Introduction: bleaching herbicides

Many structurally unrelated bleaching herbicides cause either white or yellow chlorosis of leaves, which is obviously a consequence of the total or partial absence of the normal chloroplast pigments, i.e. chlorophylls and carotenoids. The chlorosis may result from the inhibition of pigment biosynthesis or from the destruction of existing pigment. As a general rule, biosynthesis inhibitors only give rise to chlorosis in newly developing leaves, and are most effective as herbicides when given as a pre-emergence treatment. On the other and, the inhibition of photosynthesis and photosynthetic electron transport by other herbicides frequently leads to the destruction of existing chloroplast pigments. Although this chapter is intended primarily to discuss the inhibitory effects of herbicides on pigment biosynthesis, these effects can be difficult to distinguish from pigment destruction.

Chlorophylls and carotenoids in photosynthesis

To appreciate the importance of pigment biosynthesis as a target for herbicide activity it is necessary to understand the fundamental roles that the pigments play in normal photosynthesis. The chlorophylls and carotenoids are located specifically in the pigment–protein complexes (PPC) within the thylakoid membranes. Each functional PPC has its own distinctive pigment composition. Thus the reaction centre core complexes of photosystems I and II (PSI and II) have chlorophyll α and are enriched in β-carotene whereas the light-harvesting chlorophyll-proteins (LHCP) associated with PSI and PSII contain both chlorophylls a and b together with the xanthophylls, Iutein, violaxanthin and neoxanthin (Siefermann-Harms, 1985).

Introduction

The chemical industry is continually finding it harder and far more expensive to develop new selective herbicides. Conversely, there are already many herbicides in existence with excellent spectra of weed control which would be useful in a given crop, except for a minor problem; they also kill the crop. The chemical industries biorational approach to this problem is to study the herbicide degrading enzymatic pathways specific to that crop. The herbicidal moiety could then be modified by adding a chemical group that will be recognised by those enzymes, thus aiding in the herbicide degradation by the crop. Another biorational approach is to make current herbicides more effective by adding synergists that either prevent their breakdown, or prevent detoxification of toxic products in some weeds (cf. Gressel & Shaaltiel, 1988).

The approach taken by the biotechnologists in industry and the public sector is diametrically opposed to that of the chemist: to make crops genetically and biochemically amenable to herbicides by conferring resistance. There are vast differences in the levels of sophistication of the biotechnologies involved. The biotechnological approaches have the advantage of often dealing with time-tested herbicides with well elucidated weed control properties and toxicology. Registration requirements can be rather simple if it can be shown that the metabolites and residues of the herbicide match those found in crops where the herbicide is already used. Where this is not the case, more extensive research provisions might be required.

Introduction

Herbicides recommended for the selective control of unwanted weeds have been developed to exploit a difference in phytotoxicity between species adequate to kill competing weeds without significantly reducing crop yields. In some cases the margin of selectivity may be quite modest and can be rendered inadequate when the timing of application coincides with unfavourable climatic conditions, as was reported to be the case for some of the phenylureas in winter wheat in Autumn 1983. There are a number of factors which can contribute to herbicide selectivity including soil placement, rates of absorption and subsequent translocation, localisation (both within the plant and at the sub-cellular level) and transformation to products of modified phytotoxicity. In addition studies made with atrazine-resistant biotypes of such weed species as Chenopodium album and Amaranthus hybridus L. (Steinback, Pfister & Arntzen, 1982) have highlighted the importance of differences in sensitivity of the target site, in this case the 32 kD protein component of photosystem II. It is likely that other examples of reduced target site sensitivity will be encountered when biotypes resistant to herbicides with different modes of action emerge. Differences in target site sensitivity as a basis for selectivity has been considered previously by Gressel (1985) and is also referred to elsewhere in this volume (Chapters 2, 11 and 12).

The selective properties of herbicides often result from a complex interaction of a number of the factors listed above though there are many examples where one dominant factor has been implicated.

Introduction

Photosystem I (PSI), like Photosystem II (PSII) considered in the previous chapter, is an integral part of the chloroplast electron transport system. Although PSI may act independently of PSII in cyclic flow, both are required for the continuous maintenance of electron flow from water to NADP+ for CO2 incorporation. Whereas with inhibitors of PSII (Chapter 2) interaction with a protein component resulted in the indirect cessation of electron flow, herbicides considered in connection with PSI intercept electrons directly.

