Authors: Eycott AE1,2, Wilfahrt PA3
Reviewers: Vandvik V2, 4, Tielbörger K5
Measurement unit: proportion; Measurement scale: plot; Equipment costs: €; Running costs: €; Installation effort: low; Maintenance effort: low; Knowledge need: low (viability and germinability) to high (dormancy); Measurement mode: manual
Seeds are the sexual regeneration stage of plants. They are a means of plant dispersal in space and in time, allowing plants to exploit suitable habitat when and where it is available (Venable & Lawlor, 1980; Fenner & Thompson, 2005). Seed dispersal in space and in time can also reduce sibling and parent–offspring competition (Nathan & Muller-Landau, 2000). We include here all seed-like structures, for example, achenes from apomictic plants.
Seed germination can occur whenever the germination requirements are met, such as suitable environmental conditions (i.e. temperature and moisture: some species require rather specific light or temperature regimes). However, a seed or dispersal unit lying on the soil may not germinate for one of four reasons: i) the seed is inviable because the seed never formed an embryo and is in effect an empty case; ii) the seed is inviable because it died at some point; iii) the seed is viable but conditions are not suitable for germination; or iv) the seed is viable but dormant, that is, the seed has some innate mechanism that inhibits germination under conditions that are otherwise suitable. Dormancy is geographically and phylogenetically widespread (Baskin & Baskin, 2014). Dormancy can be imposed by external conditions or be an innate property of the seeds themselves, for example via an undeveloped embryo or an impermeable seed coat (hard-seededness). It can be broken by, for example, light (the red:far-red ratio is particularly important), scarification of the seed coat, temperature cycles, or extrinsic chemical signals such as smoke (Keeley & Fotheringham, 2000; Pons, 2000; Baskin & Baskin, 2014). Some species produce a mixture of dormant and non-dormant seeds, the ratios of which may be affected by climate (Wagmann et al., 2012). Other forms of global change such as nitrogen deposition can also affect the proportion of dormant to non-dormant seeds (Chen et al. 2019).
Understanding seed viability, dormancy, and germination is important for understanding plant community responses to disturbance, population dynamics, and competitive interactions. If the dormancy mechanism is known, then the proportion of seeds germinating before and after a dormancy-breaking mechanism has been applied can be compared. In the context of climate-manipulation experiments, changes in the proportion of seeds which are viable can tell us about reproductive fitness and changes in the proportion which are dormant tell us about adaptation to changing environmental conditions (Ooi et al., 2009; Shetsova et al., 2009; Walck et al., 2011). Treatment conditions mimicking a range of climate projections may be replicated either in the laboratory (e.g. Ooi et al., 2009) or in the field (e.g. Meineri et al., 2013). The effect of increased nitrogen can interact with climate effects: for example, Longas et al. (2016) show that increased nitrogen widens the thermal range at which Buglossoides arvensis will germinate. For precise climate manipulations, laboratory experiments may be preferred over the imprecise or noisy conditions of field sites. In this section we outline methods to investigate in the laboratory whether a seed is alive, alive but dormant, or dead. These methods link to protocols on reproductive success (see protocol 4.1 Sexual plant reproduction), demography (protocol 4.3 Plant demography), propagule rain (protocol 4.7 Propagule rain), and the seed bank (protocol 4.8 The soil seed bank (buried seed pool)).
4.2.1 What and how to measure?
Seed germinability
Seed germinability is usually measured using emergence tests. Seeds are sown onto growth medium (e.g. 1% agar or moist filter paper) or damp compost, kept in appropriate conditions for germination, and the proportion of seeds which produce a radicle is recorded. The number of seeds used for germination experiments depends on availability and varies between studies (Hobbie & Chapin, 1998; Shetsova et al., 2009; Ooi et al., 2014), but numbers around 100 per treatment and species are advisable. Experiments of this nature require a good understanding of the behaviour of the seeds of your study species and a good deal of forward planning (Thompson & Booth, 1993). Baskin & Baskin (2014) provide a list of guidelines for laboratory studies of germination.
