Introduction

The successful conservation of threatened species necessitates the protection and management of their habitats. If species abundance is low, i.e. the species under consideration is locally rare, management regimes must be developed and applied with extreme care if local extinction is to be avoided. The vulnerability of such populations to interference will severely restrict the extent to which experimental management trials can be used to develop measures to ensure conservation. Under these circumstances, population modelling may provide a valuable alternative to field trials. In this paper, the development and use of a simple model of the population behaviour of the early spider orchid, Ophrys sphegodes Mill. is described, and used to predict future population trends.

O. sphegodes is a rare species which occurs in ancient, species-rich chalk grassland. Its distribution and abundance in the British Isles have declined considerably over the past 50 years. It is considered to be part of the European element of the British flora (Summerhayes 1951), and its distribution is currently virtually confined to the South East corner of England. Rosettes appear above ground in autumn, persist over winter and may produce flower spikes in April or May in the following year. Shortly after flowering the above ground parts senesce (Lang 1980; Hutchings 1987a). Most plants have short life-spans, and reach sexual maturity rapidly. Few plants survive for more than three years after their first emergence above ground (Hutchings 1987a; and Hutchings, chapter 8 this Volume).

Of the estimated 191 species (Baumann & Künkele 1982) currently accepted for Europe, The Middle East and North Africa, only 53 are native to the British Isles. One of these, Spiranthes aestivalis (Poir.) L.C. Rich., is generally considered to be extinct.

Many Continental species, e.g. Epipactis muelleri Godfery, Limodorum abortivum (L.) Sw., Orchis coriophora L. and Serapias cordigera L., were prevented from ever reaching our shores by the formation of the English Channel around seven thousand years ago. Others, such as most Ophrys species, e.g. O. scolopax Cav. and Serapias species, e.g. S. lingua L., require a warmer, drier climate and only extend as far as south-west and south-central France. Although the majority of British orchids may be seen in greater numbers across the Channel, it is a mistake to assume that our native orchid flora is merely a poor representation of Continental Europe. Several species, particularly those of chalk grassland, are as widespread in Britain as elsewhere. Among these may be included Anacamptis pyramidalis (L.) L.C. Rich., Dactylorhiza fuchsii (Druce) Soó and D. maculata (L.) Soó, Epipactis helleborine (L.) Crantz, Gymnadenia conopsea (L.) R.Br., Orchis mascula L. and Platanthera chlorantha (Custer) Reichb.

A few species have their main European distribution in the British Isles, e.g. Epipactis phyllanthes G.E. Sm., a variable plant, some forms of which have cleistogamous flowers. On the Continent it is only found in western France and Denmark. Spiranthes romanzoffiana Cham. is an example of a North American species which, in Europe, is confined to Ireland, Western Scotland and Devon.

Introduction

As an adjunct to seed storage for genome preservation, orchid pollen storage has much to offer: to the hybridist wishing to overcome flowering asynchrony in species and/or to introduce wild genome contributions into cultivated taxa; to the gene bank manager seeking to preserve a large quantity of genetic material in a small facility; and to the conservationist anxious to preserve species, even if this can only be achieved through the pollination of plants held ex situ in orchid collections with stored pollen. Pollen storage may also be of considerable future importance to the biotechnologists, if the process of haploid plant production via embryo development from pollen grains (i.e. androgenesis) can be extended to orchids.

The literature on orchid pollen germination is surprisingly limited with previous studies concentrating on optimising the composition of the germination medium, particularly the sugar level. Pfundt (1910), working with two European species and a range of sugar levels from 5–20%, observed a preference for 5–10% sugar. Miwa (1937) similarly found orchid pollen generally germinates best when using cane sugar at around 3–10%. Although more exacting studies on the optimal chemical composition of the germination medium have been performed, particularly with reference to plant hormones (Curtis & Duncan 1947; Rao & Chin 1972), sugar level remains the most important single chemical factor in stimulating orchid pollen germination on artificial medium; its action being osmotic, preventing grain bursting whilst avoiding plasmolysis, rather than as a heterotrophic source of carbon (see Stanley & Linskens 1974).