In the generation of PSI, electrons expelled from P700 are raised to a negative potential (possibly –900 mV) to electron acceptors A0 and A1, that are forms of chlorophyll a. The donor and acceptors are associated with 70 kD pigment proteins that span the thylakoid membrane lipid bilayer. The subsequent electron acceptor is the hypothetical X, possibly an iron–sulphur centre with a potential of around –700 mV, and then two iron–sulphur centres A and B (Fig. 1) with potentials of around –590 mV and –530 mV. These are associated with two subunits of 18 and 16 kD and are probably identical to the former acceptor P430–. These two centres function either in series or in parallel, and are almost certainly bound ferredoxin, and link to soluble ferredoxin and ferredoxin-NADP+ reductase and hence NADP+ (Fig. 1) (Haehnel 1984).

Introduction

The growth and development of most scientific and artistic disciplines can be traced back through history and spans centuries or even millenia. The development of conservation, a subject largely based on scientific as well as ethical principles is still in its infancy, having only commanded serious academic attention in recent decades. There are now several international wildlife treaties and conventions and fortunately for both professionals and non-professionals interested in the subject the current status and legal background have been brought together by Lyster (1985), to whom much of the foregoing information is attributable.

Control on international trade in wildlife, its products and derivatives is not a recent concept. Initial public demand for such controls was made as early as 1911 by the Swiss conservationist, Paul Sarasen, who claimed that the vogue for plumed hats was having a serious effect on populations of wild birds. Sarasen was one of the most influential figures behind the establishment of the Consultative Commission for the International Protection of Nature at Berne in 1913, which had delegates from seventeen European countries. The progress of the Commission was halted by the outbreak of War and it was not until the late 1940s when the foundation of a similar international body was under discussion, that the Commission had any legal existence. Furthermore it set a precedent, being the first intergovernmental agency concerned with nature protection (Boardman 1981).

Introduction

The coastal rain forest of Brazil is a long, narrow strip of tropical forest which has been deeply eroded by small-scale and large-scale agriculture over many years. Around the large conurbations of Rio de Janeiro, Sao Paulo and Belo Horizonte the damage is especially severe, but pockets of virgin forest still remain which are of interest and in need of conservation. We have been working over the past ten years on a 1000 hectare estate near to Novo Friburgo in the upper Macae valley. The object of the part-time project has been to create a self-supporting reserve with little or no destruction of natural resources except the use of specific woods for building purposes. To achieve this, three main fund raising methods have been used:

1. A farm has been constructed for the laying, hatching and rearing of pheasants, duck, partridge and guinea fowl.

2. Tour groups from the UK and USA have been invited to stay and study orchids in their natural habitat with ourselves as guides.

3. A business has developed in the UK to germinate, grow and sell orchid seed and seedlings, and to provide information collected in the field about lesser known orchid species.

Game Farm

After numerous problems, the farm now produces around 1000 guinea fowl and 1000 pheasants per annum. The problems arose from the lack of electricity for hatching large quantities of eggs and the unreliability of the substitute, gas.

Introduction

The Nature Conservancy Council (NCC) is the official government body concerned with the policies and practices of nature conservation. Since the passing of the Wildlife and Countryside Act in 1981 it has had a legal duty to fulfil its role in protecting sites and species of special interest in Britain.

Listed under Schedule 8 of the Act are 93 plants which are so rare that they are considered worthy of special protection. Nine of these are orchids (Table 1).

In Britain we have about 50 native, terrestrial orchid species. We say ‘about’ as there is constant debate regarding the exact status of several of our species. Twelve of these are nationally rare and are listed in the Red Data Book (Perring & Farrell 1983). Two other species, Spiranthes aestivalis and Ophrys bertolonii, are extinct and a third, Hammarbya paludosa is threatened in Europe, but thankfully more widespread in Britain.

In 1978, Lynne Farrell transferred from the Biological Records Centre, Institute of Terrestrial Ecology, at Monks Wood Experimental Station to take up a post entitled ‘Botanist’ in the Chief Scientist's Team of the NCC based at Huntingdon. This post had several facets, including grasslands and heathlands, as well as rare plants. Since then, the work has been directly concerned with rare plant conservation and particularly with orchid protection as they are a group of plants which have, and will continue to attract a great deal of attention.

