Interference in Plant Cells

starch-grains (potato) in water ; examine as described above. It will be seen that when the field is dark the grain is bright and presents a well-marked dark cross ; when the field is bright, the dark cross is replaced by a bright cross. It will be observed that in examining sections in polarised light thick stratified coil-walls (particularly sclerenchymatous cells) are coloured this ; is most apparent when the field is dark. This coloration is due to interference of light. MICRO-PHYSICS OF THE CELL. 41 The phenomena of interference can be best studied by introducing a plate of selenite between the polariser and the analyser; it is to be placed on the stage of the microscope beneath the object. Various kinds of selenite-plates may be used ; it is assumed here that the plate shows red and green tints. Mount a section of a twig or of a leaf-stalk ; rotate the analyser so that the field is red or green. The interference colours will not be well seen in the thin cell-walls ; they will appear merely red or green. The thickened cell-walls will exhibit a play of colours which differs in different cases. Mount a section of part of a succulent leaf (Aloe, Crassula, Sedum, &c.). Observe that the interference colours in the ctiti- cularised external layer of the outer walls of the epidermal cells are complementary in position to those of the subjacent cellulose layers ; this indicates differences of tension in the cuticularised and uncuticularised layers. The relation of the interference colours can be more definitely made out in starch-grains. Mount some starch-grains (potato) in water ; rotate the analyser so that the field is red. Assuming that the starch-grain under examination is so placed that its long axis is directed away from the observer, it will be seen that there is a red cross on the grain corresponding in position to the dark cross mentioned above, that the two lateral segments of the grain are coloured yellow, and that the anterior and posterior segments are coloured blue. 2. Spectrum of Chlorophyll. In order to observe this, an alcoholic solution must 42 PRACTICAL BOTANY. A be prepared. quantity of fresh grass is to be taken and freed as far as possible from decayed leaves ; it is then to be boiled in water, pressed so as to get rid of as much water as possible, and spread out on a sheet of paper to dry in a dark place ; when dry it is to be put into a flask and alcohol is to be poured over it, and it is to be left for some hours in a dark place. When it is seen that the alcohol is coloured green, it is to be poured off and filtered ; the solution is now ready for use. The following is a convenient mode of examining the solution spectroscopically : The tube of a microscope is withdrawn (this may be easily done with the smaller forms of Zeiss', Hartnack's, and Crouch's microscopes), and it is replaced by a glass tube, the bottom of which covers the opening of the stage of the microscope ; the sides of the tube must be made opaque by wrapping round them a sheet of black paper ; the solution is then poured into the tube, and into the opening of the tube a microspectroscope is introduced ; the mirror of the microscope is to be so inclined that it reflects a beam of light onto the bottom of the tube. The advantage of this method is, that it enables the observer to vary the thickness of the layer of the solution to be examined. It is best to use a dilute alcoholic solution. Beginning with a column of the solution about f of an inch in height, the spectrum will present a single rather narrow absorption band (band I.), in the red, about the line of the solar spectrum, extending towards B\ if the height of the column be about doubled, band I. will be seen to have become broader, a faint narrow band (band II.) will be seen to the right of it, between the MICRO-PHYSICS OF THE CELL. 43 lines C and D, at the beginning of the orange, another faint narrow band (band IV.) in the green a little to E the left of the line t a broad faint band (band V.) in the blue to the right of the line F, a still broader faint band (band VI.) in the blue and indigo just to the left of the line G-, and finally a broad faint band (band VII.) at the extreme violet end of the spectrum. On increasing the height of the column to about six inches, the bands I., II., IV. will be seen to have become broader and darker, and the bands V., VI., VII. to have coalesced so as completely to cut off the spec- F trum to the right of the line in the blue a new ; band (band III.) rather broad but faint, will be seen at the junction of the yellow and of the green a little to the right of the line D. By this means it is possible to ascertain that the spectrum of chorophyll presents seven distinct absorp- tion-bands. PHANEROGAMS. I. ANGIOSPERMS. VEGETATIVE ORGANS. (A) DICOTYLEDONS. EMBKYO AND GERMINATION. I. EXAMINE the ripe fruit of the Sunflower (Heli- anthus annuus). N.B. The " seeds " sold for sowing are really achsenia, includ- ing the products of development of both ovary and ovule. It is a dry inferior achsenium, with narrower basal, and broader apical end : at the latter is a scar, where were inserted the style and other floral organs. Compare fruits in situ on the floral receptacle. Dissect off the brittle Pericarp, from the anatropous and exalbuminous seed, which it incloses. Note the delicate Testa, and, within this, the straight Embryo, of which the Radicle is directed towards the micropyle (i.e. towards the base of the fruit), and the two Cotyledons towards the apex of the fruit. II. Compare plants, which have been germinated for different periods from one day to one week, and observe the following points in the process : 1. The internal parts of the fruit swell, and cause the brittle pericarp to split longitudinally. SUNFLOWER. STEM. 45 2. The radicle protrudes, and curves downwards. 3. The hypo-cotyledonary stem elongates, so that the pericarp and testa are carried upwards by . the cotyledons, which remain inclosed by them for a con- siderable time. 4. The coats of the fruit fall from the cotyledons, which soon turn green, and expand as assimilating leaves, with the plumule seated between them. 5. The plumule develops leaves, which expand in succession. 6. The radicle has meanwhile elongated and produced lateral roots. Notice that when the young root is removed from the soil, many particles adhere to it, especially at some distance from the apex ; these are held by the roothairs (cf. infra}, which attach themselves closely to the particles of soil. The internal changes accompanying the process of germination and more especially the redistribution of the reserve materials stored in the embryo, may be studied by cutting sections of the seedling at different stages of the process, and comparing the cell- contents in the corresponding tissues. HERBACEOUS TYPE. * Mature. Observations with the Naked Eye. I. Examine the whole of a well-grown plant of the Sunflower. The main axis or Stem is stout, herba- ceous, and erect : it often develops to a considerable length without branching: it is cylindrical, slightly striated below, while the higher parts of it, where the 46 PRACTICAL BOTANY. lateral branches are developed, are polygonal. Its sur- face is studded by stiff hairs, which are especially obvious on the lower portions of the internodes. The stem bears laterally numerous Leaves, which are simple, 1 petiolate, cordate-acuminate, the margin slightly serrate, ciliated, venation palmate-reticulate, the surface hirsute. The arrangement of the leaves at the lower part of the plant (and including the cotyledons which wither at an early stage), is opposite, or in whorls of three ; higher up this arrangement merges into the alternate, the complication increasing constantly upwards. The stem is terminated by a bud, which may con- sist only of closely aggregated foliage leaves (or it may inclose the reproductive organs, which are contained in numerous flowers, closely aggregated so as to form a characteristic inflorescence the capitulum, (cf. infra). Similar buds, in earlier stages of develop- ment, may be observed in the axils of the leaves (axillary buds). Wash the roots and examine them. They are fibrous, and branch profusely. The primary (tap) root and earlier developed lateral roots are thicker than the later developed roots of a higher order (cf. secondary thickening of roots), the latter being successively thinner. II. Cut the stem of a well-grown plant transversely at its thickest part, and smooth the surface with a razor. The most prominent object in the section will be the massive, white, spongy Pith which occupies the centre. 