A procedure for standardizing comparative leaf anatomy in the Poaceae . I . The leaf-blade as viewed in transverse section

Descriptive “keys”, including definitions and diagrams, for standardizing and simplifying the description of grass leaf structure as seen in transverse section are given. Over 500 characters are included with the possibility for expansion to 999. Notes on variation and taxonomic importance of the characters are also included.


INTRODUCTION
Anatom ical investigations of the grass leaf-blade have long provided valuable taxonom ic inform ation.In fact, nowadays, it is generally accepted that anatomical details, especially of the leaf-blade and embryo, when used in conjunction with a wide spectrum o f other diagnostic characters, are an essential ingredient of any satisfactory treatm ent of grass taxonom y.Furthermore, in the Poaceae (= G ram ineae), with their highly specialized and reduced flowers, very fine morphological distinctions are often necessary to define differences between taxa.Anatom ical data is, therefore, regarded as being of undoubted importance in the jigsaw of complete systematic evidence in this numerically large and im portant family.
The im portance of anatomy in agrostological studies has resulted in the rapid accum ulation of an extensive body o f literature with attendant problems of lack o f uniform ity with definitions and descriptions.Valuable data is, therefore, commonly not applicable to the family as a whole and comparisons cannot be drawn with any degree of assurance.This problem was greatly ameliorated by the publication in 1960 of Anatomy of the Monocotyledons.1. Gramineae by C. R. Metcalfe and the present paper is a further attem pt to stabilize this terminology, and, at the same time to present a system whereby description and com parison of grass leaf anatom y will be simplified and standardized.

DESCRIPTIVE KEYS
In an attem pt to achieve the necessary uniformity, descriptive " keys" have been compiled for use as a framework for anatomical descriptions of the grass leaf-blade as viewed in transverse section.The " keys" , which incorporate both definitions and diagrams, have been designed to enable the user to standardize descriptions and the hierarchical layout has been chosen to facilitate the speed and ease at which complete, comparative descriptions can be compiled Anatomical characters, and all other information considered to be of diagnostic or taxonomic impor tance, and gathered from an extensive survey of the relevant literature, have been included.The keys should, therefore, prove adequate for all tribes of the Poaceae except for the Bambuseae where additional data on the fusoid cells must be included to satis factorily distinguish between certain genera.
The hierarchical tabulation of the characters has been used to expedite their use but they do not in any other way conform to any acknowledged key format or design.However, if a statement, at any level of the hierarchy, does not apply to the specimen being examined, no subsequent statements or characters of a lower rank are relevant.The user, therefore, merely proceeds to the next character of equal rank or indentation, and, if applicable, works inwards noting all the relevant numbered end points before working back outwards until the same level or rank as that of the originally chosen statement is reached.Thus, the " keys" are not dichotom ous or true indented keys, but by using this type of format all the possible characters are recorded in a constant descriptive sequence.Each character is assigned a constant num ber and the recording of these numbers effects a saving in time and space.Furtherm ore, this system enables easy conversion to edge punched and feature cards as well as for electronic data processing by computer.In addition, it is ensured that all possible structures are rapidly and routinely noted in a rational sequence which simplifies compilation of descriptions of the various taxa.By employing a standard sequence significant differences become more readily evident.
In order for the standardization of terminology and descriptions to be effective it is essential that com parative material be examined.Therefore, in this study, all descriptions refer to transverse sections taken at a point about halfway between the blade apex and the ligule of mature basal leaves.Flag leaves on flowering culms were avoided where possible.By standardizing on the material studied in this way intraspecific differences may be assessed.

OUTLINE OF PERMANENTLY INFOLDED LEAVES
211* -Two furrows on either side of median vascular bundle only .
219* Grooved at a p e x .No second order bundles present in section .The following presents a brief conspectus o f the nature of the various structures used in describing grass leaf anatomy, as well as their range of structural variation.Where relevant, an account is given of the function or development of these structures and the factors which contribute to their variability.Their importance as either taxonomic or diagnostic characters is stressed.

Outline o f the lamina in transverse section
The foliage of the grass plant is comprised of the sheath, the ligule and the leaf-blade.In the following account, the leaf-blade, the expanded and free portion of the leaf as distinct from the sheath, is termed the lamina or the blade.

Outline o f the lamina of open leaves
In many grasses with open leaf-blades the two halves of the lamina on either side of the median vascular bundle, midrib or keel, are relatively thin and wide, of equal width and usually symmetrically arranged about the median region.Infrequently, however, in certain species the portion of the lamina to one side of the midrib may not be symmetrical with that on the other half or perhaps the leaves may be relatively narrow and rather thick.These are exceptions to the general rule but are nevertheless useful for indentification purposes.
The lamina of most grass leaves in transverse section appears flattened and expanded and a straight line connects both margins and the median bundle or keel.However, expanded laminae may become variously altered by involution usually by becoming inrolled or infolded.Infolding results in the appearance becoming V-or U-shaped because the two lateral halves of the lamina tend to become folded adaxially towards each other on either side of the median vascular bundle which may, or may not, be adaxially grooved or incorporated into a keel.Grooves or keels serve to accentuate the V-or U-shape which characterizes many species.Inrolled leaves may appear variously U-shaped or may show either convolute inrolling from one margin only, or involute inrolling where the leaf-blade rolls inwards from both margins.
The degree of infolding or inrolling varies with the environmental conditions and thus is not of much value diagnostically (Metcalfe, 1960).However, leaves of grasses that are fully expanded under optimum conditions invariably exhibit a characteristic type of involution in response to environmental stress.Therefore, it is the nature, and not the degree, of the infolding or inrolling that is of importance taxonomically and diagnostically.Furthermore, it must be noted that some leaves remain flat on drying and in others the leaf folds inwards a little near the keel but the margins remain reflexed.Other responses are shown by the corrugations becoming more pro nounced in species with plicate blades, and the pro duction of filiform leaves in species which normally possess expanded blades.Further details of the involution process are included under bulliform and colourless cell function.
Expanded leaves of different species thus exhibit various, but constant, movements in response to adverse environmental change, the resultant shape being of specific or generic diagnostic value.Various members of the Andropogoneae (Hyparrhenia, Cymbopogon and Dichanthium) often show limited infolding near the keel with the margins remaining reflexed and several species of Cymbopogon produce both expanded and filiform leaves (Vickery, 1935).
Some Brachiaria and Oplismenus species do not alter shape at all under conditions o f water stress and remain flat and expanded.Certain grasses even show nyctinastic movements as described for Leersia hexandra by Goebel (Bor, 1960) in which the leaves are folded in dull weather but flatten in bright sunlight.
The variability exhibited by the leaf outline dem onstrates the necessity for a sampling method which will adequately reveal the extremes o f this variation.This should be achieved if adequate numbers of specimens o f each species are collected, and fixed immediately, from different areas, over an extended period of time and from as many habitats as possible.
It appears reasonable to postulate that the type of folding adopted by the m ature leaf in response to environmental conditions, as well as the symmetry of the leaf, may be correlated to the way in which the leaf is folded in the bud.Thus, Amedei (1932) has shown that Chaetochloa palmifolia (=Panicum palmifolium) is folded conduplicately in the bud and the m ature leaf is more or less pleated depending on circumstances.The blades of grass leaves may be either folded, convolute or pleated in the bud (Bor, 1960).Therefore, the bud arrangem ents may simply be linked to the m ajor types o f involution exhibited in m ature leaves except, perhaps, involute inrolling which is difficult to explain.Does it happen then, that a leaf which is convolute in the bud will exhibit definite V-shaped movements as a m ature leaf instead of the expected convoluted inrolling?This possibility merits further investigation.
A part from the characters of the shape of the outline, under optimum and adverse conditions, quantitative data o f leaf width and thickness are also of value in describing and identifying leaves of different grasses.De W inter (1951) mentions the extreme thinness (about 0,05 mm) of the leaf of Prosphytochloa prehensilis ( =Potamophila prehensilis) where the leaf is only about four cell layers thick.These quantitative characters are subject to considerable variation, and categories must be broadly defined to accommodate this variability.Detailed measurements have not been included in the keys and measuring is done by estim ation of the leaf dimensions in relation to the known diam eter o f the field o f view of the microscope used in the study.These broad categories are, nevertheless, useful for com parison with measurements given by other authors and can be easily modified as required.

