Scanning electron microscopy in the study of plant materials

Micron, 1969. 1:1-14 with XI plates

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Scanning electron microscopy in the study of plant materials V. H. HEYWOOD

Department of Botany. The University, Reading, U.If.

The value of the scanning electron microscope in the study of plant materials is discussed. Applications to wood structure, epidermal features, algae and fungi, soil micro-organisms, seeds and fruits, pollen and spores and palaeobotany are reviewed. The importance of scanning electron micrographs as a means of illastration is stressed and it is suggested that these should supplement conventional taxonomic descriptions. The concept of the microhabitat provided byfruit surfaces is discussed and the term carposphere is proposed for this purpose. New developments are outlined and there is an extensive review of relevant literature.

INTRODUCTION The scanning electron microscope is already proving to be the most exciting and significant tool so far produced for the study and evaluation of surface topography of solids. During the past three years a small number of workers, notably Echlin (1968a), have obtained encouraging results using the instrument in the study of surface features of a wide range of plant materials. Undoubtedly the non-availability of instruments has seriously restricted the number of botanical applications so far, but much work is now in progress and one can confidently predict that within a few years a scanning electron microscope will become as standard a piece of equipment in botanical laboratories as is the transmission electron microscope today. There are several reasons which explain the large potential importance of the scanning technique in the study of plant materials. The main one is that surface features (of seeds, fruits, pollen, fungal spores etc.) still occupy a major role in systematic, morphological and diagnostic botanical work. The restricted magnification range of the light microscope, its limited depth of focus and, despite recent technical advances, difficulties of obtaining adequate photographs, have restricted or prevented accurate representation and interpretation of cellular microtopography or substructure. The scanning electron microscope permits a depth of focus hundreds of times greater than the light microscope and consequently provides a life-like almost three-dimensional image of the object being scanned. Resolution is comparatively low--20nm (200A) being normal with both the commercially available models in widespread use today ° (the J E O L JSM-2 and the Cambridge Instrument Co. Stereoscan Mark 2A) although better resolution (about 15 nm, 150A) can be regularly obtained on both. In practice many objects are scanned at much lower resolutions, particularly at low magnifications where, in any case, resolving power is not such an important factor. This compares with a best resolution for biological materials of 300 nm (3,000A) obtainable with a light micro*There is a third commercial instrument, the Materials Analysis Company model MAC 700 but I have no o n its u s e in practice.

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scope and 1-10 nm (10-100A) with a transmission electron microscope. Detailed discussion of these factors and of the construction, mode of operation and processes of image-formation are given by Oatley et al. (1965), Thornton, (1965), Oatley, (1966) and Kimoto and Hashimoto, (1968). We must consider further the nature of the images obtained with the scanning microscope, since it is due to their obvious information-content that much of the success of the instrument's used in biology is due. As Everhart (1968) puts it, basically the machine "looks" at the actual object and presents the viewer with what is essentially a magnified image of it, so that what one sees is readily comprehensible in most cases (there are exceptions which I shall discuss later). This contrasts with much transmission electron microscopy where interpretation of micrographs is often difficult and requires extensive experience. The normal method of coating the object with a thin layer appears not to obscure appreciably the surface topography. The similarity between image formation as observed directly by size and by scanning is also discussed by Oatley (1966) who points out that hollow regions in objects appear dark while projecting areas cast shadows so that consequently the human eye which is used to the interpretation of two-dimensional pictures in terms of three-dimensions is readily able to interpret the scanning images obtained. An important difference, however, is that in the scanning microscope the image represents a magnified view of what would be seen by looking along the incident beam and, more important, details of structure inside deep crevices or holes are revealed since the very narrow incident beam can produce secondary electrons from the inner walls of such cavities. The wide range of magnification obtained with the S.E.M. is particularly valuable for botanical studies which cover such a diversity of size ranges. At the lowest end of the range, at low kV., it is possible to view objects down to x 10-100 thus overlapping the hand lens and dissecting microscope. This is useful in viewing whole objects such as seeds as is discussed below. The upper limit is x 60-100,000 although satisfactory images have been obtained of suitable plant materials at nearly × 120,000. The various methods of preparing material for examination by scanning have been reviewed recently by Echlin (1968a) and while further techniques are being tried out they will not be discussed in detail in this paper. Likewise, work continues on coating techniques. Most of the work reported has been on robust or fairly robust specimens such as seeds, fruits, fossils, with a low moisture content requiring little preparation other than further desiccation, but much research is in progress on means of preparing labile material for examination. APPLICATIONS Wood Structure

Not surprisingly scanning electron microscopy has been widely used in the study of wood structure in connexion with the pulp and paper industry. A review is given by Rezanowich (1968) who notes that during the past ten years about 25,000 micrographs, mainly of wood or paper surfaces, have been accumulated by the Pulp and Paper Research Institute of Canada. It is interesting to note that about three-quarters of these micrographs are in the magnification range of x 100 to x 1,000, the depth of field providing useful information not otherwise obtainable by optical means or by repficas. Much useful information on vessel and tracheid structure, wall thickening and pit

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structure as well as on the effects of chemical and physical treatment on fibres has been obtained from this applied research. Cell wall structure and pits in Salix and Pinus have been studied by Resch and Blaschke (1968) and structure of bordered pits in soft wood tracheids by Ishida & Ohtani (1969). Various other micrographs of wood structure have been published as "demonstration" pictures. There is also a great amount of unpublished work. Scanning electron microscopy was used by Wattendorf (1969) along with other techniques to study the structure and development ofsuberized calcium oxalate crystals in the bark of Larix. By cutting sections obliquely and by using a tilting or goniometer stage it is possible to examine the internal structure of vessels and other cell elements by "looking" into them directly. This allows one to obtain good information about wall thickening and pit structure. The didactic value of scanning micrographs of wood and its components should not be overlooked: students often have difficulties in understanding wood sections in different planes and relating them to each other, and the apparently three-dimensional micrographs provide readily comprehensible information which is more acceptable than drawn representations or reconstructions.

