Unpacking seeds and checking the paperwork

Seed collections arrive by natural research centers all around the country or seed collection teams from NRGB, are immediately unpacked in a clean area.

To safeguard against the possible escape of live insects, the unpacking area situated in a closed laboratory. If any insects are found the seed-lot is dried for one week in sealed cloth bags before freezing at -20°C for two weeks, to kill the insects. Collections are checked for signs of damage (for instance damage caused by insects) and to assess whether immediate cleaning is required, as with wet fruits. The Seed Information Database is used to determine likely storage characteristics and those that might pose storage problems are removed from the batch for drying tests. The majority of collections are placed in the dry room immediately, provided they are in sealed cloth or non-waxy paper bags. Seeds arriving in waterproof plastic or foil packaging are repacked prior to drying. Herbarium specimens that accompany the seed material are also dealt with at this stage.

Find out how scientists at Research Institute of Forests and Rangelands's Herbarium help our experts at the NRGB to identify the seeds they collect to the correct plant species. Botanical keys in floras and monographs facilitate identification.

Identifying a species from the herbarium specimen taken at the time of a seed collection

 The three components of a seed collection are

• the seeds themselves
• the associated field data (including accurate geo-references and photographs)
• representative herbarium specimens

It is the herbarium specimens that enable us to accurately identify the seeds to a particular species.

 Herbarium specimens are usually cuttings, including leaves, fruit and flowers if available, taken from a representative plant from the population from which the seeds were collected. The specimens are pressed and dried in newspaper soon after they are collected. They are sent to the Herbarium with labels detailing the field data (date, locality and plant description) and any associated material, such as photographs. When identified, the specimens are mounted and accessioned into the Herbarium and contribute to reference collection. Botanical keys in floras and monographs facilitate identification if these are available. The final identification is made by visually matching the herbarium specimen against the reference collections at NRGB.

The Herbarium of research institute of forests and rangelands houses over 15000 flowering plants, 10000 non-flowering plants herbarium specimens and 800 fosils. Some of the NRGB collections are identified in the field by specialists.

Data for each Natural Resources Gene Bank collection is held in the Gene Bank Database.

Data in the Gene Bank Database (GBD) is organized into four main sections: 

Logging data into the Gene Bank Database

Data in the Gene Bank Database (GBD) is organized into four main sections: 

1. Collection data

Gene banks can hold viable seeds for many decades so it is essential that collection data is meaningful to scientists in the future. To this end, Geographical Positioning System (GPS) data is used to pinpoint the geographical location of the collection site. Such accurately-recorded locations can also be used in a Geographical Information System (GIS), allowing linkage to climatic and other data.

• Geographical Data - includes country, province, specific location, latitude, longitude and altitude
• Environmental Data - includes habitat type and associated species
• Site Notes - notes on landform, aspect, geology and soil
• Plant Name - identification by the collector, followed by any further verification details and plant descriptions
• Sampling Data - number of plants sampled / found in the population and the approximate area sampled

Information on the plant's identification is also included. This is important as it links the collection to everything else that is recorded in the scientific literature about the species. Identifying this wild material is equivalent to the process of 'characterization' for crop genetic resource collections.  Accuracy of data entry is important at this point and particularly with respect to collectors' names, the plant names assigned in the field and field collection numbers. A serial number will be given for the identification of every seed-lot arriving for storage at the NRGB.

2. Processing data

A record of the results of procedures carried out on the seed collection once it has arrived at the Natural Resource Gene Bank. The process is sequential so the progress of a collection through the system can be tracked.

3. Seed distribution data

Seed samples can be made available to students, universities and researchers for bona fide research.

4. Taxon data

This includes accepted names for species and their synonyms, conservation ratings and inclusion within plant health and CITES (Convention on International Trade in Endangered Species) legislation.

Gene Bank Database

Our Gene Bank Database (GBD) is constantly evolving to better assist in collection management, to meet the needs of research and conservation programmes and to ease seed exchange.