1 N.B. The form of the leaves varies, the lower leaves of the plant being cordate, the upper ones lanceolate with winged petiole. SUNFLOWER. STEM. 47 Around this will be seen, arranged more or less regularly in a circle, and near the periphery, a series of more solid-looking masses of tissue, these are the Vascular Bundles. III. In order to obtain a clear idea of the course of these bundles, and of their connection with those of the leaves, cut off a piece of the stem, so as to include the insertion of a leaf or node, and about two or three inches of stem above and below that point. Bisect this longitudinally in a plane perpendicular to the median plane of the leaf. Clear away the pith with some blunt instrument, taking care not to injure the vascular bundles. This process will be made easier if the stem be boiled in water for about ten minutes. Now dissect out carefully the course of the several vascular bundles, clearing away as much of the internal parenchyma as possible. Treat the whole preparation with aniline sulphate and sulphuric acid for about five or ten minutes (cf. p. 22). The vascular bundles will be stained yellow, and their course may then be more readily followed. As in Dicotyledons generally, there are here no cauline but only common bundles (cf. Apex). It will be apparent that in the internodes the bundles run parallel to one another, and as a rule without lateral fusion. This regularity is disturbed at the nodes (a) by lateral fusions of some of the bundles, but not of all of them, and (&) by the entry of fresh bundles from the leaves (usually three from each leaf), into the vascular ring. IV. In a longer piece of the stem, follow carefully the course of several of the bundles entering from the 48 PRACTICAL BOTANY. leaves, as far as they can be traced independently and without fusion. This will be possible at least for one internode, and usually for two or three; but the distance through which this independent course can be traced is variable in this plant. Further, the lateral fusions do not occur only at or near the nodes, instances may not unfrequently be found $f fusions occurring at various points in the internodes. That the arrangement and course of the vascular bundles in the dicotyledonous stem are connected with the arrangement of the leaves is an obvious fact. It may be seen in Helianthus, but is more prominently shown in plants with regularly decussate leaves (cf. Cerastium, Clematis, Stachys}. Still the arrangement of the bundles may differ radically from that of the leaves, and is to a certain extent independent of them. This may be seen in such a case as that of Iberis amara, where the bundles do not run longitudinally, but in tangential spirals which have no direct relation to the arrangement of the leaves (Nsegeli). The arrangement of the bundles in the normal dicotyledonous stem in a cylinder is due to the fact that each bundle as it enters from the leaf passes towards the centre of the stem for a certain distance only, which is approximately equal for all, each then curves gradually into a longitudinal direction. As regards the bundle-arrangement, Helianthus is not a very good type of an herbaceous Dicotyledon, still it illustrates the most essential points ; e.g., (1) the ring of vascular-bundles as seen in transverse section ; (2) the entry of the bundles of the leaf-trace between the bundles connected with the higher leaves ; (3) the lateral fusion of the several bundles at the node. Since the fusions often occur at points other than the nodes, and since the independent course of the bundles of the leaftrace is of variable length it cannot be regarded as a perfect type. We therefore recommend a series of types for investigation, in which the vascular system has been carefully traced by Nsegeli. In most of these it may be seen how closely the arrangement of the bundles is connected with (1) the arrangement of the leaves and (2) the number of bundles entering the stem from each leaf. Iberis amara, leaves alternate, leaf-trace with 1 bundle. Lnpimis. SUNFLOWEK. STEM. 49 leaves alternate, leaf-trace with 3 bundles. Cerastium leaves t opposite, leaf-trace with 1 bundle. Clematis, leaves opposite, leaftrace with 3 bundles. Stachys, leaves opposite, leaf-trace with 2 bundles. The method which we have adopted in Helianthus is a coarse one, and only available in stout herbaceous Dicotyledons. When such a method is used we should always check our observations by comparisons of longitudinal sections of the apical bud (cf. infra} As a rule the subject should be studied in the first instance by making such longitudinal sections. These should be thick, and be cleared by treatment with dilute potash. Where the bud is not too bulky Naegeli adopted the method of bisecting the bud, clearing with potash, and drawing the bundle-arrangement in the two halves ; hence the whole bundle-arrangement at the apex can be deduced from two such sections. As a further control, series of transverse sections should be cut through the apical bud ; the order of these A and their relative position must be accurately marked. diligent comparison of these (with drawings) will supply the data for deducing the whole bundle-system. Finally, the results obtained by these two methods should coincide, if the observations be correct. Microscopic Observation. The material should he kept in spirit for some time to remove resin, and air, and to harden the tissues. This is not, however, indispensable, and fresh material may be used. I. Cut transverse sections of a stem of a well-grown plant of Heliantlms, i.e. of a stem more than half an inch at least in diameter. Mount some of these in glycerine or glycerine jelly (these may be kept as permanent specimens), and others in Schulze's solution. Examine these first with a low power (1 in.), and observe the following tissues in succession starting from the exterior. E 50 PRACTICAL BOTANY. 1. The Epidermis, a single peripheral layer of cells, not very well defined from the underlying tissues : it completely covers the surface. N.B. The margin is not perfectly regular, but is here and there extended outwards at the regions surrounding the bases of the large multicellular hairs, which may be recognised as being products of the epidermis. Since these hairs are usually injured in cutting the sections, the width of their bases being greater than the thickness of a fine section, in order to see them well thick sections should be made specially, care being taken that the hairs shall not be previously injured before the sections are cat. They will then be seen to be long conical hairs with pointed ends, consisting of many cells, uniseriate : their bases are imbedded in cells of the epidermis and underlying tissue, which together form at that point a small emergence, on the apex of which the hair is borne. Other smaller hairs also occur. Compare the description of the apical bud (p. 64). Beneath this single epidermal layer lies A 2/ band of tissue, several layers of cells thick, the walls of which are thickened at the angles where three or more cells meet, the cell-cavity being thus made oval or circular in transverse section this is the ; chief characteristic of Collenchyma, of which this is a good type. Below this lies 3. A band of thin-walled Parenchyma, in which are dotted here and there resin-passages. Within these tissues of the Cortex (a general term including the tissues described under the headings 2 and 3) lie 4. The Vascular bundles, which are wedge-shaped and are arranged in a ring : according to the stage of SUNFLOWER. STEM. 51 development of the stem, and the point at which the section is taken, the bundles may be more or less completely joined laterally with one another. In old stems, and at or near the nodes this lateral fusion is most complete : still, under any circumstances the originally separate bundles can easily be recognised. Centrally, i.e., within the ring of vascular bundles is 5. The parenchymatous Pith, consisting of thin- walled cells, which have for the most part lost their cell-nature (i.e. have no protoplasmic contents), and are filled with air: hence the whiteness of the fresh pith. (N.B. In material, which has been a long time in spirit, the air may have been removed by the alcohol, but this is usually a slow process.) II. Choose out the thinnest of the sections, and examine it with a higher power (one-sixth inch or one-eighth inch), starting as before from the periphery of the stem. 1. The Epidermal Layer will be seen to consist of cells contiguous with one another, without intercellular spaces (excepting occasional stomata, which are, however, rare ; cf. infra). The walls, and especially the external and internal walls, are thick, highly refractive, and show a stratified structure. In Schulze's solution they are blue (cellulose) with the exception of the outermost layer the cuticle : this is a continuous, well-defined layer, which stains yellow, and may thus be easily recognised. The granular protoplasmic contents of these cells (brown, Schulze's solution) are not plentiful, but form a thin layer lining the somewhat rounded cell-cavity. E2 52 PRACTICAL BOTANY. Chlorophyll grains (cf. infra) may be found in them : this is an exceptional case, as they are usually absent from cells of the epidermis. The cells surrounding the bases of the hairs