The Genus
Cymbidium >><< Propagation from
Seeds
The Genus
Cymbidium >>
Anatomical Research Cytology Pollination and Breeding Systems Seed and Pollen Storage
The Jodrell Laboratory
Many kinds of orchid research are undertaken in the Herbarium, the Jodrell Laboratory and in the Living Collections Department at Kew. The techniques that are traditionally carried out in each area have often been combined to elucidate particular problems, for example, to provide more evidence in taxonomic studies. A monographic study that was carried out recently on the genus Cymbidium (see next chapter) involved the study of both living and preserved specimens in many different ways.
Anatomical and cytological studies have always depended on the accurate identification of the plants in the Living Collection. The preparation of voucher specimens is a routine procedure. They provide a permanent record in the Herbarium of the material, often from the Living Collection, studied in the Jodrell Laboratory. Specimens are also prepared, or collected for the Spirit Collection, from the living plants which are used as a germplasm resource for propagation and conservation. The accurate identification of this material is of critical importance.
There are also close associations between scientists in other sections at Kew. The techniques of micro propagation are used in studies of pollen and seed longevity and breeding systems, while the regular routines of plant physiology are used to prepare media and determine seed viability in the course of work on germination.
Many other examples could be cited, but these are enough to show that work on and with the orchids at Kew does not fall neatly into separate departments. There is much interdependence between scientists and horticulturists and between scientists of different disciplines.
Material is also in demand from scientists in universities and other institutes, both in the United Kingdom and overseas. Orchid leaves and roots can be supplied fairly easily and quickly, but it may be a year or more before flowers or their pollinia become available, and seeds take even longer.
Cut sprays of flowers are also required as decoration for special functions at Kew and for occasions such as Remembrance Day when Kew orchids, usually Cattleya bowringiana, adorn the wreath laid at the Cenotaph by the Foreign Secretary in remembrance of armed forces from the Dependent Territories. The Orchid Supervisor maintains a list of requests for orchid material and tries to fulfill all those which merit attention. In 1985 the Sainsbury Orchid Fellow was appointed to coordinate the use of the collection for research, conservation, education and display.
The Sainsbury Orchid Fellow also supervises the Sainsbury Orchid Conservation Project which was set up in 1983. This project was the brainchild of Phillip Cribb, who has been advisor to several conservation organizations in Britain on various aspects of orchid conservation. Finding ways to grow some of the rare British orchids from seeds, especially Cypripedium calceolus, which was reduced to a single wild plant, seemed to him an urgent necessity. In 1981 he was in Durban for the 10th World Orchid Conference where he heard a paper presented by Mark Clements, an Australian orchid biologist from Canberra, who had succeeded in growing some of the rare Australian terrestrial orchids from seeds and in introducing the seed‑raised plants to the wild. Returning to Kew, Cribb approached the Director and Curator about the possibilities of a similar project here, in conjunction with the Nature Conservancy Council. Orchid enthusiasts Lisa Sainsbury and her husband Sir Robert Sainsbury provided the finance and Mark Clements was seconded to Kew for 18 months to initiate the project. With continued funding for staff and a glasshouse to hold the research collection and seedlings produced, the project has become increasingly successful. In 1989, Sir Robert and Lady Sainsbury provided an endowment to ensure the future of both the project and the Sainsbury Orchid Fellowship at Kew.
To the orchid enthusiast who is enthralled by the complexity and beauty of orchid flowers, and to conservationists trying to protect the ever‑decreasing numbers of rare plants, the detailed study of the structure and function of individual orchid cells, minute parts of the plant, may seem irrelevant. But to the serious student of the orchids, these scientific disciplines, and others, are very important, and all can add information to the complex of knowledge that is being gradually accumulated at Kew.
Several sections of the Jodrell Laboratory have, over the
years, incorporated orchid studies into their routine work. Cytology, especially
work on chromosomes, anatomy of leaves and roots, and studies on the physiology
of seed germination and storage have been carried out for many years. More
recently, studies of the reproductive biology of certain orchids have begun,
combined with investigations of the morphology and anatomy of the stigma, style
and ovary of the flowers. The surface topography of pollen grains and pollinia
and the storage of pollen for future use has also been investigated in recent
years.