Outline o f permanently infolded leaves
In many grasses the lamina is very strongly and often permanently infolded or involuted.In such leaves the stom atal bands are invariably restricted to certain areas of the adaxial surface, usually on the sides of adaxial furrows within the channel.A thick cuticle and extensive sclerenchyma development is characteristic although this is not universally true as shown by Lewton-Brain (1904) for Mibora minima (=M ibora verna) which is entirely parenchym atous.However, these leaves can be distinguished by the fact that they are extremely narrow and permanently infolded to the extent that the internal structure is altered.
Num erous leaf-types defined on m orphological criteria fall into this group.Examples are setaceous (wiry and bristle-like), filiform (thread-like), acicular (needle-like), junciform and cylindrical leaves.Anatomically these different types cannot be separated with any degree o f certainty and a term inclusive of all permanently infolded leaves is required.
Acicular leaves invariably fulfill the requirements of the definition of being permanently infolded to the extent that the internal structure is altered.In filiform leaves, often produced under adverse conditions by species which normally have open, expanded leaves, however, the blade is reduced to an enlarged midrib structure, with a mass of colourless parenchyma above the bundles and the lateral laminae being reduced or absent.Some species of Cymbopogon exhibit this reduction regularly.The extreme case of this specialization is seen in Miscanthidium teretifolium which is cylindrical in transverse section and the adaxial surface can be recognised only by the presence of a minute groove on the adaxial side of the cylinder (Metcalfe, 1960).In these cases, it must be stressed, the internal structural alteration of the leaf does not occur as a result of the leaf being permanently folded but rather as a result of loss of the lateral portions of the blade and a corresponding development of the midrib.
The cylindrical condition can probably arise by one of two different processes: the loss of the lateral regions and a concurrent development of the keel or by parenchyma development in a permanently infolded leaf excluding the adaxial channel.For convenience these two types of narrow leaf are included together under permanently infolded leaves.This further illustrates the need for a single term describing all reduced, permanently infolded and/or structurally altered grass leaves.
Transverse sections of these leaves often exhibit characters that are of taxonomic value because in assuming the permanently infolded, acicular form, the leaves of grasses of different affinities have not acquired precisely the same arrangement of vascular and mechanical tissue (Metcalfe, 1960).The outline and the shape of the adaxial channel and ribs and furrows may be distinctive as well.Burbidge (1946a) was able to separate ten species of Triodia, all possessing acicular leaves, using characters of the outline, the adaxial channel and adaxial and abaxial furrows.For descriptive purposes it is, therefore, necessary to describe the shape o f the blade as outlined by the abaxial surface, to count the number of vascular bundles present in the section, to describe the adaxial channel and to include leaf width and thickness dimensions.Ribs and furrows and sclerenchyma are described in later " keys" .With these descriptive criteria the differences present in permanently infolded leaves are of significant specific diagnostic value.
It is absolutely essential to standardize on the portion of the blade to be sectioned in permanently infolded leaves as the shape can vary from the base to higher up the leaf.Arber (1923Arber ( , 1934) ) noticed that folded leaves may approach radial symmetry and become cylindrical in the apical regions.In Triodia (Burbidge, 1946a), towards the apex of the lamina the lateral veins disappear one by one, commencing with the marginal pair.There is a corresponding progressive reduction in the am ount of mesophyll so that finally the pungent point is formed o f the median vascular bundle surrounded by sclerenchyma.This progressive change, of many characters, along a single blade is equally im portant in open, tapering leaves and is the prime reason for standardizing on material from the central region of the lamina from ligule to tip.
In the cases o f some narrow, folded leaves it is often debatable whether they in fact should be described under open or under permanently infolded leaves.In such cases it is perhaps safest to describe them under both o f these arbitrary categories.

Longitudinal ribs and furrows
The adaxial and abaxial surfaces of the grass leaf blade may be either flat or longitudinally ribbed.The ribs are usually developed in association with and adjacent to the larger vascular bundles.They are generally characteristic of and more fully developed on the adaxial than the abaxial surface (Metcalfe, 1960).Ribs have also been termed ridges (Arber, 1934;Wilson, 1971) and if ribs are present there must be corresponding furrows between adjacent ribs.Furrow, in the context used here, is a term reserved exclusively for the depressed intercostal zone between adjacent ribs and which, with few exceptions, occur between the vascular bundles.If furrows are present in any other situation they are termed grooves to avoid possible confusion.Examples are the grooves present at the apex of the well-developed ribs of Sporobolus artus (Goossens, 1938) or those described immediately abaxial to the larger veins described by Sabnis (1921).In permanently infolded leaves the area bounded by the infolded lamina is characterized by being referred to as a channel.
In different species ribs and furrows may vary in height or depth, transverse shape, spacing and location.The ribs of a single grass blade can be of one or more distinct sizes or shapes, the ribs of each type being consistently associated with a specific order of vascular bundle.It is possible to measure the inclination of the ribs by measuring the angle formed by the sides of the two adjacent ribs at their base in the furrow (Wilson, 1971).Consequently, leaves with obtuse angles would have flatter surfaces than those with acute angles.The abaxial ribs may sometimes be taller than those of the adaxial surface.Where welldeveloped abaxial and adaxial ribs and furrows are similar and occur opposite each other the leaf section resembles a string of beads and is termed moniliform.
There are numerous examples of species of grasses possessing ribs or furrows that do not conform to the generalized definition outlined above.Lewton-Brain (1904) illustrates the enormous triangular ribs of Aira caespitosa where three vascular bundles are included in a single rib.Glyceria fluitans, another example illustrated by Lewton-Brain (1904), possesses low adaxial ribs situated between the vascular bundles which are situated beneath the 'furrows'.The ribs are occupied by copious air spaces.The flat-topped abaxial ribs of Digitaria macroglossa are exceptional because the ribs are in fact sclerenchyma caps and are not linked to the vascular bundles in any way.Metcalfe (1960) comments on the correlation between prominent ribs and a marked capacity for involution, and states that this ability has been evolved in response to ecological conditions.Furtherm ore, this has occurred independently in various grasses and con sequently leaves of this type are found in grasses between which there are no close taxonomic affinities.Rib development is nevertheless, often useful for specific diagnostic purposes and G ordon-G ray and W ard (1970) have contrasted the consistent flatness of the leaf surfaces of Phragmites mauritianus with the slight ribs developed on com parable surfaces of leaves of the closely related P. australis.
A paper by Wilson (1971) shows that the adaxial ribs o f Lolium perenne appear to be under the control of a few genes as indicated by the rapid and immediate response to selection achieved in this study.Thus, in a species which appears to exhibit a wide range of phenotypic variation with respect to rib development, this variation may in actual fact be the direct mani festation of the genotype.This fact should be borne in mind when variation patterns of adaxial ribs are studied.

Median vascular bundles, midribs and keels
In order to eliminate any possibility of ambiguity arising from the use of these terms it is necessary to define them in the context that they are to be used here.The median vascular bundle is so termed only if the centrally situated vascular bundle of the leaf is structurally indistinguishable from the other basic or first order vascular bundles of the leaf, and if this median bundle is not associated with any parenchyma development or thickening of the leaf.On the other hand, a midrib is centrally positioned but structurally distinct from the other first order bundles with respect to size, bundle sheath, sclerenchyma girder or vascular structure and lacks associated parenchyma.When parenchyma or bulliform cells are developed in association with the median bundle or bundles, the whole structure, often incorporating many bundles, is termed a keel.The median bundle of a keeled leaf may be a midrib, and thus distinguishable from first order bundles, or a median vascular bundle, being indistinguishable from the other first order vascular bundles.A keel usually projects either abaxially, adaxially or both, but this is not necessarily so and in V-shaped leaves, unless there is a marked thickening in relation to the rest of the lamina, the median bundle alone is considered to comprise the keel, if it is accompanied by parenchyma.
This distinction of the three distinct types is con sidered necessary because in the past these terms have been interchanged by various workers.Metcalfe (1960) considers a projecting midrib, whether associated with parenchyma or not, to constitute a keel.Thus, no distinction is made between leaves with or without parenchyma in association with the median vascular bundle.
The size of the keel in an individual leaf varies, usually becoming progressively larger towards the base of the blade so that, in transverse section, the extent to which a keel is prominent depends on the level at which the section is taken.Therefore, the central part of the leaf must be used for comparative purposes. Vickery (1935) states that, in addition to variation at different points along a single leaf, the size and degree o f development of the keel varies tremendously in different leaves of the same species, so that the appearance in transverse section can only be of importance when taken in conjunction with the macroscopic appearance of the keels of a number of leaves.This is definitely not necessary in some groups and Fisher (1939) found the keel-size, am ount of parenchyma and sclerenchyma and the bulliform cells-to be a valuable diagnostic character in separating four species of Chloris.
Keel structure, although subject to variation, is im portant diagnostically because the various types of keel that are recognised are easily distinguished except that the inconspicuous keel with one vascular bundle merges into that with three bundles.Thus, size may vary but shapes are constant and distinguisable.In addition there are numerous associated characters such as sclerenchyma, parenchyma and bulliform cells of the keel, air spaces and the nature of the adaxial side o f the keel.
Bambusoid and oryzoid grasses display distinct keel structure with prominent lacunae, with or without diaphragms (Holm, 1896), and with a complex system of bundle arrangement.The keel is comprised of two or more vascular bundles distributed near both the abaxial and adaxial surfaces and sometimes in the interior as well.This characteristic structure has been discussed by various authors such as Holm (1892, 1895, 1896), Arber (1934), Jacques-Felix (1955), Schweickerdt & M arais (1956), Tateoka (1965) and Launert (1965).In addition to the Bambusae & Oryzeae, the Streptochaetae and Prosphytochloa prehensilis ( = Potamophila prehensilis) in South Africa (de Winter, 1951) possess superposed adaxial bundles in the keel.Air spaces developing in the keels of older leaves are common in many other genera.Sporobolus artus var.lysigenatus and S. pyramidalis (Goossens, 1938) are examples.