are extended radially (as regards the stem), and the whole epidermis is at these points pushed outwards owing to luxuriant growth of the underlying tissue : in fact the hairs are each seated at the apex of an emergence, The nature of the hairs themselves will be studied later in connection with the apical bud. 2. In the Collenchyma the protoplasmic body resembles that of the epidermis: chlorophyll grains are numerous. The cell-walls also are highly refractive, and stain blue with Schulze's solution (cellulose) : they are specially thickened at the angles, where three or more cells meet in the thickened mass the lines of ; stratification are well seen. There is no sharp internal limit to the collenchyma, but it merges gradually into 3. The thin-walled Cortical parenchyma, which differs from the preceding (a) in the thinness of its walls, (&) its less copious cell-contents, (c) the larger size of the cell-cavity. Observe carefully the resin-passages, which occur in the cortical parenchyma. (N.B. The resin, being soluble in alcohol has been removed. To see it in its original condition sections may be cut from the fresh stem, and stained with tincture of alkanet.) They are inter-cellular spaces, formed by the splitting of cellwalls. The cavity thus formed is surrounded by small, thin-walled, epithelium, the cells of which divide both radially, and tangentially as regards the passage. SUNFLOWER. STEM. 53 The development of the resin-passages may be observed with great ease and certainty in transverse sections of the stem of Ivy (Hedera Helix). Cut transverse sections from a young succulent stem, mount in glycerine. Scattered through the cortex and pith will be found passages already well developed, and having a structure similar to those in Helianthus. If the soft bast, which lies immediately outside the cambium, be examined carefully, resin-passages will be found in various stages of development, starting from a group of four cells, with no intercellular space. In older stages the cell-wall will be found to have split at the angle where the four cells meet, while in older stages again the intercellular space appears larger ; meanwhile divisions (radial and tangential, the former more frequent) occur in the epithelial cells. Note that in (1), (2), and (3), there occur, especially in stems growing apace, divisions of the cells in a radial direction. Compare the girth of the stem at the upper with that at the lower part of the plant, or that of a young plant with that of an old one. The conclusion will naturally be drawn that the stem increases in girth as it grows older, and since the outer tissues neither peel off, nor do the individual cells increase greatly in width, longitudinal radial divisions of the cells are the only alternative. Before leaving the cortical tissue it must be noticed that the Bundle-sheath, which is the inmost layer of the cortical tissue, and which is easy of observation in the younger stem (cf. Hypocotyledonary stem) may be identified also in these sections, though with difficulty. The layer of thin-walled cells abutting directly on the thick- walled sclerenchyma fibres (yellow with Schulze's solution) show in their radial walls the characters of a bundle-sheath i.e. (i.), they are coloured brown with Schulze's solution; (ii.), they resist the 54 PRACTICAL BOTANY. action of sulphuric acid ; (iii.), they have the character- istic black dot (see p. 63). This layer may sometimes be traced as continuous round the ring of bundles, but this is difficult, owing to divisions in the cells of the bundle-sheath, similar to those above noticed in the cortical tissue and epidermis. Treat some thin sections with sulphuric acid. The bundlesheath and cuticle resist its action, and since they retain their sharp contour, they are thus brought into prominence. Within this are (4.) The Vascular bundles. Select one of the largest of these for more minute examination : it will be found to consist of two well marked masses of thick-walled tissue (peripheral and central as regards the stem) with a transparent thin-walled portion be- tween them. Further, on examining the latter more carefully it will be seen that the external part of it has thicker walls, and is less regularly arranged than the central portion, and must thus be distinguished We from it. have thus four portions of the bundle which, taking them in succession from the periphery to the centre, are named as follows : A. Phloem. f ^ { (i.) Sclerenchyma. ]'/. (n.) fSoft Bast. B. (iii.) Cambium. C. (iv.) Xylem. Taking first (A) the Phloem examine (i.) The Sclerenchyma. This appears as a halfmoon shaped mass of tissue consisting of elements with rounded cavity, in which may be recognised the remnants of protoplasmic contents. The walls are SUNFLOWER. STEM. 55 thick, and lignified (yellow with acidulated aniline sulphate, or with Schulze's solution). They also show differentiation into layers, of which the most prominent is the bright-looking middle lamella. Perpendicular to the internal surface of the walls may be seen pits. (ii.) The soft bast consists of elements of very different structure and function : these are : (a.) Sieve-tubes, which appear in transverse section as the larger cavities of the soft bast : their walls are rather thin and consist of cellulose (blue, Schulze's solution). Occasionally these cavities will be found traversed by transverse septa, having a punctate appearance. These stain dark brown with iodine solution. They are transverse sieve-plates. (Cf. below, description of sieve-tubes in Cucurbita.) (6). Abutting directly on the sieve-tubes, and appearing as though they had been cut off from the sieve- tube by a longitudinal wall, may be seen smaller cells. These are the companion cells. (c). The remaining elements resemble the sieve- tubes in transverse section except in their smaller size, and absence of sieve-plates. These are cambiform cells, or phloem parenchyma. Passing inwards, the distinction of these several constituents of the soft bast becomes more difficult, while the walls are thinner, and the arrangement of the elements is more regularly in radial rows, till, in the band of thin-walled tissue which borders imme- diately on the xylem, these characters become very obvious. This band is B. The Cambium, or active formative layer. Its 56 PRACTICAL BOTANY. constituents are cells arranged in radial rows, with thin cellulose walls (blue Schulze's solution), and plen- tiful protoplasmic contents : the tangential walls are the thinnest, hence we may conclude that the most recent divisions have been in this direction, and have been repeated. Occasionally traces of recent radial division will be found, but this is less common. The form of the individual cells varies from oblong to square, as seen in transverse section : in the former case the longer axis is tangential. Trace the radial series outwards into the phloem, and inwards into the xylem : they may often be followed for a considerable distance with certainty. Note how, in passing from the cambium to the phloem or xylem the cells divide, and how the form of the individual cells is modified. Hence we may draw conclusions as to the development of the different tissue-elements of the mature xylem and phloem from the originally uniform cells of the cambium. For further details cf. the Elm and Pine, which, being lignified stems, and having more definite secondary increase, are better types for the study of cambium. C. The Xylem also consists of elements of various structure : of these the most noticeable are a. The Vessels, easily recognised by their large cavity: they are arranged in radial rows, the indi- viduals decreasing in size towards the central limit of the bundle. The walls are thick and lignified H (yellow with Schulze's solution, or with 2S04 and aniline sulphate), they have no protoplasmic contents ; their further distinctive characters can only be seen in longitudinal sections. Thyloses may be observed, [cf. SUNFLOWER. STEM. 57 infra, p. 61], especially in more central vessels. The vessels are embedded in a mass of tissue composed of two tissue-forms, which, however, are not readily distinguishable in transverse sections : they are b. Xylem-, or wood-fibres^ which appear irregular and polygonal in transverse section, and have thick lignified walls : cell-contents not prominent, or absent. c. Xylem-parenchyma cells which retain their protoplasmic contents ; their cell-walls are lignified, or of cellulose : the latter is the case with those cells which surround the more central vessels. This con- stituent of the bundle is often absent, and is not characteristically represented in this case (cf. stem of Elm, infra). 