The Micropropagation Unit   Seed Collection and Storage   Nutrient Solutions   Sterilising   Sowing   Weaning   Some Successes

Propagation from Seed

At Kew the Living Orchid Collection is augmented every year by seedlings raised in the Micropropagation Laboratory. The seeds from which the plants are grown come from a variety of sources, including from plants grown at Kew which are pollinated by hand as required. Many species which are rare or approaching extinction in the wild are increased in this way, as are particularly decorative plants for use in displays. Seeds are also obtained from plants used in breeding experiments, both species and new crosses. They are also used simply to increase the number of plants of species which are hard to grow or represented by only one specimen in the collection.

Kew also receives donations of seeds from many sources. Botanists on expeditions are encouraged to collect seeds rather than plants, thus causing less damage to wild populations. Private growers who have rare plants in their collections are often prepared to provide seeds from plants which cannot be divided, while commercial orchid growers sometimes share a crop of seeds. Other botanic gardens in many parts of the world, and organisations like the Australian Orchid Foundation, also exchange seed with Kew. From all these sources, new species are added to the orchid collection every year as young seedlings, the stage at which they are most adaptable to glasshouse culture.  Sending seeds rather than plants around the world has many advantages. They are extremely light and several thousands can be packed in a small envelope. CITES controls apply to seeds of orchids listed in Appendix 1 (see chapter 16), while European Community Regulations affect the movement of seeds of most European orchid species; seeds of all other orchids are exempt from CITES control. When they are received at Kew, seeds can be stored in a domestic refrigerator for short periods but should be sown as soon as possible.


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The Micropropagation Unit

It is now some 30 years since Peter Thompson started working in the Jodrell Laboratory on a new formulation for a medium for the germination of orchid seeds. In the course of this work he succeeded in germinating seeds and raising seedlings of a wide range of genera. His book Orchids from Seed is still widely used as an introductory guide to micropropagation. In 1974 a Micropropagation Unit was set up at Kew to make in vitro propagation techniques available to the gardens for a wide range of plants including orchids.

The Micropropagation Unit occupies the whole of the ground floor of Aiton House, situated in the Lower Nursery area of the Gardens. The main laboratory has offices, preparation, autoclave and store rooms adjacent to it. There are separate rooms where the laminar flow cabinets are kept at which all sterile techniques are carried out. Next to these are the air-conditioned growth rooms with racks of illuminated shelves where the young plants are grown in vitro. Growth cabinets where light and temperatures can be carefully regulated for small batches of flasks are also available. The unit has its own range of glasshouses where all the transfers of young plants to compost are made and the seedlings can be weaned under very humid conditions.


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Seed Collection and Storage
Seeds of most orchids take a long time to mature and it is not unusual for many months or even a year to pass, after pollination of the flower, before the capsule is ready. As the green capsules ripen they often change colour, becoming yellow or brownish. Narrow slits appear along the sides, or along the ventral surface during the ripening process, and the seeds are usually released gradually over a period which can range from several days to weeks or months. It is therefore important to watch capsules ripening in the glasshouse and harvest them before they split. Each capsule is cut off and stored separately in a small labelled jar until all the seeds can be tapped out in the laboratory. In all cases capsules which have not yet dehisced are preferred and the technique for using them is described later in this chapter.
 Orchid fruites are allowed to ripen and dry in closed containers before the seeds are collected and stored
An important feature of the arrival of new seeds for sowing is the opportunity to record information about them. As much as possible is recorded, from the donor's name to provenance data, while recording the date received and state of the seeds on receipt may prove important later. Accurate identification is of paramount importance. Where possible, seeds from the wild are accompanied by herbarium voucher specimens. Seeds obtained from the glasshouses are backed up by the information already on record for the parent plants. Seeds may be loose and dry or may arrive in the fruit or capsule, sometimes split and sometimes still intact. The latter is always preferable.

Loose seeds from the wild or glasshouse have often been collected under warm, moist conditions and thus may be contaminated by bacteria, fungi or algae. It is therefore important to dry the seeds or capsules as soon as possible after harvest. They are placed in glass vials closed with tissue paper in a desiccator at room temperature and then sown as soon as possible. This method seems to be quite adequate for periods up to three months. For longer term storage the seeds are separated from any debris of the capsule and, after drying, are kept in cold store at 4ºC The reason for the drying period is not only to impede the germination and growth of any fungal spores which may be present on the seed surface, but also to enhance seed longevity.