Introduction

The application of in vitro techniques to the propagation of plants at Kew originated from Dr Peter Thompson's work in the 1960s on the formulation of a medium for orchid seed germination. By 1971 the commercial and research potential of plant propagation by aseptic culture was becoming more apparent and the Director of Kew at the time, Professor Heslop-Harrison, determined that it was time to make the techniques available to the living collection. Plans were made for a unit to provide in vitro propagation services to the sections of the Living Collections Division at Kew. The Unit based at Aiton House was set up and opened in 1977, under the present Curator, Mr John Simmons.

The living orchid collection at Kew comprises approximately 3,500 species represented in 375 genera. Each year the resultant seed from a pollination programme is germinated at the Micropropagation Unit. Seed is also given to the Unit by botanists from field collections, from private and commercial orchid growers and from members of the general public. The main function of the Unit is to supply the orchid collection with new species and in many cases provide a back-up of viable seedlings for those species which present special cultivation problems. As much information as possible is recorded about the seed including such details as the site and habitat type where the collection was made. This type of information helps decide the type of culture conditions used.

Summary

The understanding of the host–fungus relationships in the mycorrhizas of orchids is important in relation to the application of symbiotic methods to seed germination and seedling development, and also for re-establishment in natural conditions either from seed or tissue culture as one of the contributions to conservation. Despite extensive progress in knowledge of the role of fungi in the early stages of germination, the inherent difficulty in the use of the symbiotic technique has inhibited its application.

Orchid fungi are extremely variable and relatively few root inhabitants are true mutualistic symbionts. The outcome of the relationship between the partners is a finely balanced one and many fungi isolated from orchid mycorrhizas may, after a period in culture, become incompatible with, or even pathogenic to, orchid protocorms.

Mutualistically symbiotic fungi enhance the nutrition of germinating seeds by the transference of carbon compounds. Photosynthetically active seedlings and mature plants, however, may be quite independent of their fungal partners. Evidence suggests that in conditions of nutrient stress the fungal partner may mediate in the movement of phosphate and/or nitrogen compounds, as in other mycorrhizal systems.

Introduction

Orchids, whether epiphytic or terrestrial, generally grow and thrive in conditions of nutrient impoverishment. As with other higher plants growing in similar environments this may only be possible by the association of fungi with the roots and other subterranean parts – the mycorrhizal association.

In all homosporous ferns, except those included in the Marattiaceae and Osmundaceae, the protonema initial generates a uniseriate, elongate filament composed of a varying number of chlorophyllous cells. All division planes leading up to the formation of the filament are oriented perpendicular to the long axis of the cell and thus lie parallel to one another. At each division, new walls are laid down between cells to hold them together as an elongating filament. When the filament has produced a certain number of cells, as determined by the conditions of growth and other factors, its terminal or subterminal cell divides by a wall oblique to the long axis to give rise to a bidirectionally dividing plate of cells. This marks the beginning of planar or prothallial growth. By the continued meristematic activity of this cell, aided later by the establishment of a pluricellular meristem, a flat, often cordate or exceptionally, ribbon-shaped, structure is formed. Rarely, as in Schizaea and Trichomanes (Hymenophyllaceae), no change ever occurs in the plane of cell divisions of the germ filament, which thus retains the basic filamentous morphology throughout the gametophytic phase. In this book, the terms ‘germ filament’, ‘protonemal filament’ or ‘protonema’ (plural, protonemata) are applied to the filamentous gametophyte and the term ‘prothallus’ is used to denote the structure with the planar morphology.

It is now reasonably clear that the dormant fern spore is a thick-walled, tetrahedral or bilateral cell enclosing a partially dehydrated cytoplasm with a centrally placed nucleus surrounded by other organelles and storage granules. The relationship of this basic structure to the first asymmetric division during germination is the subject of this chapter in which we shall also define those inherent and experimentally detectable aspects of spore polarity that may serve as a basis for the morphological differentiation that follows. The cytology of spore germination leaves little doubt that identical genetic information is transmitted to the two cells which are born out of a simple mitotic division of the spore nucleus. Yet, these cells follow dissimilar pathways of differentiation – one gives rise to the rhizoid, the other to the protonema initial – suggesting that each nucleus is exposed to a different milieu. Thus, it seems likely that cell differentiation during spore germination may be understood in terms of visible structural or cryptic physiological or biochemical differences in the cytoplasm.

