Seed biology research

There is no question that conserving species in the wild ‘in situ’ is critically important to sustaining life on earth, but there is a strong case for conserving species ‘ex situ’, or ‘away from the site’. For plants, this has traditionally been by seed banking.

The seedbanking process

Seedbanks have always been important resources for developments in agriculture, horticulture, medicine and other plant uses. Most of our agricultural crops are held in seed banks, and are used for breeding and other research purposes.

The critical feature of effective seed banking is that seeds can be dried to low moisture content and then stored at low temperature for later use. If the moisture content is too high, the seeds would explode, much like a bottle of beer in the freezer! Typically seeds would dried to less than 10% moisture and stored at -18 degrees C (in a freezer). In a general sense, the cooler the seed, the longer its life.

However, some species may not tolerate drying and/or freezing and it is these that cause us great concern because alternative conservation techniques might be required. Fortunately, most dryland plant species have seeds that can be banked.

Seedbanking has many advantages:

• Many seeds, and a large amount of genetic diversity, can be stored in a small space
• Many species can be stored for long periods of time
• Little maintenance is required (although seed viability needs to be checked at regular intervals)
• Maintenance costs are relatively low, compared to other methods of ex situ conservation

There are also some disadvantages to seedbanking:

• Seed collections do not have the opportunity to adapt to changing environmental conditions over time, unlike plants in their natural habitat. Seed collections therefore provide a 'snapshot' of genetic diversity at the time of collection. This is particularly important to consider for those plant species with a short life cycle, such as herbs.
• In order to use a seed collection after storage, we need to know how to germinate the seed. Some seeds are difficult to germinate, so at PlantBank we research methods to improve the germination of these species.
• Species that are difficult to store as seeds are targeted for other methods of ex situ conservation, such as tissue culture and cryopreservation. This applies to plant species that:
  • do not produce seeds or produce very few viable seeds
  • have seeds that are very short-lived, e.g. orchids
  • have seeds that do not tolerate the drying process required for seedbanking. These seeds are known as 'desiccation sensitive' or 'recalcitrant'.

Further reading on seedbanking and alternative conservation methods:

1. Offord CA and Meagher PF. Plant Germplasm in Australia: strategies and guidelines for developing, managing and utilising ex situ collections. Australian Network for Plant Conservation Inc., Canberra. Available from the Australian Network for Plant Conservation.
2. Offord CA, Guja LK, Turner SR and Merritt DJ (2017) Seeds at the forefront: synthesis of the inaugural National Seed Science Forum and future directions in Australian seed science. Australian Journal of Botany 65 (8): 601-608. 

PlantBank scientists test seed collections to assess the proportion of seeds that are available to grow into healthy plants. Critical components of this assessment are seed fill, viability and germination.

Seed fill

When seed collections come into PlantBank, seed fill is checked first, to measure the proportion of seeds with an intact endosperm and embryo. This can be done by cutting the seeds in half with a scalpel and examining them under a dissecting microscope. This is called a 'cut test'.

In some species, seed fill can be determined by floatation: filled seed will sink while empty seed will float. At PlantBank, seeds are X-rayed to determine seed fill, although the success of this technique depends on the size and structure of the seeds.

Seed viability and germination

If the seed is filled, scientists then need to find out whether the seed is alive (viable). This can be difficult, as intact living and dead seeds look exactly the same! The simplest test of viability is the germination test, as seeds that germinate are definitely alive. This works well if we know how to germinate the seed. But if we don't know the optimal growing conditions for that species, or if the species is dormant, there may be a proportion of viable seeds that do not germinate. This can lead to a significant underestimate of viability.

A cut test can also be used to estimate seed viability, as the endosperm and embryo tissue in viable seeds is usually firm and white. The cut test is simple, quick and inexpensive.

Biochemical tests such as the tetrazolium test can also be used. The tetrazolium test stains viable tissues red, while dying or dead tissues remain unstained or pale pink. The tetrazolium test is more accurate but is time consuming and requires experience and skill to interpret the results. For example, fungal infection of seeds can also lead to red staining, as the fungal mycelium is alive too! Other biochemical stains, such as fluorescein diacetate and Evans Blue, are used to determine the viability of orchid seeds.

Seed viability varies with environmental conditions, collection season and seed maturity at collection. Some species may also have inherently low viability. After collection, viability is eventually reduced by seed ageing, even under ideal storage conditions.

Further reading on seed quality and viability:

1. Gosling PG (2003) Viability testing. In Smith RD et al. Seed Conservation:Turning science into practice. Chapter 24 pp. 445-481. Royal Botanic Gardens, Kew, UK.
2. Martyn AJ, Merritt DJ and Turner SR (2009) Seed banking. In Offord CA and Meagher PF. Plant Germplasm in Australia: strategies and guidelines for developing, managing and utilising ex situ collections. Australian Network for Plant Conservation Inc., Canberra. Available from the Australian Network for Plant Conservation.
3. Terry J, Probert RJ and Linington SH (2003) Processing and maintenance of the Millennium Seed Bank collections. In Smith RD et al. Seed Conservation: Turning science into practice. Chapter 17 pp. 307-325. Royal Botanic Gardens, Kew, UK.

