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
Development of seed quality Germination or vigor(%) 100 80 60 40 20 germination desiccation tolerance emergence vigour Seed maturation 0 0 20 40 60 80 100% Relative time after anthesis
Seed maturity and chlorophyll Dry seeds may contain chlorophyll development maturation Chlorophyll content Chlorophyll breakdown After Jalink, PRI Days after pollination
Spectral properties of chlorophyll Fluorescence Laser fluorescence Detector Absorbance Chlorophylla 400 450 500 550 600 650 700 750 800 After Dr. Henk Jalink, WUR Wavelength [nm]
CF sorter in action Source: Dr. Henk Jalink, WUR
1 2 3 1 2 3 4 5 6 4 5 6
Germination curves of 6 cabbage CF-fractions Germination [%] 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 Time [days] control After Dr. Henk Jalink, WUR 1 2 3 4 5 6
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
Damage to the mitochondria In non-germinating embryo s from rice, corn, rye and pea the inner membrane is damaged Control 22 h imbibition CD 22 h imbibition A. Benamar et al. 2003
Mitochondria: Respiration / Fermentation Glycolysis Citric acid cycle 2 ADP C 6 H 12 O 6 glucose 2 NAD+ 32 ATP O 2 2 ATP 2 NADH 2 C 3 H 4 O 3 pyruvate Electron transport chain cytoplasm mitochondrion
Mitochondria: Respiration / Fermentation Glycolysis Fermentation 2 ADP 2 ATP C 6 H 12 O 6 glucose 2 NAD+ 2 NADH C 2 H 6 O Ethanol 2 C 3 H 4 O 3 pyruvate cytoplasm
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
Seed maturity and ethanol production Headspace ethanol at 20 C ( ) and 40 C ( ) (µ g/l) 2500 2000 1500 1000 500 0 1 2 3 4 Maturity (fraction) 5 6 100% 80% 60% 40% 20% 0% Normal Seedlings ( ) and total germination ( ) 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
Relation natural aging and ethanol production germination after 5 d (%) 100% 80% 60% 40% 20% Carrot (Daucus carota) 300 200 100 Headspace ethanol (µg/l) 0% 0 0 5 10 15 20 25 30 35 Positive correlation aging and headspace ethanol after 3 hour incubation. Ethanol produced in the first hours is degraded in the following hours aging time (months) germination Eth 40 C 3 hrs Eth 40 C 8 hrs Ethanol Assay: 0.5 g, 30% MC G135
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)
Seed research at Wageningen UR 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