Introduction

Although epiphytic orchids have been grown routinely from seed for more than sixty years using the asymbiotic method developed by Knudson (1922), relatively little interest has been attached to techniques for the storage of such seed. This is surprising, especially in view of the rapid loss of many orchid habitats, and in particular the loss of tropical moist forest, with the imminent threat of extinction of a large number of orchid species in the wild (Myers 1979, 1980; Koopowitz & Kaye 1983; Hagsater & Stewart 1986; Koopowitz 1986; Stewart 1986). Knudson (1934) indicated the desirability of storing seed to insure against either failure to germinate or the accidental loss of seedlings. The development of such techniques would also allow an assessment of the commercial merits and potential of a particular cross while still retaining a proportion of the seed.

The cryopreservation of seed shows considerable potential. Thus seeds of Encyclia vitellinum have been stored at a temperature of –40°C for 35 days without loss of viability (Koopowitz & Ward 1984). Svihla & Osterman (1943) reported that Cattleya hybrid seed survived freezing at –78°C, and Ito (1965) successfully stored seeds of Dendrobium nobile and Cattleya hybrids for periods of up to 465 days at –79°C. Pritchard (1984; 1985) reported that seeds of a number of terrestrial and epiphytic species with seed moisture contents below 14% were not damaged by storage in liquid nitrogen (–196°C).

Introduction

The living orchid collection at Kew consists of nearly 10 000 accessions (many of them represented by more than one plant) with around 370 genera and 3500 species represented. The collection comes under the Tropical department and is housed in the Lower Nursery, where eight separate environments are maintained for growing orchids. These range from high temperature/high humidity regimes for growing tropical species, to low temperature environments for growing temperate and high altitude species, with varying degrees of temperature, light and humidity inbetween. The plants are cared for by a team of four members of staff, together with a horticultural student and occasionally international trainees.

We attempt to grow as wide a range of genera and species as possible, to illustrate the extraordinary diversity of the family. Of course we can only hope to grow a small representation of the huge number that exist in the wild, so we must be careful that those we do find space for are fulfilling a useful purpose and are not simply grown ‘for the sake of it’.

Each plant is labelled with an accession number (a ten figure number) which is recorded on the Kew computer together with data such as the donor or collector, its country of origin and range, flowering time, habit of growth, etc. This data is easily retrieved and is of use both to botanists' and horticulturists' research programmes.

Introduction

There is a growing awareness among plant ecologists that the size of an individual is more important in determining its behaviour than its chronological age. Rabotnov (1950) was among the first to demonstrate that in any closed community there is likely to be a distribution of plants in different age classes. He noted that there would be seedlings, juveniles, immature adult plants, reproductive plants, vegetative adult plants and senescent non-flowering plants of great age, but he was unable to identify the factors which contributed to a plant switching from a vegetative to a reproductive state. More recently, Werner (1975), Baskin & Baskin (1979) and Gross (1981) have shown that for a number of biennials a minimum size must be reached before flowering can be induced and above a minimum size the probability of an individual flowering increases directly with rosette size.

This study focuses on the behaviour of rosettes of Ophrys apifera L. over a six year period, with particular reference to the fate of rosettes (flowering or remaining vegetative) relative to their age, size and number of leaves in any particular growing season.

Site details

The study area was a gentle, north-facing slope situated in Com's Field at National Grid Reference (NGR) 52/200795, about 600 m west of Monks Wood Experimental Station. Prior to 1960, this field had been part of a mixed farm.

Introduction

Considerable interest attaches to the control of growth and development of orchid protocorms and seedlings. A series of experiments were conducted which compared the relative merits of different culture vessels for the germination and growth of seedlings of Cattleya aurantiaca and then attempted to identify some of the changes which occurred in culture vessels during growth.

Materials and methods

Procedure used to surface-sterilize and sow seed

Seed was surface-sterilized using 5% commercial bleach (Domestos: Lever Bros., UK) for 1.5 minutes before sowing onto Thompson's medium (Thompson 1977). All vessels were placed in a Warren Shearer growth cabinet at a fluence rate of 142 μmol m–2 s–1, a temperature of 22.5 ± 2 °C and a relative humidity of 90% (Seaton & Hailes 1989). Continuous light was used unless otherwise stated.