Epidermalfeatures and Stomata The wealth of features shown by the epidermis has long been used by taxonomists and morphologists. These features include cuticular protuberances, glands, pits, indumentum (hairs, scales), wax, and stomata with their associated cells. A review of the use of cuticular characters in plant taxonomy is given by Stace (1965, 1966) but this does not extend to the use of scanning microscopy, which has an important role to play here as has already been shown by various studies. A preliminary survey is given by Amelunxen et al. (1967) who scanned a range of plant epidermal surfaces and proposed a classification of the multiple forms of wax excretions into six major wax types which although incomplete appears to have some general validity when applied to other samples in this laboratory. In a study of isolated plant cuticles using optical and scanning electron microscopy, Lange (1969) summarised the categories of micro-relieffound and suggested that certain concepts previously proposed on the basis of light microscopy be re-examined. He concluded that the interpretation of surface detail requires direct observation of the surface as such and a two-stage terminology based on transmitted light examination and surface observations. Verdus (1969) has made a detailed study of the upper epidermis of Euphorbia corsica Req. seedlings by both light and scanning microscopy. Scanning revealed, even at low magnifications ( x 110-1500), much clearer detail of the epidermal papillae and wax deposition, than could be seen by light microscopy and the use of cellulose-acetate replicas. The wax covering was seen to be made up of semi-crystalline platelets of a type noted earlier in transmission electron microscope studies. Similar wax platelets have been found to comprise the greater part of the epidermal covering in some 0.uercus species (Heywood, unpublished). Although the various types of hairs and other forms of indumentum have been extensively used in taxonomic descriptions, they have often been superficially or

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incorrectly observed, with the result that there is much confusion of terminology and interpretation. An example of the problems involved in such detailed studies is the work of Cowan (1950) on Rhododendron where different species show a bizarre array of hairs and scales. These are, however, very difficult to observe under the light microscope and in most cases have to be dissected out and removed from the leaf surface. This inability to see the details of the indumentum in situ is one of the main reasons for the inaccurate descriptions of hairs, or failure to describe them in detail at all. It is here that the scanning microscope has unique advantages since fragile hairs can be prepared and examined without appreciable damage or distortion. Scanning of indumentum details can be expected to become a standard technique in taxonomy; not only can accurate information be obtained rapidly with minimum preparation but the micrographs obtained will frequently have sufficient detail and clarity to serve as a permanent record instead of time-consuming, even if not inaccurate, drawings. This point is discussed in detail by Sandberg and Hay (1968) in connexion with the illustration of microfossils. Many epidermal features, including wax, can be studied by transmission microscopy using replica techniques and shadowing, and a better resolution can be obtained than by scanning. Replicas are time-consuming to prepare and need considerable skill; moreover, despite recent developments and techniques, replication is impracticable with deeply indented and complex fragile structures. Clowes and Juniper (1968) discuss this point and note that in comparing the techniques of carbon replication with metal shadowing and scanning on a similar specimen there is no significant difference between them except the higher resolution of the former technique. Plate 78 in their book, of the adaxial leaf surface of Tulipa sp. showing a shadowed carbon replica should be compared with Plate 7b of Amelunxen et al. (1967) of a scanning micrograph of Tulipa gesneriana. Although the replica is clearer and of higher resolution than the scanning micrograph, as Clowes and Juniper point out, the probably solid rods of crystalline wax appear in the former to be hollow tubes while in the latter they are evidently solid. Moreover better resolution by scanning can be obtained than that in this particular scanning micrograph. Many problems concerning the mechanisms of wax deposition (and the chemistry of the waxes) remain to be solved (cf. Clowes and Juniper, 1969: 277-280, Hall, 1967; Eglinton & Hamilton, 1967) and scanning microscopy will be a useful technique to apply in such research. Polystyrene replicas have been used for scanning studies (Chapman, 1967, Echlin and Chapman, 1968) of leaf surfaces with a view to overcoming the difficulty of damaging the specimen during coating. The replicas can be handled and coated without difficulty and show significant detail. Thus, Chapman (1967) has published a scanning micrograph of a polystyrene replica of a salt gland in Limonium vulgare complementing the transmission electron microscope studies by Ziegler and Luttge, (1966, 1967), and by Thomson and Liu (1967). Excellent scanning micrographs of stomata and associated cells have been published by Amelunxen et al. (1967) and by Verdus (1969), the latter showing details of the positioning of the cuticular "beaks," folds and stomatal cells in the open and closed position of the stomatal pore. The problems of investigating stomatal apertures using replication techniques are discussed by Idle (1969) who employed cellulose-acetate reconstructions and silicone-rubber intermediates, viewing them by scanning.

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Comparative scanning micrographs of stomata in contemporaneous and fossil Ginkgo species have been taken in this laboratory and a remarkable similarity between them noted. Opaline silica deposits (phytoliths) of intracellular origin, in Sieglingia decumbens have been studied with the scanning microscope by Sangster (1968). He found that the structural details revealed were in general agreement with the model postulated by earlier workers using light microscope studies, although there appeared to be some variation in surface topography between phytoliths coming from identical cell types.