5. The Pith consists of cells, which have for the most part lost their cell-contents : they are very thin- walled; the walls are slightly pitted: intercellular spaces small. The cell-cavity is usually filled with air, which replaces the protoplasm, especially near the centre ; hence the whiteness of the pith. III. Cut radial longitudinal sections of an old stem of Helianthus, and choosing such as have passed through a vascular bundle (easily recognised with the naked eye), trea.t them as above. Bear in mind the observations already made on the transverse sections, and compare those results with the observations about to be made. To complete the study of the tissues it would be necessary also to cut tangential sections, and, in the case of tissues in which the radial differ from the tangential walls, such sections must be made, and the comparison drawn between them and the transverse and radial sections (cf. stem of Pinus). In the present case, however, 58 PEACTICAL BOTANY. this is hardly necessary, since the components of the several tissues of this stem appear almost uniform in their tangential and radial aspects. Starting as before from the periphery, note successively the following tissues 1 : 1. The Epidermis, consisting of oblong cells, whose walls and contents present the appearance already observed in the transverse sections. Note the dis- turbance of their normal arrangement around the bases of the larger hairs. Beneath the epidermis lies 2. Collenchyma, consisting of oblong cells with thick longitudinal cellulose walls (blue, Schulze's solution), and thin transverse ends : the contents are protoplasm, with a nucleus and chlorophyll- grains. Below each of the larger hairs the collenchyma gives place to short, thin-walled parenchyma, which, together with the epidermis covering it, forms those emergences on the summit of which the hair is seated. Within this is 3. Thin-walled Cortical parenchyma, the cells of which are shorter, but wider, than those of the collenchyma ; there is however no sharp limit between them : observe transitional forms. Cell-contents re- semble those of (2), but there is less chlorophyll. Note the resin-passages, the course of which is directly longitudinal; they therefore appear as longi- 1 It is but rarely possible to see all the tissues here enumerated satisfactorily represented in a single radial section, therefore the study of the tissues and their relative positions should be conducted by comparison of a number of sections one with another. SUNFLOWER. STEM. 59 tudinal bands of small, oblong, thin- walled cells (epithelium). The Bundle-sheath may occasionally be recognised as the layer of cells immediately outside the bundle. Very commonly starch grains may be detected in its cells. 4. The Vascular bundle. Supposing the section to have been approximately median through the bundle, the following components will be found to be included in it : A. Phloem, which is made up of i. Hard Bast, Sclerenchyma, or bast fibres. These appear in longitudinal section as long prosenchymatous cells, occasionally divided by more or less oblique septa. Walls thick, lignified (yellow with Schulze's solution, or with acidulated aniline sulphate), and pitted : remnants of the protoplasmic contents may be found, especially if the stem, cut be not very old. ii. Soft bast, consisting of tissues with cellulose walls (blue with Schulze's solution) and abundant pro- toplasmic contents : its several constituents are a. Sieve-tubes, long tubular structures with thin walls and transverse or oblique septa (sieve-plates), the structure of which is the chief characteristic of the sieve-tubes ; they are readily recognised in sections treated with Schulze's solution (or iodine solution) by the deep brown coloration of the protoplasm, which is collected round the sieve-plates. Treat some sections with potash : the protoplasm, and mass of callus surrounding the sieve-plates, swells, and the perforated or sieve-like character of the septum, which does not swell, is then easily recognised. Sieve-plates occur occasionally on the lateral walls, 60 PRACTICAL BOTANY. where two sieve-tubes are contiguous. The sieve-tubes will be more easily recognised in sections which have been stained with Eosin (see p. 12). For more accurate study of these structures, see sieve- tubes of Cucurbita (p. 84). 5. Side by side with the sieve-tubes may be found the Companion cells which are smaller sister-cells of the segments of the sieve-tubes, cut off during development : these are, however, difficult to distinguish, but their presence is proved by the transverse sections. ';. Bast-parenchyma, or Cambiform cells. These are oblong parenchymatous cells with thin cellulose walls (pitted, but not very distinctly) and protoplasmic contents. B. The Cambium, a band (here very narrow) of oblong cells with very thin walls, and dense protoplasmic contents. As the tissue in this case differs in no essential point from that in other plants treated elsewhere, and as it is here difficult to study, its description will be deferred, though its presence here must not be forgotten. C. The Xylem, consisting of a. Vessels, which are its most prominent constituent. They are structures with lignified walls (note reactions), which are variously marked ; they have no protoplasmic contents, their wide cavity containing water or air. The cavity is continuous, owing to the partial or complete absorption of the transverse or oblique septa. Note instances of this partial or complete absorption. According to the various markings, or thickenings, of their walls, the vessels may be grouped under the following heads, the first named being the nearest to the periphery of the stem : SUNFLOWER. STEM. 61 (a.) Pitted vessels, which are the largest, having very large cavity, walls with pits which appear oval in surface view, and which have the same characters as the round bordered pits of Pinus. Having observed the pits in surface view, focus so as to obtain a longitudinal optical section of one of the walls (or better, find a place where the preparation is so thin as to show this in real section). Compare this with what was seen in surface view. (/3.) Spiral vessels found in the more central part of the xylem, those most central having the spirals more closely coiled. Note transitional forms (irregularly reticulated) between spiral and pitted vessels. (y.) Annular vessels found at the central limit of the xylem, the thickening is here in the form of rings ; in mature stems these vessels are usually more or less disorganised. 5. Fibrous cells (wood fibres), which are long and pointed : it is difficult to follow one individual fibre throughout its whole length, owing to its taking a sinuous course, the fibres being interwoven one with another : their walls are lignified and pitted : the cell contents are reduced or absent. c. Parenchyma, which is to be found more especially around the vessels near the central limit of the bundle. The phenomenon of thyloses is the result of the encroachment of these cells on the cavity of the vessels. The normal individual cells are oblong with square ends, they have cellulose walls (reactions), and retain their protoplasmic contents. The cells termed thyloses (Tullen) are properly included under the term xylem parenchyma, being derived directly from this 62 PRACTICAL BOTANY. tissue in the following way. When fully developed the vessels have lost their protoplasmic contents and their turgescence ; their = walls are unevenly thickened, at some points being thin ( pits) at others strongly thickened. If thin-walled tissue, the elements of which are active and turgescent, abut on such a wall, it is obvious that but slight resistance to the internal tension will be offered at the pits, where the wall of the vessel is thin. As a result the wall bulges at these points, and the cells encroach as papillae upon the cavity of the vessel. Cell-divisions may occur in these papillae, and the whole process be continued till the cavity of the vessel is completely filled with a cellular tissue. Look in the longitudinal sections of the old stem of Helianihus for instances of such encroachment of cells upon the cavity of the vessel. Good results may be obtained from the old stem, or root, of Cucurbiia, and from the stems of Robinia, or Vitis. 5. The central Pith is composed of parenchymatous cells, with thin walls consisting of cellulose (reactions) slightly pitted : they have lost their protoplasmic cell- contents in many cases, and especially near the centre of the stem. Occasional resin-passages may be found in the pith. * * Young Stem. IV. Cut transverse sections of a young stem, i.e. not more than one-eighth of an inch in diameter. If the sections be cut from the hypocotyledonary stem, though they will correspond in all important points to the following de- scription, they will differ in some minor details ; e.g. hairs will be absent, the bundle-sheath will be more obvious, &c. Mount in glycerine, and passing from the periphery inwards observe successively under a low power 1. The Epidermis as before a single layer, with SUNFLOWER. STEM. 63 hairs of various complexity and shape (cf. apical bud). Beneath this 2. Cortical tissue, which is more or less clearly differentiated into a. Collenchyma. /3. Cortical Parenchyma. 