Vascular bundle arrangement
The vascular bundles, or parallel longitudinal veins, are usually arranged in a single row embedded at various positions in the mesophyll as seen in section.
Certain grasses, such as Oryza coarctata (Tateoka, 1963) and Porteresia (Tateoka, 1965a) have a norm al abaxial vascular bundle in each rib, but in addition, a superposed, adaxial, amphivasal bundle is situated immediately anterior to each o f the abaxial bundles.This type of arrangement is also found in the keels of some bambusoid and oryzoid grasses.
The arrangement most commonly found in grasses* is for all the bundles to be centrally positioned in the vertical plane of the blade.However, all bundles may be located closer to the abaxial or to the adaxial surface.Irregular arrangements, with bundles of different sizes or orders situated at different levels within the mesophyll are also found, In these instances, a regular pattern o f arrangem ent of the different orders of bundles in the mesophyll is usually dis cernible.Cases where the bundles are located at irregular and inconsistent levels are rare, such as Stipa tenacissima (Metcalfe, 1960).This positioning of the vascular bundles in the blade appears to be a useful diagnostic character above the genus level that has been largely overlooked in the past.
The total num ber of vascular bundles in a section through a leaf blade taken halfway between the sheath and apex is inconsistent, varying with the width of the leaves.Little reliance can be placed on the total num ber of vascular bundles in a leaf as Vickery (1935) correctly stresses.She found that Themeda avinacea and Cymbopogon exaltatus may have flat expanded leaves 5-7 mm wide but under certain conditions filiform leaves consisting essentially of an enlarged keel are formed.This reduction in the width of the blade involves a reduction in the num ber of vascular bundles.W ider leaves do not necessarily have more vascular bundles than narrower leaves, however, as the bundles may be more or less crowded in different specimens o f the same species.The total number of vascular bundles in a section is only considered to be useful in permanently infolded leaves, where there has been a reduction in the total num ber of vascular bundles.
The relative proportion and the alternation of the various sizes of vascular bundle along leaves o f any one species of grass is remarkably constant.Slight variations in this respect may be found in leaves taken from different levels on a single plant, the variation being linked with differences in the width of the blades.For this reason basal leaves have been standardized upon for comparison.However, the proportion of first order bundles and smaller bundles in grasses belonging to different genera and species show marked differences.
The distribution patterns o f bundles of different orders, and their variation, are im portant diagnostic characters.Thus, Holm (1891a) found a pattern of 1 first order bundle, 1 third order bundle, 1 second order bundle, 1 third order bundle, 1 first order bundle, etc., in Uniola latifolia.He notes that this formula is not strictly constant but that it gives the general features concerning the relative num ber and arrangement of bundles.For other species of Uniola he was unable to give a formula because of excessive variation, especially in leaves from different localities.Leigh (1960Leigh ( , 1961)), in a study of a few Eragrostis curvula strains distinguished two groups both with a basic pattern of 3 or 4 second order bundles between successive first order bundles repeating itself either four or eight times along the width of the leaf.
The use o f a formula to describe the pattern of arrangement o f the various orders of vascular bundle is only suitable when a regular pattern from median bundle to margin is present.Often no pattern is obvious with progressively fewer first order vascular bundles and more third order vascular bundles nearer the margin or more first order vascular bundles and fewer second and third order vascular bundles laterally situated.For this reason the use o f formulae to describe these patterns is not suitable.On the other hand, if a constant part of the blade is described, e.g. the central part of the lamina between the median bundle and the margin, these observations on bundle alternation will be comparable, even for leaves where the pattern changes from median bundle to margin.
Vascular bundle structure Metcalfe (1960) visualizes the vascular bundle as consisting solely of the xylem and phloem elements, the bundle sheaths not being treated, for descriptive purposes, as if they constitute part o f the bundle.For consistency and convenience this principle has been adopted in the present work and thus, when a bundle is described as being circular or angular in outline, these terms refer only to the outline of the xylem and phloem tissue.
The vascular bundles of the Poaceae are divided into three orders or ranks for descriptive purposes.These orders are not necessarily distinct but may intergrade and intermediate types are sometimes present.However, within a given leaf, three, and sometimes even four, classes o f bundle are usually evident even although their structure may not con form exactly with that of the generalized definitions given below.
The three different orders in a single leaf are usually immediately evident because they differ in size.However, the relative diameters of the various orders cannot be used satisfactorily for the grass family as a whole, because equivalent orders of bundle differ in size and structure from one species to the next. Bowden (1964) classified bundles using the diameter as a criterion, but in species where all bundles are approximately the same size, but structurally different, this system is inadequate.
The first order bundles, or basic type (Metcalfe, I960) are characterized by having a metaxylem vessel on either side of the protoxylem which may be non functioning, and replaced by a lysigenous cavity or lacuna.A few well-developed protoxylem vessels may be present in addition to the lysigenous cavity, or both cavity and vessels may be absent.In the second order bundles the xylem and phloem are easily distinguishable, but the large metaxylem vessels are lacking as is the lysigenous cavity.The third order vascular bundles are often small bundles in which the xylem and phloem is reduced to a few elements or may even be indistinguishable using light microscopy.
In first order bundles the metaxylem vessels may appear to be paired, but these are merely the oblique overlapping ends of otherwise solitary vessel elements.Thus the num ber of metaxylem vessels in an individual bundle is unreliable as a diagnostic character because it varies with the level at which the sections are taken (Metcalfe, 1960).For this reason, only the size and shape of these vessels are mentioned in the descriptive keys.
The phloem of the first order vascular bundles may exhibit varying degrees of sclerosis.This is especially common in grasses from dry localities (Metcalfe, 1960).In Triodia (Burbidge, 1946a) and Merxmuellera ( -Danthonia) (de Wet, I960) the phloem may be divided into two or three groups by intrusion of small fibres.The phloem may adjoin the inner or parenchyma, bundle sheath or it may be completely surrounded by thick-walled fibres which jsolate it from the xylem and the bundle sheath (Jefferies, 1916).
Bundles of the second order commonly resemble the first order bundles in shape and size and are only recognisable by the absence of the metaxylem vessels.Some authors have pointed out that second order bundles may show a tendency to develop into first order bundles (Vickery, 1935;Goossens, 1938).
In third order vascular bundles the metaxylem vessels are always lacking and in the smallest bundles of this order the xylem and phloem elements may be indistinguishable.These bundles, in fact, form a reduced conducting system and sometimes only a few lignified cells are present together with the phloem elements. Fisher (1939) notes that in these instances neither the protoxylem nor the metaxylem is present.In some genera the third order bundles are not conspicuously smaller than the basic type bundles, but are then distinguishable by the absence of sclerenchyma girders, or even strands, and/or the presence of bulliform cells adaxially.
The shape of the third order bundles may be classified as angular, inconspicuously angular, circular or elliptical in outline.They exhibit these shapes more clearly than do the larger bundles.This shape is taxonomically an im portant character, linked with the size and number of bundle sheath cells.Thus, small, angular bundles are, on the whole, characteristic of the panicoid, and non-angular bundles of the festucoid grasses.
Various classifications of the orders of vascular bundles employ the extent of development of the sclerenchyma strands and girders.Thus, Breakwell (1914, 1915) distinguishes primary bundles, in which the sclerenchyma is in direct contact with the bundle, and secondary bundles which possess an entire bundle sheath. Bowden (1964) also groups first and second order bundles as those possessing sclerenchyma girders and third and fourth order bundles as those with strands.As was pointed out above, the nature of the sclerenchyma developed in association with the various orders of vascular bundle is especially im portant in recognizing the larger types of third order bundle, but as bundles are defined here, bundle sheaths and sclerome are not included in vascular bundle descriptions.
Surprisingly few detailed studies have been con ducted on the phloem of the Poaceae and these only recently.Thus, the mature phloem of Avena was studied by O'Brien & Thinman (1967), Buvat (1968) studied the sieve elements of Hordeum and Singh & Srivastava (1971) the phloem of Zea.
These studies have shown that the first-formed protophloem elements are located abaxially within a bundle and subsequent sieve elements differentiate successively in an adaxial direction to within a couple of cells of the differentiating xylem elements.Thus, in a mature bundle, all the major features of I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION differentiation of the sieve elements and companion cells are visible.The sieve elements of the protophloem often lack companion cells and may differentiate without lateral expansion.The later-formed sieve elements, as a rule, are accompanied by one or more com panion cells and extent laterally as a first step in differentiation.Furtherm ore, the protophloem is usually thin-walled and may become crushed in m ature bundles.The metaphloem has thicker walls and may even be thick-walled as in Distichlis stricta (Roy, 1969).
The xylem consists of primary xylem only.In first order bundles it can be divided into the protoxylem, which is that area in which the tracheary elements have either annular or helical thickenings or both, and metaxylem, which is the remaining primary xylem.This metaxylem can be sub-divided into the early and the late metaxylem; the late metaxylem being the two large vessels so characteristic of these bundles.
The structure of these late metaxylem vessels is an indication of their specialization. Cheadle (1955specialization. Cheadle ( , 1960) ) has shown that specialization in the Angiosperms proceeds from long vessel members, that are angular as seen in transverse section, and with long scalariform plates with many bars and perforations on oblique end walls, to short vessel members that are circular in section, with simple plates, with single perforations on transverse end walls.In the Poaceae no great variation occurs in the late metaxylem which is very specialized.Thus, simple plates are common and scalariform plates rare.A character that is variable, and revealed in section, is whether or not the vessel members are circular or angular.