Algae and Fungi The preparation of algae for examination by scanning microscopy requires special preparation such as freeze-drying. Techniques are discussed by Echlin (1967, 1968a, Echlin and Chapman, 1968) who have published micrographs of various species of Cyanophyta. Much work is in progress at the British Museum (Natural History), and at the Botany School. Cambridge, and papers are in the press. Calcite-secreting fossil algae are illustrated by Dwornik (1966). Various fungal structures have been examined with success. A preliminary note by Jones (1967) considered the surface features of the conidia of Aerimoniella sp. and the hyphae of a sterile fungus. Williams and Davies (1967) used the method for studying surface details ofreproductive and vegetative structures of Actinomycetes at high magnifications. They found that the ease of preparation both obviated disruption due to staining and immersion oil needed for optical microscopy and permitted rapid examination of morphological details of taxonomic importance. They suggested that the scanning electron microscope could be most useful for routine examination and identification of specimens. It also allowed surface ornamentation to be observed at magnifications previously only possible by transmission electron microscopy. The potential value of the scanning electron microscope in association with transmission studies for studying developmental processes was also stressed. Beautifully detailed micrographs of conidiophore branches and conidial scars were obtained by Greenhalgh (1967) in RoseUinia and Hypoxylon spp. (Xylariaceae), supplementing knowledge ofthese features observed by light microscopy. Willetts and Calonge (1969) studied spore development in Sderotinia spp. using both transmission and scanning electron microscopy, illustrating micro- and macro-conidia. Schwinn (1969) surveyed the spore surfaces of various parasitic fungi by scanning electron microscopy and found many unexpected details. A survey of surface structure of fungal spores by scanning is also given by Akai et al. (1969) : they also consider morphological differentiation of the spores during germination and the behaviour of some pathogenic fungi on leaf surfaces. Further examples of ultrastructural surface details of fungal spores given by Jones (1968) and Hawker (1969) included details obtained by scanning electron microscopy in her description of the basidiospores of a species of Hysterangium, complementing features revealed by light microscopy. An elegant study of the wall ornamentation of ascospores in Elaphomyces species as shown by scanning electron microscopy was made by Hawker (1968). Hawker and Gooday (1968) used both transmission and scanning electron microscopy in their study of the development of the zygospore wall in Rhizopus sexualis and discuss the value of these techniques in the light of earlier work with the light microscope. Barnes and Neve (1968) discuss the advantages of using scanning

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electron microscopy over other techniques in the study of the microbial complex at the leafsurface. In a study of Erisyphepolygoni on red clover leaves it was possible to show the relationship of the fungus to the topography of the leaf surface. The authors suggested that it should be possible to build a reference collection of photographs of surface characteristics of microorganisms in situ to aid in identification. The surface structure of yeast cells both untreated and fixed for 1 hour with 5 °/ /0 glutaraldehyde is illustrated by Osumi (1968). The micrographs show an apparently smooth cell, surface and details of the bud scars which will assist in studying the mechanisms of budding. Many other fungi have been observed by scanning e.g. Streptomyces spp. (Hamada, 1969) and there is a great amount of unpublished work. Although special methods of preparation are often necessary before examination, it is remarkable how robust many kinds of mycelia and fungal spores are in resisting collapse and consequent distortion under vacuum. The specimens examined by Greenhalgh, Williams and Davies mentioned above were not submitted to any special treatment prior to coating.

Soils and Soil Micro-organisms Scanning electron microscopy has been used by Gray (1967) to study soil microorganisms in situ together with the topography of individual soil particles. The advantages of the scanning electron microscope in this kind of study are that the micro-organisms neither have to be removed from the soil particles nor sections made. The results were promising and it was possible to recognise various micro-organisms including, apparently, bacteria. Because of dehydration and distortion, the technique has serious limitations which it may be possible to overcome by preparative techniques. Even so, it has provided for the first time pictures of a microhabitat previously unattainable by other direct means. Bacteria in three different soils were viewed by Hagen et al. (1968) with considerable success, although concentration of cells was shown to be critical, the minimum number of micro-organisms required with the scanning technique being between 10~and 10~° cells per gram of soil. The scanning electron microscope is also being used in this laboratory tostudy spatial relations between the particles in various soil materials as an adjunct to pedological and plant physiological research. Details of this work will be published later. Seeds and Fruits Surface details of seeds and small fruits are extensively used in taxonomic and biosystematic studies (Davies and Heywood, 1963:163 seq.) while seed identification is of major practical importance in agriculture, horticulture, pharmacology, forensic studies and several other fields. Identification manuals rely on descriptions, drawings and sometimes photographs: accurate drawings are very time-consuming to prepare and do not always convey a realistic impression of the seeds while most photographs have insufficient depth of focus with a consequent lack of clarity. Scantling micrographs provide a rapid and life-like means of illustrating seeds (Plate II) and will undoubtedly become a routine technique in the future, largely replacing other forms ofiUustration. In addition, scanning often permits the resolution of taxonomically important details not previously