Some of the information that has emerged is of great interest to the herbarium botanist, and when allied to morphological and biological studies may lead to the development of a more useful system of classification. Other details will be of particular value to the breeder or hybridist of cultivated plants, and to the conservationist trying to save a rare plant on the brink of extinction. To the specialized scientists who carry out these investigations, the information they yield is of interest not only for its own sake but also for the interesting comparisons that can be made between orchids and other flowering plants.
Cytology Pollination and Breeding Systems Seed and Pollen Storage top
The orchid family presents a daunting prospect to the plant anatomist. Not only are there so many species, worldwide, but many of them are rare, so good material for anatomical work is hard to obtain. Because anatomists prefer to work with live material, they are generally not welcomed in glasshouses with valuable collections. However, at Kew, the collections are principally for scientific study, and providing the requests are not excessive, leaves, stems and roots are usually made available. No permanent damage is inflicted on the plants by their removal.
In the orchids, leaf anatomy is often typical of the genus rather than the species and there are rather large groups of genera that share quite similar leaf structure. At first sight this would appear disappointing, since it would seem to reduce the chances of using leaf anatomy as an indicator of relationships, and hence of evolution. However, the immediate appearance is rather misleading, and when detailed studies are made, involving both leaf surface and sections, a useful range of character syndromes, or sets, emerges. Silica bodies, often found in orchid leaves, are small opals which generally have a characteristic shape. Their function is uncertain, but they are very hard, and may damage the mouthparts of animals that try to eat the leaves.
The flower stems of orchids do not vary as much in their structure as those of some other monocotyledons. However, on the large scale, trends in evolution can be seen, and close relationships can be detected.
Unlike the stems, it is the roots that have provided a large range in variation, which is unusual among the monocotyledons. This is due partly to the fact that so many orchids are epiphytes, and thus have interesting adaptations in their aerial roots for water capture and retention. The fine details of the velamen (a multiple layered epidermis) and the nature of the exodermis (a layer or layers of thick‑walled cells bordering the cortical parenchyma), provide useful characters for the taxonomist.
Plants that have to withstand drought on a regular basis
often show xenomorphic characters in addition to a thick, sculptured cuticle. If
the leaves are retained, they are usually adapted so that they do not readily
lose scarce water. In the orchids, as in other plant families, it is the
xerophytes that show the widest range of anatomical variation, since there are
many different tissues involved to a greater or lesser extent in the job
of conserving water. For example, the cell walls of the epidermis may become
thickened in a characteristic way. The stomata (pores through which water vapor
moves from the leaf into the air during transpiration) must be able to close
tightly at times of water shortage and are often protected by over‑arching
flanges, or may be sunken into the leaf surface. The leaves may be very fleshy,
and contain water‑storing cells, or they may be tough and reinforced with strong
fibers and sclereids (cells that are shorter than fibers but have thick,
strengthened walls). The chlorenchyma cells, which contain the chloroplasts, may
become modified into upright, closely packed, narrow cells. The vascular bundles
(veins), which have special cells for transporting water (xylem) and others for
transporting soluble food material (phloem), can also be modified in xerophytes,
and may be sheathed by one or more layers of cells with thick or thin walls.
With the various permutations and combinations of these different tissue types, it is easy to see how individual species or genera may have a particular set of characteristic features.
Leaf surfaces also have many interesting characters. These may be of taxonomic importance, but they also provide information about the normal habitat in which the plants occur. The shapes of the epidermal cells and the sorts of stomata (pores) are typical for a species. Often the cuticle, the outer waterproof layer of the leaf, has very fine surface sculpturing. This can be seen with the light microscope, but is easier to understand using the scanning electron microscope. This outer layer plays an important part in the survival of the orchid. Different habitat preferences are often reflected in the different sorts of surface sculpturing on the cuticle. Plants that grow in dry, or periodically dry conditions, such as epiphytic orchids, often have very thick cuticles. In plants that grow in moist, humid conditions the cuticle is often thin and smooth.
