Richard M. Holaian fclOJLOGr A COURSE OF PRACTICAL INSTRUCTION IN BOTANY. A COUESE OF PKACTICAL INSTRUCTION IN BOTANY BY F. 0. BOWER, M.A, F.L.S., LECTURER ON BOTANY AT Till NORMAL SCHOOL OF SCIENCE, SOUTH KENSINGTON AND SYDNEY H. VINES, M.A., D.Sc, F.L.S., FELLOW AND LECTURER OF CHRIST'S COLLEGE, CAMBRIDGE, AND READER I{J ,BOTANX jy, THE UNIVERSITY. WITH A PREFACE BY W. T. THISELTON DYER, MA., C.M.G., F.R.S., F.L.S., ASSISTANT-DIRECTOR OF THE ROYAL GARDENS, KEW. PART I. PHANEROGAM^PTERIDOPHYTA. MACMILLAN AND CO. 1885. Tlie Eight of Translation and Reproduction is Eeserved. BIOLOGY LIBRARY RICHARD CLAY AND SONS, PRINTER^, . BREAD STREET HIpL, T^PON, E.G., IN MEMORIAM PREFACE. A FEW words may be said to explain the origin of the work of which the present portion is a first instal- ment. In 1873 I was invited by the Science and Art Department to conduct a course of instruction in what is now the Normal School of Science at South Ken- sington. It was a condition of the undertaking that the instruction should be carried on continuously from day to day and throughout the working hours of each day. My friend Mr. Lawson, late Professor of Botany at Oxford, was so good as to give me his assistance. We had the use of Professor Huxley's convenient and well-appointed laboratory, and we determined to attempt a course of instruction which should embrace the lead- ing morphological facts of every important type in the vegetable kingdom. We, in fact, resolved to adopt exactly the same plan of work as Professor Huxley in his own teaching had found convenient for the animal side of morphology. At this time, as far as I am aware, no previous at- tempt had been made in this country to give an extended 981914 VI PREFACE. course of botanical instruction of this kind. Professor Lawson and myself found our own difficulties scarcely less considerable than those of the students. The interest, however, which the novelty of the new method of work excited in the class soon became very obvious. The enthusiasm of the more skilful students at once stimulated and assisted us, and at the conclusion of the course we found that there was scarcely anything of importance in the rather comprehensive range which had been attempted which the students had not been able to study, examine, and draw for themselves. This course was an experiment. It was repeated at irregular intervals during the next few years. It gradually took a more systematic shape, and with the appointment of Mr. Bower . as Lecturer on Botany in the Normal School, it is likely, I think, to settle down into a permanent system of instruction. I had always hoped to put together the results of the experience in teaching methods acquired at South Kensington in the form of a handbook, which should save teachers who wished to follow our example from much of the trouble and difficulty which I, and those who, at different times, have taught in this way, have had to face. But, in the meanwhile, I had been drawn off to administrative duties which have left a steadily diminishing leisure for purely scientific work. For- tunately, my friend Mr. Bower was willing and with far greater competence to take up the task which I was unable to perform, and to him are entirely due the PREFACE. Vll laboratory instructions for studying the different types selected. Dr. Vines Las very kindly supplied the chapters on methods and on the morphology of the cells. But besides this he has at every step given the assistance of his own extensive experience in practical teaching. It had been our intention to preface the directions for the study of each type with a short account, in language fairly intelligible to the general reader, of its salient morphological facts. This would have repre- sented the brief lecture with which the work of each day began in the course as originally organised. To carry out this intention would have postponed the publication of the teaching directions already prepared by Mr. Bower, and, in justice to him, it has seemed the best course to issue what is already finished without delay. It is intended to follow the present part with another, which will comprise the remaining types of the vegetable kingdom. Should the book be found as useful to students as it is hoped may be the case, I look forward to seeing the original scheme upon which it was planned still carried out in a future edition. W. T. THISELTON DYER. ROYAL GARDENS, KEW, December 1884. TABLE OF CONTENTS. INTRODUCTORY CHAPTERS. I. MET BODS AND REAGENTS. PAGE A. Making Preparations 1 B Micro-chemical Reagents 17 II. STRUCTURE AND PROPERTIES OP THE CELL. A. General Structure 24 B. Micro-chemistry of the Cell 29 C. Micro-physics of the Cell 37 CONTENTS. PRACTICAL DIRECTIONS FOR THE STUDY OF TYPES. PHANEROGAMS. I. ANGIOSPERMS. t VEGETATIVE ORGANS. A. DICOTYLEDONS. PAGE EMBRYO AND GERMINATION 44 STEM HERBACEOUS TYPE. * Mature 45 ** Young 62 Apical Bud 64 Node 67 STEM ARBOREOUS TYPE 68 STEM AQUATIC TYPE. ....... 