Vascular bundle sheaths
In the Poaceae every vascular bundle is surrounded, either completely or partially, by one or two single layered bundle sheaths.These bundle sheaths are taxonomically very im portant, and as a general rule single sheaths are characteristic of the panicoid and double sheaths o f the festucoid grasses.Structural variations of the bundle sheath are also im prrtan t for diagnostic purposes, as well as constituting evidence for im portant physiological differences between the various groups of tribes of the Poaceae.As would be anticipated in a tissue undergoing extensive changes, even within genera, to almost every generalization there are exceptions.Notwithstanding, the bundle sheath is undoubtedly of the utm ost importance from a comparative anatomical viewpoint and fortunately numerous ultrastructural, physiological and taxo nomic studies of the structure and functions o f the sheath have been undertaken.
Recent terminology, to avoid reference to physiolo gical activities and anatomical structure, as well as to eliminate the concept of specific functions for the two layers (Lommasson, 1957), has been restricted to the terms double (comprised of an inner and outer) and single sheaths.Various other terms have been used in the past, but none of them are applicable to the grass family as a whole.
The single sheath, and usually the outer sheath, when there are two, commonly consists of a single layer o f large, thin-walled or slightly thickened cells.In section they are generally inflated and conspicuous, being larger than the adjoining mesophyll cells.Panicoid grasses, in general, have more inflated single o r outer sheath cells than those o f the outer sheath o f the festucoid species (Metcalfe, 1960).This sheath has often been referred to as the parenchyma bundle sheath but other terms include starch sheath, border parenchyma as well as mestome sheath.None of these terms are generally applicable, as for example Aristida species have an inner bundle sheath con sisting of larger cells than the outer sheath, which may be composed of thickened cells.
The typical single or outer sheath cells are either translucent, without chloroplasts, or they may contain green pigment in plastids similar to, or differing from, the cholorplasts of the chlorenchyma of the mesophyll.Many early workers ignored the contents of these sheath cells and represented them as being empty (Lewton-Brain, 1904;Lohauss, 1905;Prat, 1936;Vickery, 1936).However, the nature of these contents, whether typical chloroplasts or specialized plastids is undoubtedly a character of systematic importance as Brown (1958) correctly stresses.In this connection, it is im portant that in older leaves of wheat few or no plastids are present in the bundle sheaths (Percival, 1929).Thus, age of leaf sampled must be standardized for this character to be reliable.The detailed structure and function of these bundle sheath cells will be elaborated on later.
The arrangement of the chloroplasts in the bundle sheath cells has been used for diagnostic purposeswhether they display a horse-shoe shaped arrangem ent against the inner or outer tangential wall, or an even distribution in the cells.However, Roth (1968), has shown in xerophytic species such as Sporobolus virginicus that the horse-shoe shaped arrangements occur under low-intensity illumination, such as in the mornings, afternoons, at night and in rolled up leaves, while on sunny days the chloroplasts are evenly arranged all over the walls.This is probably an adaptation to efficient light utilization, but cannot carry much weight as a diagnostic character.
The single or outer sheath may possess girder-like extensions which are of diagnostic importance.The extensions either consist of colourless cells resembling those of the bundle sheath or there may be a gradual transition to the sclerenchyma fibres o f the adjacent strand.The extensions may be uni-seriate to very wide, and sometimes flank narrow sclerenchyma strands or girders as in the first order bundles of Garnotia scoparia (Tateoka, 1958).Burbidge (1946) and Decker (1964) discuss the unique outer bundle sheath of Triodia leaves where the sheath of one bundle is continuous with those of adjacent bundles.
The adjacent sclerenchyma girders may interrupt the single, or outer, sheath to a greater or lesser extent, either adaxially, abaxially or both.Thus, the outer or single bundle sheath may be entire, horse-shoe shaped or even reduced to two lateral arcs.In Arundinella leptochloa (Tateoka, 1958) this sheath is only developed adaxially adjacent to the xylem, not being replaced by a sclerenchyma girder abutting on the lateral parts o f the phloem.Schweickerdt (1941) has used the num ber of cells comprising this sheath to distinguish the closely related genera o f Monelytrum and Tragus.This cell num ber appears to exhibit excessive variability in most instances, but it is relatively constant for the third order bundles of groups such as the Paniceae.
Various degrees of possible reduction o f the vascular bundles, as perhaps evidenced by relic bundle sheath cells have been observed in Arundinella (Vickery, 1935) and in Garnotia (Tateoka, 1958).In these grasses the first order vascular bundles have well developed outer sheaths, Garnotia even having abaxial extensions.In smaller bundles these extensions are reduced, and only a few cells, resembling those of the bundle sheath, are irregularly present on the sclerenchyma girders or strands.The extreme situation is found between successive vascular bundles o f some Arundinella and Garnotia species, where solitary, or groups of two to six cells, similar to those of the bundle sheaths are present, scattered in the mesophyll. Tateoka (1958) calls these groups of sheath-like cells, lacking associated vascular tissue, distinctive cells. Carolin, Jacobs & Vesk (1973) have shown that these distinctive cells are in fact isolated files of parenchyma sheath cells connecting with the bundle sheath proper.They consider this to be a specialization to the wide expanses of mesophyll between adjacent vascular bundles found in these species.These distinctive cells ensure that all mesophyll cells are ultimately in contact with the bundle sheath parenchym a cells.
The inner sheath normally consists o f living, thickwalled, chloroplast-free cells which are elongated parallel to the vascular bundles.In section the cells of this sheath are nearly always smaller in diameter than the outer sheath and possess thickened walls, the thickening often being more conspicuous on the radial and inner tangential walls.Exceptions to these generalizations do occur as in Aristida, Elytrophorus (Schweickerdt, 1942) which has a thin-walled inner bundle sheath, and the Meliceae (Decker, 1964) where the inner sheath cells are uniformly thickened.
This sheath often resembles an endodermis and it has been shown to have similar physiological activity (van Fleet, 1942).It has been variously called the endodermis or mestome sheath in the past.Unfor tunately, the term mestome sheath, has been used for parenchym atous sheaths as well and modern authors (Metcalfe, 1960;Barnard, 1964), therefore, refer to it merely as the inner sheath.
The inner sheath is sometimes difficult to recognise, especially in cases where the cells are not distinct from those o f the fibres of the vascular bundle ground tissue.However, cells of this layer are always in contact with the metaxylem vessels (Vickery, 1935).Thus, if large inflated cells are immediately adjacent to the metaxylem vessels it can be assumed that no inner sheath is present.
In addition to sometimes not being sharply differentiated from the bundle ground tissue, in some smaller bundles the inner bundle sheath is sometimes only developed adjacent to the phloem (Fahn, 1967).For this reason Metcalfe (I960) proposed the intro duction o f a third bundle sheath category-inter mediate-in addition to single and double sheaths.
The inner bundle sheath cells o f Aristida are anom alous in two ways: the diam eter of the inner bundle sheath cells is greater than those of the outer sheath, and they contain numerous chloroplasts.These larger inner sheath cells may also have uniformly thick walls.Holm (1901) was the first to record this double bundle sheath, Vickery (1935) interpreted this condition correctly and Lommasson (1957) considered it to be of taxonom ic importance, so much so as to w arrant the placing o f Aristida in a separate tribe. Theron (1936) attached little taxonom ic im portance to the sheaths of Aristida, probably having misinterpreted the specialized bundle sheath.Lommasson (1957) was further able to show that, in the leaf sheath, where both bundle sheaths lack chlorophyll, on the adaxial side against the culm, the cells o f the outer bundle sheath were larger than those o f the inner bundle sheath.The transition to the opposite condition, as in the leaf blade, occurs where chlorophyll is formed in the bundle sheath cells and mesophyll.He advanced the explanation that this anomalous condition represents the result of special activities occurring at the chlorophyllvascular interface.
The structure and function of the parenchyma cells of the outer, or single sheath, and their dimorphic chloroplasts have been extensively studied. Zirkle (1929) recognised that the cells of the bundle sheath in Zea contained specialized chloroplasts concerned with starch storage.In 1944, Rhoades and Carvalho reported that in panicoid grasses, such as Zea and Sorghum, the single parenchyma sheath contained large specialized plastids, functioning in starch formation and storage.In the outer parenchyma sheath cells of the festucoid grasses, as represented by barley, wheat and oats, they noticed the plastids to be smaller than those in the mesophyll.In these species the plastids of the chlorenchyma, as well as those of the bundle sheath cells, form starch.
Rhoades and Carvalho further demonstrated that in festucoid grasses the mesophyll cells photosynthesise and accumulate starch during daylight.In these grasses the outer parenchyma sheath cells have numerous plastids which, however, are smaller than those of the mesophyll.Small amounts of starch, lying in the central region of the plastid were found in the sheath plastids as well as those of the mesophyll.
In Zea and Sorghum the chlorenchyma cells photosynthesise, but do not store starch at all.Thus, no trace of starch was found in the chloroplasts even if abundant starch was present in the bundle sheath plastids.The plastids of the single sheath appear to elaborate, and temporarily store starch, but have little photosynthetic activity.These workers showed, that the starch found in these plastids was derived from soluble carbohydrates made in the mesophyll plastids, and translocated to the sheath cells where starch synthesis occurred, and not from sugars synthesised in the plastids of the bundle sheath, which could be capable o f photosynthesis since they contain a green pigment.
This transformation of soluble carbohydrates to starch in the bundle sheath plastids occurs only when the rate of movement o f sugars into these cells is greater than the translocation from the bundle sheath cells into the vascular bundles.Starch, which accumulates throughout the day, is transformed back into soluble carbohydrates during the night, so that by morning the bundle sheath plastids are devoid of starch. Roth (1968) considers the possibility that these cells function in water storage as well, especially in xerophytic species.
Grasses with this specialized type of bundle sheath structure and function have been termed the eupanicoid subtype of the panicoid subfamily (Brown, 1961).The other subtype, the chloridoid grasses have a dark green parenchymatous sheath and a single layer of pale green radial chlorenchyma.Here the sheath cells photosynthesise in addition to accumulating starch. Brown (1961) postulates that the chlorenchyma may have assumed some unknown function to replace the activities of photosynthesis and starch storage which it has more or less lost. Otieno (1967) is of the opinion that the radiating mesophyll cell layer, of these chloridoid grasses, constitutes part of the bundle sheath which is then composed of three layers.This is not acceptable because all intermediate types between irregular and radiate chlorenchyma are found.
The starch-free bundle sheath plastid has numerous, small, colourless areas, resembling vacuoles, contained within a peripheral rim.It is in these vacuolar-like regions that starch is deposited.Thus, each plastid I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION contains numerous simple starch grains embedded in, but protruding from, the surface of the plastid.
Ultrastructural studies of these dimorphic chloro' plasts of the panicoid grasses, by Laetsch & Price (1969) on Saccharum, Downton & Pyliotis (1971) on Sorghum bicolor and Andersen et al . (1972) on Zea show, that the bundle sheath chloroplasts loose their grana during ontogeny.Young bundle sheath chloro plasts have well-developed grana, but mature plastids are agranal whereas mesophyll chloroplasts contain grana irrespective of their developmental stage.They conclude that the structure of the specialized chloro plasts in bundle sheath cells is a result of reduction and that this chloroplast dimorphism is a specialization of labour.There are various degrees of reduction in the sheath chloroplasts.Zea, Coix and various members of the Paniceae and Andropogoneae have rudimentary grana and Carolin, Jacobs & Vesk (1973) have shown that the Eragrostoideae in fact have well-developed grana. Johnson (1964) suggests the possibility that the degree of specialization of these chloroplasts has phylogenetic significance.
The chloroplast-containing sheath cells contain, in addition, a remarkably high concentration of additional cellular organelles, such as mitochondria, endoplasmic reticulum and perioxisomes, but vacuoles are difficult to locate.The differences in the ultrastructural features of the cells of the bundle sheath and those of the chlorenchyma further suggest the existence of differences in the functions of these cells (Dobychina, 1970;Carolin, Jacobs & Vesk, 1973).
This detailed discussion of the chloroplasts and the bundle sheath has been considered necessary because in recent years this aspect has received considerable attention from the point of view of the Kranz syn drome.Thus, grasses with bundle sheaths containing specialized chloroplasts are typical of the high ph o tosynthethic capacity grasses and those with no, or normal chloroplasts are characteristic of low photo synthetic capacity grasses (Black, 1971).
At present the advantages of highly developed sheath cells still remain unclear. Black (1971) infers that the anatom y of the high photosynthetic capacity plants results in more rapid rates of translocation, and perhaps higher concentrations of translocates, which may both prevent a feedback type of inhibition o f photosynthesis by a product such as starch.N utrients may, in addition, be supplied to non photosynthetic parts o f the plant such that high growth rates are facilitated.
The possibility of the inner bundle sheath, or the single sheath, when the inner sheath is lacking, having functions and characteristics similar to an endodermis has received considerable attention in the literature.In grasses with a single bundle sheath, the cells of this sheath do not have the anatom ical characteristics associated with an endodermis, such as Casparian strips, thicker radial and inner tangential walls or suberised walls.This is applicable to a num ber of inner sheaths as well.
Van Fleet (1950) demonstrated, with a variety of histochemical reactions, however, that the cells of these sheaths share common substance reactions and may be induced to develop typical endodermal characteristics.Thus, normally the single sheath cells contain chlorplasts and do not exhibit characteristics of an endodermis.But in variegated and albino leaves, or in etiolated leaves, Van Fleet showed that those areas lacking chlorophyll show characters of a typical inner bundle sheath, i.e. endodermal characteristics.
In Pennisetum villosum and Oplismenus hirtellus he further showed that casparian deposits are developed on the radial walls prior to the unilateral desposition characteristic of the mature inner sheath.Endodermal characteristics, therefore, appear at varying stages in the development of the leaf in at least some of the cells of an atypical inner sheath.Thus, in any sheath bounding the vascular tissue, some or all, attributes of a true endodermis can be detected.Van Fleet points out that the endodermal characteristics are always more pronounced opposite the phloem than opposite the xylem.Once again for comparative work it is essential to standardize upon leaves o f equivalent developmental stages.
Schwendener (1890) stated that the walls of the parenchymatous sheath of Zea contained suberized lamellae, something Van Fleet (1950) was not able to show for normal maize leaves.O 'Brien & C arr (1970) also described a suberized lamella, which is probably the site of suberin deposition, in the walls of the inner sheath of Triticum and Avena as well as in the parenchyma sheath of maize.The suberized lamellae modify the plasmodesmata of the num erous pit-fields connecting the cells of the inner sheath with those of the outer sheath and vascular parenchyma, or between cells of the single parenchyma sheath.
Using these ultrastructural details O 'Brien & C arr (1970) propose a function for the inner bundle sheath and a further function, in addition to photo synthesis and starch storage, for the single sheath.These workers are of the opinoin that the suberized lamellae are relatively impermeable to water and thus there is the possibility that in grass leaves water loss is regulated at the vascular bundle as well as by the stom ata.This control of water loss at the vascular bundles is probably essential for an adequate supply of solute to be maintained to the leaf tip, because, under conditions of water stress the lower part of the blade could conceivably transpire all the water available to the leaf.If the suberized lamellae restrict passive loss of water from the vascular bundles to the mesophyll, forcing the water to follow a symplastic route through the sheath cells, the water flow across the sheath could be regulated by the activity of the sheath cells.
Further circumstantial evidence for this possibility is that transfer cells, which are believed to help regulate solute exchange between tissue systems, are absent from the leaves of grasses.In dicotyledon leaves these transfer cells are thought to assist exchange between xylem and phloem, and between these and the adjacent mesophyll.O 'Brien & C arr reason that if passive loss of water is restricted from the vascular bundles because of the suberized lamellae, and if the bulk of solute transfer must pass through the plas modesmata, then transfer cells are unnecessary.

