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Dr Karen Sommerville, Scientific Officer, John Siemon, Senior Technical Officer, and Dr Cathy Offord, Senior Research Scientist
A large number of the terrestrial orchids in NSW are currently listed as threatened under the NSW Threatened Species Conservation Act (1995); some of these species require ex situ conservation as part of an integrated plan to promote their recovery. As a mycorrhizal association is needed for the germination of many terrestrial orchids in their natural environment, ex situ conservation of the orchids also requires the conservation of their fungal partners. At the Australian PlantBank, we tested a technique (encapsulation-dehydration) that enables the simultaneous storage of orchid seeds and the fungus required for germination. Encapsulation-dehydration was initially developed for the cryopreservation of Solanum shoot tips and was subsequently modified at the Royal Botanic Gardens Kew for terrestrial orchids. The technique consists of mixing the seeds and fungus together in a solution of sodium alginate, then pipetting the mixture drop-by-drop into a solution of calcium chloride to form individual beads. The beads are soaked in a sucrose solution and are then dehydrated prior to storage.
We conducted an experiment to determine the effect of storage temperature and storage duration on the ability of the fungus to grow, and the seeds to germinate, when stored in this manner. To date, we have successfully germinated beads held for up to two years at -196°C. The seedlings produced during the experiment proved to be quite robust and were able to be transferred directly from the laboratory to pot culture, a procedure that often incurs heavy losses in orchid propagation.
This initial experiment was conducted using seeds and fungi of two threatened NSW orchids - Pterostylis saxicola and Diuris arenaria (Sommerville et al., 2008). Shorter term experiments have also been conducted successfully using seeds and fungi of Diuris flavescens, Diuris tricolor and Pterostylis gibbosa.
What are terrestrial orchids?
Simply put, terrestrial orchids are orchids that grow in the ground as opposed to orchids that grow on trees (epiphytes) or rocks (lithophytes). Some terrestrial orchids are evergreen but most temperate species die back and lie dormant over summer, then re-shoot from an underground tuberoid the following autumn.
A large number of the terrestrial orchids in NSW are currently listed as Vulnerable or Endangered under the NSW Threatened Species Conservation Act (1995). These species are at risk of extinction as a result of one or more of the following: land clearing (e.g. for agriculture or residential developments), habitat fragmentation, habitat degradation (e.g. through grazing, weed invasion or pollution), illegal collection and inappropriate fire regimes. The small size of some populations means that they are also at risk from chance environmental events such as prolonged drought.
The NSW National Parks and Wildlife Service has prepared a Priorities Action Statement that lists the actions required to promote the recovery of individual threatened species and communities. For a small number of terrestrial orchids, ex situ conservation has been listed as a priority.
What is ex situ conservation?
Ex situ conservation is the conservation of plants outside their natural habitats. This may incorporate such activities as growing the plants in pots or garden beds and storing seed in a seed bank. Ex situ conservation is more difficult for terrestrial orchids than for other plant species due to the manner by which the seeds germinate.
The seeds of most plants contain endosperm, a food reserve that enables the embryo to develop till it is able to produce food for itself. Orchid seeds don’t have this readily available food store and so another supply of nutrients is needed to start the germination process. In natural environments, the nutrients are obtained via a fungus that resides in the soil and is able to grow into the cells of the orchid embryo. For some terrestrial orchids, this association (termed a mycorrhizal association) continues throughout the life of the plant. In terms of ex situ conservation, the need for a mycorrhizal association means that storing orchid seed is not useful without also isolating and storing the appropriate fungal symbiont(s).
What are we researching?
Much of our work on terrestrial orchids at the Australian PlantBank involves researching ways to conserve the fungal symbionts as well as the orchids themselves. This includes a number of related activities, each of which is outlined below.
Collection of seed and soil samples
We collect seed, under license from NSW National Parks & Wildlife Service, for as many threatened orchids as possible. In order to develop good seed collections that will be useful for future restoration programs we have begun using the following protocol:
Once seed is collected, half of each collection is sent to the Royal Botanic Gardens, Kew to form part of the Millennium SeedBank collection. The remaining seed is stored in the seed vault at the Australian PlantBank, and a small proportion is utilised for research purposes.