Statistics from the GBD allow progress towards NRGB collecting targets to be monitored and are useful when planning future collecting expeditions. Seed viability monitoring data is used to make decisions about whether to recollect or regenerate a collection if viability has dropped to 85% of initial seed viability.

The seed collection data is not available publicly,

Our scientists collect data at every stage of the seed conservation process, adding value to the collection and aiding its use.

We collect data about the seeds we safeguard

throughout the conservation process

The processing stages that yield key processing data are as follows:

• Seed quantity determination
• Storage
• Initial viability test

At each of these stages, data is recorded on the Gene Bank Database. In addition, the confirmation of identification of the species is recorded, as are the results of moisture content and tetrazolium tests (if carried out). There are also fields for noting the location and details of herbarium specimens and the purpose and outcomes of sending seeds to be grown into plants (usually for the production of a voucher or for regeneration).

Processing data can be used to assess the status of a collection or batch of collections through the various stages and the results employed to evaluate the quality of collections received from different research centers. The processing data field categories are:

Seed weight:  Number of seeds per sample weight and standard error.
Seed quantity: Original quantity (usually derived from seed weight data) and current quantity.
Germination test data:  Date tested, conditions used and result.
Moisture content test data:  Date tested, conditions used and result.
Tetrazolium test data:  Date tested, conditions used and result.
Duplicate locations:  Location (where collection split).
Banking data:  Date banked, container type and number and location in the bank.
Voucher data:  Wild or cultivated pressed specimen, herbarium notes and location of specimen(s).
Glasshouse data: How the plants performed when grown out.

Not all seeds store easily, so before the Natural Resource Gene Bank (NRGB) undertakes seed storage any likely problems need to be identified.

Most of the trees produce recalcitrant seed and seeds of harbaceus plants are usually Orthodox

Most species possess 'orthodox' seeds that remain viable when dried to the low moisture levels necessary for long-term storage. ‘Recalcitrant’ seeds are more or less completely intolerant of drying and rapidly die when they are dried. As the name suggests, species with ‘intermediate‘ seeds can withstand partial drying but are very short-lived if they are stored at sub-zero temperatures. Recalcitrant seeds are often large and fleshy with thin seed coats and the NRGB seed collectors will be cautious when confronted by such seeds on collecting missions. The different seed information databases can be used to assess whether seeds from a particular species, genus or family are likely to possess recalcitrant or intermediate seeds. If a species has been collected that is closely related to a known recalcitrant or intermediate species or has large fleshy seeds, NRGB scientists will test for dessication tolerance before transferring the seeds to a dry room. This test involves viability tests before and after drying on a small sample of the collection. To Identify desiccation-sensitive seeds more information can be found in different seed information databases.

All seed collections need to be cleaned before storage to remove dust, debris and unnecessary plant parts.

Seeds are carefully cleaned before storage

Seeds are dispersed from their parent plants in a wide variety of ways and some germinate years later and great distances away. Some seeds are carried into the air in winged fruits or are blown great distances at the end of a feathery parachute. Many seeds may set out on their journey within a brightly-coloured wet fruit that is eaten by birds. Other seeds develop inside hooked fruits that are whisked from the maternal plants and onto the fur of passing mammals. This diversity of appendages and covering structures pose a challenge for collectors and seed processing staff and often need to be removed to assist processing and germination or simply to reduce the bulk of the collection for storage. On the other hand some species produce fruits that dehisce when they are ripe and for these it is often possible to collect clean seed in the field, for instance by simply tapping seed capsules into a collecting bag.

After a seed collection has been dried it is cleaned to remove empty and poorly-developed seeds, debris and to reduce bulk for effective packaging and storage. Seed cleaning also reduces the risk of disease. Releasing the seed unharmed from the diversity of fruit types requires great care and expertise. To limit damage, much of the work is done by hand using sieves. However, equipment such as an aspirator is used to winnow the debris from the heavier, filled seeds.