Sclerenchyma o f the leaf
In the grass leaf blade, the sclerenchymatous tissue, or the sterome, includes all fibres as well as other thick-walled cells in certain instances.The scleren chyma is commonly found in association with the vascular bundles, with the midrib or keel and in the margin.
Xylem and phloem fibres, and thickened vascular parenchyma, constitute part of the ground tissue of the vascular bundles.The mestome, or inner bundle sheath, is also fibrous, the fibres appearing, in some instances, ontogenetically the same as those of the hypodermal sclerenchyma, but differing in that they possess numerous pits which are lacking in the true sterome (Artswager, 1925).This tissue is included in the description of vascular bundle structure and will not be discussed further here.
The sclerenchyma associated with the vascular bundles is in the form of sub-epidermal longitudinal bands following the course of each vascular bundle.In transverse section this circumvascular sclerenchyma may surround, be in contact with, or may be situated above or below, but not connected to the bundle or it's sheath.This sclefenchyma tissue is termed a strand when it does not extend sufficiently deeply into the mesophyll to make contact with the bundle sheath cells (Metcalfe, 1960).These strands, therefore, appear in section as small hypodermal " islands" of thickened tissue situated above and below, or on one side only of each vascular bundle.When the inner face of a group of fibres is in contact with, disrupts, or envelopes the bundle sheath, it is termed a girder (Metcalfe, 1960).It resembles a girder in transverse section, extending from either or each epidermis to the bundle sheath.When girders are continuous from the vascular bundle to the epidermis on either side it is termed an I-beam construction (Gould, 1968).
All distribution patterns of the circumvascular sclerenchyma occur either as strands or girders.In addition, preliminary observations in the present study suggest that a further distinction between girders in contact with, and those disrupting the parenchymatous sheath, may be useful taxonomically.
In leaves possessing both strands and girders, the former are usually associated with the third order vascular bundles and the latter with larger bundles.Other leaves may have strands or girders only developed in association with all the vascular bundles of the leaf.The distribution and arrangement of the sclerenchyma associated with the bundles is, therefore, useful diagnostically.Furtherm ore, the am ount of sclerenchyma present varies from species to species (Metcalfe, 1960).This can be misleading, however, because marked intraspecific variation will be found in specimens from different localitities or from different seasons as the work of Burduja & Toma (1970) on Deschampsia flexuosa and other species has shown.They found that on flowering tillers there was less sclerenchyma in the leaves than on non flowering tillers where the fibre walls were also thicker with the lumen smaller.Taxonomically sclerenchyma distribution is seldom of more than specific diagnostic importance.
The distribution of sclerenchyma associated with the vascular bundles can be correlated with ecological factors as well.Grasses from arid areas are thus characterized by well developed sclerenchyma tissue while many tropical grasses often have a high proportion of the smaller bundles not accompanied by sclerenchyma.
The function of all the sclerenchyma present in the leaf is undoubtedly to provide mechanical support for the softer tissues.The development of scleren chym atous tissues makes possible the withstanding of the physical stresses and strains imposed on an elongated, straplike leaf such as is predominant in the Poaceae.An additional function of the fibres is seen by their frequent silicification, especially in older leaves (Parry & Smithson, 1964) and they, therefore, can act as a depository for excessive silica.Silicified fibres result in elongated, pointed needles of silica.
The thickening of the fibre cell walls is usually by lignification, but in many specimens the staining reaction varies across a single sclerenchyma girder, or in girders from different parts of the lamina.In these cases, when stained with safranin and fast green, there is a gradation from typically red-staining fibres to structurally similar fibres which attract the blue or green dye. Hoefer (1941-42) also found variations in the reaction of fibres in different parts of the leaf of Stipa tenacissimci when treated with lignin stains.In addition, lignin tests with phloroglucin and hydro chloric acid and tests with Maules's reagent did not give identical results.Hoefer's tests showed, in addition that there is zonation in the degree of lignification in different layers of the cell wall of individual fibres as seen with transverse section.It appears, therefore, that the degree of lignification varies, but in addition, lignins of various chemical constituents appear at present to be grouped under the general term " lignin" .
In certain grasses sub-epidermal longitudinal strands are found between successive vascular bundles, either alone or in addition to the circumvascular strands or girders.These supernumary strands are usually abaxial and situated opposite the bulliform cell groups or furrows (Lewton-Brain, 1904).When sclerenchyma is found between the bundles in expanded leaves it is of special importance diagnos tically.
Continuous abaxial hypodermal bands are found in certain species, especially those with acicular leaves.These bands may be a regular, narrow strip, 2-4 cells deep, located immediately beneath the epidermis, or may result from lateral extensions of the fibrous tissue comprising strands or girders.
Mechanical tissue of the leaf margin may occur in the form of a cap or hood.When the marginal sclerenchyma is not in contact with the lateral bundle, it is termed a cap, and a hood is formed when this sclerenchyma extends above or below the lateral bundles.The fibres of the hood may, or may not, be in contact with the bundle sheath of the ultimate bundle, but always extend inwards from the margin, as far as, or further than, this bundle.This lateral fusion with the sterome tissue associated with the lateral bundles forms the hood-like structure (Goossens, 1938).In some instances, there is no sclerenchyma development at the extreme margin but the ultimate and penultimate lateral bundles may be intimately associated with specialized sclerenchyma development.Immediately interior to the fibrous cap may be situated normal mesophyll cells, small groups of enlarged, colourless parenchyma cells or a lateral intercellular duct may be developed.
Sclerenchymatous tissue is also found developed in association with midribs and keels.This has been described together with the median vascular bundles, midribs and keels.
In reduced, permanently infolded leaves the distribution of the sclerenchyma follows that outlined above in basic pattern, but appears symmetrical and distinctive because of the nature of the leaf blade.In these leaves this arrangement of the mechanical tissue is of taxonomic importance because, in assuming the permanently infolded form, the leaves of different grasses have not acquired precisely the same sclerenchyma tissue arrangements (Metcalfe, 1960).