Collect seeds at maturity. How to collect seeds depends on the architecture of the seed-bearing part of the target species and descriptions of the plants can be found in floras. The timing of seed collection is important and should be done at the time when the seeds have ripened, but ideally before they disperse. The outer part of the dispersal unit will often dry out or change colour when the seeds start to ripen, particularly for animal-dispersed fleshy fruits. For annuals, biennials, and perennials which make seasonal stems, the whole flowering structure or even plant is likely to be dying back. For seeds dispersed from capsules, the capsules split open or the seeds start to rattle. For seeds that are wind dispersed and have a pappus (e.g. Asteraceae), the inflorescence can be covered with netting to prevent the seeds dispersing before collection because dispersal can be very sudden and happen immediately after maturity is reached (Figure 4.2.1). Add the bag only after the flower has been pollinated and begins to senesce. In wet areas the seeds can start to become infected by fungus if they are covered so the bag technique may not be appropriate. In species where dormancy is broken by a dry or cold season, or by fire, it is possible to collect seeds after the dormancy-breaking event has passed provided that they are large enough to find (or remain on the parent plant, e.g. Pinus sylvestris or other serotinous cones; Goubitz et al., 2002), but note that some mortality may have occurred during that time.
Check seeds for the presence of an embryo. This is tricky to do non-invasively but seed cases which are completely empty shells, or are relatively easily squashed between the fingers in comparison to healthy seeds, should be discarded.
Test and use seeds immediately after harvesting. This is not always possible, for example in the middle of a field campaign, in which case the seeds should to be stored under optimal conditions. It is important to know the suitable storage conditions for different species as some seeds do not tolerate drying: these are called recalcitrant seeds (this can be checked in Baskin & Baskin, 2014 or on the Kew Seed Information Database). Keep non-recalcitrant seeds cool (ideally below 5 °C but at least below 25 °C), air dry, and in the dark. Recalcitrant seeds should be kept cool and dark and slightly damp (but not soaking wet). Longer term storage can be attained for non-recalcitrant seeds by drying to 5–7% moisture and freezing (Band & Hendry, 1993).
Check that seeds can imbibe water. If your seeds sit in water without any hint of softening or swelling, they may either be dead or be hard-seeded (physical dormancy). If they are physically dormant, the coating may need to be broken gently, for example with a needle or some sandpaper (Baskin & Baskin, 2014, p. 150).
Use intact natural dispersal units. This is because the pericarp (parts of the dispersal unit formed by the parent plant) first protects the seed, for example from drying out, and second may be rather securely attached such that detachment damages the seed. Removing the pericarp can also change the germination rate (see Baskin & Baskin, 2014, pp. 12–13 for a list of examples) – which is acceptable for viability testing but not for testing germination of the seeds under “natural” conditions (Baskin & Baskin, 2014, p. 13). It is of course difficult to access some seeds, for example, the fruit of an almond, without removing the tough endocarp but in this case collection and storage should be in the natural dispersal units, only removing them when the seed is ready to be tested.
It is vital that the conditions the seeds are placed under are suitable for their germination and that they are given adequate time. Suitable germination conditions can be implied from conditions at a site where new individuals of the species are frequent or determined experimentally. These may include fluctuations in photoperiod or temperature or both (Thompson, 1993). Regarding duration, Baskin & Baskin (2014) caution against running experiments for longer than four weeks, but this recommendation relates to finding the dormant proportions and where dormancy could be broken by germination conditions. Milbau et al. (2009) monitored their Arctic seeds for 13 weeks and Mondoni et al. (2012) incubated alpine seeds for 48 weeks, albeit with a temperature-induced period of germination suppression during the experiment. Establishment of maximum germinability (for use in population models) takes considerably longer than recording relative germinability (for example, for comparing between manipulation treatments). For maximum germinability experiments, decide beforehand a length of time during which no new germinations will trigger the end of the experiment. Ungerminated seeds can be tested for viability (see below).
If the mechanism for breaking dormancy is known, then that should be applied before starting the germination test. For example, for temperate, high-elevation and high-latitude areas, vernalisation is the most common dormancy-breaking mechanism applied – seeds should be kept slightly damp and refrigerated for four months (this can be shorter for climates with warm winters; alternatively seeds can be gathered after winter). Seeds with a hard coat can be scarified by gently rubbing the seeds with medium sandpaper on a flat surface until chips of testa (seed coat) can be seen (Thompson & Booth, 1993). Some seeds need to be kept warm and dry (Thompson & Booth, 1993). A fresh seed control can be used to determine dormancy fractions (proportion of the seeds that are dormant and non-dormant). If the mechanism for breaking dormancy is not known, some caution should be applied to subsequent germination data. Determining what kind of dormancy a species has is challenging and most probably outside the scope of climate-manipulation experiments, but a protocol is given in Baskin & Baskin (2014), along with a table of dormancy type for many species and advice on methods for breaking dormancy.