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recorded and may be ofvalue in providing a means of separating cultivars and strains in commercial crops, as preliminary observations have suggested for barley and wheat. Seed coat differences are often used to separate members of polyploid complexes and scanning micrographs may permit more certain identification of infraspecific taxa than optical methods. Differences between the seed coats of two subspecies of Arenaria dliata are illustrated by Echlin (1968a). The Caryophyllaceae in fact shows a very wide range of external features of the seed, including elaiosomes, and these are often related to reproductive capacity, germination differences and other biological problems as many studies have already shown. A detailed survey of the seeds in this family is needed and with scanning electron microscopy now available as a rapid technique it is likely that such a survey will be undertaken. Seed details tend to be imprecise in taxonomic descriptions and it is tempting to think that scanning will lead to much fuller and more accurate descriptions as a matter of routine but there are difficult problems of terminology to overcome before this will be possible, as is discussed below. Most of the above considerations also apply to small fruits. For some years we have been studying fruit morphology in the Umbelliferae as part of wider systematic and phytochemical investigation of the family. Particular attention has been paid to the tribe Caucalidae whose members possess spiny fruits, details of which have been used as the traditional basis for the recognition of genera. Many of these fruits were surveyed by scanning electron microscopy and preliminary results already published (Heywood, 1967, 1968) not only confirmed the taxonomic value of the traditionally used fruit surface details, such as the number of rows of spines on the primary and secondary ridges and their arrangement and structure, but revealed a large number of taxonomically significant microcharacters not previously recorded, such as microhairs on the primary ridges of all species of the genus Torilis examined. Remarkable information on the structure of the fine bristle-like hairs which cover the fruits of the genus Ghaetosciadium has been obtained (Heywood, 1969). Plates IV to IX illustrate very clearly not only the morphological detail of the fruit surface ornamentation of another Umbellifer, Turgenia latifolia, but suggest a number of biological problems which deserve following up. Plate IV gives a panoramic view of the surface and shows how it can be considered as a microhabitat occupied by microorganisms such as fungi, by pollen grains, and by insects and other organisms. This worms' eye view of the microhabitat cannot but be helpful in allowing us to pose more precisely all kinds of questions concerning the biological and selective value of such detailed landscapes in relation to dispersal, germination, infection etc. To what extent such microdetails have a selective value has yet to be decided, but as Stebbins has frequently pointed out, failure to find a selective role for many features is often the result of our own lack of imagination. With such detailed information now available from scanning electron microscopy it should be possible to envisage possible mechanisms much more clearly and thus to devise tests to confirm or deny our hypotheses. Without doubt this new concept of the microhabitat in fruit biology will open up a further dimension for study, analogous in some ways with the rhizosphere concept in soil microbiology or the phyllosphere (Ruinen, 1961). The term carposphereis proposed here for the microhabitat provided by fruit surfaces. The various protuberances (apart from the main spines) indicated in the accompany-

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ing micrographs are cuticular in nature and lie within the limits of a single celt. Despite this, the almost phyllotactic development shown by the apices of the micro-spine-masses shown in Plate VI raises interesting problems for the morphogeneticist as does the developmental detail shown as a whole. These fruit landscapes should provide a rewarding experimental ground for the study of gene action. How far the details of ornamentation are heritable and how far they are the result of environmental influences is not yet known. A major problem that faces one immediately is again that of terminology; no terms exist for most of the microstructures observed although the wax-types have been tentatively classified by Amelunxen et al. (1967) as mentioned above. In our present state of knowledge it would clearly be inadvisable to attempt to devise a terminology although if the features were on a larger scale, they would doubtless be described. In the absence of a terminology, reference has to be made to the micrographs but this has serious disadvantages. This problem is returned to later. Pollen and Spores

Scanning electron microscopy has so far found its widest botanical application in the analysis of the surface sculpturing of pollen grains, both in taxonomic-morphological research and in pollen analysis. Already detailed treatments have been published by Echlin (1968b), Burrichter et al. (1968) and Reyre ( 1969d ), to which reference should be made. Echlin's paper is particularly valuable in that it surveys the whole role of pollen in reproduction, the morphology of the mature pollen grains, and changes that take place in the grain during maturation. Scanning electron microscopy permits us to study the complex surfaces of the grains in unprecedented detail, as Echlin puts it, and has the additional advantage that with the ease of preparation of samples, it is possible to study a large number of specimens in a short space of time. Features of the pollen grain that have been profitably studied by scanning include gross morphology (size, germinal furrows and pores), and fine detail of wall structure, notably the form of the bacula which comprise the ektexine and which are responsible for the great diversity of form shown by the outer layer of the grains. Echlin's paper illustrates a wide range of ektexine surface patterns and discusses the role that heredity and environment play in determining them. Other scanning micrographs of pollen coats have been published by Bortenschlager (1967), Burrichter et al. (1968), Echlin (1968a), Echlin et al. (1966), Erdtman & Dunbar (1966), Fiser & Walker (1967), Godwin et al. (1967), Ockendon (1968), Thornhill et al. (1965), Reyre ( 1968a, b, c, d) and others. Additional examples are illustrated in Plate X. Following their survey of a range pollen various families of flowering plants and spores of Lycopodium clavatum, Burrichter et al. proposed a classification of pollen and spores based on the type of surface ornamentation. They proposed three major classes: (1) Pollen with macrosculpturing, more than 1 ~tm,subdivided into five subclasses, heteroreticulate, foveoloreticulate, echinoreticulate, lacunoreticulate and reticulorugulate, (2) Pollen with microsculpturing, less than 1 ~tm, subdivided into microverrucate, micropapillate, microechinate, microverrucorugulate, microbaculorugulate, (3) Pollen with macro- and micro-sculpturing, divided into reticulate]mucroverrucate, verrucate/microechinate and echinate/microfoveolate.

