Cypripedium

A circumboreal genus, Cypripedium occurs in bogs, swamps and woodland, especially in forest margins and clearings, usually not in deep shade. C. calceolus is virtually confined to calcareous soils, the topsoil being characteristically friable with a high humus content (Fuchs & Ziegenspeck, 1926a). C. candidum grows in black humus saturated with lime, the soil having a pH of 7.4 (Stoutamire, 1990). A stand of C. reginae in Canada was found growing in soil with about 20% more calcium than the average for the region (Harvais, 1980). Although most of the species thus prefer a neutral or alkaline substrate (Wherry, 1918; Curtis, 1943), C. acaule grows in acid soil (pH 3.5–5.4: Wherry, 1918; Curtis, 1943; Stoutamire, 1963).

In all species the adult plants have a horizontal rhizome and well-developed, dark green foliage leaves, and maintain this foliage from spring to autumn. They overwinter below ground. The vegetative period before flowering is assumed to be long: up to 10 years or more in American species (Curtis, 1943) and 6–10 years in C. calceolus (Kober, 1972; Fast, 1985).

In C. arietinum, C. candidum and C. calceolus var.pubescens the tips of 2 to 3-year-old roots may form buds as a means of vegetative propagation (Curtis, 1943).

Life history

C. calceolus presumably germinates in spring (Fuchs & Ziegenspeck, 1926a), although Irmisch (1853) observed very small protocorms of C. calceolus in December. C. acaule begins to germinate in spring and early summer (Curtis, 1943). The seeds usually lie fairly deep (2–5 cm), typically occurring below a layer of living mosses and partially decomposed plant debris on top of the humous mineral soil (Fuchs & Ziegenspeck, 1926a; Curtis, 1943).

All underground parts of terrestrial orchids must either accommodate the endophyte or actively reject it. The mycotrophy of the protocorm is obligate, and most roots are also mycotrophic in varying degrees, but in many species the rhizome loses its mycotrophic function while the plant is still young. Storage organs such as root-stem tubers and corms are usually not infected.

An apparently important aspect of the mycotrophic organs is their longevity; although in the tuberous species some roots function for only about 9 months, orchid roots typically continue to function for a number of years.

Protocorm

The germinating embryo develops into the protocorm. Unlike other angiosperm seedlings it has no radicle, since the suspensor end of the embryo becomes specialized to form mycotrophic tissue. Neither root cap nor meristem is formed, and the suspensor end of the embryo remains stationary in the soil (Fig. 5.6, p. 111; Fig. 6.1c, p. 114; Fabre, 1856; Stojanow, 1916). As far as is known all orchid seedlings are modified to form mycotrophic tissue in the basal part, even in Bletilla which has the most highly differentiated embryo yet observed (Bernard, 1904).

The opposite, i.e. chalazal, end with the functional meristem is solely responsible for all increase in width and length in the seedling; from this end a condensed or elongated mycorhizome develops which allows the shoot tip with leaf primordia to progress in the soil.

Strictly speaking the protocorm comprises only that part of the seedling axis that develops below the lowermost leafy appendage and corresponds to the radicle and hypocotyl in seedlings of other plants. The boundary between protocorm and mycorhizome is indistinct, however, since the first scale leaves are often inconspicuous and occasionally vestigial.

Since the young seedling of terrestrial orchids lives underground, the early life history is largely unknown. Some investigators have painstakingly dup up seedlings and underground structures around the year and endeavoured to deduce the developmental process from this material (Fuchs & Ziegenspeck 1924a, b, 1926a, b, 1927a–c). This is usually the only source of information that we have as regards the life history since many species has never been grown from seed to the adult stage in culture or their life history has not been described when cultured plants were available.

Both excavation of plants in natural populations and observation of cultured specimens have their limitations as methods of investigating the life history. Unless the same individuals are repeatedly dug up and carefully replanted this method gives information only on the sequence of organogenesis and a rough seasonal timetable. Construction of an absolute timetable requires that there is a close correspondence between size and age, but this assumption is unrealistic, since cultivated seedlings of many species show considerable individual variation under uniform culture conditions. Estimates of the duration of seedling stages that have been made on the basis of excavations, particularly by Fuchs & Ziegenspeck (1922–7), have been justly criticized for being exaggerated, but it is mainly the most extreme estimates such as for the underground phase of Orchis ustulata that have been singled out (Summerhayes, 1951) and been met with scepticism. Most of the estimates made by Fuchs & Ziegenspeck do not fall wide of the mark if they are compared with those now emerging from population studies, as can be judged from Table 10.1.