Measurement of growth and development

The percentage of seeds germinated was recorded at 14 days and 28 days, and growth was monitored at intervals by measuring the diameter of 50 protocorms, in each of 4 flasks, using a Leitz inverted microscope equipped with an eyepiece graticule. As this parameter gave no information about the development of protocorms an index of development was also employed, which was modified from that of Spoerl (1948). Seedlings were assigned to one of four different developmental stages (Figure 1). The number in each class was multiplied by the class number, and the values for the different classes summed to give the Protocorm Development Index (PDI).

This book is based on the proceedings of a national symposium on orchid conservation, which was held at the Royal Botanic Gardens, Kew, Richmond, Surrey, 12th & 13th November, 1986. It contains a series of articles on orchid conservation from three separate perspectives: in relation to physiology, ecology and management.

The intention of the symposium was to exchange viewpoints and to foster collaboration between scientists involved with experimental physiology and ecology, and members of the various national conservation organisations mainly concerned with management. The subject matter encompassed storage and germination of seeds and pollen, tissue culture, population biology, reserve and living collection management, and international trade regulations.

With this diversity of topics covered in this book it is hoped that it will be a useful starting point for those involved in all aspects of conservation, not just with orchids, providing an outline of the modern methods which are now available to the conservationist.

I would like to express my gratitude to all my colleagues at Wakehurst Place for their support and help in running the symposium. Thanks also to Mrs P. Bloomfield for secretarial services and Mrs J. Peschiera for help in preparing the artwork for the book. Finally, thanks to the contributors for their co-operation throughout, and to the staff of Cambridge University Press for their assistance in the production of this volume.

Introduction

The discovery, about thirty years ago, that orchids could be asexually multiplied by a tissue culture technique (Morel 1960; 1964a,b) has led to an enormous increase in the number of plants, mostly artificial hybrids, in cultivation. Individual clones have been multiplied on a wide scale in many parts of the world either because their flower production can be controlled precisely, to meet heavy seasonal demand, or because of the quality, colour or longevity of their flowers. Since the first successful adaptation of in vitro techniques for the multiplication of Cymbidium clones, many other orchids have been investigated and many selected plants in more than 30 genera have been propagated in this way (Holdgate 1974; Murashige 1974; Morel 1974; Arditti 1977; Sagawa & Kunisaki 1982; George & Sherrington 1984) (Table 1). The reviews of Rao (1977) and Hughes (1981) list nearly twice as many genera but they included reports of orchids grown in vitro from seeds as well as tissue and organ cultures.

More recently it has been shown that, as for other plants, protoplasts can be isolated from the roots, stems, leaf tissue, petals and protocorms of orchids (Teo & Neumann 1978a,b; Pais et al. 1982; Price & Earle 1984; Loh & Rao 1985; Seeni & Abraham 1986) (Table 2). To date orchid protoplasts have been induced to grow and divide in culture but they have not stayed alive long enough to regenerate tissue or protocorms.

Introduction

The project at Kew is concerned primarily with the symbiotic method of raising European orchids from seed. However, asymbiotic sowings have occasionally been made for direct comparison of the relative effectiveness of the two methods, and in an attempt to raise seedlings where the symbiotic method has proved unsuccessful. This paper describes a comparison between asymbiotic and symbiotic germination of three species of Orchis, for which both methods were successful. In addition, the effective asymbiotic methods for germination of rare British species, where symbiotic methods have failed, and the germination response of three orchid species from each of the genera Orchis, Ophrys, Dactylorhiza and Serapias to nine vigorous and eight less vigorous orchid symbionts are reported. The pattern of orchid/fungus compatibility is also discussed in relation to the raising of seedlings beyond initial germination stages, and the routinely used orchid/fungus combinations for bulk propagation of certain species are recorded and illustrated.

Materials and methods

All sowings were made on to agar-based media in Petri dishes using aseptic techniques (Muir 1987). For symbiotic germination the media used were Modified Oats Medium (O3) (Clements et al. 1986) and G4 – a modification of O3, using 1.2 gdm–3 amylopectin in place of the oats, on the recommendation of P. Milon (Laboratoire de Recherches Horticoles, 78570 Chanteloup-les-Vignes, Paris). The media of Harvais (1973), Mead & Bulard (1975), Norstog (1973) and Curtis (1936) were used for asymbiotic germination.