As preparation for anatomical study, comparable parts of leaf, stem, pseudobulb and the various root types are removed from the plants and fixed, or pickled, in a noxious mixture of formalin, acetic acid and alcohol ‑ FAA. It is best to use fresh material, so that the resulting specimens are as lifelike and unwrinkled or unshrunken as possible. If it is absolutely impossible to obtain live material, particularly of very rare plants, then a technique known as reviving herbarium specimens may be necessary. The dried plant parts have to be gently boiled in water, to try and make them regain the shape they had in life, but not boiled for too long so they cook. As soon as they appear reasonably expanded, they are cooled, drained and transferred to FAA. Often the material has been damaged by drying, and only rather poor sections can be made from it.
After a minimum of 48 hours in the fixative, the material can be removed and thoroughly washed in running water, prior to sectioning. Because the sections have to be drawn or photographed and fine details studied the sectioning is carried out mechanically. Two methods are used. The easiest, which produces good although fairly thick (20‑25 micro meters) sections from firm material, involves the use of a sliding, or sledge microtome. The leaf, stem or root is carefully supported in a clamp in the microtome, and slices are cut with a very sharp blade fixed to a head which runs smoothly along tracks. After each section is cut, returning the knife to its start position raises the specimen by a pre‑set amount, ready for the next cut.
The second sectioning method involves infiltrating the delicate tissues with wax, or a wax‑like substitute in a solvent, and gradually adding more and more wax, eventually allowing it to set. The wax supports the tissues, and slices of the embedded material can be cut on a rotary microtome. This produces thinner sections (about 10‑15 micrometers thick). It can also produce a series of sections in sequence. One limitation is that the plant material to be sectioned should be reasonably soft, or at least of uniform texture. Unfortunately, many orchid leaves have rather hard fibers set in the middle of very soft tissues, so they tend to tear rather than cut evenly.
When sections have been cut they can be looked at
immediately, particularly for the examination of cell contents. Usually,
however, it is the types of cells and their arrangements within the tissues that
are of most interest. So the cell contents are flushed out with domestic bleach
solution. Then the sections are washed thoroughly to remove all traces of the
bleach. Finally the sections are put in special dyes or stains that color the
different sorts of cell walls in distinctive ways. Nowadays the dye Alcian blue,
mixed with the red stain, safranin, is mainly used. The blue goes into the thin
cellulose walls of the chlorenchyma, parenchyma and phloem, while the safranin
stains the thicker, lignified cell walls of fibers, stone cells and xylem.
After staining, the sections are carefully dehydrated and then mounted on microscope slides under cover slips, in a medium which sets hard when fully dry. The sections retain their colors for many years if the slides are stored in the dark.
Rather like fingerprints, sections can be used to help in identification. They contain a lot of microscopical information, which, taken as a whole, provides a unique story for each of the species from which they are prepared. It is true that the structure of plants that have grown in different conditions shows some minor variability, but this is slight. Anatomists have learned which features may be unreliable indicators of identity or relationship, and these are avoided. Consequently, a section can provide a good set of characters to the tutored eye.
When these are added to those details of flowers, fruits, seeds and leaves which are commonly significant in taxonomy, or evolution, a complete picture of each plant begins to emerge.
The main involvement of the Kew plant anatomy staff in the orchids is currently in co‑coordinating the preparation work and editing of the major volume on Orchidaceae in the series Anatomy of the Monocotyledons. This will be a major contribution to a series of important volumes recording anatomical features of monocotyledonous plants which was initiated by a former Keeper of the Jodrell Laboratory, Dr C R Metcalfe. His monumental work on the grasses, Gramineae appeared in 1960, and other volumes have followed so that there are now seven in the series. Two of the present Kew anatomists, David Cutler and Mary Gregory, have succeeded Dr Metcalfe on the editorial board. Preparatory studies for the Orchidaceae volume were started by Edward Ayensu and are now being continued by William Stern and his staff at the University of Florida, with help from Alec Pridgeon. Work will probably take at least another three years, as many species still remain to be studied and described anatomically in order to provide even an overview of this vast family. Clearly it would be the task of a lifetime to study representatives of all the genera thoroughly. A careful, considered selection has to be made, based on information from other sources, and then the manuscript must be edited so that it conforms with the style of the rest of the series.