82 SIEVE TUBES 84 LATICIFEROUS TISSUES 87 LEAF PETIOLE 90 LAMINA Bifacial Type 91 Centric Type 97 ROOT 99 Apex of the Hoot 102 B. MONOCOTYLEDONS. EMBRYO AND GERMINATION . 104 STEM HERBACEOUS TYPE 107 STEM ARBOREOUS TYPE 112 LEAF 114 ROOT 118 Apex of Root 120 tt REPRODUCTIVE ORGANS. DEVELOPMENT OF THE FLOWER .122 STAMEN 125 CARPEL AND OVULES 129 FERTILISATION 131 DEVELOPMENT OF THE EMBRYO. i. Dicotyledon 132 ii. Monocotyledon 134 CONTENTS. XL PHANEROGAMS (continued). II. GYMNOSPERMS. t VEGETATIVE ORGANS. PAGE EXTERNAL CHARACTERS 136 HISTOLOGY OF THE STEM ... 138 LEAF .149 ROOT .151 tt REPRODUCTIVE ORGANS 154 RIPE SEED AND GERMINATION 160 PTERIDOPHYTA. A. LYCOPODIN.E. I. SELAGINELLA. SPOROPHORE , 162 OOPEORE 173 II. LYCOPODIUM. SPOROPHORE 175 13. FILICINE.E. ASPIDIUM. MATURE SPOROPHORE. EXTERNAL CHARACTERS 186 ANATOMICAL CHARACTERS TO BE OBSERVED WITH THE NAKED EYE 188 MICROSCOPIC OBSERVATIONS. Stem 190 Root ....... 198 Leaf . . \. ."..'. ; 201 Sporangia .... 202 OOPHORE 204 YOUNG SPOROPHORE 210 C. EQUISETINE^l. EQUISETUM. SPOROPHORE 212 Sporangia 224 OOPHORE. .... 226 PKACTICAL BOTANY. i. METHODS AND REAGENTS. A. Making Preparations. Preservation of Material. In many cases it is possible, and even preferable, to use fresh material, but it is often convenient to keep it for a time ; the best liquid for this purpose is ordinary methylated alcohol, in such quantity as to completely cover the material. It must be remembered that this will extract the green colouring matter (chlorophyll) from the material im- mersed in it, as well as resin and other substances. Hardening. It is not necessary, for the general study of the histology of plants, to harden them, for the tissues are usually sufficiently firm to admit of their being cut satisfactorily. In the case of exclusively parenchymatous tissues, especially those of cellular plants, it is necessary to harden them somewhat, and for this purpose dilute alcohol (50 per cent.) may be used. B 2 PRACTICAL BOTANY. When it is desired to study the structure of the protoplasm and of the nucleus, special methods must be employed for hardening them, or rather, for fixing them as nearly as possible in the condition in which they were during life. For this purpose the fluids mentioned below must be used. Care must be taken that the objects are of small size, that the quantity of hardening fluid is very large relatively to the bulk of the object, and that the fluid has ready access to all parts of it. The following are the best fluids for this purpose : 1. Absolute alcohol. 2. Picric acid (saturated solution in water). 3. Chromic acid (O'l 0'5 per cent, solution in water). 4. Osmic acid ('1 - 1 per cent, solution in water). These reagents can only be applied to fresh material. The following is a useful method for preparing sea-weeds : to a quantity of saturated solution of picric acid in sea-water add three or four times its volume of sea- water, and treat the tissue with it for | hr. 2 hrs. : then treat successively with 30, 50, 70, and 90 per cent, alcohol. When absolute alcohol is used, the object may be kept in it for an indefinite period. Such treatment generally makes the object brittle ; this may be reme- died when the object is to be mounted in glycerine by placing it, for at least twenty-four hours before it is to be cut, in a mixture of glycerine and absolute alcohol in equal parts, leaving it exposed to the air so that the alcohol may gradually evaporate. The glycerine slowly saturates the object and restores its consistency. This METHODS. 