7. Resin-passages. 8. Bundle-sheath. These severally hold the same position, and have the same characters, though less strongly developed, as were above observed in the older stem. The bundle-sheath in the young stem is more easily recog- nised than in the older stem. It is a continuous layer of cells, whose radial walls have a characteristic dark dot on each radial wall, due to reflection of light from the peculiar sinuous waves of the central part of the radial walls. The oblique part of each wave acts as a reflector, so that the greater part of the light is diverted before it reaches the eye. Hence the origin of the dark dot. The bundle-sheath lies immediately outside the vascular bundles, curving slightly towards the centre of the stem in the spaces between the bundles. It is more prominent in the hypo- cotyledonary stem, and especially when this is young. The cells are then filled with starch, and the layer may be readily recognised in sections treated with iodine. Under ordinary circumstances it is brought into greater prominence by treatment of the sections with potash. "Within the bundle-sheath, and arranged in a ring, lie 3. The Vascular bundles, which are wedge-shaped, of variable size, composed of similar elements to those described above in the older stem. Note that, if the stem be young enough, the bundles are not joined laterally as in the older stem, but are separated from one another by broad bands of ground 64 PRACTICAL BOTANY. tissue. In slightly older stems the cells of this tissue may be found actively dividing, by tangential and occasionally by radial walls. An Interfascicular Cambium is thus formed, and by the tissues derived from it the vascular ring, as seen in the older stem, is completed. Centrally lies 4. The Pith, consisting of thin-walled cells, with sparing cell-contents. These, then, have not yet lost their cell-nature ; compare the older stem where the protoplasmic contents are replaced by air. We Note on Interfascicular Canibium. have seen that in the Sunflower the bundles are quite separate in the young stem, being isolated by masses of quiescent ground tissue. Later, the cells of the latter tissue begin to divide actively as an interfascicular cambium layer, lying between the originally separate bundles. This interfascicular cambium joins the margins of the fascicular cambium, and a complete cambial cylinder is thus formed. But here in the Sunflower, as in most herbaceous annual plants, the interfascicular cambium is not very long active ; the product of its activity being but a narrow band of secondary fascicular tissue : the identity of the original bundles can thus be recognised at a glance. In some stems (Ranunculaceai) the interfascicular cambium is completely absent. Compare this with the case of most ligneous perennial plants, e.g. Elm, Pine. Apical Bud. V. Take the apical bud of a young plant, or of a young lateral branch of the Sunflower, and cut longitudinal median sections : treat with potash, and mount SUNFLOWER. STEM. 65 in glycerine : examine with low power, and then observe 1. That the axis ends in a naked, broadly-conical Apex (punctum vegetationis), which is surrounded and enveloped by 2. Leaves : these may be observed in various stages of development, the youngest being nearest to the apex (i.e. their order of development is thus acropetal) ; the surfaces of the older leaves are covered with 3. Hairs, which are absent from the apical cone and the youngest leaves (i.e. the hairs are developed subsequently to the leaves themselves). Note (with a higher power) that the apical cone itself consists of thin-walled cells with plentiful protoplasm, which are smaller than the cells of the mature tissues already studied, and are in a state of active division (i.e. are meristematic). The whole meristematic mass is differentiated into parts, which maybe distinguished more or less clearly from one another, and their continuity may be traced with the several tissue-systems of the stem and leaves, of which in fact they are the formative We layers. may thus distinguish the following : 1. The Derm ato gen, as a single continuous layer of cells, which divide only in a direction perpendicular to the external surface of the organ (stem or leaf), which it covers completely : it is easily seen to be continuous with the epidermis, of which it is the formative layer. Within this is a solid mass of tissue, which looks for the most part dark, owing to its being permeated by intercellular spaces filled with air. It is traversed at a short distance from the external surface by transparent, longitudinal bands of F 66 PRACTICAL BOTANY. 2. Procambium, which is the formative tissue of the vascular bundles. Trace its continuity with these. Between the procambial bands and the dermatogen lies 3. The formative tissue of the Cortex, which is (partially at least) characterised by dark-looking intercellular spaces. 4. Centrally lies a dark bulky cylinder, which is continuous with, and formative of, the Pith. Observe carefully the mode of origin of the leaves. They appear at the periphery of the cone as protuberances of the dermatogen and the subjacent cells. As they increase in size their internal tissues become differentiated into (1) procambium, which is subsequently connected with that of the stem, and (2) tissue with intercellular spaces, which is continuous with the cortex. At the same time single cells of the dermatogen grow out, and divide, so as to form the conical multicellular hairs, which cover the surfaces of the leaves (cf. leaf-section). In the older leaves of the bud the development of the emergences around and below the bases of these hairs may be traced. Note on passing back from the apex towards the more differentiated part of the stem a gradual increase in length of the cells, corresponding to the gradual extension of the internodes, while in the stem (internode) below the bud this is very marked. Observe also the various stages of the process of vacuolisation of the protoplasm. In cases where the apical cone is broad, as in Helianthus, the tissues, with the exception of the dermatogen, are usually not sharply denned from one another at a point immediately below SUNFLOWER. STEM. 67 the apex ; but the various tissue-systems appear to originate from a common meristem. In some cases, however (especially water plants), the definition is more marked. As an instance may be cited the apex of Hippuris (cf. infra, p. 82). Node. VI. Cut moderately thick longitudinal sections through a young node of the Sunflower, so as to include the median plane of the leaf (or of both leaves if they be opposite, as they often are in the lower part of the plant). Treat with potash and glycerine, and warm for a few minutes [or better treat with very dilute potash for twenty -four hours or more]. Mount in glycerine, and examine with a low power. The course of the vascular bundles, which appear dark, is easily followed through the more transparent parenchyma. Note 1. The continuity of tissues of the stem and petiole ; there is no definite boundary between these two parts. 2. That the bundles from the petiole . pass into the stem, and, curving at first inwards, they soon assume a longitudinal course. 3. That no bundle of the upper internode lies in the same vertical plane as the bundle which enters from the petiole, i.e. the bundle from the petiole enters between two successive bundles of the vascular ring. 4. If axillary buds be present, note how their bundlesystem is inserted on the bundles of the main axis, as well as on those entering from the petiole. Observe the large multicellular hairs seated on the apex of small emergences as before seen (p. 50). F2 68 PRACTICAL BOTANY. STEM ARBOREOUS TYPE. I. Note the following external characters of a twig of Elm (Ulmus campestris) of the current year. It is cylindrical, hirsute, green or brown according to age, the latter colour being due to the formation of cork (cf. infra, p. 70). Small brown excrescences are scattered over its surface these are lenticels. ; The arrangement of leaves is bilateral, phyllotaxis ^, branching axillary. II. Cut transverse sections of a twig of the current year ; mount in glycerine, and examine with a low power. [Other sections may, for comparison, be treated with Schulze's solution, others again with aniline sulphate and sulphuric acid.] Observe the general arrangement, of tissues in con- centric layers, which will be found to succeed one another in the following order, starting from the outside : 1. Epidermis : a single layer of small cells : many of them have grown out, as conical hairs, perpendicular to the surface. 2. Cork : consisting of one or more layers of square cells : it will be more strongly developed in older twigs, while it is completely absent in very young twigs (for development cf. infra). Here and there a lenticel may have been cut through : in which case it will appear as a lateral extension of the band of cork. 3. Cortical tissue : parenchyma with chlorophyll, and cellulose walls, and intercellular spaces ; here and there are large transparent cavities (mucilaginous cells). ELM. STEM. 69 4.1 Thick-walled masses of Sclerenchyma (hard bast), which form an irregular broken ring (walls brownish-red with Schulze's solution). 