Seed viability
Seed viability is a more specific test of whether the seed embryo is still alive. There are three main groups of methods: chemical, physical, and non-invasive. The most common chemical method is to expose the embryo to tetrazolium solution. The method works best on large grass and tree seeds, but is challenging with very small seeds due to difficulties in handling and visual assessment (although not impossible, see van Waes & Debergh, 1986). The seed coat is cut open very carefully with a scalpel, without damaging the embryo (practice on spare material). Then 2,3,5-triphenyl tetrazolium chloride (usually 0.1–1% w/v) is dropped onto the embryo. If the embryo goes pink or red, there is living tissue in the embryo which has reduced the tetrazolium chloride (colourless) to formazan (pinkish red) via dehydrogenase enzymes. The International Seed Testing Association protocol (ISTA, 2018) is the gold standard for viability tests, but that protocol requires 2500 seeds per test. Physical methods involve poking the seed to see if the embryo is fleshy rather than hollow inside, although note that some plants produce hard and hollow seeds (Baskin & Baskin, 2014 p. 10). It is therefore critical to cut the seed open and examine the embryo under a stereo microscope (see Pake & Venable, 1996) and so poking and cutting could be considered the bronze standard viability test, although note that the method is destructive and that the structure of the seeds of the study species should be studied and understood beforehand. Non-invasive methods such as the use of X-rays (Foucat et al., 1993; Dell’Aquilla, 2007) are either under development or very expensive and outside the scope of this protocol.
Where to start
Baskin & Baskin (2014)
4.2.2 Special cases, emerging issues, and challenges
In all cases where material is handled in the laboratory, care must be taken to minimise artefacts of the laboratory conditions, such as by accounting for soil temperature differences relative to air temperature, or properly replicating the diurnal cycle of light and temperature (Ooi et al., 2009, 2014). Likely, warming is the easiest global change variable to manipulate, which is appropriate given the propensity toward temperature as the dominant control of dormancy and germination (Baskin & Baskin, 2014). Germination tests from different treatment populations in identical laboratory conditions may fail to account for altered dormancy effects from treatments, but can still capture population-level changes in other seed traits that influence viability (Walck et al., 2011).
4.2.3 References
Theory, significance, and large datasets
Baskin & Baskin (2014) contains a list of known dormancy types. Seed Information Database at Kew Gardens (data.kew.org/sid/) is an extensive source of information on dormancy, dispersal, germination requirements, and more.
More on methods and existing protocols
Baskin & Baskin (2014), ISTA (2018)
All references
Band, S. R., & Hendry, G. A. F. (1993). Seed collecting, cleaning and long-term storage. In G. A. Hendry, & J. P. Grime (Eds.), Methods in Comparative Plant Ecology: A Laboratory Manual. Springer Science & Business Media.
Baskin, C. C., & Baskin, J. M. (2014). Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination (2nd ed.). Elsevier.
Chen, Y., Zhang, L., Shi, X., Ban, Y., Liu, H., & Zhang, D. (2019). Life history responses of spring-and autumn-germinated ephemeral plants to increased nitrogen and precipitation in the Gurbantunggut Desert. Science of the Total Environment, 659, 756-763.
Dell’Aquila, A. (2007). Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis. Biologia Plantarum, 51(4), 777-781.
Fenner, M., & Thompson, K. (2005). The Ecology of Seeds. Cambridge: Cambridge University Press.
Foucat, L., Chavagnat, A., & Rennou, J.-P. (1993). Nuclear magnetic resonance micro-imaging and X-radiography as possible techniques to study seed germination. Scientia Horticulturae, 55(3-4), 323-331.
Goubitz, S., Werger, M. J. A., & Ne’eman, G. (2002). Germination response to fire-related factors of seeds from non-serotinous and serotinous cones. Plant Ecology, 169(2), 195-204.
Hobbie, S. E., & Chapin, F. S. (1998). An experimental test of limits to tree establishment in Arctic tundra. Journal of Ecology, 86(3): 449-461.