3 can of course only be done when the sections are to be mounted in glycerine. When picric or chromic acid is used, the object should be immersed in it for several hours ; the length of time varies with different material, and, in the case of chromic acid, with the strength of the solution used, from a few minutes to twenty-four hours. The objects must then be washed with dilute alcohol (50 per cent.), and then placed in stronger alcohol (70 per cent.), and finally in absolute alcohol (or 90 per cent.), which must be changed so long as any colour is still extracted from the objects. They may be preserved in this for future use. When osmic acid is used, the fixing effect is pro- duced much more rapidly; in the case of simple structures, such as unicellular or filamentous Algae a few minutes (5 15) generally suffices; in the case of more complex structures, such as ovules, sporangia, growing-points, &c., the object may be left in the acid till it looks black on the exterior : it must be then well washed with dilute alcohol (50 per cent.), and left in it for \some time, and be then removed to 70 per cent. The sections are best mounted in dilute glycerine. In some cases osmic acid produces an excessive blackening of the cells, which can be removed by treatment with chlorine-water. It is advisable in cases in which the cell-walls tend to swell up excessively (as in many Algas) to use solu- tions of picric, chromic, and osmic acids, to which an equal volume of absolute alcohol has been added. Of these hardening reagents the most serviceable are absolute alcohol, or 90 per cent, alcohol, and picric acid. B 2 4 PEACTICAL BOTANY. Cutting Sections. A sharp razor is the best cutting instrument. Care must be taken to keep the object and the razor wet during the process of cutting, in order to avoid the entrance of air into the tissue, and to prevent adhesion of the section to the razor. When fresh material is cut, water or very dilute alcohol may be used for this purpose, but if material which has been hardened is cut, it is advisable to use alcohol of the same strength as that in which the material has been preserved. When a successive series of sections of an object is required, a microtome may be used. Imbedding. The objects are frequently so large that they may be held in the hand whilst they are being cut. If they are too small for this it is convenient to imbed them in some substance. The simplest method is to fix the object into a slit in a piece of pith. Elder-pith is the best. When the sections are to be made with a microtome, it is more convenient to imbed in some easily fusible substance ; by this means also the position of the object is less likely to be distorted in the process of cutting. Many mixtures of waxy and fatty substances are used for this purpose, of which the following is perhaps the best : Solid paraffin (melting-point about 58 C.) : 2 parts. Yaselin : 1 part. These must be melted together and well stirred. The resulting substance is sufficiently transparent to enable the exact position of the object to be ascertained ; it is easy to cut, and it is readily soluble in carbolic acid and turpentine. The relative proportions of paraffin and METHODS. 5 vaselin may be varied somewhat to suit the object ; a softer mixture is produced by increasing the proportion of vaselin. For soft objects cacao-butter, which has the advantage of being soluble in ether or chloroform, is useful. The method of imbedding is to make a cavity in a piece of the substance sufficiently large to contain the object, which must have been previously washed with alcohol to remove all traces of water from its surface ; a small quantity of the substance is then melted and poured into the cavity so as to surround and cover the object. When it is cold it may be cut. Another method of imbedding is to moisten the object in water, and then suspend it by means of a pin attached to a thread in some white of egg, which has been previously well shaken up, and then strained through muslin. The white of egg should be in an evaporating dish. The object should be left thus sus- pended for some hours, so that the white of egg may come into close contact with all parts of it. Heat is then applied by means of a water-bath, and the white of egg coagulates. The part surrounding the object is now cut out and hardened in alcohol for some days. This method is useful for making sections of buds and flowers. It is important to keep the imbedded objects wet with alcohol during the process of cutting, in order to prevent the drying-up of the object, and its consequent contraction away from the substance in which it is imbedded. A third method of imbedding is very useful when it is desired to obtain sections of very small objects, such 6 PRACTICAL BOTANY. as spores, pollen-grains, &c. This is effected by means of gum. A thick layer of strong clean gum is laid on the flat surface of a piece of pith ; this is allowed to become nearly dry ; and then the pollen grains or