5. Soft bast : a transparent tissue with cellulose walls, and plentiful protoplasm. 6. Cambium : a misty layer of thin-walled tissue with plentiful protoplasm : cells in radial rows. 7. Xylem : a broad band of thick-walled lignified tissue, with crenated inner margin ; centrally lies 8. The Pith or medulla : round-celled parenchyma, with thin pitted walls : mucilage cells here and there. The crenated appearance of the inner margin of the xylem is due to the presence of the wedges of primary xylem (forming the so-called medullary sheath), separated from one another laterally by parenchymatous bands, which may be followed outwards in a radial direction through the whole thickness of the vascular ring : these are the primary medullary rays : other rays will also be seen following a similar course, but extending only part of the way from the cambium to the centre and periphery of the vascular ring : these are secondary medullary rays. Compare with the vascular arrangement of Helianthus. Cut transverse sections through the axis of a bud, or of a young twig, during the process of extension in spring ; treat with potash, and mount in glycerine. In these sections the vascular system will be found to be much less developed, but even here the primary bundles will not be found to be as clearly distinct from one another as in the young stem of Helianthus. In ligneous Dicotyledons the interfascicular cambium begins to be active at an earlier period than in those which are herbaceous. } 4, 5, 6, 7, together form the vascular ring. 70 PKACTICAL BOTANY. Examine the several tissues, above enumerated, in detail with a high power : 1. Epidermis : a single layer of cells, with the outer wall thickened and cuticularised (test with the usual reagents) : Stomata will be found in a normal position in young twigs, in older ones they are found at the apices of the lenticels (cf. infra, p. 72). Note the form of the conical hairs, the walls of which are silicified. To obtain proof of the latter fact, treat tangential sections of the surface of the stem with potassium chlorate and nitric acid ; dry them with blotting paper and ignite on a cover slip, or plati- num foil ; mount the ash in water, and treat with nitric acid. Silicified walls will after this treatment present the same out- line as they originally did. In this case complete skeletons of the conical hairs will be found. 2. The Cork (when present) lies immediately below the epidermis : it consists of cubical cells, with thin walls, and little or no cell-contents : they are arranged, in radial rows, without intercellular spaces. Select a thin part of the section for special study of these radial rows, and note in each the following succession of tissues, passing from without inwards : A a. series of Cork cells as above described : walls stained yellowish-brown with Schulze's solution (Peri- derm). 6. At least one cell with very small radial diameter, and with protoplasmic contents and thin cellulose walls Cork-cambium or Phellogen. c. Cells with thick cellulose walls, and protoplasmic contents with chlorophyll : no intercellular spaces : this is the Phelloderm, which is also derived from the cork-cambium. ELM. STEM. 71 Treat a thin section with concentrated sulphuric acid : the walls of all the tissues will swell, and gradually lose their sharpness of outline, with exception of the cuticularised outer wall of the epidermis, and the cork. N.B. The cork is sometimes developed to an extraordinary extent on the twigs of the Elm, so that it appears externally as thick radial plates of tissue. By comparing sections of twigs of various ages, starting from such as have just escaped from the bud, the following facts may be established i. The cork-cambium appears in the layer of cortical cells immediately below the epidermis. ii. These cells divide parallel to the surface of the stem. iii. The result of successive divisions in this direction is the formation of secondary tissues, which develop externally as cork, internally as phelloderm. iv. The true cork-cambium consists of only a single cell in each radial row, from which, by successive division, all these secondary tissues are derived (cf. cambium of vascular bundles). v. The cells of the cork-cambium occasionally divide radially. As stems grow older, layers of cork appear successively further and further from the external surface : not only the cortex but also the outer and older portions of the phloem are thus cut off from physiological connection with the inner tissue ; the term Bark is applied to tissues thus cut off, together with the cork which forms the physiological boundary. As a good example of such successive layers of cork may be mentioned the stem of Vitis. Examine points where a lenticel has been cut through, or make median sections through a lenticel. Note that here the cork layer widens out laterally so as to form a hemispherical mass (semicircular in section), which is covered by the extended epidermis ; if the section be median, there will usually be seen a 72 PKACTICAL BOTANY. stoma at the apex of the lenticel : the whole mass of tissue consists of cells of a corky nature, with inter- cellular spaces. By comparison of sections of twigs of various ages it may be seen that lenticels originate below the stomata, by divisions of the subjacent cortical tissue by walls both radial and tangential ; secondary lenticels are also formed later ; these appear at points independent of the stomata. 3. The Cortical tissue is a broad band consisting of parenchymatous cells, with intercellular spaces. According to their various characters they may be thus grouped: a. Ordinary parenchyma cells, with cellulose walls and protoplasmic contents, with nucleus, chlorophyll, and starch-granules. The two latter are not constant. b. Cells (idioblasts) with large crystals. c. Large cells whose mucilaginous walls almost or entirely obliterate the cell-cavity. Note that the cells (a) are subject to radial division, and that the whole cortical tissue is tangentially extended, so as to keep pace with the increasing bulk of the internal tissues. N.B. No obvious bundle-sheath is present in this stem. 4. The Sclerenchyma consists of cells with walls so; thickened that the cell-cavity is often obliterated ; the walls are differentiated into two or more strata. Reactions with aniline sulphate, light yellow ; with Schulze's solution; brownish red. 5. The Soft bast is, as in the Sunflower, composed of several different thin-walled tissue-elements, which ELM. STEM. 73 are, however, difficult to distinguish in transverse sections. They are : a. Sieve - tubes, which are nearly circular in section, and usually of larger cavity than the other constituents. 1). Bast-parenchyma : cells often arranged in more or less regular radial rows : certain of the cells differ from the rest in containing one or more crystals. The nature of these several tissues will be more successfully studied in longitudinal sections. 6. The Cambium consists of thin-walled cells ar- ranged, as in the Sunflower, in radial rows, which may often be traced outwards into the phloem, and in- wards into the xylem : the cells have copious protoplasm, in which a nucleus may often be observed. Note that the tangential walls are thinner than the radial walls ; also that the radial diameter of the cells is less than the tangential. These facts, together with the arrangement of the cells in radial rows, point to a sequence of divisions, by walls parallel to one another, in a tangential direction. If careful comparisons of a number of different radial series be made, it will be found that the arrangement is such as would result from the action of Sanio's law of cambial division (compare Pinus, p. 141). 7. The xylem also consists of several different tissue- forms, all of which have lignified walls (cf. reactions). They are: a. Vessels, easily recognised by their large cavity, and by the absence of any protoplasmic body. They occur, singly or in groups, scattered through the xylem. It may be found that the cavity of some of the vessels is filled with a cellular tissue. This is especially frequent in the part of the xylem-ring nearer to the centre. The name thylose is given to such cells (see above, p. Cl). 74 PRACTICAL BOTANY. b. Xylem-fibres or Wood-prosenchyma, elements with much smaller cavity, little or no protoplasm, and thick walls. c. Xylem-parenchyma, recognised by the presence of a protoplasmic body, and (at all events in autumn) of starch grains. The cells of this tissue are usually grouped round the vessels, and often form bands connecting two consecutive medullary rays laterally. The cells of the Medullary rays are in the xylem thick-walled (lignified) and pitted ; they have protoplasmic contents and starch. They are elongated radially. Note that they have special cambium cells, differing in form from the ordinary cambium. In the phloem the cells are thin-walled (cellulose), and have plentiful protoplasm. 