Seeds require specific temperature, moisture and light cues to germinate, to ensure that they begin to grow when temperatures are not too harsh and rainfall is sufficient to keep the tiny seedlings alive. If moisture, appropriate temperature and light conditions are provided, but seed still does not germinate, then two things may have happened - the seed is dead or the seed is dormant.

What is dormancy?

Dormancy is a characteristic of the seed that defines the environmental conditions required for germination. It is influenced by genetics and by the environment, both during seed maturation and following seed dispersal. Dormancy ensures that seeds germinate under favourable conditions and often, over an extended period of time rather than all in one flush. This minimises the risk that all seedlings will be destroyed soon after germination and ensures species survival into the future.

Breaking dormancy

Cues to break dormancy include seasonal temperature cycles, heat from fires or hot days, smoke, stratification (exposing moistened seed to a period of warm or cold), after-ripening (maturation in dry conditions following dispersal) and scarification (to break the seed coat). Understanding which cues break dormancy in the natural habitat helps researchers choose which conditions will help seeds to germinate in the laboratory. In the laboratory, germination may also be stimulated by chemicals such as gibberellic acid. Seeds may be scarified by chipping the coat with a scalpel or abrading with sandpaper. Storage in the seedbank may also change the germination response to particular cues.

Germination and dormancy in Australian species

Some Australian species have unknown dormancy and germination mechanisms, which hampers efforts to germinate seeds from conservation seed banks. It also means that many popular ornamental Australian species are propagated by cuttings rather than from seed. Australian plant families with known dormancy include Cyperaceae, Dilleniaceae, Ericaceae (Epacridaceae), Goodeniaceae, Lamiaceae, Restionaceae, Rutaceae and Violaceae.

Further reading on seed dormancy and germination:

1. Baskin, C.C. & Baskin, J.M. (1998) Seeds. Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego, USA.
2. Baskin JM and Baskin CC (2004) A classification system for seed dormancy. Seed Science Research 14: 1-16.
3. Bell DT (1999) The process of germination in Australian species. Australian Journal of Botany 47:475-517.
4. Turner SR and Merritt DJ (2009) Seed germination and dormancy. In Offord CA and Meagher PF. Plant Germplasm in Australia: strategies and guidelines for developing, managing and utilising ex situ collections. Australian Network for Plant Conservation Inc., Canberra. Available from the Australian Network for Plant Conservation.
5. Offord CA, Guja LK, Turner SR and Merritt DJ (2017) Seeds at the forefront: synthesis of the inaugural National Seed Science Forum and future directions in Australian seed science. Australian Journal of Botany 65 (8): 601-608. 

Each seed has a 'life span' or 'shelf life' which is influenced by its genetics (e.g. plant family), conditions during seed development and handling after harvest. It is critical to understand which seeds are likely to survive for long periods in storage and which seeds have a short shelf life. 

Understanding seed longevity is useful for prioritising which seeds must be processed and stored first - a key task at the end of a busy seed collection season. A knowledge of seed longevity helps curators set appropriate schedules for testing the viability of seeds in the PlantBank vault and work out which species will need to be regenerated or replenished with fresh wild-collected seed.

Seed banking is well-established as a suitable method for storing seeds of Australian plants. But like many aspects of seed biology, the expected lifespan of seeds of Australian species is still largely unknown. The Millennium Seed Bank has used a rapid ageing experiment to rank species longevity, with both short and long-lived representatives from the Australian continent. The Australian Seed Bank partners applied the same methods to seed of 172 Australian species and found wide variation between the estimated lifespan of seed of  different species. While Australian species from Mediterranean, temperate and arid climates are among the longest-lived in the world, others such as orchids are very short-lived.
 
At PlantBank, we use the rapid ageing experiment to estimate the ‘shelf life’ of rainforest species, as many species are expected to be viable for only a short period of time following dispersal.

Further reading on seed longevity:

1. Millennium Seed Bank ‘How long can seeds live?’
2. Offord, C.A., Mckensy, M.L., Cuneo, P.V. (2004). Critical review of threatened species collections in the NSW Seedbank: Implications for ex situ conservation of biodiversity.  Pacific Conservation Biology 10(4): 221-236.
3. Crawford, A. D., Steadman, K.J., Plummer, J.A. Cochrane, A. and Probert, R.J. (2007) Analysis of seed-bank data confirms suitability of international seed-storage standards for the Australian flora. Australian Journal of Botany 55:18-29.
4. Probert RJ, Daws MI, Hay FR (2009) Ecological correlates of ex situ seed longevity: a comparative study on 195 species. Annals of Botany 104(1): 57-69.
5. Merritt DJ, Martyn AJ, Ainsley P, Young RE, Seed LU, Thorpe M, Hay FR, Commander LE, Shackelford N, Offord CA, Dixon KW and Probert RJ (2014) A continental-scale study of seed lifespan in experimental storage examining seed, plant, and environmental traits associated with longevity. Biodiversity Conservation 23(5):1081-1104.