Isolation and testing of fungal symbionts
In our laboratory, fungal symbionts for each orchid species are isolated by first sowing seed on soil taken from the root zone of the adult orchid (a process known as ‘ex situ baiting’). Fungi residing in the soil then infect the seeds, providing them with the nutrients required for germination. When a germinating seed has developed into a protocorm, the protocorm is rinsed in sterile water, then placed on an agar-based medium that encourages the growth of the fungus while inhibiting the growth of bacteria. The fungus is sub-cultured as needed till a pure culture is obtained. Each fungal isolate is then tested to determine whether it is capable of initiating germination.
Storage of fungi
Fungal isolates that do initiate germination are placed in storage on oatmeal agar at 15°C. As the fungi are actively growing during storage, the isolates require sub-culturing every four to six months. This process involves taking a small section of the original culture and placing it on fresh oatmeal agar.
Simultaneous storage of orchid seed and fungi
In addition to the above storage method, we have been testing a technique that enables the simultaneous storage of orchid seed and the fungus required for germination. The technique, known as encapsulation-dehydration, was initially developed for the cryopreservation of Solanum shoot tips (Fabre and Derreudre, 1990), and was subsequently modified by Wood et al. (2000) for terrestrial orchids. The technique consists of mixing the seed and fungus together in a solution of sodium alginate, then pipetting the mixture drop-by-drop into a solution of calcium chloride to form individual beads. The beads are soaked in a sucrose solution and are then allowed to dehydrate before storage. We conducted an experiment to determine the effect of storage temperature and storage duration on the ability of the fungus to grow, and the seeds to germinate, when stored in this manner. The experiment was initially conducted on two threatened species - Pterostylis saxicola and Diuris arenaria. We successfully germinated beads held for two years at -18°C and -196°C for P. saxicola and for two years at -196°C for D. arenaria. The procedure has also been successfully tested on D. flavescens and D. bracteata. Seedlings produced in this way have proven to be quite robust and have been successfully transferred directly from the laboratory to pot culture (Sommerville et al., 2008).
Storage of fungi in site soil samples
In many laboratories, fungal symbionts are isolated through the extraction and culture of pelotons (coils of fungal hyphae) obtained from the cells of orchid roots, stem collars or other underground parts. This method, however, requires the disturbance of adult plants, and fungi isolated from adult plant parts do not always initiate germination (Warcup 1971, Ramsay and Dixon 2003, Bonnardeaux et al. 2007). Consequently, in our laboratory we have been concentrating on isolating fungi from protocorms germinated on site soil samples (ex situ baiting - see 'Isolation and testing of fungal symbionts' above). This method minimises the disturbance of adult plants - particularly important in the case of threatened species - and might also provide a better representation of the genetic diversity present in the associated fungal populations.
As ex situ baiting was found to be an effective way to germinate several species in our laboratory, it was thought that the banking of soil samples could prove a useful addition to existing seed banking procedures, providing the soil could be stored without loss of viability of the resident fungi. As a preliminary test of the feasibility of ‘soil banking’, samples of potting mix were taken from the root zone of a potted collection of the Sydney Plains Greenhood Pterostylis saxicola and stored under varying combinations of relative humidity and temperature. The samples were then tested at regular intervals to determine whether the resident fungi were still able to initiate germination. The results of the experiment indicated that soil samples could be stored for several months, at a variety of temperature and humidity combinations, and still be used effectively in ex situ baiting (Sommerville et al., 2009). Fungal viability gradually declined, however, over a period of 12 months.
Native Orchid Societies
If you are passionate about orchids and would like to find out more about them, or would like to learn how to grow your own, then why not consider joining a local Orchid Society? The Australian Native Orchid Society (ANOS) has groups right across the country. Please refer to their website for more information.
This research has been funded by the Hermon Slade Foundation.