8. The Pith. In the peripheral part the cells have thick, lignified, pitted walls, and a protoplasmic body with starch (at least in autumn). Tissue of this nature merges gradually into the central tissue with thin walls (lignified and pitted) and no protoplasm. Mucilage cells occur here and there. III. Cut a four-year-old twig of Elm transversely, and smooth the cut surface with a razor. Note, the age of a twig may be judged externally by counting backwards the annual increments of growth from the apex. The limits of each annual increment of growth may be recognised by the closer aggregation of the scars of the leaves or scales at those points. Examine with a lens, and observe : 1. The Pith, which occupies the organic centre of the stem. [Its position does not, as a rule, coincide ELM. STEM. 75 with the geometrical centre.] Externally to this lies : 2. The Xylem, which is here a broad yellowish band, clearly marked off into a succession of concentric rings; these, as a rule, correspond in number to the years of the twig (annual rings). 3. The Phloem, which is a much narrower band than the xylem, is also marked off, though less distinctly, into concentric rings of equal number. Outside this lie : 4. The Cortical tissue and Cork, which are of insignificant bulk, compared with that of the vascular tissues. Note the medullary rays. Some of these (primary rays) may be traced the whole distance from pith to cortex; others (secondary rays) only part of that distance. The latter have been entirely formed by the cambium. IV. Cut transverse sections from the above cut sur- face, so as to include all the bands of tissue from the pith to the cortex : moisten them with alcohol, and mount in water or dilute glycerine. Examine with a low power. Note that the constituents of the several tissues, produced during the later years, are similar to those already observed in the first year's stem ; also that they are arranged, more or less regularly, in radial rows. This is best seen in the xylem : this points to their origin from the cambium. Observe that the constituents of the autumn-formed xylem are smaller, and have slightly thicker walls than those formed earlier in the year, also that vessels of large cavity are absent from it. Hence arises the appearance of the annual rings. 76 PRACTICAL BOTANY. V. Cut radial sections from a four-year-old stem of Elm ; soak them for ten minutes or more in alcohol (to remove the air bubbles), and mount in glycerine. Use a low power. It will be found difficult to cut good sections so as to include the whole radial surface ; it is therefore better not to attempt it, but to study the several structures in a number of successive sections, each extending over only a part of the radial surface. Starting from the outside, observe the same succes- sion of tissues as already seen in the transverse sections, viz. : 1. Epidermis, which is often dried up and dis- organised. 2. Cork (including the cork-cambium and peri- derm), with the short cells arranged in radial rows. 3. Cortical tissue, with large mucilage cells. 4. Hard bast, consisting of long fibres. 5. Soft bast, thin-walled elements with much protoplasm. 6. Cambium, a misty band ; cells not easily defined. 7. Xylem, with thick lignified walls, the vessels appearing as large tubular cavities. 8. Pith, parenchymatous ; its appearance as in transverse sections. Note the medullary rays, which appear as narrow bands of parenchyma, following the plane of section. Examine these several tissues in detail with a high power. 1. The Epidermis, when still persistent, shows the same characters as are observed in transverse sections. ELM. STEM. 77 2. The Cork is composed of square cells arranged in radial rows, which are continuous through the cork- cambium to the periderm, the latter presenting much the same appearance as in transverse sections. 3. The Cortical tissue, which is parenchymatous throughout, also appears much the same as in transverse sections. 4. The Hard bast consists of long fibres, with thick walls, and very small cell-cavity: they are distributed in irregular groups among 5. The Soft bast, characterised by thin walls and protoplasmic contents, and composed of a. Sieve-tubes, which are best seen in the part of the phloem nearest to the cambium. They resemble, in the main, those of Cucurbita (p. 84), but are not so wide ; the sieve-plates are oblique, and face the radial planes. This is the usual arrangement of sieve-plates in secondary phloem ; but their structure is often more complicated, e.g. in Vitis, Tilia. The sieve-tubes may easily be recognised in stems cut in autumn by the masses of callus which surround the sieve-plates : this stains brown with Schulze's solution. For the reactions of the callus, see p. 31. Companion cells are not easily seen. I. Bast-parenchyma: oblong cells with cellulose walls, some contain protoplasm and starch. (More or less of the latter according to the season.) Others contain crystals : note the medullary rays as before. Passing inwards the differentiation of tissues of the phloem is lost in 6. The Cambium, which appears here as a narrow band of cells with thin walls, and abundant protoplasmic 78 PRACTICAL BOTANY. contents. The form of the cambial cells may be better studied in tangential sections; here it is difficult to make it out. 7. In the Xylexn (excluding for the present the medullary rays), observe the following structures, all of which have lignified walls (a). Vessels of various orders, which may be grouped as (i). Spiral vessels (protoxylem) found at the central part of the xylem, i.e. next the pith : they are usually more or less disorganised, being often filled with thyloses. (ii). Pitted vessels, the lateral walls of which are crowded with bordered pits, of essentially the same structure as those in Pinus (p. 142). These vessels are usually of large cavity. Vessels (iii). with both pitted and reticulate marking, superposed on one another on the same lateral walls : these vessels usually occur in groups, and are of small bore. Note in all these, but especially in (iii.) points where transverse or oblique septa have been partially or completely absorbed. (5). Fibrous cells, which occur in large groups, between the vessels : they are long, and prosenchymatous, and are intertwined, so that it is difficult to follow them through their whole length. Little or no cell- contents : walls not pitted. (c). Xylem-parenchyma : oblong cells with pro- toplasmic contents, and starch : walls thick, lignified, and pitted : they occur in longitudinal bands : note their close contact on the one hand with medullary rays, on the other with vessels. ELM. STEM. 79 Examine the medullary rays in the xylem : they are composed of oblong cells, with their longer axes horizontal, arranged like bricks in a wall : in characters they resemble xylem parenchyma. 8. The Pith presents in radial section, for the most part, the same characters as already noted in transverse section. VI. Treat some small pieces of the wood of the Elm with Schulze's macerating fluid (potassium chlorate, and nitric acid), and warm gently till the tissues break up, and the several constituents begin to separate : then wash with water, and mount in water or glycerine. Some at least of the constituents will be found lying separately, or may be detached by slight pressure on the cover slip : the true form of the wood-fibres will now be seen. Note also vessels, and xylem-paren- chyma. VII. Cut tangential sections through the xylem of a 4-5 years' old stem of Elm, treat with solution of iodine, and mount. Observe first with a low power 1. The Medullary rays of lenticular appearance, easily recognised as masses of small thick-walled cells, filled with starch, which appears dark blue. (This is best seen in stems cut in autumn.) In close connection with these 2. The Xylem-parenchyma, the cells of which also contain starch, and are thus easily recognised : note that it more or less completely surrounds 3. The Vessels, the walls of which are stained yellow, and present those characters already observed in radial sections. The interspaces are filled by 80 PEACTICAL BOTANY. 4. Masses of Xylem-fibres, wlricli appear as before. VIII. Cut tangential sections of the phloem of a similar stem : treat as before, and observe 1. The form and arrangement of the medullary rays as in the xylem, but the walls of the cells are thinner, and not lignified : copious protoplasm is to be found. 2. Phloem-parenchyma, the cells of which differ in their cell-contents (a). Some containing crystals. (&). Others with copious protoplasmic contents. Both forms will be seen to have been derived by division from original elongated cells with pointed ends, since they are arranged in groups of this form. (cf. cambium.) 