Mesophyll
In the Poaceae, the term mesophyll is generally applied to the ground tissue occupying all the space in the leaf not occupied by the vascular bundles, the bundle sheaths and the sclerenchyma.The mesophyll can be subdivided into the assimilatory chlorenchyma I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION and the colourless parenchyma which consists of translucent cells often in close association with the bulliform cells.For this reason further details of colourless parenchyma will also be found under the discussion of bulliform cells.
Grass leaf blade chlorenchyma seldom exhibits a distinct differentiation into pallisade and spongy regions as seen in transverse section.In some species the adaxial chlorenchyma cells are more regularly and vertically arranged than the remainder.Metcalfe (1960) correctly stresses that this distinction is rare and at best unclear.Statements in the literature often imply that there is greater contrast between these zones than there actually is.
In dicotyledons, as Watson (1942) has shown with Hedera helix, the English ivy, the leaves respond to strong light by the production of pallisade tissue.On the same plant shade-grown leaves have no pallisade tissue at all.These cell differences are caused by factors affecting the processes of vacuolation and enlargement of the cells.Thus, the uppermost layer of mesophyll cells has an increased osmotic value due to a change in the starch-sugar ratio.This, according to Watson (1942), results in the absorbtion of water, increased vacuolation and expansion, with the resultant formation of pallisade tissue.
In the Poaceae, the above does not appear to apply and in certain instances the opposite may be the case.In the genus Panicum, the shade-loving forest species often have the upper layer of chlorenchymatous cells more or less vertically arranged in a pallisade manner in contrast to most other species of this genus where the assimilatory tissue is more of less radiate.
The arrangement of the chlorenchyma cells appears to be of fundamental taxonomic significance.Thus, in the festucoid type, the chlorenchymatous tissue is more or less homogenous without being arranged in any definite pattern in relation to the vascular bundles.In grasses o f the panicoid type the assimi latory cells are arranged in a radiating manner around the vascular bundles as seen in transverse section.Each bundle is, therefore, situated in the centre of a regular circle, or partial circle of chlorenchyma.The former irregular arrangement is, in addition, generally associated with leaves in which the vascular bundles are widely spaced, whereas the radiate condition and closely placed vascular bundles are correlated (Vickery, 1935).
The division o f the Poaceae into two sub-families with radiate or non radiate chlorenchyma is an oversimplification. Brown (1958) shows that within the panicoid grasses the Chlorideae and related tribes have the regularly radiate condition, whereas this radiate condition is less regular in other panicoid genera such as Andropogon and Panicum.Thus, the radiate condition is of more than one kind, and grasses with partially or incompletely radiate chloren chyma also occur, as Metcalfe (1960) stresses.Nevertheless, the distinction between radiate and non-radiate chlorenchyma is im portant taxonomically provided that the limitations are recognized.For example, both conditions may occur in the same genus, as in Sporobolus where many species have regular, elongated chlorenchyma cells radiately arranged around the bundles, but in S. panicoides the cells are irregular in shape and arrangement (Goossens, 1938).
Early workers correlated the non-radiate condition with grasses from temperate regions, whereas radiate assimilatory tissue was linked with tropical grasses.
The geographical pattern is not so distinct, however, with many exceptions and Metcalfe (1960) remarks that the mesophyll arrangement is of more funda mental significance taxonomically than geographically.
In transverse section the individual chlorenchyma cells of most panicoid grasses appear elongated, narrow and tabular in shape.Their long axes are at right angles to the bundles.In festucoid grasses the cells are often irregular in shape and size.In some species, such as Poa annua, the largest cells are found nearest the vascular bundles (Bobrov, 1955) or the chlorenchyma cells abutting on both epidermides may be somewhat elongated and regular as in Triticum vulgare (Hayward, 1948).The irregular condition may be characterized by small, isodiametric cells tightly packed together or by irregularly shaped cells with many intercellular air spaces between them.The largest intercellular air spaces are commonly seen subtending the stom ata and projecting deeply into the mesophyll. Pool (1923) illustrates such distinct sub-stomatal chambers in Andropogon furcatus.Slade (1970) in a study of sun and shade-grown leaves of Poa alpina has shown that in sun leaves the mesophyll cells are more or less isodiametric and not longitudinally elongated as in shade leaves.The intercellular spaces are relatively inconspicuous in sun leaves but extensive in shade leaves as seen in longitudinal section.Slade also demonstrated that in transverse section there was no noticeable change in the horizontal widths o f the intercostal zones of chlorenchyma between adjacent vascular bundles, but that the depth or thickness of the mesophyll was greatest in sun-grown leaves.This is as a result of reduction in cell size and intercellular spaces in shade leaves seen in transverse section in contrast to the situation in longitudinal section above.
The overall effect of these anatomical changes in sun and shade leaves is to physically weaken the leaf blade.Slade (1970) has shown, in addition, that in low light intensity the cell walls are thinner and a well defined cuticle may be absent.
It appears, therefore, that the light intensity under which a leaf develops has a profound effect on the arrangement o f the assimilatory cells into pallisade and spongy mesophyll, especially in dicotyledons (Watson, 1942), as well as the size and shape o f the individual cells as shown by Slade (1970).The applicability of these findings to the chlorenchyma cell structure and arrangement in the Poaceae as a whole does not appear clear at present.Thus, if the radiate and non-radiate conditions are of funda mental phylogenetic significance it appears unlikely that major differences of tissue arrangement will occur under varying light intensities.Some generalizations do seem to apply and most forest dwelling species examined are characterized by very thin mesophyl tissue.Prosphytochloa prehensilis is an extreme example with the whole leaf being only four cell layers thick (de Winter, 1951).
Intercellular air spaces occur between the chloren chyma cells of many grasses belonging to tribes of both the major subfamilies.These spaces may be very conspicuous in hygrophilous species (Arber, 1934;Vickery, 1935).In other aquatic species there are, in addition, distinct air cavities or lacunae present.All the species of Elytrophorus have these lacunae situated between adjacent vascular bundles (Schweickerdt, 1942) and in Vetiveria they occur over the smaller bundles (Kanm athy, 1969).The lacunae are traversed by colourless aerenchyma cells which are often stellate in shape and represent diaphragms in the air cavities.Kanmathy (1969) reports sclerotic strands interspersed in the stellate cells.
Cavities developing as a result of breakdown of parenchymatous tissue, especially in the region of the keel, and in the keel itself, are found in many grasses.These differ from lacunae in being indistinct with the breakdown of the cells continuing.This type of cavity has been used diagnostically for Molinia caerula (Lewton-Brain, 1904) and Sporobolus artus var. lysigenatous (Goossens, 1938).
A further modification of the mesophyll tissue is found in certain markedly infolded leaves where the chlorenchyma is confined to small bands adjacent to the adaxial and abaxial cleft-like furrows which contain the stomata.The tissue between the groups of assimilatory tissue is made up of colourless parenchyma.This unique type of anatom y is dis cussed by Burbidge (1946Burbidge ( , 1946a) ) for Triodia species.Burbidge believes that this reduction of the chlorenchymatous tissue results in a corresponding narrowing of the width of the furrows and consequent pro tection o f the stomata. De Wet (1956) found localized bands o f chlorenchyma cells around the abaxial grooves in species of Merxmuellera ( =Danthonia) and suggests that this may indicate relationships.
In some species, groups or isolated, bundle sheath like cells with chloroplasts, but lacking associated vascular tissue are found scattered in the mesophyll.These are termed distinctive cells (Tateoka, 1958) and are described under vascular bundle sheaths.
Bamboos, and a few other grasses, are characterized by the presence of fusoid cells in the mesophyll of their leaves.These cells have been termed enlarged parenchyma cells (Page, 1947) and in transverse section have a fusiform or pyriform outline, being elongated transversely and alternating with chloren chyma cells and the vascular bundles.Adjacent fusoid cells may be separated by a single vertical column of chlorenchyma or many chlorenchyma cells may separate them.The bulliform cells are located imme diately adaxially to these chlorenchyma cells separating adjacent fusoid cells.
The fusoid cells are, in fact, narrow and plate-like with the long axis of the cells lying at right angles to the long axis of the lamina.Longitudinal sections of the blade show these cells to have very narrow lumina in transverse section, the cells having collapsed in such a way that the tissue resembles a row of " I's" .Between the dead, collapsed, mature cells there are large spaces which connect with the inter cellular spaces of the chlorenchyma.In certain species the fusoid cells fail to collapse, but become rounded and separated from one another.The regularity and form o f these collapsed cells seems to indicate that some force operates on the whole leaf at the same time (Page, 1947).
In addition, the Bambusae, as well as the Oryzeae, are characterized by having chlorenchyma com prised o f what, in the grass anatom y literature, have become known as arm cells.These have also been called irregularly lobed or cleft cells (Page, 1947) or plicate mesophyll (Esau, 1960).These so-called arm cells possess inwardly directed projections or folds which may be continuous across the cell as seen in section and completely divide the cells into elongated compartments.The infoldings may end blindly in the lumina as well.These projections may be developed from the upper or the lower wall of the chlorenchyma cell, or from both the upper and lower, or from all walls (Brandis, 1907).Chih-Ying Wu (1958) has shown that in some bam boo species the arm cells located above the fusoid cells have projections from the lower walls, whereas the cells below the fusoid cells and cavities have folds projecting from the upper walls.This phenomenon is mentioned by Arber (1934), but in the Oryzeae, even when fusoid cells are present, the arm cells illustrated by Tateoka (1963) have infoldings from all directions and of equal length.
It is not certain how these projections arise.They could be infoldings of the cell walls as an illustration of Carolin, Jacobs & Vesk (1973) indicates, but in some grasses their appearance suggests that they arise as fine partitions that at first traverse the cell completely, but subsequently become broken as the cells become enlarged (Metcalfe, 1960).Haberlandt (1884) called these " armpalisadezellen" and explained the signi ficance of the infoldings as increasing the inner surface area of the cell, thereby creating space for more chloroplasts.Infoldings are common in many grass genera, and are termed arm cells or peg cells, and in the bamboos and their relatives it is possible that the pegs become " fused" , eliminating the air spaces.
Fusoid and arm cells are im portant taxonomic characters.Thus, fusoid cells are especially charac teristic of the Bambusae (Metcalfe, 1960), and when they occur in other genera, such as Oryza (Tateoka, 1963), affinities between these genera and the bamboos may be indicated.Arm cells, although characteristic of the Bambusae and Oryzeae, may also show variations in their occurrence.Thus, Oryza tisseranti is exceptional amongst the Oryzeae in not having arm cells (Jacques-Felix, 1958).Gordon-Gray & Ward (1971) noticed mesophyll cells with invaginated walls in Phragmites and suggest that this may reflect closer relationships to the Bambusae and Oryzeae than was previously thought possible.The extreme case is that o f Saccharum, where Merida (1970) found cells with internal folds situated below the stomata in some varieties.This apparent occurrence of arm cells in a member o f the Andropogoneae is certainly surprising.
Bambusoid and oryzoid grasses are very poorly represented in South Africa.For this reason, the accompanying descriptive key is not concerned w ith the details of arm and fusoid cells to any great degree.As presently constituted it cannot be expected to differentiate between the various bam boo genera.
The colourless parenchyma constituent of the mesophyll, especially that intimately associated with the bulliform cells is described, together with these bulliform cells, under the description of bulliform cells.

