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The Sainsbury Orchid Conservation Project Seeds and Fungi Fungal Isolation Media for Seed Sowing and Germination Seed Collection and Sowing Infection and Development Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
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A wide range of European terrestrial orchids have been raised from seeds by the asymbiotic methods of micro propagation that are widely used in horticulture for tropical orchids, especially epiphytes. However, progress is very slow when this technique is used for many of the terrestrial species, and it has proved difficult with others. The transfer of seedlings developed in vitro to a soil‑based medium in pots is often a particular problem.
The symbiotic methods of seed germination pioneered by the French botanist Bernard at the beginning of this century, and redeveloped in Australia in the last 30 years, more closely resemble what happens in nature. The seeds are invaded by an appropriate fungus which develops a mycorrhizal association in the protocorm and later in the roots of the seedling. Young plants already infected with a symbiont are more easily transferred to pot culture and eventually to the wild.
About 50 species of European orchids are currently recorded in the British Isles, but 10 of these are listed as endangered, and two are reduced to a single locality only. In other parts of Europe a similar situation prevails.
The project initiated in 1983 at the Royal Botanic Gardens, with the co‑operation of the Nature Conservancy Council and the generous backing of Sir Robert and Lady Sainsbury, was intended to propagate from seeds those British and European orchids which are threatened in the wild.
The work began with a wide range of orchid species, rare and relatively common, in an attempt to formulate the best methods to follow. Now, seven years after the first attempts at isolating fungi from orchid plants in cultivation, both symbiotic and asymbiotic methods are used.
Since 1985 the project has been supervised by the Sainsbury Orchid Fellow who also liaises with the various conservation bodies which support and take great interest in it. Five orchid species have been reestablished in wild situations where they have flowered and set seed; 35 species have been raised and transferred to pots in the greenhouse; and a total of 46 species have germinated in the laboratory where the plantlets are at various stages of growth and development. The techniques learned from Mark Clements and developed by Harriet Muir and Robert Mitchell are used and refined by Margaret Ramsay and Grace Prendergast on an ever‑increasing range of species. There is still much to do, but this chapter describes the considerable progress that has been made so far.
Fungal Isolation Media for Seed Sowing and Germination Seed Collection and Sowing
Infection and Development Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
Orchids produce a very large number of minute seeds which are as fine as dust and among the smallest seeds of all flowering plants. Each seed has a tough, transparent testa, or seed coat, and an embryo composed of only 100‑200 cells. The cells contain some lipid and starch but there is no food reserve such as endosperm that comprises the bulk of many other seeds. The difficulties of germination and early development of such a small and fragile embryo have been overcome by an intimate symbiosis between the orchid and an appropriate fungus.
The dispersal of orchid seeds by wind ensures that some will land not too far from the parent plant. These have an excellent chance of encountering the right fungus. Suitable fungi are widespread in the soil, though research has indicated that some orchids are specific in their fungal partner. Others appear to be capable of using various fungi, and several fungi have been isolated which will assist the germination of a variety of orchids.
The fungus penetrates one end of the embryo through its large suspensor cell. As the fungal hypha grows into the inner cells of the embryo it forms coils called pelotons which, in turn, are gradually digested by the orchid plant. As the infection proceeds, the orchid embryo grows rapidly to form a protocorm which, in due course, develops a shoot from which the leaves and roots arise. The fungal hyphae also infect the young roots and form pelotons in the cortical cells of the root.
In wild orchid plants the mycorrhizal fungi are found as pelotons in live and healthy roots which appear cream or yellowish. Usually the coils of hyphae are linked via root hairs to the soil or substratum. It is believed that the fungus digests organic materials in the surrounding medium and the resulting nutrients diffuse into the pelotons from which they are obtained by the orchid.
The underground parts of terrestrial orchids are quite varied, but the fungus is usually present in the roots rather than the tubers or rhizome. The degree of infection varies in different species and it also appears to be somewhat seasonal. In many species there appears to be heightened activity of the fungus while the orchid is at the peak of its vegetative growth. Orchids with tubers usually do not hold the fungal infection through the dormant season, and the new roots of these plants become infected from the soil when vegetative growth resumes each year. Orchids with rhizomatous growth, like Cypripedium, seem to harbor fungal activity after the leaves have died down, while for much of the year their roots contain masses of starch but no pelotons. The extent of the dependence of the mature orchid plant on the fungus is still unknown, but it has been interesting to find that the fungal isolates that are more effective for germination have been obtained from the most vigorous orchid plants.
Seeds and Fungi Media for Seed Sowing and Germination Seed Collection and Sowing
Infection and Development Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
Mycorrhizal fungi have been isolated at Kew from the roots and, rather rarely, the protocorms of European orchid species in cultivation. A few roots have been obtained from wild plants, with the landowner's permission, and from three of the scheduled endangered species under license from the Nature Conservancy Council. Fungal isolation is not a destructive technique, as a sample of root can be collected with minimal disturbance to the plant.