Visual checking or cut-testing at the seed cleaning stage will not determine for certain whether seeds are viable and therefore likely to be able to germinate. However, such tests can provide important clues to the overall quality of the seeds. For example, we can determine the proportion of empty or incompletely-formed seeds and those that have been damaged by insects. Seeds that are empty or where insects have damaged key parts of the seed embryo are ‘incompetent’ and will not germinate. Tests are carried out on a small sub-sample of the main collection by means of a cut-test. If it reveals a high proportion of ‘incompetent’ seeds then it may be possible to reduce the proportion by re-cleaning. If ‘incompetent’ seeds cannot be removed from the collection, the proportion of healthy-looking seeds is recorded to help interpret the results of subsequent germination tests. For instance, if there are 50% incompetent seeds in a seed collection yet 50% germinate, then 100% of the competent seeds germinated. The NRGB approach to recording germination results ignores the incompetent seeds when calculating germination and viability. In the commercial testing of crop seeds the proportions of incompetent seeds are usually included in calculations so that growers can more easily work out how many seeds need to be sown.

Several methods are used to estimate the number of seeds within collections. If fruits are to be stored, then the number of seeds per fruit must be recorded.

Weighing seeds on a three decimal place balance

For most collections, a three or a seven decimal place balance (dependent upon seed size) is used to weigh five samples of 50 seeds or fruits. If seeds are exceptionally small (e.g. those of Orchidaceae and Orobanchaceae), a sample of 250 seeds is used. Once the weight of the sample has been measured, the remainder of the collection is then weighed and the total quantity of seed calculated. The total is deliberately underestimated to ensure that the collection is not exhausted earlier than expected. For small collections (less than about 300 seeds), they are counted individually. If the collection is clean and has suitably sized seeds/fruits it may be counted by a seed counting machine, but this method is too slow for very large collections. The determination is recorded on the Gene Bank Database (GBD) which calculates the final seed quantity.

Aluminum foil bags are used to keep seeds dry within the cold rooms of Natural Resource Gene Bank.

Packing seeds before transferring to the cold rooms

After final drying, seed collections are packaged into airtight containers within the drying room and then transferred to the cold rooms for long-term storage at a temperature of -20°C(base collection) or 4°C(active collection ). All collections are divided into a 'base' collection that is infrequently accessed and an 'active' collection from which samples are taken periodically for viability testing and seed distribution. The base collections are always double packaged to reduce the risk of moisture ingress to an absolute minimum. In both cases silica gel indicator sachets (previously equilibrated with the dry room conditions) are placed inside the containers and periodically checked for evidence that moisture has entered the containers. All collections are carefully labeled with moisture-proof labels.

The majority of the world's seed-bearing plants produce ‘orthodox’ seeds that can be dried to a low moisture content and then frozen. Drying, sealing and freezing will often lead to at least a 100-fold increase in seed storage life.

A seed drier unit

Air dried using desiccant-impregnated dryers to 15% relative humidity is fed into the initial dry room which is maintained at 15^oC. Seeds lose water to this dry air by diffusion until they are in equilibrium, a process that can take from a few days to several weeks depending on the size of the seeds and other physical characteristics. When equilibrium has been achieved, the seed is checked for dryness by non-destructive means using a hygrometer. Once dried and processed, the collections are packaged in a variety of different air-tight containers prior to cold storage. For small seed lots seed dryer is used.

The seeds are stored at -20 degrees centigrade in base collection and may live for hundreds of years.

placed in cold storage

Once the seed has been dried to equilibrium with 15% relative humidity it is sealed in a aluminum foil bags and placed in a cold room where it is kept at a temperature of -20°C. A small sachet of silica gel impregnated with an indicator that changes from orange to green when moist, is placed in each container to monitor the effectiveness of the container seal during storage. As an extra precaution, small samples from high conservation priority collections or from species expected to be short-lived are also stored in liquid nitrogen vapour at approximately -196°C.