Bulliform and colourless cells
Bulliform cells are single, translucent cells, or groups of colourless cells, constituting part of, or the entire epidermis, but differing from other epidermal cells in being larger and more inflated.These cells occur most commonly, but not exclusively, at the bases of adaxial furrows (Metcalfe, 1960). Shields (1951) restricts the term bulliform cell to inflated epidermal cells present in the adaxial furrows.However, structurally similar cells may be present in the abaxial epidermis as well as, or instead of, the adaxial epidermis.O ther terms used to describe these cells are hinge cells and m otor cells, but the I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION descriptive term bulliform, denoting their inflated appearance, is preferred to these terms referring to their disputed function in leaf involution.
Bulliform cells often form longitudinal, parallel, intercostal bands, and are commonly associated with underlying groups of structurally similar cells, which usually form vertically elongated units in the meso phyll when viewed in transverse section.Shields (1951) designated these associated cells, hinge cells.Here they are grouped with the colourless cells, a term incorporating all the translucent cells present in the mesophyll excluding bundle sheath cells devoid of chloroplasts, fusoid cells of bamboos and epidermal cells.The bulliform and associated colourless cells may appear in transverse section as deep girders, or may be restricted and superficial.The colourless cells associated with the bulliform cells are variable in shape and size, but all colourless cells, whether associated with the bulliform cells or not, are always without chloroplasts.
Bulliform cells have long been recognised as valuable taxonomic characters.As early as 1907, Brandis reviewed the distribution of bulliform cells in the Poaceae as a whole.The major character subdivisions he used were the presence of bulliform cells in the adaxial epidermis only, the presence of bulliform cells in both adaxial and abaxial epidermides, and the absence of distinct bulliform groups.The adaxial bulliform cell distribution was further sub divided into those grasses where bulliform cell groups alternated with all vascular bundles, those where the bulliform cells were present between successive first order bundles, but located over the third order bundles, those with bulliform cells on either side of the midrib and at the margins, those with bulliform cells only located on either side, or above the midrib.Using these distributional criteria of bulliform cells Brandis (1907) found uniform distribution in the tribes Bambuseae and Maydeae only.In all the other tribes studied various genera had different bulliform cell arrangement in some of their species.
The arrangement, frequency, distribution, as well as the relative size and shape of the bulliform cells and colourless cells are of taxonomic importance especially at the specific level.Thus, in Elytrophorus, Schweickerdt (1942) found the presence of conspicuous bulliform cells flanking the midribs only, to be the the oustanding diagnostic character of E. africanus, whereas in E. interruptus bulliform groups are present throughout the leaf width, but decrease in size towards the margin.Furthermore, in one species, the shape of the individual bulliform cells in trans verse sections is rectangular, whereas in the other species they are trapezoidal with the outer wall being shorter than the inner wall.In Leersia hexandra there are groups of bulliform cells between all the vascular bundles and on both epidermides but other Leersia species have only two groups of bulliform cells on either side of the midrib (Holm, 1895). Goossens & Theron (1934) found the bulliform cells to be of significance in classifying the different varieties of Themeda triandra.The arrangement of the colourless cells in relation to the bulliform cells can be of specific diagnostic importance.In Imperata cylindrica (Vickery, 1935), where the bulliform cell groups occur over third order bundles, two rows of colourless cells are produced towards the abaxial surface, one on either side of each bundle.
The function of the bulliform cells has been the subject of much controversy in the literature and a totally satisfactory explanation of their function has, as yet, not been forthcoming.Duval-Jouve (1875) was the first to describe these cells and believed that the opening and inrolling of the blade resulted from changes in the turgor pressures of these cells.Some workers, such as Breakwell (1915), have carried this even further by postulating that rolling occurs only at times of water shortage and results in the leaf becoming tubular, thus protecting the greater number of adaxial stom ata from excessive transpiration.This teleological viewpoint is not supported by the fact that many grasses, including drought resistant types which exhibit inrolling, have more stom ata on the exposed abaxial surface than the protected adaxial surface (Vickery, 1935). Hayward (1948) mistakenly states that the outer surfaces of the bulliform cells are not cutinised, and thus readily lose water through diffusion.Metcalfe (1960) points out that in many grasses exhibiting involution the outer wall o f the bulliform cells may be considerably thickened.
The conventional explanation of bulliform function thus accepts that the bulliform cells regulate the movement of the blade when it opens and closes.Goossens & Theron (1934) draw a comparison between bulliform cells and stomatal guard cells and the changing of cell shape and turgidity with turgor changes.However, whether the bulliform cells actually cause the rolling movements by collapse due to water loss, or whether their size and plasticity merely permit them to be compressed and so allow rolling to occur, has been the subject of some con troversy.
Numerous facts point to caution in explaning the function of these cells. Brandis (1907) remarks on the fact that in mature leaves the bulliform cells may become entirely filled with solid silica making the leaf rigid. Bor (1960) quotes Goebel (1926) as describing nyctinastic movements in Leersia hexandra in which the leaves fold up in dull weather and flatten again in full sunlight.These movements obviously are not dependant on different water regimes within the bulliform cells.In addition, involution may occur in the absence of bulliform cells and often the thin lateral walls of bulliform cells are not collapsed or distorted in naturally rolled or wilted grass leaves (Shields, 1951).
Tschirch (1882) challenged this theory that move ments depended only on turgor changes in the bulli form cells.While he agreed that rolling and unrolling of grass leaves was influenced by the loss and uptake of water, he thought that these movements were due, at least in some cases, to changes in the leaf fibres rather than the epidermal bulliform cells.He explained that the adaxial, subepidermal schlerenchyma fibres have a high capacity for imbibition and a marked tendency to shrink when dehydrated.This results in contraction of the adaxial ribs causing inrolling because the inflexible lower epidermis, reinforced by sclerenchyma gives the abaxial surface an unbroken rigidity.This contraction, coupled with cohesion among the chlorenchyma cells and among the bulliform cells may result in leaf involution.Thus, involution has also been defined as a cohesion and shrinkage phenomenon, the result of a differential contraction of the mesophyll and the rigid lower epidermis.As the mesophyll cells shorten in drying, they become stretched around the vascular bundles, causing the leaf to roll. Shields (1951), in a detailed study of grass leaf involution, has shown that differential shrinkage of the adaxial and abaxial rib surfaces occurs.This shrinkage was more pronounced in all the species studied on the adaxial rib surface.This illustrates the lateral shrinkage in the adaxial sclerenchymatous fibres, where present, and in the mesophyll.In leaves lacking adaxial sclerenchyma girders or strands, large adaxial mesophyll cells collapse in the wilted leaf and the bulliform cells show varying degrees of buckling.Shields found that lateral and vertical contraction in the bulliform and associated colourless cells was no greater than in the adjacent mesophyll.In wilted leaves the abaxial ribs appear more pro minent as a result of the smaller am ount of shrinkage in the vascular tissue than in the surrounding cells.Shrinkage of the bulliform and colourless cells appears to play no part in involution except to facilitate the turning inward of the upper leaf surface.
The fact that the bulliform cell walls are hygro scopic and, therefore, may assist in movement was reported by Goossens & Theron (1934).This was supposedly shown by the fact that dead, detached leaf segments roll more tightly than in normal reversible involution and, conversely, increasing their water content leads to the partial unfolding of the dead leaf segment. Shields (1951) comments that this partial expansion of a dried leaf through adsorption by all the cell walls (which are also dead in the living blade) suggests that decreased hydration of the cell wall may play a passive part in reversible wilting.Since the bulliform cells are dead in this case, absorbtion of water by the protoplasm and large central vacuole cannot occur.
Involution does not result from plasmolysis.Plasmolysis, through vertical contraction of the mesophyll, may cause an increase in width, probably by relieving tissue strains through shrinkage of the protoplasts (Burstrom, 1942).As a result vascular bundles become more widely separated, and the thick-walled, stiff abaxial epidermis, which does not change in width, tends to curve backwards in plas molysis so that the underside of the leaf becomes concave.Since plasmolysis involves turgor change the diametrically opposed characteristic o f inrolling in wilted leaves implies that involution does not result from turgor changes.
Loss o f water from the bulliform cells to the chlorenchyma in particular, may further contribute to the collapse of these cells.With a cuticle on their outer surface these epidermal cells are more resistant than the mesophyll cells to the drying effects of the atmosphere.However, Haberlandt (1928) showed that when an organ is transpiring rapidly, the epidermis loses water to photosynthetic tissue with its higher osmotic pressure.Shields (1951) examined the bulliform cells in unwilted but tightly rolled leaf buds.They are small but turgid, rather than flaccid and collapsed, as in the wilted blades o f mature leaves.Furtherm ore, un folding from the developing bud involves general growth, particularly in the adaxial mesophyll, and does not result primarily from the enlargement of the bulliform cells, although these enlarge by stretching growth due to turgor changes.The rolling mechanism is, therefore, not related to the unfolding of the bud.Rolling involves cohesion and shrinkage in both living and non-living parts, and bud unfolding, turgor and stretching movements at a time when all cell walls are highly elastic.
To summarize, it has been shown that structural elements other than bulliform cells contribute to involution. 1. Rolling in drying is characterictic of certain grass leaves entirely lacking in bulliform cells, and if bulliform cells are present they may show no buckling or other distortion in the wilted leaf.2. Since average measurable lateral shrinkage (which is negligible on the abaxial surface o f wilted leaves) amounts to 7-12 per cent on the adaxial rib face (Shields, 1951) in naturally wilted leaves, subepidermal sclerenchyma and other adaxial elements of the mesophyll must contribute to involution.3. Since dried leaves unfold partially when placed in water, involution must result in part from decreased water content in non-living cell walls which may or may not contain protoplasts.Rolling cannot result entirely from turgor movement, which, by definition, is a reversible change in the water content of living cells.W ater loss by protoplasts alone is insufficient to cause involution in a leaf composed largely of non-living mechanical tissue.The form of the wilted leaf is determined by a number of elements in the mesophyll, the buckling of bulliform and colourless cells, where it occurs, being in part from passive compression.
Anatomical variations in the structure and distri bution of bulliform cells in different genera exhibiting involutionary movements preclude the universal application of any one explanation of this pheno menon in wilting grass leaf blades.W hat is im portant from a descriptive point of view is the fact that the outline of the lamina can be markedly altered by this phenomenon.It appears to be constant within a species and, therefore, it is not the degree of infolding or unrolling that is im portant but rather the type of movement.
This is confirmed by the work of Dunlop (1913) on the curling of the leaves of different varieties of sugar cane.Involute curling, where the upper surface is protected, is characterized by the following anato mical structures: the bulliform cells are not prominent and the cells immediately interior to these cells are comparatively large and thin walled; the upper epidermis is not greatly lignified.Varieties of sugar cane exhibiting revolute curling, whereby the lower surface is protected, have large prom inent bulliform cells attached to the vascular bundles by lignified cells.The vascular bundles are closer together and the upper epidermis is greatly lignified. Dunlop (1913) is of the opinion that this revolute curling is a per manent characteristic of the varieties showing it.
A further possible function of the bulliform and colourless cells has been advanced by Breakwell (1915).He states that the arrangement and distri bution of the colourless cells allows light to penetrate to the chlorenchyma cells, even in the rolled up position of the leaf bud and thus aids in the develop ment of the leaf.The rapid development of leaves with large bulliform groups and colourless parenchyma, such as Astrebla pectinata, is cited as circumstantial evidence for this possible function.

