

Sapindaceae

3) Fundamental - Recalcitrant seeds die during slow drying above unfrozen water content

Nadarajan & Pritchard (2014) PLOS One 50

Variable unfrozen

water content

Laurus nobilis

3). Fundamental - PVS2 permeation and optimisation

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

51

Laurus nobilis axes

Nadarajan and Pritchard (2014) PLOS One

Summary

• Long history of seed cryo • Numerous policy instruments to

support seed banking • Fundamental aspects that inform

approaches – • Seed types • Control water (all seeds) • Control temperature (all seeds) • Control cryoprotection (Recalcitrant

seeds) • Control lipid crystallisation

(‘Intermediate’ seeds)

Funding

53

Documents

Seed cryobiotechnology fundamental aspects - NBPGR€¦ · 3). Fundamental - Measuring seed moisture •Moisture content by weighing after forced air oven drying (103°C, 17 h; 130°C,