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Nutrient Solutions

Orchid seedlings have been grown on many different substrates but best results are obtained where the correct balance of nutrients is present. This varies from species to species, but all the media used contain a carbohydrate source, a range of mineral salts and agar, which solidifies the medium. In addition, many media contain other additives including vitamins, amino acids or plant extracts such as banana pulp or potato extract. Some of the standard media are now available as powdered mixes, which simplifies their preparation. After mixing, the media must be sterilised, normally in an autoclave, to prevent the growth of contaminants.

The minute orchid seeds lack food reserves for their early growth and this is provided by the carbohydrate. Ordinary sugar, or laboratory refined sucrose, is normally used. The young seedlings will only grow satisfactorily if they are provided with a complete and balanced mineral solution as well. The different elements are necessary in various amounts with much higher concentrations of nitrogen, potassium, phosphorus, calcium, magnesium and sulphur, rather less of iron and minute quantities of manganese, copper, zinc, boron and molybdenum. Where prepared mixes are not available, media are made up from the pure chemicals. Where only very small concentrations are used, stock solutions prove to be the most convenient method.
For full detail for preparation medium read here...
Or read Peter Thompson's book, Orchids from Seed for full details for the preparation of the medium will behe developed and other nutrient solutions, some of which are still used routinely at Kew.


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Sterilising

Orchid seeds are very small. Unlike the nutrient solutions, they cannot be sterilised by heating, because this would kill them, so a chemical method is usually used instead. Sodium hypochlorite, the active ingredient of most domestic bleach, makes a good sterilant. Alternatively, water can be added to fresh calcium hypochlorite which is then filtered after five minutes. A strong solution containing chlorine is thus obtained, which must be used with care. Orchid seeds are tiny, like specks of dust, each seed has a minute, ovoid embryo inside the net-like coat. Magnification x 250)
Small quantities of seeds are sterilised in filter paper packets so that none is lost. A small filter paper is folded twice and stapled shut with the seeds inside. The packets are immersed in the chlorine solution together with a wetting agent for 10 minutes, and then rinsed in sterile water. The seeds can then be transferred to the medium on the opened paper or pressed on the surface of the medium from the paper.

Seeds from young or intact capsules need not be sterilised. In this case only the outside of the capsule is treated. It is then opened in the sterile conditions of the air-flow bench and the seeds, which are naturally sterile, removed and sown. Better results are obtained in this way for many species, and especially in the genera Cypripedium and Paphiopedilum. A growing volume of evidence suggests that these species germinate better when the seeds are immature, harvested about 50 days after pollination for Cypripedium and 120 days for Paphiopedilum.


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Sowing

The sowing of seeds has become much more reliable since the advent of the laminar flow bench. This is a sterile environment where the air is filtered free of contaminants and the worker can open sterile containers and manipulate sterile seeds without risk of reinfection.
Various culture vessels have been tried at Kew, including test tubes, Petri dishes, conical flasks, Kilner jars and honey jars. For sowing, pre-sterilised Petri dishes are often used because their flat shape means they can be examined easily under the dissecting microscope. Their disadvantage is that the medium they contain dries out more quickly than in a larger container. When germination has begun, the protocorms are replated to a larger container for further growth. Small honey jars with plastic caps are frequently used at this stage.
 Seedlings of Orchis laxiflora raised in the laboratory and almost ready for transfer to the greenhouse
At Kew, the culture conditions which seem to be suitable for a wide range of species are a 16-hour photoperiod and a temperature range of 22º- 25º C.  Light appears to be useful for the germination of epiphytic species. Terrestrial species are often germinated in the dark, by keeping the cultures in dark polythene bags, and only moved into light conditions once a green shoot has appeared.

The dishes and jars are examined regularly and any which become infected with bacteria or fungi are removed. Sometimes very precious seeds of a contaminated culture can be saved by opening the vessel and moving the seeds to a fresh medium, but this can only be done successfully during the very early stages of infection. The contents of vessels must also be watched for drying out. If the agar shrinks away from the sides, the seedlings must be transferred to fresh medium in a new vessel.

