Non-Invasive Techniques for Diagnosing Seed Viability
Conventional methods in analytical chemistry and molecular biology are invaluable to study chemical traits of seeds, but they are usually destructive. Recent advances in the development of non-invasive techniques now enable us to study metabolic changes in organisms, although often not with the same level of detail as invasive techniques. In our research into seed traits, we aim to find an appropriate compromise between the use of invasive and non-invasive techniques. The use of non-invasive techniques to diagnose seed viability would allow seed collections to be screened for viability, obviating the need for germination tests, which are seed- and time-consuming and often require specialist knowledge for breaking dormancy. Therefore, a non-destructive test would protect valuable seed collections, and may also serve as an early warning of impeding viability loss to enable intervention such as regeneration or re-collection of seeds.
We tested the use of infrared thermography to detect the temperature changes that occur during imbibition and germination. After constructing a library of “thermal fingerprints” viable and dead Pisum sativum seeds can be distinguished in less than two hours. When a dry seed takes up water, the sugar within the seed dissolves, and this process cools the seed down. Viable seeds maintain cool temperatures because they break down storage reserves into sugar. Aged and dead seeds fail to break down their reserves, or can only break them down after a phase of repair, delaying the thermal profile. So far, such processes were studied with destructive methods whereas the new infrared-based method is a breakthrough in the non-invasive diagnosis of seed quality. Using infrared thermography, viable and dead seeds can be distinguished within three hours of water uptake, a time period short enough to re-dry the seeds and store them again. Importantly, the opportunity to select live and dead seeds prior to germination is a useful tool to improve studies into the fundamental principles of ageing and cell death, which are similar in plants, animals and humans.
We have also used Differential Scanning Calorimetry (DSC) to determine melting properties of seed oil, producing another type of thermal fingerprint based on enthalpy. Compositional variation in oil between species contributes to thermal properties varying by c. 50 °C with implication for storage stability (see ‘Thermal fingerprint and seed storage’ project).
Headspace gas chromatography – mass spectroscopy (GC-MS) is another non-invasive technique that is currently being developed to relate the volatile fingerprint of seeds to the mechanisms of viability loss during seed ageing. Seed volatiles have been studied during artificial ageing of several orthodox seed species, and during desiccation of one recalcitrant seed species. Future work will include more species, and also a comparison with different storage conditions to determine the effect of temperature and relative humidity on the mechanisms of seed ageing.
Finally, together with collaborators at the German Federal Gene Bank, the IPK Gatersleben, we have started assessing the use of high-throughput Nuclear Magnetic Resonance (NMR) to non-invasively measure seed water and oil content, both of which are essential determinants of seed longevity in storage.
Project partners and collaborators
University of Graz
University of Bergen
Food and Environment Research Agency, York
University of Sheffield
Key papers published since 2006
Colville, L., Bradley, E.L., Lloyd, A.S., Pritchard, H.W., Castle, L., Kranner, I. (2012) Volatile fingerprints of seeds of four species indicate the involvement of alcoholic fermentation, lipid peroxidation, and Maillard reactions in seed deterioration during ageing and desiccation stress. Journal of Experimental Botany 63: 6519-6530.
Kranner, I., Kastberger, G., Hartbauer, M. & Pritchard, H.W. (2010) Noninvasive diagnosis of seed viability using infrared thermography. Proceeding of the National Academy of Sciences of the USA 107: 3912-3917.
Saatgut als Kulturerbe – Produktion, Nutzung und Erhaltung (2010). In Förster, K., Lohwasser, U. and Börner, A. Berichte der Gesellschaft für Pflanzenbauwissenschaften 5: 1-12.
Hamilton, K., Ashmore, S.E. & Pritchard, H.W. (2009) Thermal analysis and cryopreservation of seeds of Australian wild Citrus species (C. australasica, C. indodora and C. garrawayi). CryoLetters 30: 268-279.
Nadarajan, J., Marzalina, M., Krishnapillay, B., Staines, HJ., Benson, EE. & Harding, K. 2008. Application of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds. CryoLetters 29: 95-110.
Daws, M.I., Pritchard, H.W. (2008). The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance. CryoLetters 29: 189-198.
Conferences and workshops
Since 2006, 7 conference presentations have been delivered, including the 10th Conference of the International Society for Seed Science (ISSS) (Bahia, Brazil, 2011), a thermography workshop (Eugendorf, Austria, 2010) and Seed Ecology III, Salt Lake City (Utah, USA, 2010). In addition, a lecture on non-invasive techniques was given at the 8th Master Class on Seed Technology at the Wageningen University, designed for participants from the seed industry.