The areas of discussion delineated for this chapter have their roots in classical morphological investigations on the germination of spores of diverse ferns made during the past hundred years or so. However, only recently has it been possible to pose the relevant questions in physiological terms.

Fertilization which heralds the diploid or sporophytic phase, is a normal feature in the life cycle of ferns and partly accounts for the typical alternation of generations. A number of fern species also exhibit a way of life in which a sporophyte with the gametic number of chromosomes is born out of the gametophytic cell without fertilization. From both developmental and cytological perspectives, the parenchymatous cell of the gametophyte, dedifferentiating as it does during transformation into a sporophyte, is an obvious target for experimental analysis. With the accounts of sexual reproduction presented in previous chapters as a background, we will explore here the methods and mechanisms by which a sporophyte is regenerated from the gametophyte, while keeping innocent of sex.

Apogamy is the preferred term used to designate the developmental and reproductive adaptation of gametophytic cells that are deflected into the sporophytic pathway without sexual union. The term as used generally includes the regeneration of any organ of the sporophyte from the cells of the gametophyte. In some early accounts of apogamy, the term was extended to include production within the gametophyte of characteristic tissue elements of the sporophyte such as tracheids. However, as gametophytes that do not regenerate apogamous sporophytes differentiate tracheids, when supplied with sucrose or hormones in the medium (see chapter 9), it would seem logical to consider apogamous regeneration and tracheid differentiation as separate processes.

Gametophytes of homosporous ferns acquire the potential to form antheridia and archegonia during a period of growth and maturation. A striking aspect of sexuality in fern gametophytes is the complexity of the division sequences giving rise to sexual cells and the simplicity of the final products. Initiation of sex organs (gametangia) on the gametophyte thus poses important developmental questions inasmuch as certain cells in a homogeneous population respond to reprogramming cues and differentiate into gametes adapted for sexual recombination. What is the trigger that starts off cells of the gametophyte on a particular course of metabolism and behavior which will turn them into antheridia and archegonia? Analysis of this question has been an important thrust in the developmental biology of ferns and as a result there is strong evidence to show that antheridium formation on the gametophyte occurs in response to hormonal signals. The controlling factors in the initiation of the archegonium have as yet been hardly considered, but the challenge is great.

In this chapter we emphasize the ontogeny of the antheridium and archegonium and follow it up with a discussion about the physiological control of their differentiation. Beginning with a survey by Näf (1962b) which was partially devoted to the physiology of antheridium formation in fern gametophytes, new knowledge gained in the field has been incorporated into periodic reviews (Näf, 1963, 1969, 1979; Näf, Nakanishi and Endo, 1975; Voeller, 1964a; Voeller and Weinberg, 1969).

In the gametophytic generation of homosporous ferns, the germ filament represents a transient phase in anticipation of a major morphogenetic event, as, sooner or later, its terminal cell characteristically divides by an oblique or longitudinal wall to initiate planar morphology. The division wall also occasionally appears in the subterminal cells; in other cases, after the terminal cell is partitioned longitudinally, most of the cells in the filament may also follow suit. For purposes of discussion in this chapter, the essential mystery of transition of the germ filament to a planar gametophyte is considered to revolve around the orientation of the mitotic spindle in the terminal cell from a position parallel to the long axis of the cell to one perpendicular to it. This and the formation of the crosswall itself are engineered by a complex series of developmental processes of which we have only a rudimentary understanding.

Induction of planar growth in the fern protonema represents the beginning of a morphogenetic process that prepares it for the production of gametes. The major axioms governing morphogenesis in complex systems where a change in the plane of cell division initiates a change in the pattern of growth, can be analyzed with relative ease in the protonema.

In this chapter we will take a close look at the manner in which the antheridium and archegonium give rise to gametes and at the act of sexual recombination. Formation of sex organs and the various physiological and genetic interactions between gametophytes represent one phase of the reproductive biology that terminates in sexual fusion and gene exchange. The final acts in this drama are gametogenesis and fertilization. In ferns, the function of gametogenesis is to construct two specialized reproductive cells, the spermatozoid (sperm), produced by the antheridium and the egg, produced by the archegonium. Fertilization involves the union of the sperm with the egg which is generally housed in a privileged location in the archegonium. In the first part of this chapter we will examine spermatogenesis, the origin of the sperm and follow it up with oogenesis, the formation of the egg and conclude our traverse of the gametophytic landscape with an account of fertilization.