The root is first washed gently under running water to remove most of the external debris. A soft paint brush is sometimes used to remove the more persistent particles. Pieces about 5‑6 mm long are cut from any yellowish regions of the root and transferred to a dissecting microscope.
The epidermis is sliced away from the pieces of root and thin sections are cut from the cortex. These are examined for the presence of pelotons and, if found, the infected tissue is excised and placed in a few drops of sterile distilled water in a Petri dish. With the aid of a fine scalpel and needle, individual pelotons are teased out of the tissue. After all extraneous material has been removed, the pelotons are covered with cool fungal isolating medium (FIM)
Fungal isolating medium (FIM)
g/l
Calcium nitrate (Ca(N03)2.4H20) 0.5
Potassium phosphate (KH2P04) 0.2
Potassium chloride (KCl) 0.1
Magnesium sulphate (MgS04.7H20) 0.1
Yeast extract 0.1
Sucrose 5.0
Agar 8.0
Distilled water to make up to 1 litre of medium
The cultures are sealed with laboratory film and maintained at room temperature.
Within 12‑24 hours the fungi that are likely to be mycorrhizal begin to grow. They are removed in blocks of medium and subcultured until free from contamination. This is achieved by the use of 'window' plates. FIM is poured so as to leave an area free from medium in which the blocks of inoculum are placed. As the fungus grows across the plastic surface to reach the new medium, contaminants are left behind in the block, which can be removed later.
The fungal isolates are maintained as cultures in Petri dishes of FIM which are kept at room temperature.
Stock cultures of fungi growing on FIM are maintained at 4ºC in the refrigerator.
So far the fungi have been identified rather tentatively as they have only been seen in the vegetative state. In the past they have all been referred to the genus Rhizoctonia, but it appears that at least two different genera of Basidiomycetes are active as mycorrhizal symbionts with the European orchids. For the time being all the fungi at Kew are known by numbers only, and F414, isolated from Dactylorhiza iberica growing in the gardens, is the most useful and vigorous that has been isolated.
Seeds and Fungi Fungal Isolation Seed Collection and Sowing
Infection and Development Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
Media for Seed Sowing and Germination
The major differences between the symbiotic method of raising orchids from seeds and the asymbiotic techniques described in propagation from seed chapter relate to the presence of the fungus in the culture. For symbiotic sowings the medium must contain sufficient nutrients for the fungus as well as the developing orchid seedlings. One of the best media, which is used most frequently, is the basic oats medium, but it can be enriched with sugar and the salts of FIM medium if required. A small cube of agar from the chosen fungus isolate is added to the surface of the medium
Basic oats medium
g/l
Powdered oats 3.5
Yeast extract 0.1
Agar 6.0
Distilled water to make up to 1 litre of medium
The seeds must be sown thinly, to allow each protocorm space to develop and to ensure that there are sufficient nutrients for both fungus and seedlings. Cultures must be watched carefully so that transfers to new media are made at the optimal time for continued growth.
For asymbiotic sowings, which are often made as a control, the media used for tropical orchids appear to be too rich for the terrestrial species, but some success has been achieved with half and quarter strength of several well‑known media. A commercially available medium called TGZ‑N is particularly useful. For the immature seeds from green capsules, which has been successful with Cypripedium calceolus, a new medium called Kew‑A has been devised. It is dilute but complex and contains peptone and potato extract.
Seeds and Fungi Fungal Isolation Media for Seed Sowing and Germination Infection and Development
Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
Small quantities of mature seeds are sterilized in paper packets as described in growing from seeds chapter. Larger amounts of dry seeds are surface‑ sterilized for 3‑20 minutes in a 2‑10 % solution of sodium hypochlorite (bleach: 10‑14 % available chlorine) to which a wetting agent, such as Tween 80 has been added. The seeds are removed by vacuum filtration, rinsed twice in sterile distilled water by suspension and vacuum filtration and then resuspended in sterile water. Small aliquots of this suspension are poured on to filter papers, under vacuum, and sowings are made by inverting these squares on to the surface of the oats medium in Petri dishes. The medium is inoculated at the edge with a cube of agar from an appropriate fungal isolate and the dishes are sealed with laboratory film. The cultures are maintained at 20‑22ºC in the dark.
Seeds and Fungi Fungal Isolation Media for Seed Sowing and Germination
Seed Collection and Sowing Weaning the Seedlings Establishing the Seedlings in the Wild Future Work
The fungal hyphae grow out from the cube of inoculum across the new medium and very soon reach the seeds. They first enter the seeds through minute gaps in the seed coat called micro pores, and, once inside, appear to be attracted to the suspensor region of the embryo where they penetrate the cell wall. When they reach the cortical region of the embryo they begin to form pelotons within the cells. This usually happens within five to six days of sowing and is the first sign that the orchid/fungus pairing is a compatible one and that germination will proceed. Rather little is known of the seeds of most terrestrial orchids ripen within a few weeks or months of pollination. At maturity, the capsule usually begins to change color, from green to yellow or brown. The capsules are taken off the plant before they split and dried in glass vials, covered with muslin or nylon net, in a desiccator containing anhydrous calcium chloride or silica gel. Drying takes place more uniformly like this, and the seeds are less likely to be contaminated by micro‑organisms in dry conditions. The vials are sealed and moved to the refrigerator if the seeds cannot be sown at once. At 4ºC they may remain viable for two years or more.
