Ethanol production an early indicator for

seed vigour loss

DanSeed Symposium, 10 March 2015

Steven P.C. Groot and Jan Kodde

Seed vigour

� The ability to provide a healthy and well growing

seedling, even under sub-optimal conditions

● What determines seed vigour?

● How to analyse that?

Seed maturity

� Seeds gain maximum quality on the moment of natural shedding

� But it is often not possible to wait for that moment

0 20 40 60 80 100%0

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Relative time after anthesis

Ger

min

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)germination

desiccationtolerance

emergence

vigour

Development of seed quality

Seed maturation

Seed maturity and chlorophyll

Dry seeds may contain chlorophyll

Days after pollination

Chlo

rophyll c

onte

nt

maturationdevelopment

Chlorophyllbreakdown

After Jalink, PRI

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Wavelength [nm]

Absorb

ance

Spectral properties of chlorophyll

Chlorophyll-a

fluorescenceFlu

ore

scence

Detector

Laser

After Dr. Henk Jalink, WUR

CF sorter in action

Source: Dr. Henk Jalink, WUR

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4 5 6

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32

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control

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Germination curves of 6 cabbage CF-fractions

After Dr. Henk Jalink, WUR

Seed vigour tests

� Soil test

� Cold test

� Germination speed

� Conductivity (electrolyte leakage)

� Ethanol analysis

� …

Seed vigour tests

� Speed of germination or emergence

� Cold test for maize (emergence in soil at low temperature)

� Electro conductivity test (membrane leakage)

� Controlled deterioration test

� Tetrazolium test (seed viability)

� Ethanol test

� …

Ethanol as marker for seed quality

Relation seed vigour and membrane integrity

� Seed deterioration can be accompanied with membrane

oxidation

� Inner membrane mitochondria contains a high proportion

poly unsaturated fatty acids

� Mitochondria are prone to deterioration

� In non-germinating embryo’s from rice, corn, rye and pea the inner membrane is damaged

Damage to the mitochondria

Control 22 h imbibition CD 22 h imbibition

A. Benamar et al. 2003

Mitochondria: Respiration / Fermentation

Glycolysis

C6H12O6

glucose

2 C3H4O3

pyruvate

2 NAD+

2 NADH

2 ADP

2 ATP

O2

mitochondrion

Citric acid cycle

Electrontransport chain

32 ATP

cytoplasm

Mitochondria: Respiration / Fermentation

Glycolysis

C6H12O6

glucose

2 C3H4O3

pyruvate

2 NAD+

2 NADH

2 ADP

2 ATP

Fermentation

C2H6OEthanol

cytoplasm

2 NAD+

2 NADH

Wayne Buckley (Brandon Research Station, Canada) 2006

Wayne Buckley (Brandon Research Station, Canada) 2006

Wayne Buckley (Brandon Research Station, Canada) 2006

Wayne Buckley (Brandon Research Station, Canada) 2006

Ethanol assay with breath analyser

� Method Wayne T. Buckley

(Agriculture and Agri-Food Canada,

Brandon)

• Seeds + water + air in a closed vial

• Incubation

• Ethanol detection in the head space

with a modified breath analyser

(Dräger Alcotest 6810)

� Simple, fast and inexpensive

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rma

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Maturity (fraction)

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Maturity (fraction)

Seed maturity and ethanol production

• Seed (white cabbage) sorted based on chlorophyll content. Fraction1: High chlorophyll, lesser maturity, low vigour.

Ethanol assay: • Seeds in a GC vial

with water (final MC= 30%). Headspace ethanol measured.

• 20°C: 24 hours incubation

• 40°C: 6 hours incubation

40°C more sensitive and faster

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Hea

dspa

ce e

than

ol (

µg/l)

germ

inat

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afte

r 5

d (%

)

aging time (months)

Carrot ( Daucus carota)

germination Eth 40°C 3 hrs Eth 40°C 8 hrs

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aging time (months)

Carrot ( Daucus carota)

germination Eth 40°C 3 hrs Eth 40°C 8 hrs

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Hea

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Carrot ( Daucus carota)

germination Eth 40°C 3 hrs Eth 40°C 8 hrs

Relation natural aging and ethanol production

Positive correlation aging and headspace ethanol after 3 hour incubation.

Ethanol produced in the first hours is degraded in the following hours

G135

Ethanol Assay: 0.5 g, 30% MC

Relation ethanol production and quality

� Asparagus

� Arabidopsis

� Barley

� Cabbage

� Canola

� Carrot

� Chinese cabbage

� Lettuce

� Onion

� Sugarbeet

� Tomato

� ...

N.B. Within species genetic variation. Sometimes also variation

between seed lots in response. But assay works very good for

comparing effect of treatments with a single lot.

Conclusions ethanol assay

� Clear correlation seed quality and ethanol levels

� Different types of reduced quality correlate with increased

ethanol production

● Less maturity

● Heat treatment

● Natural aging

● Conditional deterioration

Further reading

� International Seed Testing Association. 2003. Working Sheet on

Tetrazolium Testing. Vol 1. 1st Edition. CH-Switzerland.

� International Seed Testing Association. 2007. Biochemical Test for

Viability : The Topography. Tetrazolium Test International Rules for

Seed Testing 2007. ISTA. CH-Switzerland.

� http://umanitoba.ca/afs/agronomists_conf/2003/pdf/buckley_ethanol

_test.pdf

� Buckley, W.T. and Huang, J. (2011) An ethanol-based seed vigour

assay for canola. Seed Science and Technology, 39(2), pp. 510-526.

� Kodde, J., Buckley, W.T., de Groot, C., Retiere, M., Zamora, A.M.V.

and Groot, S.P.C. (2011). A fast ethanol assay to detect seed

deterioration.", Seed Science Research, 22(1), pp. 55-62.

Acknowledgements

The results were obtained in the project ‘seed vigour’

supported by the Netherlands Ministry of Economic Affairs and in

collaboration with several Dutch seed companies and Dräger (Germany)

Contact information:

Steven P.C. Groot, Dr.

Researcher Seed Science

Business unit Bioscience

Wageningen UR (University & Research centre)

PO box 16, 6700AA Wageningen, The Netherlands

Wageningen Campus, building 107 (Radix)

Droevendaalsesteeg 1, 6708 PB Wageningen

Tel. +31 317 480 833 (office) / +31 620 846 816 (mobile)

E-mail: steven.groot@wur.nl

Skype: steven_groot

Vcard: www.vcard.wur.nl

Websites: www.pri.wur.nl/UK/

www.seedcentre.nl

www.internationalseedacademy.com

www.researchgate.net/profile/Steven_Groot

Seed research at Wageningen UR