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They regard such a classification as a useful aid for taxonomic-morphological studies and for practical pollen analysis. A different classification has been proposed by Reyre (1968d) based on a scanning survey of 42 species of Gymnosperms (Pinaceae, Podocarpaceae, Araucariaceae, Taxodiaceae, Cupressaceae, Cephalotaxaceae, Taxaceae, Welwitschiaceae, Ephedraceae, Gnetaceae). His system, which has some similarities to that of Burrichter et al., is illustrated in Fig. 1 of his paper. It comprises three main classes, each of which is divided into a number of types: 1. Sculpture simple (elements all at the same level): rugose, grumous, conicalprickly, embossed, mammilliform, verrucose. 2. Sculpture compound (different elements not forming distinct levels) : pustules with verrucae, mammillate with verrucae, rough with verrucae. 3. Sculpture double (elements forming two distinct levels): with verrucae and glomerules, mammiUate and glomerules, rugose with glomerules. 4. Sculpture grainy (elements arranged like the grains of plaster or rough casting) : heterometric (elements ofvarying dimensions) with balls, isometric (elements all the same size) with verrucae, heterometric with verrucae. (Note: the various terms have been translated literally and are largely self-explanatory). Reyre in this and in other papers (1968 b, c,) stresses the need for accurate descriptions of pollen grains in palaeobotanical research and comments on the inadequacies of morphographic nomenclature established by light microscopy. He found that the detailed descriptions of the exine made possible by scanning electron microscopy allowed a much more precise definition of the palynological species, corresponding to the species or species group of palaeobotanists. In another paper Reyre (1968a) discusses the structure and morphology of the pollen of the form genus Classopollis and indicates the way in which scanning studies have helped to clarify their evolutionary status and relationships. An example of the taxonomic value of scanning electron microscopy in the study of fresh pollen is given by Ockendon (1968) in his work on the Linum pcrmne group. He found that the pores of the grain which are difficult to see with the light microscope are clearly visible in scanning micrographs and that details of the exine which had been previously reported could be analysed in more detail. Thus pollen morphology could be used to distinguish diploids from tetraploids and inbreeders from outbreeders, a fact which is of great importance to palynologists. Moss spores have also been studied by scanning electron microscopy. Bonnot (1967) surveyed the ornamentation of the exospores ofBruchia vogesiaca (Dicranales) with some success and suggested that the technique would be of use in distinguishing between many of the spores previously reported as being similar. Fern spores too have been studied byJermy (1968) in the Dryopteris aemula group and were found to be of value in elucidating hybrid relationships. Further work on fern spores is in progress at the British Museum (Natural History) and elsewhere.

Palaeobotany Various applications of the scanning electron microscopy in palaeobotany work have already been mentioned and a short review is given by Taylor (1968). The value of the

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technique in Quaternary studies needs no stressing. In addition to the pollen examples already given, the paper by Hibbert (1967) on carboniferous microspores should be consulted. Very little work on macrofossils has so far been published but several investigations are in progress. Plate I illustrates a sample of fossil wood showing clearly tracheid structure and thickening as well as fossil fungal spores. New Developments Although the technique of scanning electron microscopy is relatively recent, new developments have been rapid. The present tendency is to regard the scanning electron microscope as a basic unit or module to which other devices such as a transmitted electron detector or an X-ray detector can be linked. An exciting new technique is ion-etching as used in metallurgical work, involving controlled ion bombardment on to the surface of the material being studied. In biological materials this has the effect of removing the outer layers of the cell and revealing its inner structure. It has been applied to red blood ceils which are then examined under the scanning electron microscope (Osborn et al. 1969), and is being used by Echlin to study the different layers of pollen grain walls (Echlin, unpublished). Attention is also being paid to the nature of the images produced by the scanning electron microscope and to methods of computer processing of the information provided. As White et al. (1968) points out, 'the scanning electron microscope raster image is ideally suited to digital recording and subsequent computer processing for rapid quantitative measurement of textural parameters such as grain size, shape and orientation.' Techniques have been devised for ceramic materials and it may prove possible to find some way of applying similar methods to biological material which would permit the large amounts of character information provided by scanning electron microscope images to be more easily handled than by conventional descriptive means, the problems of which have already been alluded to. By means of the transmitted electron detector which can be fitted to both the J E O L JSM-2 & the Cambridge Stereoscan Mk 2 A microscopes, the observation of internal structure of specimens can be achieved as with a conventional transmission electron microscope. The technique has been little applied so far and is described in detail by Kimoto and Hashimoto (1968). Important features are (1) the high contrast and brightness possible at lower accelerating potential than is possible for scanning a specimen of comparable thickness with a transmission microscope, (2) negligible damage due to electron bombardment (3) a different principle ofimage formation so that the sharpness of the image is not related to chromatic and spherical aberrations of the objective lens as is the case in the conventional electron microscope. Comparative images of cotyledonary cells of soybean are given by Kimoto and Hashimoto.

