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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 Botanic Garden, Mount Annan, we have been testing a technique (encapsulation-dehydration) that enables the simultaneous storage of orchid seed 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 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 then soaked in a sucrose solution and allowed to dehydrate prior to storage.
An experiment has been underway for the past 12 months to determine the effect of storage temperature and storage duration on the ability of the fungus to grow, and the seed to germinate, when stored in this manner. To date, we have successfully germinated beads held for twelve months at -18 and -196 °C. In addition, seedlings produced in this way have proven to be quite robust and able to be transferred directly from the laboratory to pot culture, a procedure that often incurs heavy losses.
To date, we have tested encapsulation-dehydration on two threatened NSW orchids - Pterostylis saxicola and Diuris arenaria (results in press). Future work on the technique will involve testing other species, testing germination after storage for 2 years, and determining whether the beads can be used for direct sowing in the field.
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 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 recently 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 food reserve and so an external supply of nutrients is needed to start the germination process. In natural environments, these nutrients are obtained via a fungus. The fungus grows into the cells of the embryo and supplies it with sugars obtained by breaking down organic matter in the soil. 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 Botanic Garden 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 are currently collecting seed, under license from NSW National Parks & Wildlife Service, for as many threatened orchids as possible. The seed is collected as part of the SeedQuest NSW program and thus 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 NSW Seed Bank at the Australian Botanic Garden, and a small portion is utilised for research purposes. Where possible, soil samples are taken from a site at the same time the seed is collected; these are subsequently used for the isolation of fungal symbionts for that species.
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. We are currently storing each isolate in three different ways - on oatmeal agar at 4°C, on oatmeal agar covered with sterile water and on oatmeal agar covered with sterile mineral oil. Fungal isolates stored under water or oil are currently held at room temperature.
Simultaneous storage of orchid seed and fungi
In addition to the above storage methods, 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. An experiment has been underway in our laboratory to determine the effect of storage temperature and storage duration on the ability of the fungus to grow, and the seed to germinate, following storage in this manner. To date, the experiment has chiefly been conducted on two threatened species - Pterostylis saxicola and Diuris arenaria - and we have successfully germinated beads held for two years at -18oC and -196oC for P. saxicola and for tw years at -196oC for D. arenaria. The procedure has 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 to pot culture (Sommerville et al.,2008). Future work on the technique will involve testing other species and determining whether the beads can be used for direct sowing in pots or in the field.
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. Fungal viability gradually declined, however, over a period of 12 months. Future experiments in this area will investigate whether it is possible to increase the longevity of the fungi by manipulating storage conditions.
Native Orchid Societies