After a few months the developing seedlings become crowded inside the vessels and grow less well as a result. It is important to move them on before growth begins to slow down, either to a fresh medium in another vessel, or into compost in the glasshouse. In either case, the gentle separation of the entangled seedlings is a task requiring much time and patience to ensure they are not damaged.


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Weaning

Large, well-grown seedlings are removed from their flasks in the laboratory and established in the glasshouse environment. This process is known as weaning, and every precaution is taken to ensure that the seedlings suffer as little stress as possible.

The glasshouse used for weaning seedlings is maintained at a minimum temperature of 18ºC (64ºF) at night and 23ºC (73ºF) during the day. Heavy shading is used to prevent the temperature rising above 28ºC (82ºF) even during the summer. An overhead humidifier fills the air with water vapour and, because it is large in relation to the size of the glasshouse, provides strong air movement.

Deflasking is carried out from February until May. Seedlings then come into an environment when the days are lengthening and they have a full growing season ahead of them in which to become established. The largest seedlings are selected from each group in the laboratory growth room. It is essential that the seedlings are large enough to grow new roots from their existing reserves in case of damage during deflasking. The smaller seedlings can be kept in the laboratory to provide a useful back-up should the first batch fail, although this is a very rare occurrence.

The flasks of seedlings selected for establishment stand out on the staging in the glasshouse and begin to acclimatise while they await attention. Each seedling is lifted carefully from its agar substrate and put straight into lukewarm water containing a dilute fungicide solution. Any remaining agar is gently rinsed away and the fungicide prevents any fungal growth in the traces which may adhere too closely to be removed completely. The seedlings are dried and potted singly in 5-6 cm square plastic pots.

Two different seedling composts are used. For epiphytes the mixture is: 5 parts fine orchid bark, 1 part perlite and 1 part fine charcoal. For the tropical terrestrial species the compost consists of: 3 parts grit, 1 part loam and 1 part fine bark.

Terrestrials and epiphytes are arranged separately on the staging as they need different kinds of management. After watering﷓in, the terrestrials require rather little additional water until they are well established and growing rapidly. The epiphytes are also watered-in and then misted-over thoroughly several times each day for the first few weeks after deflasking. This is necessary to prevent desiccation for the leaf cuticle of the seedlings is poorly developed while they remain in the flasks. The pots are watered frequently, too, just before the compost dries out. As the seedlings root more strongly, the misting becomes lighter and less frequent, until, by the late autumn, misting once a day in the early morning is sufficient. Seedlings are fed each week with a dilute balanced fertiliser.

Most of the seedlings remain in the seedling house for about a year. However, some epiphytes from high altitudes require a cool, dry winter, and these are moved in the autumn so that they can experience conditions similar to those of their natural habitat at an early stage. All the rest of the orchid seedlings are moved to the main orchid collection at the end of the winter. Then the seedling house soon fills up with the new season's young plants.


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Some Successes
The species Cattleya dowiana is rare in cultivation because it requires more humid conditions than many other cattleyas, and because plants imported from the wild have been difficult to establish. It has become rather rare in the wild because its habitat, along cliff edges in humid forests at medium altitudes, always where there is high humidity and plenty of air movement, has become greatly reduced by development and because plants in accessible places have been collected for sale. On a short visit to Costa Rica in 1986, Joyce Stewart was given a small packet of seeds by Clarence Horich, a resident of Costa Rica who has sent many specimens to Kew.
 Cattleya dowiana
Cattleya dowiana, a species which is greatly reduced in Costa Rica because of loss of habitata  and over-collection; wild collected plants have always been difficult to grow in cultivation
The seeds were sterilised and sown on standard Vacin and Went medium. They germinated without difficulty and many bright green protocorms developed very quickly. Seedlings were transferred to fresh medium to which homogenised banana was added. They grew rapidly and 12 months after germination husky young plants were potted into a compost of fine bark, charcoal and vermiculite. Some are now growing strongly at Kew and several hundred others have been distributed to other botanic gardens and experienced individuals.