Staff in the Anatomy Laboratory also maintain, on computer, the world's most comprehensive set of literature references to the anatomy of the Orchidaceae. This is invaluable in the editorial work and is added to on a weekly basis. When the Orchidaceae volume is complete, a set of all the slides described will be housed in the Jodrell Laboratory for future reference.
Anatomical Research Pollination and Breeding Systems Seed and Pollen Storage top
Like other plants and animals, orchids are made up of millions of cells. In a mature plant there is a variety of dead cells, which often provide supporting tissue, and many living cells, some of which are actively dividing. Although these are usually transparent or translucent, and difficult to see except with a microscope, their study has become a fascinating and complex subject known as cytology. It is made easier by the technique of killing and preserving the cells at certain stages of development and then staining them with particular dyes before examining them microscopically.
To those who study orchid cells, the structures of greatest interest are the chromosomes, so called because they are bodies which take up certain dyes very readily. They are important to all living organisms because they carry genes, or hereditary information. They thus control the development of the plant and also its breeding potential.
In a normal, non‑dividing cell, the chromosomes are in an expanded state and mass together inside a special membrane to form the nucleus. Just before each cell divides, in a process called mitosis, the chromosomes contract and become more readily visible under the microscope. Thus tissues that are actively growing can be prepared for examination so that the chromosomes can be studied and counted.
Several kinds of tissues can be investigated with relative ease. Epiphytic orchids produce aerial roots which are clean and easily accessible. In many terrestrial orchids, too, the root tips are easily extracted from the loose compost in which the plants are grown because they will often be found in the part of the pot where there is most air, near the edge or bottom of the compost.
Apart from the root tips, there are several parts of the flower worth investigating. These have the advantage that their removal does no harm to the plant. The development of the individual pollen grains in an orchid pollinium takes place synchronously. Provided a flower bud is harvested at the right moment, many thousands of dividing cells can be obtained with their chromosomes suitably presented for counting. Similar success can be obtained by studying ovary material. Here, however, the developing ovules need to be examined some days or even weeks after pollination. Obviously it is much easier to get results from a long inflorescence such as an Oncidium or Eulophia, where there are many flowers opening successively over a long period, than from orchids which normally have only single flowers, such as many of the Paphiopedilums. But with observations and records of more than one flowering season, and perhaps a little trial and error, even these will yield their secrets.
Chromosome counts are of particular interest because closely related species and genera often have the same number and hence a similar amount of genetic material in their tissues. They may interbreed easily. Hybrids made from parents with the same chromosome number are likely to be fertile, whereas those with widely different parental numbers are usually not.
The number of chromosome sets a plant contains is sometimes referred to as its ploidy level. Most wild orchids are diploid and contain two sets (2n) having inherited the haploid number (n) from each of its parents, just as humans do. Diploid plants usually grow easily and flower well under good cultural conditions. Sometimes, however, plants can contain four sets of chromosomes. These tetraploid (4n) orchids usually grow rather slowly and produce fewer flowers than diploids, although these may be unusually large and of superior form. Tetraploids are fertile, however, and those which have been bred with other tetraploids, sometimes over several generations, have produced some superior and famous lines of hybrids. Tetraploids can also be crossed with diploids to form triploid (3n) progeny with three sets of chromosomes. Triploids are often very fine, easy to grow and free‑flowering with larger flowers than the diploids, but they are usually or almost infertile. Thus a knowledge of chromosome numbers and how to discover them is often of interest to the orchid grower and breeder.