ISTA (2018). International Rules for Seed Testing. [https://www.seedtest.org/en/home.html].
Keeley, J. E., & Fotheringham, C. J. (2000). The role of fire in regeneration from seed. In M. Fenner (Ed.), Seeds: The Ecology of Regeneration in Plant Communities (2nd ed., pp. 311-330). Wallingford: CABI.
Longas, M. M., Chantre, G. R., & Sabbatini, M. R. (2016). Soil nitrogen fertilisation as a maternal effect on Buglossoides arvensis seed germinability. Weed Research, 56(6), 462-469.
Meineri, E., Spindelböck, J. P., & Vandvik, V. (2013). Seedling emergence responds to both seed source and recruitment site climates: a climate change experiment combining transplant and gradient approaches. Plant Ecology, 214(4), 607-619.
Milbau, A., Graae, B. J., Shevtsova, A., & Nijs, I. (2009). Effects of a warmer climate on seed germination in the subarctic. Annals of Botany, 104(2), 287-296.
Mondoni, A., Rossi, G., Orsenigo, S., & Probert, R. J. (2012). Climate warming could shift the timing of seed germination in alpine plants. Annals of Botany, 110(1), 155-164.
Nathan, R., & Muller-Landau, H. C. (2000). Spatial patterns of seed dispersal, their determinants and consequences for recruitment. Trends in Ecology & Evolution, 15(7), 278-285.
Ooi, M. K., Auld, T. D., & Denham, A. J. (2009). Climate change and bet‐hedging: interactions between increased soil temperatures and seed bank persistence. Global Change Biology, 15(10), 2375-2386.
Ooi, M. K., Denham, A. J., Santana, V. M., & Auld, T. D. (2014). Temperature thresholds of physically dormant seeds and plant functional response to fire: variation among species and relative impact of climate change. Ecology and Evolution, 4(5), 656-671.
Pake, C.E., & Venable, D. L. (1996). Seed banks in desert annuals: implications for persistence and coexistence in variable environments. Ecology, 77(5), 1427-1435.
Pons, T. L. (2000). Seed responses to light. In M. Fenner (Ed.), Seeds: The Ecology of Regeneration in Plant Communities (2nd ed., pp. 237-260). Wallingford: CABI.
Shetsova, A., Graae, B. J., Jochum, T., Milbau, A., Kockelbergh, F., Beyens, L., & Nijs, I. (2009). Critical periods for impact of climate warming on early seedling establishment in subarctic tundra. Global Change Biology, 15(11), 2662-2680.
Thompson, K. (1993). Germination at alternating temperatures. In G. A. Hendry, & J. P. Grime (Eds.), Methods in Comparative Plant Ecology: A Laboratory Manual. Springer Science & Business Media.
Thompson, K., & Booth, R. E. (1993). Dormancy breaking. In G. A. Hendry, & J. P. Grime (Eds.), Methods in Comparative Plant Ecology: A Laboratory Manual. Springer Science & Business Media.
van Waes, J. M., & Debergh, P. C. (1986). In vitro germination of some Western European orchids. Physiologia Plantarum, 67(2), 253-261.
Venable, D. L., & Lawlor, L. (1980). Delayed germination and dispersal in desert annuals: escape in space and time. Oecologia, 46(2), 272-282.
Wagmann, K., Hautekeete, N. C., Piquot, Y., Meunier, C., Schmitt, S. E., & van Dijk, H. (2012). Seed dormancy distribution: explanatory ecological factors. Annals of Botany, 110(6), 1205-1219.
Walck, J. L., Hidayati, S. N., Dixon, K. W., Thompson, K., & Poschlod, P. (2011). Climate change and plant regeneration from seed. Global Change Biology, 17(6), 2145-2161.
Authors: Eycott AE1,2, Wilfahrt PA3
Reviewers: Vandvik V4, Tielbörger K5
Affiliations
1 Faculty of Biosciences and Aquaculture, Nord University, Steinkjer, Norway
2 Department of Biological Sciences, University of Bergen, Bergen, Norway
3 Department of Disturbance Ecology, BayCEER, University of Bayreuth, Bayreuth, Germany
4 Department of Biological Sciences and Bjerknes Centre for Climate Research, University of Bergen, Bergen, Norway
5 Institute of Evolution and Ecology, Plant Ecology Group, University of Tübingen, Tübingen, Germany