DISCUSSION AND CONCLUSIONS Although development of the scanning electron microscope continues, more people are nowadays concerned with applying it in their own special fields as an established, if not quite routine, technique and with devising new methods of preparing material for examination. That it is now no longer necessary to describe the capabilities of the

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scanning electron microscope in each paper published in which results obtained with the instrument are reported, may be regarded as a sign of its having come of age. The main advantages of scanning electron microscopy for the botanist are: (1) Examination of solid objects (such as seeds, pollen and spores) within the ranges of magnification possible with the light microscope but with vastly better resolution and depth of focus. (2) Examination of solid objects (such as seeds, pollen, and spores) at magnifications beyond the range of the light microscope and still with good resolution and depth of focus. (3) Examination of surface structures at high magnifications with minimum preparation which previously required time-consuming replicas for viewing by transmission electron microscopy or which because of their complicated or delicate nature could not be replicated. (4) Ease of interpretation of the micrographs produced because of the method of image-formation. (5) Provision of detailed, accurate, almost three-dimensional micrographs which are a better form of illustration of many botanical materials than conventional photographs or illustrations. A technique which allows us to see objects in a way that has not previously been possible obviously presents problems and a certain amount of re-education is necessary. We can legitimately talk about the scanning electron microscope opening up a new dimension for us. Micromorphology will now be able to enter into a new phase of development. ACKNOWLEDGMENTS I wish to thank Dr. D. M. Moore for the material illustrated in Plate X; Dr. B. Pickersgill for the seed in Plate III; Mr. R. Polhill for the seed in Plate II; and Dr. R. M. Wadsworth for the fossil wood illustrated in Plate I. My gratitude is also due to Mr. S. K. Irtiza-Ali for his skilled technical assistance and photography. I am also grateful to the J E O L laboratory in Tokyo for assistance with the micrographs in Plates IV-IX. This investigation has been supported in part by the Science Research Council (grant B/SR/2460) and in part by the Agricultural Research Council (grant towards the cost of purchasing the JSM-2 microscope). REFERENCES AKAI, S., FUKUTOMI, M. & SHIRAISHI, M., 1969. Application of a wanning electron microscope to the studies in phytopathological and mycological ficlds.oTe0lNews,7B: 22-25. AMELUNXEN, F., MORGENROTH, K. & PICKSAK, T., 1967. Untersuchungen an der Epidermis mit den Stcrcoscan-Elcktronmikroskop. Z. Pflanzenphysiol., 57: 79-95. BARNES, G. & NEVE, N. F. B., 1968. Examination of plant surface microflora by the Scanning Electron Microscope. Trans. Brit. Mycol. Sot., 51: 811-812. BONNOT, E.-J., 1967. Etudes sur le Bruchia vogesiaca Schw~igr. (Mousses, Dicranales). VI: L'ornementation sporale en microscopic dlectronique A balayagc. Bull. Soc. Bot. Fr., 114: 361-70. BURRICHTER, E., AMELUNXEN, F., VAHL, J. & GIELE, T. 1968. Pollen- und Sporcnuntcrsuchungen mit dcm Obcrfl~iehen- Rasterelektronenmikroskop. Z. Pflanzenphysiol., 59: 226-237.

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CHAPMAN, B. 1967. Polystyrene replicas for scanning reflexion electron microscopy..Vature, 216: 1347-1348. CLOWES, F. A. L. & JUNIPER, B. E. 1968. Plant Cells. Oxford & Edinburgh: Blackwell. COWAN, J. M. 1950. The Rhododendron Leaf. Edinburgh & London : Oliver & Boyd. DWORNIK, E.J. 1966. Use of the scanning electron microscope in geologic studies. U.S. Geol. Survey Prof. Paper, 550-D, D209-213. ECHLIN, P. 1967. The use of the scanning reflection microscope in the study of the algae. Brit. Phycol. Bull., 3: 413. ECHLIN, P. 1968a. The useofthe scanning reflection electron microscope in thestudyofplant and microbial material. 07. Roy. Microscop. Soc., 88: 407-418. ECHLIN, P. 1968b. Pollen. Scientific American, 118(4): 80-90. ECHLIN, P. & CHAPMAN, B. 1968. Scanning reflection electron microscopy of plant surfaces. 4th European Regional Conference on Electron Microscopy: 49-50. ECHLIN, P., CHAPMAN, B., GODWIN, H. & ANGOLD, R. 1966. The fine structure and the development of the pollen in Helleborusfoetidus L. Electron Microscopy, 2: 315. EGLINTON, G. & HAMILTON, R.J. 1967. Leafepicuticular waxes. Science., 156: 1322-1335. ERDTMAN, G. & DUNBAR, A. 1966. Notes on electron micrographs illustrating the pollen morphology in Armeria maritima and Armeria sibirica. Grana Palynologica, 6: 338. EVERHART, T. E. 1968. Reflections on scanning electron microscopy. In: O. Johari (ed.), Scanning Electron Microscopy~1968: 1-12. FISER, J. & WALKER, D. 1967. Notes on the pollen morphology ofDrimys Forst. Section Tasmannia (R.Br.) F. Muell. Pollen et Spores, 9: 229. GODWIN, H., ECHLIN, P. & CHAPMAN, B. 1967. The development of the pollen grain wall in Ipomaea purpurea (L.) Roth. Rev. Palaeobot. and Palynol., 3: 181-195. GRAY, T. R. G. 1967. Stereoscan electron microscopy of soil micro-organisms. Science, 155: 1668-1670. GREENHALGH, G. N. 1967. A note on the conidial scar in the Xylariaceae. New Phytologist, 66: 65-66. GREENHALGH, G. N. & EVANS, 1968. The developing ascospore wall of Hypoxylonfragiforme. J. Roy. Microscop. Soc., 88: 545-56. HAGEN, C. A., HAWRYLEWICZ, E. J., ANDERSON, B. T., TOLKACZ, VIVIAN K. & CEPHUS, M A R J O R I E L. 1968. Use of the scanning electron microscope for viewing bacteria in soil. Appl. Microbiol., 16: 932-934. HALL, D. M. 1967. The ultrastructure of wax deposits on plant leaf surfaces. II Cuticular pores and wax formation. J. Ultrastr. Res., 17: 34-44. HAMADA, M. 1969. Fungi observed through J S M scanning microscope. Jeol News, 711: 26. HAWKER, L. E., 1968. Wall ornamentation of ascospores of Elaphomyces as shown by the Scanning Electron Microscope. Trans. Brit. Mycol. Soc., 51: 493-498. HAWKER, L. E., 1969. A species of Hysterangium from Idaho attributed to H. separabile. Mycologia, 61:115-119. HAWKER, L. E. & GOODAY, M. A., 1968. Development of the Zygospore Wall in Rhizopus sexualis (Smith) Callen. aT. Gen. Microbiol., 54: 13-20. HEYWOOD, V. H. 1967. Plant Taxonomy. London: Edward Arnold. HEYWOOD, V. H. 1968. Scanning electron microscopy and microcharacters in the fruits of the Umbelliferae-Caucalideae. Proc. Linn. Soc. Lond., 179: 287-289. HEYWOOD, V. H. 1969. Fruit structure and the affinities of the genus Chaetosciadium. (in press).

