Botanic Gardens Trust, Sydney, Australia

Australian Botanic Garden




Plant banking

Tissue culture at the Australian PlantBank

Tissue culture is a collection of techniques used to grow whole plants from very small pieces of plant tissue. The plants are grown under sterile conditions on liquid or solid medium that contains nutrients and one or more plant growth regulators. Tissue culture can be used to produce large numbers of identical plants for commercial sale, but is also useful in the following situations:

  • To conserve highly threatened species, particularly when there is a shortage of material available for conservation
  • To reproduce plants which donít produce seed or donít germinate well
  • To conserve plants not suited to seedbanking (e.g. rainforest seeds)
  • To germinate seeds of species which require a mycorrhizal partner in the wild (e.g. terrestrial orchids)
  • To enable the transport of plants to other states or countries without transporting pathogens or pests
  • To prepare material for cryopreservation and to recover it after freezing.

The three main steps of tissue culture are initiation (sterilising the plant material), growth and multiplication (producing multiple shoots from the original plant tissue) and deflasking (moving the new plants from sterile conditions to a glasshouse). The techniques used at each step may differ slightly among laboratories but can also be dependent on the starting material used. The following paragraphs outline the techniques most commonly used at the Australian PlantBank.

Initiation

  • Shoots and nodes are collected from a plantís fresh new growth
  • The material is washed in soapy water, then surface sterilised
    • The sterilant most commonly used is sodium hypochlorite (bleach) with a surfactant added
    • The plant material needs to be as clean as possible in the beginning to limit bacterial and fungal contamination
  • The material is rinsed in sterile water then cut into small pieces (each containing a single node) in a sterile environment
  • The material is then placed on an agar-based medium containing sucrose and nutrients
    • Sucrose is added at a fairly high level so that any contaminated material can be identified and eliminated quickly
    • Macro and micronutrients and vitamins are included to promote plant growth
  • The material is monitored for 4-6 weeks for contamination
    • Clean material is subsequently transferred to a medium containing growth regulators
    • Any material that shows signs of contamination is eliminated.

Growth and multiplication

  • Clean material is placed on a medium containing growth regulators to encourage shoot growth
    • The relative proportion of cytokinins (growth regulators which encourage shoot multiplication) and auxins (growth regulators with encourage root formation) determines whether shoots or roots are produced. A higher proportion of cytokinins is required to generate shoots
    • Some experimentation can be required at this stage to determine the best combination of growth regulators to use for a particular species
  • Once a suitable medium is found to promote plant growth, subculturing is required at regular intervals.
    • Subculturing involves removing newly generated shoots and placing them on fresh medium
    • This step allows plant numbers to be increased and is carried out frequently in commercial laboratories

Deflasking

  • Deflasking is the transfer of tissue cultured plants from sterile culture to potting mix
  • New shoots can be treated as cuttings and transferred directly to potting mix at this stage, or roots can be generated prior to removing the shoots from the sterile environment
    • If roots are required, the shoots are transferred onto a medium that contains a higher proportion of auxins than cytokinins
    • Again, some experimentation can be required to determine the best combination of growth regulators to use for a given species
  • Material removed from tissue culture is very delicate and usually requires careful handling and high humidity till the tissues have had an opportunity to harden.

At the Australian PlantBank, tissue culture techniques are regularly used to maintain cultures of flannel flower and waratah cultivars. Tissue culture is now also used to produce material for cryopreservation for those species not suited to seedbanking (e.g. rainforest seeds).

Seedbanking at the Australian PlantBank

The seed vault at the Australian PlantBank is a facility for storing seed collected from plants throughout Australia, with a focus on NSW native species and threatened species. There are currently more than 10,035 collections of fully documented wild-sourced seed held in the Seedbank, containing more than 100,000,000 individual seeds. These collections are from 4669 species, of which 44 per cent are from New South Wales.

Seeds are stored under various conditions. 

  • At 2 to 4ºC for short term requirements
  • At -18 to -20ºC for long term storage
  • At -196ºC in cryogenic storage for species with special requirements. 

The seeds are vacuum sealed in aluminium foil packets and housed in walk-in cold rooms. These cold rooms have thick insulation panels surrounded by a concrete shell to insulate and protect the whole collection.

The seed vault has been built in stages so we only refrigerate those sections required. On opening, the seed vault has 76 cubic metres of storage. This can be expanded to 190 cubic metres as the seed collection grows.

Role in conservation

Through partnerships with universities, other government land management agencies and NGO conservation agencies such as Greening Australia, the Australian PlantBank will act as a focal point for conservation research. It will therefore complement the existing functions of the Herbarium and living plant collections and will give greater service to native plant conservation by our organisation.

Well-sampled and documented seed collections provide a very cost effective means of conserving genetic diversity for future conservation work that may include reintroduction into the wild. Under good storage conditions, some species may retain viability for hundreds of years.

New South Wales comprises many biogeographical regions, supporting over 6000 vascular plant taxa. However, continued pressure on the landscape for human use has altered the native vegetation, and there are now more than 600 threatened plant species and 81 ecological communities are listed as threatened under NSW state legislation. The diversity of vegetation and the need to conserve what we can, provides the impetus for the Australian PlantBank to continue collecting and storing seed as an important aspect of plant conservation. The seed vault holds collections of both threatened and non-threatened native species.

Recognising the conservation role that ex situ seedbanking can provide, the Global Strategy for Plant Conservation has a target of 75 per cent of threatened plant species in accessible ex situ collections, preferably in the country of origin, and 20 per cent of them included in recovery and restoration programs by 2020. Seed collection, storage and research are also strategies identified in the NSW Threatened Species Priorities Action Statement (Office of Environment and Heritage), to complement in situ (on-site) conservation measures. Building on many years of experience in seedbanking for threatened species recovery plans, the Australian PlantBank is expected to play a key role in the implementation of ex situ actions identified in the Priorities Action Statement. However, it is important to note that not all types of seed will survive conventional seedbanking conditions, and so other methods for conservation of these seeds must be investigated.