Topics covered in this chapter have been reviewed by Duckett (1975), Bell and Duckett (1976), DeMaggio (1977), and Bell (1979b) and the reader is referred to these sources for additional information.

Spermatogenesis

Our knowledge of spermatogenesis in ferns is of recent vintage, derived largely from some careful work on Pteridium aquilinum, Marsilea vestita, and Ceratopteris thalictroides, which has provided a rich heritage of new information and some excellent electron micrographs.

While the gametophyte featured in our previous discussions is born out of a reduction division of the sporocyte, in this final chapter we will pay some attention to the development of gametophytes directly from sporophytic tissues bypassing meiosis.

The reproductive strategy which results in the generation of gametophytes without meiosis or sporulation is a facultative property of sporophytic tissues of certain homosporous ferns and is known as apospory. Aposporously formed gametophytes grow without any restrictions and behave in every respect like those evolved from germinated spores except that their germ cells display the same chromosome number as the sporophyte. For this reason, apospory is considered to account for the natural polyploidization in ferns, although other factors might also be involved. Unlike apogamy, apospory is of sporadic occurrence in nature and only a few ferns have been shown to display this phenomenon consistently in their life cycle. However, aposporous regeneration of gametophytes is readily induced when different parts of the sporophyte are challenged by simple manipulative or cultural techniques. The early work on apospory has been reviewed by Steil (1939, 1951).

Although Steil (1939) considers apospory in a broad sense to include the formation, without meiosis, of rhizoids, gametophytic cells, sex organs or sperm on the sporophyte, the term will be used in this book in a restricted sense to include regeneration from the sporophytic tissue of a more or less complete gametophyte that perpetuates a persistent diploid genome in its cells.

With the description of the landmark stages in the early development of fern gametophytes behind us, we are now in a position to consider the factors that regulate their growth and maturation. Studies in this area have been largely aided by the ease with which gametophytes of homosporous ferns can be raised from spores in large numbers under aseptic conditions. For the most part of its growth, the gametophyte is only one cell layer thick, a property which makes determination of cell numbers a less formidable task; the structural simplicity of the gametophyte also makes it possible to study a range of morphological expression of cells which seem to possess unlimited potentialities. It is, therefore, no wonder that the effects of a staggering variety of chemicals on the growth of gametophytes of a number of ferns have been tested, with a view to determine the optimum nutrient or chemical environment for their growth. Surprisingly, in these studies there were relatively few examinations for the changes of biochemical constituents or hormone levels during growth. This was because it is so much easier to follow growth of gametophytes in a given nutrient milieu in terms of a few growth parameters like increase in surface area, increase in cell number or change in the dimensionality of growth (such as from filamentous to planar) than process the material for arduous biochemical determinations.

Extant ferns comprise a group of about 12 000 species of plants widely distributed throughout the world in many habitats and niches. As wide as their distribution is their range in size, with extremes such as the small water ferns with leaves less than 1 cm long and the giant tree ferns which attain heights of almost 25 m and bear crowns of leaves 30 cm or more in diameter. In many contemporary systems of classification with which developmental botanists will feel comfortable, ferns are assigned to the group Pteropsida or Filicopsida. Members of this group along with those of Psilopsida, Lycopsida and Sphenopsida constitute a major division of the plant kingdom known as Pteridophyta (pteridophytes). A distinctive anatomical feature of pteridophytes, which they share with gymnosperms and angiosperms, is the presence of a vascular system in the plant body, but pteridophytes differ from the latter two divisions in lacking the seed habit (hence the name, seedless vascular plants, for the division). During their evolutionary past, pteridophytes have stabilized and almost perfected the vascular system for a seedless plant so much so that they are also designated as vascular cryptogams. Most pteridophytes, including ferns, are trapped into a life cycle in which they are constrained by some primitive features such as the production of motile sperm and the requirement for free water for fertilization.