The chromosomes of most orchids are small and take up stains easily, but they are often not easy to count because their numbers in any one cell can be rather high. A chromosome number of 2n = 38 is a common one throughout the orchid family, whereas 2n = 14 is frequent in lilies and grasses. There is also wide variation between species, even in a single genus. The lowest number so far recorded is 2n = 10 in Psygmorchis pusilla (formerly known as Oncidium pusillum), and numbers over 200 have been recorded in several species. Many of the species of commonly cultivated genera, such as Cattleya, Epidendrum, Laelia and Cymbidium have 2n = 40, but the species of Paphiopedilum vary between 2n = 25 (P. fairrieanum) and 2n = 42 (P. venustum).
The techniques for assessing chromosomes used at Kew were developed by Keith Jones, formerly Keeper of the Jodrell Laboratory and Deputy Director, and his coworkers. Root tips are commonly used.
Orchid roots are usually quite bulky and covered on their outer surface with a layer of dead, air‑filled cells, the velamen. The tips are usually pale green or yellowish because they are free of mature velamen. The ultimate 1 cm or so of the tip is removed from the plant and placed in a reagent which will disturb the normal phasing of cell division and produce a degree of chromosome contraction. Several different chemical reagents can be used, but the two which have been found most satisfactory are 8‑hydroxyquinoline, used at a concentration of 0.002M (0.29g/litre), or a saturated solution of monobromonaphthalene. The roots are immersed for about four hours at room temperature (16ºC) or overnight at about 4ºC (the usual temperature of a domestic refrigerator). The consequences are that the chromosomes become well spread out throughout the cell and appear compact. The main problem here is to ensure that the reagent is able to penetrate rapidly into the interior of the root tip. To facilitate this, it is the practice at Kew to slit the covering of the root tip along several longitudinal planes, rather like peeling back the skin of a banana. The exposed interior part of the root is then sliced longitudinally.
Following these treatments, the roots are fixed in absolute alcohol and glacial acetic acid, 3 parts to 1, for at least 30 minutes, hydrolyzed in normal hydrochloric acid at 60ºC for about 8 minutes, and then placed in Feulgen reagent and left in a dark cupboard. The tips of the roots (which contain the dividing cells) become deep purple, usually within an hour, but are better left for two hours or more. Small pieces of the root are removed to a microscope slide and any velamen that remains is carefully dissected away. The material to be squashed is then covered with a cover slip and firmly squashed with the ball of the thumb. At this stage the preparation can be examined microscopically, or it can be made into a permanent mount after freezing with pressurized CO2 removing the coverslip, dehydrating in absolute alcohol for 15 seconds and then covering the squashed material in Euparal before replacing the coverslip.
The procedure for making preparations from actively dividing pollen grains is even more simple than examining chromosomes in root tips. Fresh (or fixed) pieces of pollinia are squashed under a coverslip in a 1 % solution of orcein or carmine in 45 % acetic acid and are gently heated over a spirit flame to aid differentiation. The slide can be made permanent, as outlined above, for later study, or counts can be made immediately.
A wide range of chromosome counts has now been reported in the literature but in taking account of these, two points have to be remembered. First, the attribution of a chromosome number to a species depends on the correct identification of the plant from which the root tip or pollinium was obtained. It is therefore important to preserve voucher specimens of the plant material investigated, which can be checked by a taxonomist if necessary. Secondly, since orchids have such high numbers of chromosomes, and contaminating bodies such as fungal spores or bacteria which stain similarly can be present, it is very important to develop a high standard of preparation and to count only those preparations which are worthy of analysis. Many observations are usually required, from more than one preparation, before one can be reasonably certain that the true number has been determined for any species.
The fascination of work with chromosomes in orchids continues and much more needs to be done. Many species are as yet uninvestigated, and, for those where only single counts have been made, more plants need to be examined. At the same time, a new facet of cytology has begun in the Jodrell Laboratory and orchids are among the first subjects for analysis. This is the technique of DNA 'fingerprinting'.