SCANNING ELECTRON MICROSCOPY IN STUDY OF PLANT MATERIALS

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IDLE, D. B. 1969. Scanning electron microscopy of leaf surface replicas and the measurement of stomatal aperture. Ann. Bot., 33: 75-76. ISHIDA, S. & OHTANI, J. 1969. Observation of bordered pits in softwood tracheid made with the scanning electron microscope, aTeolNews, 7B: 3. .JERMY, A. C. 1968. Two new hybrids involving Dryopteris aemula. Brit. Fern. Gaz., 10: 9-12. .JONES, D. 1967. Examinations of mycological specimens in the scanning electron microscope. Trans. Brit. Mycol. Soc., 50: 690-691. JONES, D., 1968. Surface features of fungal spores as revealed in a Scanning Electron Microscope. Trans. Brit. Mycol. Soc., 51: 608-610. KIMOTO, S. & HASHIMOTO, H. 1968. On the contrast and resolution of the scanning electron microscope. In: O. Johari (ed.) Scanning Electron Microscopy]1968: 63-78. LANGE, R. T., 1969. Concerning the morphology of isolated plant cuticles. New Phytol., 68: 423-425. OATLEY, C. W. 1966. The scanning electron microscope. Sci. Progress, 54: 483-495. OATLEY, C. W., NIXON, W. C. & PEASE, R. F. W. 1965. Scanning electron microscopy. Adv. Electronics Electron Phys., 21: 181-247. OCKENDON, D. J. 1968. Biosystematic studies in the Linum pererme group. New Phytol., 67: 787-813. OSBORN, J. S., STUART, P. R. & LEWIS, S. M. 1969. Reported in Scientific Research (McGraw Hill), 4(5) : 14-15. OSUMI, M. 1968. Surface structure of yeast cells, oTeolNews, 6B, No. 1 : 3. RESCH, A. & BLASCHKE, R. 1968. f0ber die Anwendung des Raster-Elektronimkroskopes in der Holzanatomie. Planta, 78: 85-88. REYRE, Y. 1968a. Pr~cisions sur la structure et la morphologie des prfipollens du genre de forme Classopollis (Pflug) Pocock et Jansonins. Consequences pal6obotanique et stratigraphique. C.R. Acad. ScL Paris., 266: 1233-1235. REYRE, Y. 1968b. Valeur taxinomique de la sculpture de l'exine des pollens de Gymnospermes et de Ghlamydospermes. G.R. Acad. Sci. Paris., 267: 160-162. REYRE, Y. 1968c. Valeur taxinomique de la sculpture de l'exine des pollens fossiles attribu~ aux Gymnospermes ou aux Chlamydospermes. C.R. Acad. Sd. Paris, 267: 488-490. REYRE, Y. 1968d. La sculpture de l'exine des pollens des Gymnospermes et des Chlamydospermes et son utilization dans l'identification des pollens fossiles. Pollen et Spores, 10: 197220. REZANOWICH, A. 1968. Some applications of the scanning electron microscope at the Pulp and Paper Research Institute of Canada. In: O. Johari (ed.) Scanning Electron Microscopy/1968: 13-27. RUINEN, J., 1961. The phyllosphore 1. An ecologically neglected milieu. Pl. Soil., 15: 81-109. SANDBERG, P. A. & HAY, W. W. 1968. Applications of the scanning electron microscope in palaeontology and geology. In: O. Johari (ed.). Scanning Electron Microscopy/t968: 29-38. SANGSTER, A. G. 1968. Studies of opaline silica deposits in the leaf of Sieglingia decumbens L. using the Scanning Electron Microscope. Ann. Bot., 32: 237-240. SCHWINN, F. J. 1969. Die Darstellung yon Pilzsporen im Raster-Elektronenmikroskop. Phytopath. ,~., 64: 376-379. STAGE, G. A. 1965. Gutieular studies as an aid to plant taxonomy. Bull. Brit. Mus. Nat. Hist. Bot., 4(1). STACE, C. A. 1966. The use of epidermal characters in phylogenetic considerations. New Phytol., 65: 304-318. TAYLOR, T. N. 1968. Application of the scanning electron microscope in palaeobotany. Trans. Amer. Microscop. Sot., 87: 510-15.

