Seed Bank Procedures
In addition to regular staff, the Australian National Botanic Gardens' (ANBG) seed-bank relies on volunteer assistance from the Friends of the ANBG and has partnerships with the Australian National University and Greening Australia.
Read the Friends Newsletter article [page 14 of 16] about the operations of the Seed Bank.
There are many procedures involved in seed banking:
- Seed collecting
- Identifying and cleaning seed
- Seed quality and quantity
- Cold storage
- Germination and dormancy
- Seed longevity
- Seeds for nursery
- Searching seed database
Seed is collected in situ by ANBG staff and collaborators under permits issued by State and Territory organisations or by written permission of private landholders.
For each seed collection, a Herbarium specimen is collected and lodged at the Australian National Herbarium and provenance details (location, vegetation information, date, time etc) are recorded and entered into the IBIS database.
It is important to distinguish seed from chaff, fillers and dividers and this is carried out under the microscope.
Seeds are extracted from fruits by mechanical means such as fire, heating, wetting or drying cycles. For instance, Acacia fruits may be opened by drying, Macadamia fruits using tools and Banksia cones by heating.
Cleaning or 'processing' seed collections is done using sieves, blowing machines or by hand and volunteers play an important role in this aspect of the ANBG's Seed Banking activities.
For each seed collection, a sample are dissected in a 'cut-test' to identify good quality seed and determine the percentage of seed fill.
Seed viability is also determined over three consecutive days with the tetrazolium chloride staining technique (known as 'the TZ test'). Living seed tissue will stain red and these staining patterns help to determine percentage seed viability.
For each collection, a sample is counted and weighed and the 'Thousand Seed Weight' (approximate weight of one thousand seeds) is recorded.
Drying is critical to ensuring long-term seed viability. Upon arrival at the ANBG, all seed collections are placed in the Seed Bank drying room which is at 15°C and 15% relative humidity. Prior to banking at -21°C, seed moisture content is checked using a Rotronics Hygropalm instrument and must be between 5 and 7%.
The time taken to reduce seed moisture to 5 – 7% depends on the size and quantity of seed and seed moisture content upon dispersal in the field.
Once each seed collection is sufficiently clean and dry, it is sealed into moisture-proof containers and provided with a registration number which is linked to the Integrated Biodiversity Information System (IBIS) database.
Short term: Seeds that are to be stored short-term (less than 5 years), such as those to be used in the ANBG nursery or for Greening Australia's Seeds for Survival Project, can be stored at 15% RH and 15°C in the Seed Bank dry room.
Long term: For long-term storage of conservation collections, seeds at 3 – 7 % moisture content can be safely stored in the ANBG Seed Bank freezers at -21°C. These procedures comply with international seed banking standards. In addition, the Seed Bank is currently experimenting with seed longevity to investigate how long Australian alpine seeds can be expected to remain viable in ex situ seed bank storage.
The viability of seed collections stored in the ANBG Seed Bank is monitored by a program of germination trials. Seeds are sown onto 1% water agar in Petri dishes, at various temperature regimes. For the majority of collections there is very little information available regarding germination requirements.
All seeds, whether for short or long-term storage, have germination trials conducted to determine the:
- Optimal germination conditions (temperature, light, pre-treatments etc)
- Germination rate
- Total percentage germination.
Mature, viable seeds that do not germinate under seemingly favourable germination conditions (that is, with water, oxygen and appropriate temperatures) may be exhibiting seed dormancy. The viability of a collection can be grossly underestimated if seed dormancy goes undetected. In cases of suspected dormancy, seed viability is assessed using the TZ test, and a series of experiments are carried out in order to uncover effective dormancy alleviating treatments. Attempts at mimicking the seed's natural in situ environment often reveal optimal ex situ germination and dormancy treatments.
Seed dormancy can be alleviated, or in some cases bypassed, by:
- dry after-ripening (warm, dry storage)
- stratification (cold/warm, wet storage)
- application of smoked water
- application of gibberellic acid (GA3)
- application of Potassium Nitrate (KNO3)
- seed coat scarification (chipping or removal)
- burying the seed in soil.
How long seeds will remain viable in ex situ seed bank conditions can be estimated by artificially ageing seeds in seed longevity studies.
Seeds are placed in a carefully controlled rapid ageing environment of high temperatures and relative humidity (RH; 45°C and 60% RH). Lithium Chloride (LiCl) solutions provide the desired RH environments. Seeds are extracted from the ageing environment at various intervals and tested for viability.
Results of subsequent germination tests generate a seed survival curve that can be compared with the longevity of 'marker' species aged under the same conditions. Comparison across species and with markers enables the ranking of species into longevity categories. This is particularly important for alpine seed collections as recent evidence suggests that seeds from cooler, wetter environments are shorter lived than seeds from warmer, drier environments (Probert et al 2009).
Longevity test results can also indicate how often a conservation collection should be replenished or regenerated. Ideally, seeds should be replaced before their viability falls below 85% of the initial value.
Reference: Probert R J, Daws M I and Hay F R (2009). Ecological correlates of ex situ seed longevity: a comparative study on 195 species. Annals of Botany doi:10.1093/aob/mcp082.
Seeds germinated for propagation in the ANBG nursery are pricked out of the agar at the cotyledon stage (after the seed leaves emerge and before the true leaves develop) and seedlings are transplanted into punnets of seed raising mixture.
Fumigation with carbon dioxide is available for bulk seeds which are intended for short term storage (six months or less). This treatment kills insects and pathogens and is used for seeds for Greening Australia's Seeds for Survival project.
For long-term seed conservation, fumigation is not necessary as drying followed by storage at -21°C kills insects and pathogens.