Under suitable aeration and temperature conditions non-dormant plant seeds need only access to water to be able to germinate, but since orchid seedlings are heterotrophic to varying degrees their seeds often have several requirements from outside besides water. These are undoubtedly met by the endophyte when germination takes place in nature, or indirectly by the substrate that the fungus lives on.

Some other requirements for germination of orchid seeds are associated with dormancy. The currently available evidence suggests that several types of dormancy, both exogenous and endogenous, occur in the seeds of holarctic orchids. In spite of the apparently delicate nature of the translucent testa its impermeability to water represents a form of physical dormancy that can be overcome by prolonged soaking or by scarification. There are different types of physiological dormancy that can be broken by darkness, temperature regime, atmospheric conditions, or certain external chemical signals. Finally, morphological dormancy, expressed as a requirement for the embryos to after-ripen, probably also occurs in orchid seeds.

The treatments and substrate additives that are necessary to obtain germination in vitro can thus be divided into those that are needed for seeds to germinate in symbiotic culture, i.e. such that break dormancy, and the more extensive set of conditions that must be met if the seeds are to germinate asymbiotically. The difference between these sets would seem to directly represent the effects of the fungus, but this conclusion may not be valid, since several external stimuli may elicit the same physiological response, for instance when different metabolites influence different steps in the same group of reactions.

Practical cultivation of terrestrial orchids beyond the seedling stage is thoroughly described elsewhere (Fast, 1980; Cribb & Bailes, 1989), but since these plants are not subjected to large-scale production the present culture methods rely largely on a relatively small amount of accumulated experience. With more effective in vitro techniques for seedling production more plants will be available for culture experiments in the future. It would be desirable to have a more objective foundation on which to construct rational production schemes.

In this brief account I will focus only on the establishment in soil of the earliest life stages and on such aspects of cultivation as soil requirements and phenology that have a relation to the mycotrophic lifeform of the plants.

Weaning of seedlings from in vitro culture

Transfer of seedlings to the soil is perhaps the most challenging step in the cultivation of terrestrial orchids (Fast, 1982). Before they are removed from the protected environment of the culture vessel the seedlings should be as large and vigorous as possible, but there is usually an economic incentive to reduce the length of time they are kept in vitro and the number of transfers undertaken.

When seedlings are transferred to a fresh medium in vitro the growth rate always increases, particularly when the seeds have been sown densely (Fast, 1982). Wide spacing allows the seedlings to develop more rapidly (Tsutsui & Tomita, 1989; Rasmussen et al., 1989). This advantage should be balanced against the greater number of culture vessels and amount of substrate necessary.

Mycorrhiza is widespread in all groups of higher plants (Stahl, 1900; Allen, 1991). In general mycorrhizal relationships increase the plant's access to soil resources, i.e. water and mineral ions, thereby increasing its drought tolerance and ability to grow in poor and leached soils. Ectotrophic mycorrhiza is thus strongly associated with conditions of mineral nutrient stress (Harley, 1969).

Orchid mycorrhiza differs in important respects from most other types of mycorrhiza (e.g. Smith, 1974; Harley, 1984; Allen, 1991). The green plant functions as the producer of energy in ectomycorrhizal systems, VAM and ericaceous mycorrhiza, but in the mycorrhiza of orchids the fungus is the source of energy and thus provides the plants with a separate nutrition. This may alternate with, supplement or entirely replace the phototrophic nutrition. Although gametophytes of certain pteridophytes and a few specialized groups of higher plants such as Monotropa have established a similar parasitism on fungi, Orchidaceae is the only large plant group that makes consistent use of this kind of nutrition. It is necessary for seedlings of most plants to develop leaves before the nutrient reserves in the seed are exhausted, and for the plants to remain phototrophic throughout their life. In contrast, orchid seedlings have an option of living for extended periods as heterotrophic organisms; this opens habitats and evolutionary pathways for orchids that would not otherwise have been accessible.

Ecological consequences

Importance of light

Since the orchid mycorrhiza represents an alternative source of energy, it is easy to explain how some species of orchids thrive in deep shade where few other plants will grow. This is most evidently true of the species with chlorophyll deficiency.