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THOMSON, W. W. & LIU, L. L. 1967. Ultrastructural features of the salt gland of Tamarix aphylla. Planta, 73: 201-220. THORNHILL, J. W., MATTA, R. K. & WOOD, W. H. 1965. Examining three-dimensional microstructures with the scanning electron microscope. Grana Palynologica, 6: 3-6. THORNTON, P. R. 1965. The scanning electron microscope. ScienceJ. November 1965:66-71. VERDUS, M.-C. 1969. L'epiderme cotylddonaire d'Euphorbia corsica Req. C.R. Acad. Sci. Paris, 268: 793-795. WATTENDORF, J., 1969. Feinbau und Entwicklung ver Verkorten CalciumoxalatKristallzellen in der Rinde yon Larix decidua Mill. Z. Pflanzenphysiol., 60:307-347 WHITE, E. W., McKINSTRY, H. A. & JOHNSON, G. G. 1968. Computer processing of SEM images. In: O. Johari (ed.) Scanning Electron Microscopy~1968: 95-103. WILLETTS, H. J. & CALONGE, F. D., 1969. Spore development in the brown rot fungi (Sclerotinia spp.) New Phytol., 68: 123-131. WILLIAMS, S. T. & DAVIES, F. L. 1967. Use of a scanning electron microscope for the examination of Actinomycetes. J. Gen. Microbiol., 48: 171-177. ZIEGLER, H. & LUTTGE, U. 1966. Die Salzdrtisen yon Limonium vulgare. I Mitteilung die Feinstruktur. Planta, 70: 193-206. ZIEGLER, H. & LUTTGE, U. 1967. Die Salzdrtisen yon Limonium vulgare. II. Mitteilung die Lokalisierung des Chlorids. Planta, 74: 1-17.

Die Niitzlichkeit des abtastenden Elektron Mikroskops in der Untersadmg von PflanzHchen Materien. Die Niitzlichkeit des abtastenden Elektron Mikroskops in der Untersuchung von pflanzlichen Materien wird besprochen. Anwendung ftir die Holz Struktur, epidermitale Merkmale, Algen und Pilze, Boden Kleinlebwesen, Samen und Frtichte, Pollen und Sporen und die Paleobotanik wird besprochen. Die Wichtigkeit des abtastendes Elektron Mikrographs als ein Mittel zur Erl~uterung ist betont und es wird vorgeschlagen, dass dieses die gew6hnlichen taxonomischen Beschreibungen erg~inzen soll. Der Begriff des Mikrohabitats versorgt v o n d e r Oberfl~iche der Frucht wird besprochen und der Ausdruck C A R P O S P H E R E ist ftir diesen Zweck vorgeschlagen. Neue Entwicklungen werden umrissen und eine umfassende Literatur Ubersicht von belangvollen Besprechungen ist angegeben.

L'utilisation du microscope~iectronique ~ balayage dans I'~tude des mtitres

v~tales. O n discute de la valeur du microscope ~lectronique ~ balayage dans l'~tude des mati~res vrgrtales. On examine les applications h la structure du bois, aux caract~ristiques ~pidermiques, aux algues et aux champignons, aux micro-organismes de la terre, aux graines et aux fruits, au pollen aux spores, et ~t la pal~obotanique, On accentue l'importance des micrographes 6lectroniques ~ balayage comme moyens d'illustration, et l'on sugg~re qu'ils pourraient remplacer les descriptions conventionnellest axonomiques. O n discute le concept de microhabitat qu'offre la surface des fruits auquel on propose le nom "carposph~re". On expose les grandes lignes de nouveaux drveloppements, et il y a une revue 6tendue de la bibliographic qui se rapporte ~ ce sujet.

PLATE I Fossil wood of Taxus from Methwold Common, Cambridgeshire; probably of subBoreal age. ( X 2,200). The bands of thickening in the traeheids and the pits are dearly visible, Two chains of fungal spores may be observed in one of the traeheids.

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Plate I

PLATE II Seed of Aotus villosa Sm. (Leguminosae)

( × 49)

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Plate II

PLATE III Seed of

(×31)

Physalis ixocarpa Brot. (Sotanaceae)

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Plate III

PLATE IV Fruit of Turgenia latifolia (L.) Hoffm. (Umbelliferae). General view of mericarp surface (part). Prominent ridged and tuberculate spines can be seen on the ridges running at an angle across the field and spine-masses and mammillate tubercles in the curved valley between the ridges ( x 180).

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Plate IV

PLATE V Fruit of Turgenia latifolia (L.) Hoffm. Detail of a single spine mass which can be seen in bottom right of Plate IV. Note the development of papillae on the apex and protuberances ( x 1,800).

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Plate V

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Plate V

PLATE VI Fruit surface of Turgenia latifolia (L.) Hoffm. Surface view looking down on the spine mass shown in Plate V. Note the regular phyllotactic-like arrangement of the papillae on the developing apex and on the lateral protuberances

( x 1,800).

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Plate VI

PLATE VII Fruit of Turgenia ( X 18,000).

latifolia (L.) Hoffm. Detail of apex of spine-mass shown in Plates V & VI

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Plate VII

PLATE VIII Fruit of Turgenia latifolia (L.) Hoffm. Detail of another spine-mass showing prolific develop ment of papillae. Note their tear-drop appearance ( x 5,400).

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Plate VIII

PLATE IX Fruit of Turgenia latifolia (L.) Hoffm. Elongate spine-mass showing similar detailed development to that of Plates V to V I I I but the whole structure elongate and resembling a ridge-spine ( x 5,400).

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Plate IX

PLATE X Pollen grains of Escallonia sp ( × 2,300)

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Plate X

PLATE XI Pollen grains of Saxifraga sp

( x 4,2o0).

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Plate X l