One capsule of the rare species Dendrobium spectatissimum was collected in Sabah, Borneo, by Phillip Cribb and Christopher Beles in 1983. The seeds germinated successfully on Vacin and Went medium and the seedlings grew very quickly. They were transferred to a modified medium as soon as they were big enough to handle and after a further 16 weeks were sufficiently large to be potted in a fine bark, perlite and charcoal mix.

As soon as they were large enough to travel, the first seedlings were returned to the National Park Authorities in Sabah. This work has been repeated with some of the additional seeds and more seedlings have been produced with equal ease. They have been distributed to botanic gardens and other collections as well as incorporated into the collection at Kew.

The beautiful species Peristeria elata sometimes known as the dove orchid, or holy ghost orchid, is the national flower of Panama. Seeds donated to Kew by David Jones in 1983 germinated on a range of media.

After nearly two years in culture, the seedlings were large enough to be potted up and many were subsequently distributed to botanic gardens and scientific institutions around the world. Growers in Britain with facilities for growing this tall terrestrial species were also offered plants.

Epidendrum ilense from Ecuador was known from only six plants in the wild when it was propagated at the Marie Selby Botanic Gardens, Sarasota, Florida, and a small plant given to Kew. The area the species came from was completely deforested and for a few years it was thought to be extinct in the wild. Recently, another small population has been discovered, but even in that area the future of this unusual species is also precarious.

Seeds were collected from the Kew plant in 1986 and 1987. They germinated on several different media. The small seedlings were transferred to half strength Murashige and Skoog medium, supplemented with banana pulp or activated charcoal. They grew well on both these media and plants have since been widely distributed.
 Epidendrum ilense

Epidendrum ilense from Ecuador was known from only six plants in the wild when it was propagated at the Marie Selby Botanic Gardens, Sarasota, Florida, and a small plant given to Kew. The area the species came from was completely deforested and for a few years it was thought to be extinct in the wild. Recently, another small population has been discovered, but even in that area the future of this unusual species is also precarious.

Seeds were collected from the Kew plant in 1986 and 1987. They germinated on several different media. The small seedlings were transferred to half strength Murashige and Skoog medium, supplemented with banana pulp or activated charcoal. They grew well on both these media and plants have since been widely distributed.

Seeds of Clowesia rosea, an endangered and very beautiful orchid, were sent to Kew by Ed Greenwood who lives in Oaxaca and who has been supplying Kew with orchid seeds from Mexico for the last 15 years. This Clowesia grew best on Vacin and Went and on half strength Murashige and Skoog medium when these were supplemented with activated charcoal.

Strong plants are now thriving in the glasshouses at Kew and have been distributed to other gardens and growers. Other successes with Mexican orchids include several attractive species of Encyclia.

The 'lost' species Cymbidium rectum was rediscovered by the botanist Tony Lamb in Sabah. It differs from Cymbidium bicolor in having an upright raceme, an unusual characteristic in this section of the genus. The habitat where a few plants were rediscovered has since been clear-felled and it is likely that the species is extinct in the area now. Seeds were sown in 1983. The resulting seedlings grew most vigorously on Vacin and Went or half strength Murashige and Skoog medium with activated charcoal. Like the other species discussed above, this Cymbidium has now been distributed to many institutions and growers.

Gynoglottis cymbidioides is the only species in this curious genus. It occurs in Sumatra at about 1550 m altitude and it was unknown in cultivation until seed capsules were collected by Jim Comber in 1988.

The species resembles Coelogyne in habit, but has rather small white flowers of distinct structure in a long, many-flowered raceme. The seed capsules were undehisced on arrival at Kew and seeds germinated within two weeks of being sown. Surplus capsules were sent to Richard Warren in Edinburgh who raises a wide range of tropical orchids from seeds for sale.

Seedlings are now growing well in vitro and have been distributed in flasks to other institutions with facilities for growing﷓on plants under in vitro conditions.

These success stories are only a sample of the routine work of orchid propagation carried out by Dickon Bowling in the Micropropagation Unit under the supervision of Dr Michael Fay. Though primarily a service to the living collections at Kew, the work is increasingly involved with conservation, either by the return of propagated plants to their place of origin, or by the distribution of surplus seedlings to growers in other botanic gardens and elsewhere. It is likely that this work will become increasingly important as the wild orchids throughout the world continue to disappear and as national and international legislation controls the movement of wild﷓collected plants.

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