For difficult species and asymbiotic sowings, immature seeds are commonly used. In this method, one has to determine the best time to harvest the seeds for each species. Usually about half the usual maturation time is best, and for most European orchids this is six to seven weeks after pollination. The capsules are surface sterilized and opened at the laminar flow bench. The seeds are full‑sized, but white, and are details of the orchid‑fungus symbiosis, but a project in the Jodrell Laboratory has been started to investigate the molecular basis of the relationship using fungi and seeds provided by the project.
The cultures are examined every week and the first clear sign of success is the swelling embryo that splits the testa and begins to develop rhizoids on its outer surface. At this stage it becomes a protocorm. The protocorms are white or cream, sometimes almost translucent, and they continue to expand in size eventually becoming top‑shaped and developing a shoot on the upper surface. The cultures are kept in the dark for germination and the early stages of protocorm development. They are moved into the light when the first leaf forms. This may vary from six to eight weeks for Ophrys and some Orchis species, to three to four months for Dactylorhiza species.
The sowing plate is often rather crowded and the protocorms are best transferred to fresh media as soon as they are large enough to handle safely. They can also be sorted at this stage and those of equivalent size grown on together. They need to be well‑spaced, usually five seedlings per jar.
The asymbiotic sowings develop more slowly and the plants exhibit a variety of growth patterns. The Cypripedium calceolus protocorms look like little pearls after eight to ten weeks of growth and then begin to elongate into a variety of curious shapes. These elongated structures become the first roots of the orchid. When small, yellowish green buds begin to appear amongst them, the cultures are moved to a refrigerator where they are kept for 10‑12 weeks at 4‑5ºC. This period of chilling stimulates leaf development when the cultures are moved into light and warm conditions again.
Recently the use of a chilling period has proved to be beneficial with some of the symbiotically grown species as well. Orchis militaris produces a round protocorm which then appears to require a cold period before there is any further development. Other species of Orchis, however, all have round protocorms that produce their first leaves very readily without a cold period. These are species which, in nature, are wintergreen, usually producing their rosettes of leaves early in the winter after a dormant period in autumn. With several of the Dactylorhiza species, chilling appears to stimulate bud growth and also tuber development underground.
Seeds and Fungi Fungal Isolation Media for Seed Sowing and Germination Seed Collection and Sowing
Infection and Development Establishing the Seedlings in the Wild Future Work
Young orchid seedlings are kept in the controlled conditions of the growth room until they are large enough to have a good chance of surviving the experience of moving to the fluctuating conditions of the glasshouse. The transfer from agar to a compost medium, and the move from an atmosphere of 100% humidity, constant temperature and artificial lighting to the diurnal changes in these environmental factors in the glasshouse can represent disaster to the young plant if it is attempted too early. Nevertheless, it is important to try to move the plants on when they are at the right stage of tuber formation or leaf development.
The easiest stage to transfer terrestrial orchids from the laboratory to the glasshouse is as dormant tubers. This works well with those species that readily produce tubers in culture, including several species of Orchis and Serapias. Excess agar is removed from the tubers and they are potted into a basic weaning compost and watered in.
Basic weaning compost
1 part beech/oak leaf mould (passed through a 13 mm sieve)
1 part perlite (fine grade)
1 part Terragreen
1 part basic terrestrial compost (see below)
Base dressing of hoof and horn meal at 0.5 ml per litre
Basic terrestrial compost
3 parts heat‑sterilized loam
3 parts coarse gritty sand or crushed grit (6 mm particle size)
2 parts beech/oak leaf mould (passed through a 13 mm sieve)
1 part composted pine bark (6 mm size)
Base dressing of hoof and horn meal at 1 ml per litre
The winter‑green species, such as Ophrys apifera, are best weaned during the autumn and winter months. The seedlings with two or three leaves are remarkably resistant to cold conditions. The jars containing plants are moved to a shaded position in the glasshouse for one to two weeks and the lids partially removed for a few days more before the plants are potted into sterile weaning compost in seed trays or community pots. It is best to wash away all traces of agar from the roots before potting, especially with the asymbiotic cultures.
The Dactylorhiza and Cypripedium cultures that benefit from a period of chilling are best weaned as soon as they come out of the refrigerator. The protocorms, or young shoots with their cluster of roots in Cypripedium, are potted into sterile weaning compost and covered to a depth of 5‑10 mm. The pots or trays are shaded and the compost is kept evenly moist. Symbiotic protocorms of Dactylorhiza fuchsii have been planted direct into the open ground at Kew, in spring, after a period of chilling, and their subsequent development and growth was normal and rapid.