Sophisticated biochemical techniques, involving the extraction of DNA from a small piece of an orchid leaf and its treatment with various enzymes, will permit the recognition of individual plants. This is intrinsically interesting, and it will be especially important in artificial propagation for conservation.
Where a species has become reduced to a very small number of individuals, it is beneficial, when hand pollinating them for seed production, to breed with individuals that are genetically different. This not only enhances the gene pool available in the resulting seeds, but usually ensures that a greater quantity of viable seeds is produced.
In Britain, for example, only one plant of the lady's slipper orchid, Cypripedium calceolus, can still be found in the wild. Several plants that are said to be of wild origin are still preserved in gardens and flower every year. Some of these are thought to be divisions from one wild source, but no one can be sure. The new technique of DNA 'fingerprinting' is about to be tried out on these plants and may show whether they are clonally related or not. It will then be possible to co‑ordinate an improved breeding programme and obtain more seeds that will germinate in the laboratory (see chapter 15).
Anatomical Research Cytology Seed and Pollen Storage top
Pollination and Breeding Systems
In functional terms, orchid flowers could be described as a mechanism to attract a pollinator, usually an insect, who will physically transport many pollen grains from the anther of one flower to the stigma of another in a single journey.
The pollen consists of tetrads of pollen grains held
together by fine longitudinal threads to form a more or less globular mass, the
pollinium. The lower parts of the sticky threads are fused to form a caudicle by
which the pollinium is attached to a sticky base, the viscidium, sometimes with
an intervening stalk or stipe The whole of this unit is called the pollinarium,
and it is usually removed en masse by a suitable insect visitor. The
caudicle or stripe sometimes both, soon dry out after leaving the flower, and, as
described by Charles Darwin more than a hundred years ago, the pollinarium shows
characteristic movements on the insect's body during flight. These movements
change the orientation of the pollinarium on the insect's body, ensuring that it
will be in the right position to come in contact with the stigma of another
flower. The fact that these movements usually take several minutes for
completion may ensure that the next flower visited is on a different plant.
The other peculiar feature of orchid flowers, from the reproductive point of view, is that at the time the ,flowers open (anthesis) the female reproductive organs, the ovules, are entirely undeveloped. In most orchids the differentiation of ovules occurs after anthesis or even after pollination. It might be regarded as an economical adaptation that energy is not used for the growth of millions of ovules if they are not going to be needed, but it also means there are other hazards, and long delays, in the pathway between pollination and the release of viable seeds to start the next generation.
The study of pollination in both temperate and tropical orchids is a fascinating one, and there is still a great deal to learn about the majority of species. What has been achieved already, by Darwin in Kent in the last century, and by Dodson, Dressler and their coworkers in Central and South America recently, only emphasises how much there is still to do. However, it is work that must be carried out in the field or forest where the orchids grow and the insects live, rather than in the glasshouse. But at Kew, hand pollination of the flowers in the nursery can be followed by careful observations, the keeping of records and the employment of new techniques of microscopy and biochemistry in the laboratory. Thus various stages of the breeding system can be studied in a wide variety of species.
There are several reasons for doing this. It is interesting to know which species will produce viable seed after pollination with their own pollen, i.e. which are self‑compatible. With very rare species this may be important for propagation and conservation. Some orchids are well known to be self incompatible, more than 80% of the species of Oncidium for example, and work in the Anatomy Section is being undertaken to discover why this should be so. It would be interesting to identify what it is that is controlling pollen tube growth from the stigma into the ovary and whether fertilisation is successful or not. It would be very useful to discover ways of overcoming such barriers, if they exist. This information could also be of great value to orchid hybridisers who already know that many of their crosses, especially in the more complex modern hybrids, will yield no seeds but do not know why. More knowledge of breeding systems might help them to overcome this difficulty.