Epidermal cells in transverse section
Typical epidermal cells in transverse section normally do not exhibit any particularly im portant diagnostic characters, although examination of them in section can be of assistance in the interpretation of many epidermal structures as seen in surface view.Sections through the epidermis can aid in establishing the nature of the papillae, the attachm ent and structure of other epidermal appendages, the positioning of the stom ata or the cuticle form and thickness.
Epidermal cells are usually square to rectangular in section, or the outer wall may be more or less arched or even papillate.The papillate appearance, as seen in surface view, may be due to a pronounced arching of the whole outer tangential wall, or more commonly (Vickery, 1935), due to the presence of a number of separate papillae arranged in a longitudinal row or rows.However, when these papillae are in a single row, and as wide as the epidermal cell, the outer wall I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION also appears as strongly arched in section.Narrower papillae appear as distinct papillae on the outer tangential wall surface.Where more than one row of papillae are present on each cell they appear as bifurcate or multiple papillae.The papillae may be inflated and thin-walled, resemble conical warts or may be sharp-pointed expansions of the outer wall (Holm, 1891a) or teeth (Sabnis, 1921).It is also common for the distal ends of the papillae to be thickened.
The level of the stom ata in different varieties of sugar cane has been correlated with differences in drought resistance by M erida (1970).They may be located at the same level as the epidermal cells or sunken below them as seen in transverse section.In certain grasses, such as Spinifex hirsutus, the stom ata are present in depressions formed by the surrounding cells being enlarged and raised above the general level of the epidermal surface (Breakwell, 1915).The presence of stom ata on both surfaces, or only the adaxial or abaxial surfaces can also be confirmed.
Micro-hairs are rarely seen in leaf sections but prickles and macro-hairs are commonly sectioned.The nature of the cushion cells surrounding the base of many macro-hairs, whether raised or not, can easily be determined.Interlocking prickles forming arches over the stom ata can sometimes be seen as in certain Danthonia species (De Wet, 1960).Burbidge (1946a) found hairs consisting of a bulbous-based cell and a small apical cell imbedded in the epidermal cells at the base of grooves of leaves of all species of Triodia.
The size of the epidermal cells may vary over and between successive bundles as well as the cells of the adaxial and abaxial epidermides being of different sizes.In many grasses, especially members o f the tribe Andropogoneae, the epidermal cells are excep tionally large, and can occupy up to half the leaf thickness.These are included under bulliform cells.
Cuticle thickness can be determined in leaf sections.Thus, it may be seen in many species that the lower epidermis is more strongly cuticularized than the upper.Slade (1970) has dem onstrated the lack of a well defined cuticle in shade-grown leaves of plants which have distinct cuticles on the sun-grown leaves.
The thickness of this cuticle is o f the utm ost importance in faecal analysis studies, because during the digestion process all the cellulose cell-walls are dissolved away leaving the cutinized cuticular mem brane behind.Thus, it is the relative thickness of the cuticular membrane, and not of the entire outer tangential wall, that is of importance in these studies of the diet of grazing animals.It should be stressed that the outer wall may be markedly thickened but lack a thick cuticle.The cuticle proper, therefore, consists of a layer of adcrusted cutin continuous over the entire leaf surface.Each cell does not have an individual cuticle but it may be individually thickened.This difference can readily be determined in transverse sections of these epidermal cells.

CONCLUSION
It is hoped that this attem pt to introduce uniform standards to the description o f grass leaf blades, as seen in transverse section, will stress the urgent need for standardization in these studies.If this can be achieved, anatomical descriptions will have a much wider applicability in the fields of comparative leaf anatom y and grass systematics in general.
ribs or ridges as well as the associated furrows or grooves commonly found on the adaxial leaf surface but sometimes on the abaxial surface as well.If ribs are present there must be corresponding furrows and vice versa.If furrows aie not found between the ribs but in any other situation they are termed grooves, e.g. the grooves present on some ribs.Ribs and the associated vascular bundles form regular associations and patterns.The furrows and the adaxial channel of acicular leaves can be distinguished by the fact that the channel is the area bounded by the permanently infolded lamina and the furrows lie between the ribs which may also be in the channel.Depth of adaxial furrows in comparison to the leaf thickness i.e. the depth of the larger ribs in the central region o f the lamina between the margin and the median vascular bundle regarded as leaf thickness: Slight, shallow furrows i.e. less than a quarter of the leaf th ic k n ess..and 2nd order vascular bundles i.e. present over 3rd order vascular bundles ......................... 209* Furrows between 1st order vascular bundles i.e. present over 3rd and 2nd order vascular bundles ......................... 210* Furrow on either side of the median vascular bundle o n l y .
either side of the median vascular b u n d le .
Shape of adaxial ribs as seen in T/S; all types present must be included: Rounded, obtuse ribs i.e. apex rounded: Situated over the vascular bundles ..
order vascular bundles with flattened tops and those over the 3rd order bundles triangular .over 1st order vascular b u n d le s.
.. 241* Composed of girder or strand of sclerenchyma in contact with the e p id e r m is....................................................low magnification.Only half the width of the lamina, from the margin to and including the median vascular bundle considered.When the section is incomplete and the median bundle is indistinguishable only those characters that are possible to determine are considered.In certain cases where the 2nd order vascular bundles are not clearly distinguishable from the 1st order bundles they are (tonsidered as 1st order bundles for the purposes of arrangement.I. THE LEAF-BLADE AS VIEWED IN TRANSVERSE SECTION 5. VASCULAR BUNDLE DESCRIPTION as being circular or angular in outline, these terms refer only to the vascular tissue and exclude the inner or single bundle sheath.DESCRIPTION: Third order vascular bundles; usually very small bundles often with xylem and phloem indistinguishable and consisting o f only a few lignified cells and a few phloem elements; when not obviously smaller than basic type bundles often distinguishable by the absence of sclerenchyma strands and/or the presence of bulliform cell groups adaxially: No third order bundles present in section ..
Basic type vascular bundles; large metaxylem vessel present on either side of protoxylem elements; lysigenous cavity commonly present; associated with sclerenchyma girders or strands: by thick-walled fibres .
or protoxylem vessel present .
structure and thus the sheaths associated with the different orders o f vascular bundles are considered individually.ORDERS OF VASCULAR BUNDLE present in section; bundle sheaths of each order of vascular bundle present completely described before continuing with the descriptions o f the sheaths of the other orders of vascular bundle present Extensions of the bundle sheath; comprised o f parenchyma cells associated with the sheaths and not part of the bulliform groups; extend to adjacent sclerenchyma girders or strands or to the epidermis: No extensions of the bundle sheath present ..; consisting o f two columns of cells .
cells gradually decreasing in size as walls increase in thickness and eventually merge into the sclerenchyma strand .
with the cells o f the single or outer bundle sheath .
radiate or arranged in a definite pattern; vascular bundles usually widely spaced; festucoid: Vertical arrangement in the mesophyll between successive vascular bundles:Occupying the major or entire area between the adaxial and abaxial epidermides abaxial half of the leaf thickness .
abaxial third of the leaf thickness .
groups; bundles close together .
traversed by colourless aerenchyma cell chains .
spaces in diffuse mesophyll with many intercellular spaces and chlorophyll-bearing aerenchyma; often subtending the stomata .
to the opposite epiderm is.
leaf consisting mainly o f colourless cells .
size and shape; tissue irregular in appearance .
.. 908* Extensive groups of large, inflated bulliform cells extending over one or many vascular bundles: Not associated with colourless parenchyma: Distribution of extensive bulliform groups: Present throughout the epidermis; may be slightly reduced opposite the larger bundles ............................... 909* Present in most of the epidermis but not present opposite the first order bundles and usually reduced over the second order b u n d le s .