There are several early nineteenth century observations of disorganized clumps of material in the cortex of orchid roots, but Reissek (1847) may have been the first to realize that these clumps were the remains of fungal hyphae coiled inside the plant cells. He studied the roots of Neottia nidus-avis and observed tissues that were so young that living hyphae were still present. Reissek also reported these coils from roots of other European orchid species belonging to Orchis, Ophrys and Gymnadenia and from several tropical orchids. Some years later, ‘une matière granulaire’ originating from decomposed hyphal coils was observed in the basal cells of orchid protocorms (Prillieux & Rivière, 1856). These observations stimulated interest in orchid mycorrhiza, particularly as regards investigating the species that display marked chlorophyll deficiency (Prillieux, 1856; Johow, 1885, 1889; Groom, 1895; Janse, 1897; MacDougal, 1899a, b; Magnus, 1900).

In a survey of more than 500 cultivated orchid species, Wahrlich (1886) managed to find the characteristic fungal infections in every one of them, and Frank (1891) concluded from observations of the infected tissue that the hyphae became lysed, and that the nuclear modification in the infected cells was a sign of the active participation of the host in this process.

A more complete understanding of the functions of fungi in the life history of orchids arose from Bernard's germination studies during the years 1899 to 1909, and from Burgeff's study that primarily dealt with the fungi (1909). Both of these investigators succeeded in germinating orchids in vitro and in observing the process of infection in embryos and seedlings. Among the early investigators opinions differed as to the nature of the relationship.

Isolation

Orchid endophytes can be isolated from naturally occurring protocorms, from roots, and occasionally from rhizomes, tubers or corms. The advantage of extracting a fungus from protocorm tissue is that its role in seedling development may almost be taken for granted, whereas organs of mature plants may house a variety of fungi (Harvais & Hadley, 1967a; Hadley, 1970b). Furthermore, infection is extensive in the basal part of the protocorm, and protocorm cells are often fairly large. In contrast, the cortical cells of roots are long and narrow and the infection tends to be patchy and irregular. The most promising places to search for infection in roots are areas of the cortex below epidermal hairs, especially those close to the root tip (Bernard, 1909). Infected parts of an orchid root tend to become faintly yellowish or opaque. The living pelotons, where present, will be found close to the surface of the organ.

When the rhizome is infected it is usually the outer cortex that is most suitable for isolation purposes. In Liparis lilifolia both the condensed rhizome and the bases of attached leaves are infected. Isolation from tubers of orchidoid species has apparently never been successful, although Fuchs & Ziegenspeck (1925) report that superficial cell layers of globose tubers may be infected as well as the extremities of palmately divided tubers. Corms are usually without infection (Chapter 7).

Care must always be taken to remove the soil organisms that are normally found on any underground plant parts. Fungi that occur in the rhizosphere can include strains of Rhizoctonia that differ from those that form pelotons inside, and their relevance for the orchid mycorrhiza is not clear (Warcup & Talbot, 1967).

Background

Surely no plant group surpasses the orchid family in its appeal to scientific curiosity and imagination. The untiring professional and public interest in the Orchidaceae is reflected in a considerable number of volumes published each year on orchids; in particular, the great structural variety in the orchid flower is well represented in the literature.

Compared with the floristic study of this large and taxonomically complex group, other types of investigation lag behind. Of the many unusual features that can be noted in the orchid family their mode of life in association with fungi has probably attracted the least attention, which is surprising since it is without doubt a fundamental aspect of orchid biology.

The orchid family is the only large group of higher plants that makes consistent use of an alternative nutritional system. Orchid mycorrhiza differs from other major types of mycorrhiza in that the fungus supplies the plant with energy. During some of its life stages the orchid can rely entirely on mycotrophy for nutrition, while during other stages the plant makes use of both mycotrophy and phototrophic nutrition, either alternately or the one supplementing the other.

The great diversity of orchid habitats, which include places where paucity of soil or light precludes most plant life, is best understood in the context of orchid mycotrophy. In fact, many of the outstanding features of the orchids, such as the complexity of floral structures, the diversity of plant–pollinator interactions and the unusual characteristics of the seeds, can all be viewed as functional adaptations in relation to or dependent on the mycotrophic lifeform (Chapter 12). This in itself justifies a treatise on orchids centred on their mycorrhiza.