Like most plant families, the Orchidaceae exhibit a full
range of breeding strategies from the fully self pollinating and self ‑fertile
species to the fully crosspollinating and self‑incompatible species. A recent survey
of a wide range of genera revealed self~ incompatibility in about 40% of the
species in the glasshouses at Kew. Many species might be termed intermediate,
that is having no self‑incompatibility system but promoting out crossing to
variable degree. Orchids are, however, best known for some of the most bizarre
examples of breeding systems that exist. The insect‑plant relationship is often
unique to species and this may mean that the likelihood of successful
pollination is infrequent or remote. Perhaps this is why one successful
pollination in an orchid flower produces so many seeds, over a million in some
cases. Such a massive fertilisation requires massive numbers of pollen tubes and
a complex structure to support and enhance their growth down the stylar canal.
Orchids are also unusual in having viable pollinia and receptive stigmas for considerable periods of time two or three months is by no means extraordinary, and some Dendrobium flowers have been known to last for nine to twelve months, still fertile and receptive, until they are pollinated. When one considers that a pollen grain of wheat or maize may only be viable for minutes, or at the most a few hours, this once again puts orchids in an exceptional class.
Studies of breeding systems in orchids were started at Kew in 1987, with representatives of the subtribe Oncidiinae. Pollination in these species sets off a series of responses in each flower, some of which appear general while others appear specifically associated with the result of the pollination, incompatible (fail) or compatible (success).
The most obvious are changes in the flower, which may
include fading of the bright colour of the lip and petals, other colour changes
or wilting of some or all of the floral parts. Less obvious is the closure of
the column wings or stigma lobes around the pollinium. This happens within one
day of the pollinium being placed on the stigma. Both these responses appear to
happen in every pollinated flower and thus might be considered to be general
responses. They are not necessarily always followed by the swelling of the
column and ovary which occurs in successful pollinations.
In most other plants the female part is fully formed and ready for fertilisation when the flower opens. Fertilisation follows rather rapidly after pollination. In the orchids there is a hidden response to pollination, which is partially indicated by the swelling of the ovary, and that is the differentiation of its internal tissues. Pollen tubes arriving in the ovary after growing down the column (or its stylar tissue) for six or seven days, may have to wait more than six weeks before fertilisation can take place. It is only during this period, and after pollination has been effected, that the ovules develop and produce the egg cells which the male gamete can fertilise. There are few other plants that have such an economical system, where the female gamete is not developed until the male gamete is already in close proximity. The presence of 45,000 pollen tubes, which were counted by Sean Clifford in the transverse section of a developing ovary of an Oncidium, is a remarkable sight and a precursor of the enormous potential for seed development in this and other orchids.
One means of controlling whether a pollination may be successful or not is a breeding system called selfincompatibility. This breeding system appears to be widespread throughout the plant kingdom, and has been recorded for a wide range of species in the Orchidaceae.
In most of the examples that have been thoroughly examined so far, self‑incompatibility (SI) is controlled by a gene which has many forms (alleles) Though pollen, stigma, style and ovary are perfectly formed and viable, pollen carrying a form of the SI gene which is the same as that in the style is unable to achieve fertilisation. The pollen grains germinate and a pollen tube is produced, but it is prevented from growing the full distance into the ovary. In the Oncidium species examined at Kew, the inhibition of pollen tube growth takes place at the top of the style. Usually this response follows a self‑pollination, but it has also been observed when plants of the same clone or some siblings are cross pollinated. Crossing genetically different plants of the same species ensures seed set.
The system, which is not yet understood, thus promotes
cross fertilisation and hence genetic diversity. It is also clear that in some
interspecific and intergeneric crosses, pollen tubes are able to grow down the
style and into the pollen tube guides of the interior wall of the ovary, but
fail to stimulate the necessary development of the female. With no egg to
fertilise, the pollination is unsuccessful.
The work at Kew is still at an early stage and there is still much to be learned. The most exciting pieces of evidence are just beginning to accumulate. They strongly suggest that chemicals held in the pollen wall, and/or the growing pollen tube, are responsible for stimulating both the incompatibility response of some species and all of the characteristic post‑pollination changes.