3. Sieve-tubes answering to the description given for radial sections (cf. Cucurbita). The sieves are oblique, the form of the successive segments oblong. The sieves are callous, and are easily recognised in sections stained with iodine or eosin. 4. Bast-fibres as before in radial sections. IX. Cut tangential sections through the cambium of the stem of Elm : treat with dilute potash, and mount in glycerine. Examine first with a low power, and note that the general arrangement is similar to that already seen in tangential sections through the mature tissues, also that the form of the cells, in each part of the cambium-zone, is like or similar to the average form of the elements of the mature portion of wood or bast, which borders on it in a radial direction. Thus the cambium is differentiated into 1. Cambium of medullary rays, which appears as ELM. STEM. 81 consisting of roundish cells, resembling cells of the medullary rays in form. . 4 2. Cambium from which all the other tissues are derived, the cells of which have a prismatic form. Taking these cells as a starting point, the several tissues above described are derived from them in the following way : (i). Phloem. (ii). Xylem. (a). Sieve-tubes, by lateral distension and conversion of the oblique walls into sieve-plates. (&). Parenchyma, by division of the cells by transverse septa, (c). Fibres (sclerenchyma), by elon- gation and interweaving of cells, the width of the cells at the same time being relatively reduced. (a). Vessels, by lateral distension, and absorption of cell-contents, and of the terminal walls. (6). Parenchyma, by division of the cells 'by transverse septa, (c). Fibres, by elongation and interweaving of the cells, while the width of the individual cells is relatively reduced. Observe intermediate stages between cambium cells, and these several mature tissues : this may best be done in sections cut from stems in early summer. X. To investigate the nature of the crystals, several times observed in the parenchyma of the stem of G 82 PKACTICAL BOTANY. the Elm, cut tangential sections of the phloem or of the cortical tissue, mount in water, and having found one or more crystals (i). Run some iodine solution under the cover slip : the crystal is not stained. (ii). Acetic acid : it is not attacked. (iii). Dilute nitric acid : it is more or less completely dissolved. These reactions, coupled with what is known from the analysis of ash, point to the conclusion that these are crystals of calcium oxalate. STEM AQUATIC TYPE. Note the cylindrical smooth stem of the Mares-tail (Hippuris vulgaris), bearing whorls of simple leaves. I. Cut transverse sections of an internode of the stem of Hippuris vulgaris; mount in glycerine and examine with a low power. Observe : A 1. well-marked Epidermis with cuticle. Here and there are to be seen radiating scale-hairs. These occur especially in the axils of the leaves. 2. Cortical parenchyma : consisting of thin-walled, chloro- phyll-containing cells, with large intercellular spaces. A 3. well-marked Bundle-sheath, with the usual characters, which immediately surrounds 4. The central Vascular Cylinder. This is composed of : A (a) basis of thin-walled parenchyma, in which are distributed (6) In the central part vessels of the xylem with lignified walls, (c) Towards the periphery elements with the characters of soft bast ; the sieve quality is in this case doubtful. II. Cut thick transverse sections of nodes ; treat with potash, mount in glycerine ; and observe, with a low power, that the HIPPURIS. STEM. 83 distribution of tissues is in the main the same as in the internode, but 1. The large intercellular spaces are divided by horizontal septa, consisting of single layers of cells. 2. Branch bundles leave the central cylinder, and pass horizontally outwards to the bases of the leaves. III. Cut median longitudinal sections of the apical bud of Hippuris, so as to pass through the elongated apical cone ; treat with potash, and mount in dilute glycerine. Examine first with a low power, and observe : 1. The Axis, which is wide, and cylindrical below, but tapers upwards to the rather elongated apical cone (punctum vegetationis). The axis is composed of the several tissues already noticed. Note especially : (a) The rectangular intercellular spaces divided transversely by septa at the nodes. (6) The axial vascular cylinder, which may be followed far up into the apical cone, and which gives out lateral branches to the leaves. 2. The leaves, diminishing in size towards the apex. Note the scale-hairs about the bases of the leaves. Put on a high power, and examine the apical cone. Note : 1. The Dermatogen, (cf. p. 65) a continuous layer of cells, which covers the apical cone externally. Trace it backwards from the apex : it will be seen to give rise to the epidermis. 2. The Periblem, consisting of 4-5 layers of cells, which may be traced backwards, and be thus shown to give rise to the cortex. A 3. central cylinder of Plerome, which is continuous with, and gives rise to, the vascular cylinder. Note that the Leaves originate from the outgrowth of the derma- togen and periblem, the plerome taking no part in their formation- Also that the vascular system of the stem is already developed We at a higher point on the axis than that of any of the leaves. have thus an instance of cauline vascular bundles, that is such as are proper to the stem, as distinguished from common vascular bundles, which terminate at their upper extremities in the leaves. G2 84 PRACTICAL BOTANY. SIEVE-TUBES. i. Cucurbita. Though the sieve-tubes of the Sunflower are fairly large, the soft bast does not occur in large masses. In the Vegetable Marrow, however, the sieve-tubes are of extraordinary size, and occur in large numbers : this stem is thus excellently fitted for the study of the sieve- tubes of the type found in herbaceous stems. I. Cut transverse sections of the stem of Vegetable Marrow, stain with eosin, and mount in water or glyce- rine. The general arrangement of tissues in this stem differs in several important points from that in the Sunflower, and, indeed, from that in most herbaceous Dicotyledons. Thus : 1. There occurs at a short distance below the epidermis a thick- walled band of sclerenchyma with lignified walls (yellow, with H Schulze's solution, or aniline sulphate and S0 2 4 ). This is quite distinct from the vascular bundles. 2. The vascular bundles are always separate and distinct : though an interfascicular cambium is formed in old stems, no secondary vascular tissue is derived from it. 3. The structure of the individual bundle is abnormal, there being in each bundle a central mass of xylem with the phloem masses lying, the one on the central, the other on the peripheral side of it. Between the xylem and the peripheral phloem mass is the cambium layer. The structure is the same in both phloem masses : either will therefore serve for the study of the sieve-tubes. In the soft bast, which resembles that of Helianthus, but has larger constituents, observe (i). The transverse, circular, punctate Sieve-plates, having the same appearance as in Helianthus, and easily recognised by their contents being stained with eosin. (ii). The Companion-cells appearing as though cut off from the side of a sieve-tube by a longitudinal wall. STEM. SIEVE-TUBES. 85 (iii.) Gambiform cells. Treat some sections with Schulze's solution all the ; walls of the soft bast turn blue (cellulose), but the sieve-plates appear yellow or brown, (cf. longitudinal sections.) II. Cut longitudinal sections through the soft bast : either radial or tangential sections will do. Mount some in iodine solution. The transverse sieve-plates will be brought into prominence by the deep yellowish brown staining of the mass of substance, which surrounds them : this may consist of A a. Callus mass, which immediately surrounds the plate, and is apparently a derivative of cellulose, thpugh it differs from it in its properties : the size of the callus mass is variable according to season, age, &c., being greater in autumn, and in old sieve-tubes. b. Protoplasm, which is usually collected in close contact with the sieve-plate (or callus if present), and more especially on its upper side. Note, i. the oblong form of the segment of the sieve-tubes. ii. The companion-cells, short, with granular protoplasm, and nucleus, iii. Cambiform cells of similar form to the segments of the sieve -tubes. Other sections should be stained with Eosin> then washed, and mounted in glycerine. The sieve-tubes will be readily seen as their contents will have stained deeply. III. Mount some sections in water, and having found a sieve-plate with callus, run some dilute potash under the cover slip. 86 PEACTICAL BOTANY. The callus mass swells : the protoplasm also swells : the section thus becomes more transparent, and the cellulose basis or true sieve becomes more apparent, and its pores can be easily seen. For further reactions of the callus, see p. 31. IV. Treat some fresh