History of the seedbank

The NSW Seedbank was established in 1986 as an integral part of the Australian Botanic Garden, Mount Annan, the Australian plant garden of the Royal Botanic Gardens & Domain Trust. The initial role of the Seedbank was to provide wild-collected seed for the development of the new Garden, particularly the Garden's major collections of wattles, eucalypts and plants in the family Proteaceae. These collections were generally in small quantity but covered a very wide range of species and localities.

Collections for the NSW Seedbank also supported a range of horticultural research projects, from plant breeding and horticultural development of flannel flowers and waratahs to highly specific conservation projects for threatened species such as the Wollemi pine. A major upgrade of the seedbank facility in 1999 and collaboration with the Millennium Seed Bank (UK) from 2003 has ensured that high quality seed collecting, processing and research is carried out for conservation and to support the ongoing development of the Australian Botanic Garden.

The new Australian PlantBank is more than just a rebuild of the NSW Seedbank. It incorporates many other living plant conservation collections other than seeds, such as the Australian Botanic Garden's living collection of tissue cultures. It may be extended in the future to include fungal cultures, fern spores and other potential regenerative entities. 

The strength of PlantBank is that, like the National Herbarium of NSW, it will form a reference collection for identification, research and restoration.   

The main function of the PlantBank, through its research activities, will be to document the biology of species through studies in the field, the laboratory and in cultivation. It will therefore enhance other conservation initiatives by serving as a repository of regenerative material and associated knowledge.

Why seedbanking?

Seedbanking is a method of ex situ (off site) conservation that complements the in situ (on site) conservation of natural populations. The method essentially consists of drying seeds to low moisture content and then storing them at low temperatures (ideally -20°C). Seeds stored in a seedbank provide an 'insurance policy' against the extinction of natural populations, as they can be used for restoration and translocation. Seed collections also provide a source of material for research into plant biology that can assist in conservation efforts.

Seedbanking has many advantages over other methods of ex situ conservation, including the following:

  • 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

In order to utilise a seed collection after storage it is necessary to know how to germinate the seed. Some seeds are difficult to germinate, however, so we have an active seed research program that aims to improve the germination response 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'.

It should be noted that 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 (herbs).

Collecting and processing

Click here to find out about collecting and processing.

Seed testing

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

When seed collections come into the Australian PlantBank, seed fill (the proportion of seeds with intact endosperm and embryo) is examined first. This can be done by cutting the seeds in half with a scalpel and examining them under a dissecting microscope (called a 'cut test'). In some species, seed fill can be determined by floatation: filled seed will sink while empty seed will float. Seeds can also be X-rayed to determine seed fill, although the success of this technique depends on the size and structure of the seeds.

Once the scientists have determined whether or not an embryo is present, they need to determine 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 the conditions required for germination are known. However, if we don't know the optimal test 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.

The 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. Ooi M, Auld T and Whelan R (2004) Comparison of the cut and tetrazolium tests for assessing seed viability: a study using Australian native Leucopogon species. Ecological Management and Restoration 5(2): 141-143.
  4. 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.

Seed germination and dormancy

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.

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.

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 (breaks in the seed coat). Determining which cues operate in the field to break dormancy helps researchers choose which conditions will help seeds to germinate in the laboratory. In the lab, 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.

Many Australian species have unknown dormancy and germination mechanisms. This hampers efforts to germinate seeds from conservation seed banks for the recovery of threatened species, and also limits our ability to predict and manage populations in the field. It also means that many popular Australian ornamental 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. These families are the focus of dormancy breaking studies at the Australian PlantBank, with the aim of promoting germination in the laboratory and providing information for management of common and threatened species in the field.

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. Finch-Savage WE and Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytologist 171: 501-523.
  5. Leubner G (2000) The Seed Biology Place.
  6. Millennium Seed Bank Partnership, Seed Science.
  7. 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.

Links to other seedbanks, seedbanking procedures and information resources

Click here to find out about the PlantBank laboratories 

 

 

Tissue culture
Jar containing tissue cultured Flannel flowers growing in a sterile environment

Tissue culture
Tissue culture is carried out in a sterile environment using sterile equipment

Tissue culture
New material is surface sterilised and small sections placed into test tubes containing agar media

Tissue culture
Once the material has grown it is removed in a sterile environment

Tissue culture
Here it is cut into smaller sections or 'multiplied'

Tissue culture
Large amounts of clonal material can be produced this way

Sorting-seeds

Richard-Johnstone-recording

Seed-test-1

Boronia-anemonifolia

Amelia-Martyn-examining
Amelia Martyn examining a seed test

Germination-tests-in-cabinet
Germination tests in cabinet

Boronia-anemonifolia
Boronia anemonifolia grown from seed

squeezing-Callistemon-seed
Squeezing Callistemon seed can help distinguish good seed from frass.

xray-Carex-fascicularis
X-ray of Carex fascicularis seed Ė 2 of these seeds would be considered empty and a 3rd damaged or inviable (Image supplied by W. Woodward, University of Queensland).

xray-Philotheca-ciliata
X-ray of Philotheca ciliata seed Ė 5 of these seeds would be considered empty or inviable, including 2 predated seed where the grub is visible inside (Image supplied by W. Woodward, University of Queensland)

Solanum-seed-germinating
Solanum seed germinating

Stacks-of-germination-tests
Stacks of germination tests

NSW-seeds

Pultenaea villifera

Acacia nova-anglica

Pittosporum revolutum