The length of time that each collection can remain alive depends on the species. Some seeds may live for only a few decades, others for centuries and, in some cases, even millennia.

A germination test is the most reliable way to measure seed viability. It also provides valuable information that can be used in the future to turn the seeds into plants for reintroduction, restoration or research.

Seeds germinating on Whatman paper disks

Most of the species conserved by the NRGB have never been germinated in a laboratory before. The conditions needed for germination vary considerably between different species and even between different populations of the same species. NRGB scientists use knowledge about the plant such as its ecology and life cycle as well as climate information to predict the best conditions for germination and any pre-treatment that may be required to overcome seed dormancy.

The viability of NRGB collections is assessed about one month after they are placed into the -20°C cold store to establish the 'initial viability' of the collection. Subsequently, collections are re-tested every five or ten years depending on whether they are expected to be short- or long-lived. For wild species germination tests can take many weeks or even months.

Seeds are kept at an appropriate

 temperature in a germinator

A container of seeds for testing is removed from the cold room and allowed to warm up for one day in the adjacent drying room. The number of seeds removed for testing depends on the number of dormancy-breaking treatments and the size of the sample. Normally 50 seeds are used for each treatment, though for very small collections, as few as 20 or even 10 seeds may be used. Seeds are sown into Petri dishes containing agar and then incubated at an appropriate temperature, depending on the local climate, where the collection came from and the time of year that germination would probably occur in the wild.

Germination is usually identified by the protrusion

 of the root end of the seed embryo radical

Each week the seeds are checked, and germinated seeds are removed, recorded and discarded. The tests are checked in a clean air cabinet, to minimize the risks of inhalation of fungal spores produced by any mould on the seeds. When germination has stopped, the test is ended. Visual inspection and a cut test is used to determine whether the remaining seeds are full, empty or mouldy. Excessively mouldy but filled seeds are an indication that seed viability has declined. Statistical tests built into the Gene Bank Database are used to check re-test results when they are entered into the database to see if viability has declined since the last test. This information assists the management of collections by informing re-rest intervals and by signalling that viability is approaching the NRGB viability standard (85% of initial viability). Decisions about whether to make a new collection or undertake regeneration are taken at this point. The presence of seed dormancy means that sometimes we are unable to get all viable seeds to germinate. Thus the NRGB germination standard (75%) is lower than our viability standard. Collections are given a ‘pass’ if the lower 95% binomial confidence interval on the germination percentage is above 75%. Germination test results are ‘accepted’ if there is no statistical difference between the number of seeds that germinate and the number of germinated seeds + the number of fresh seeds remaining at the end of a test as determined by a ‘cut test’. If the number of germinated seeds is significantly lower, then experiments will be carried out to investigate the dormancy problem if the collection is rated as high priority.

Plants are grown out for four reasons: harvesting fresher or more seed; identification; research; and display.

If viability of a stored species falls to 85% of the initial viability, seed may be grown out to harvest new seed, in a process called 'regeneration'. Where a species in Natural Resources Gene Bank (NRGB) is poorly represented or has very low seed numbers, seed may be grown out to harvest more seed, known as ‘multiplication’. This process is usually used for species that are rare, endemic or endangered in the wild. The plant material will be distributed to other ex situ conservation projects. The process of growing out leads to the development of propagation protocols which are vital for effective re-introduction and restoration projects

Natural Resources Gene Bank (NRGB) is committed to sharing seed samples with other institutions for bona fide research purposes.

 for use in research or restoration

One of the main outputs of the NRGB is the distribution of seed samples for use in projects that will contribute to human innovation, adaptation and resilience in the face of current and future environmental challenges. Seed samples from the NRGB are used for research into food, agriculture, forestry, health and eco-system repair.

The Gene Bank Database contains a list of 'customers', and through the seed ordering programme produces all the information and printouts required to fulfill this commitment. We keep records of who orders what and for what purpose.

Seed Germination evaluations (excell)

Seed Germination guide and Forms (doc)