Anatomical Research Cytology Pollination and Breeding Systems top
There has been an interest in research on orchid seed germination and storage since the inception of the Seed Unit at Kew in 1969. The unit, which subsequently became part of the Physiology Section of the Jodrell Laboratory and moved to Wakehurst Place in 1973, was set up to preserve seeds under refrigerated conditions over a number of growing seasons. Today's modern Seed Bank at Wakehurst Place operates at a deep freeze temperature of ‑20ºC and aims to store seeds at around 5% moisture for hundreds of years. In this way, seed banking can protect species against the increasing threat of extinction which is brought about by habitat destruction, and, in some orchids, by the overcollecting of plants for study or trade.
Routine germination testing to monitor viability loss is an
essential part of seed‑banking. However, with orchid seeds, germination testing
is far from straightforward. The seeds are usually less than ten millionths of a
gram in weight, and possess an embryo less than I mm in length with few
nutritive reserves for germination. In the natural environment, the energy
resources required for germination and early seedling developments are provided
through a symbiotic association with one or more Basiodomycete fungi (often
referred to as Rhizoctonia species). Reproducing such an association in
vitro is one way of checking orchid seed viability, and may be the only
method of achieving germination in some terrestrial species.
Alternatively, germination of most terrestrial and epiphytic orchid species can
be induced by sowing the seeds on a complex medium containing inorganic salts,
vitamins and amino acids, and a carbohydrate source the so‑called asymbiotic
method for germination .
One limitation of these tests, however, is the relatively slow progress of germination over many weeks. Where there is a need for a quicker assessment of viability, i.e. within 24 hours, it is possible to stain hydrated living orchid seed embryos with a fluorescent dye and observe them under the microscope.
In comparison with seeds of most other families, orchid seeds are short‑lived, even when stored dry at ‑20ºC; their longevity apparently being restricted to a few years rather than centuries. The use of ultra‑low storage temperatures, i.e. below ‑15ºC, may offer some scope for the improvement of orchid seed storage longevity, because the degenerative biochemical processes associated with seed viability loss should be reduced to a negligible rate.
Liquid nitrogen storage (at ‑196ºC) is an economically feasible alternative to conventional freezer storage systems when the seeds to be stored are in short supply or are small in volume. As approximately 50,000 orchid seeds could be stored in a 1‑cm vial, this system would appear to be ideal for long‑term storage of orchid seeds. To date, orchid seeds have shown no loss in viability after storage in liquid nitrogen at ‑196ºC over a period of a few years.
Orchid pollen storage is of obvious benefit to the hybridist, but also has an important long‑term role to play in conservation. For example, several of the rarer British orchid species are at the limits of their natural geographical range. A feature of their relative uncompetitiveness, in an ecological sense, is often the shortage of suitable insect pollinators. The controlled pollination of such populations of orchids, with stored pollen from elsewhere or from an earlier flowering, could ensure a high level of seed set and increase the likelihood of a new generation of plants developing. The orchid pollinia are easily collected and stored.
As with seed storage, pollen viability also needs to be monitored during storage. A simple sugar‑agar medium will generally suffice, with the germination process taking between one and two days.
Several experiments with British orchids have shown that pollen viability is at a peak just as the flower opens. To have the maximum chance of obtaining many seeds, pollinia from freshly opened flowers should therefore be used in hand pollinations. Compared with nonorchidaceous species, pollen longevity on the inflorescence is relatively good, however; at ambient temperatures and moisture contents the pollen remains viable for many days.
Short‑term storage, of up to three months, may be possible at normal refrigeration temperatures. For longer term storage, of more than one year, ‑20ºC or ‑196ºC storage temperatures should be used. Before storage at sub‑zero temperatures, however, some predrying of the pollen should be performed, but over drying, such as using a desiccant like silica gel crystals for about one day, should be avoided, as pollen of some British orchids has been killed at low moisture contents.
Long‑term storage of pollen in air‑filled gelatine ampoules at sub‑zero temperatures is better than storage in the presence of cryoprotectants. Stored in this way
Anatomical Research Cytology Pollination and Breeding Systems Seed and Pollen Storage