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Isolation and testing of mycorrhizal fungi

Conservation of orchid seeds also requires the conservation of their fungal partners, isolated from plant tissue or from soil where the orchids grow.

Fungal symbionts for each orchid species can be 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. Alternatively, fungi already residing in plant tissue can be isolated directly.

For ex situ baiting, when a germinating seed has started to develop a shoot, the seedling 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. If obtaining mycorrhizal fungi directly from plant tissue, the tissue is cut open in a drop of water, releasing the fungi into solution. The water is then added is then added to the agar-based medium.

The fungus is sub-cultured as needed till a pure culture is obtained. Each fungal isolate is then tested to ensure it is capable of initiating germination in the orchid it was collected from. Fungal isolates that do initiate germination are placed in storage on oatmeal agar (agar made with Uncle Toby’s Oats) at 15°C.

As the fungi are actively growing during storage, the isolates require sub-culturing every six to twelve months. This process involves taking a small section of the original culture and placing it on fresh oatmeal agar.

Seed is sown over soil from the collection site to allow isolation of mycorrhizal fungi.

Mycorrhizal fungi are isolated from seedlings using a selective agar-based medium containing an antibiotic.

Orchid seeds and fungi are incubated at 20 degrees C till shoots appear.

Orchid seedlings are transferred to potting mix for growing on.

Mycorrhizal fungi in the cells of a root section of Caladenia tessellata - Z Newby

Mycorrhizal fungi in the cells of a root sample taken from Caladenia tessellata - Z Newby