spores are dusted on to it ; these are then covered with an- other thick layer of gum, and the whole is allowed to dry. Sections are now made of the dried gum, and, on their being placed in water, the gum is dissolved, and the sections of the pollen-grains or spores are set free. Staining. It is often useful to stain sections in order to bring out certain points in their structure, which are difficult to observe under ordinary circum- stances. A great number of colouring matters have been used for this purpose, among which may be men- tioned as the most useful: Hsematoxylin, Carmine, Cochineal, Gold Chloride, various preparations of Aniline, such as Safranin, Nigrosin, Fuchsin, Methyl- green, Eosin, and Methyl-violet. Staining is best performed by placing a few drops of the staining-fluid in a watch-glass and immersing the sections in it. The exact strength of the fluid, and the time of exposure of the sections to its action varies in each case, and must be ascertained by preliminary trials. As a rule, when differentiated staining is desired, the best results are obtained by using a dilute solution, and by exposing the sections for a long time to its action. Haematoxylin. A number of preparations of this colouring-matter are in use ; of these the following are those generally employed for vegetable tissues. 1. Alum solution of Hrematoxylin. Dissolve 0*35 grammes of hsematoxylin in 10 c.c. of water, and add to it a few drops of a METHODS. 7 solution of alum consisting of 1 gramme of alum to 1 c.c. of water. 2. Kleinenberg's hsematoxylin. Saturate some 70 per cent, alcohol with calcium chloride ; let the mixture stand for twelve to twenty- four hours over alum, shaking occasionally ; add eight parts of 70 per cent, alcohol ; filter, and then add a solution of hsematoxylin in absolute alcohol until the liquid has a purple-blue colour; let it stand in a corked bottle exposed to sunlight for about a month ; it is then fit for use. The liquid is to be diluted as required with alum solution. 3. Expose a few crystals of hsematoxylin to the action of gaseous ammonia in a watch-glass under a bell-jar : then add water, and a good colouring fluid is obtained. The disadvantage of this is that it has to be freshly prepared every time it is required. The alum-solutions will stain all parts of the cell, including the cell- wall. Their especial uses are (a) to make the cell- walls more evident when they are naturally transparent and colourless ; (b) to stain the protoplasm, so as to make its intimate structure apparent ; (c) to stain the nucleus, so as to demonstrate its pre- sence and to show up its structure. The ammoniacal solution is especially adapted for differentiated staining. If a dilute solution be used, the first thing to become stained is the chromatin of the nucleus, then, after a time, the rest of the nucleus (achromatin), then the protoplasm. The cell- walls do not stain with this fluid, or only slightly. Kleinenberg's hsematoxylin stains in a few minutes, whereas the alum-solution is much slower in its action. Hsematoxylin may be used either for fresh material, or for sections which have been previously hardened with alcohol, or with picric or chromic acid. In the latter case the sections must be washed repeatedly in distilled water to remove every trace of the acid, which, if present, would interfere with the proper action of the hsematoxylin. If the section becomes too deeply stained, as sometimes happens when the alum-hasma- toxylin is used, the excess of colouring-matter may be removed by washing with watery solution of alum. 8 PRACTICAL BOTANY. Sections stained with alum or with Kleinenberg's hgematoxylin are to be mounted in Canada balsam (or Dammar). Those stained with the ammoniacal solution are to be mounted in glycerine. Carmine. The two best preparations of carmine are those of Beale and Thiersch : carmine possesses, however, but little differentiating power. 1. Beale's Carmine To prepare this 0'6 gramme of carmine is dissolved in 2 c.c. of boiling solution of ammonia ; the solution must then stand for an hour or so to cool, and to allow of the escape of the superfluous ammonia ; to the solution are added 60 c.c. of distilled water, 60 grammes of glycerine, and 15 grammes of absolute alcohol. The mixture must be allowed to stand for some time ; it is then to be filtered. 2. Thiersch's Carmine Four grammes of borax are dissolved in 56 c.c. of distilled water ; to this 1 gramme of carmine is added, and then twice its volume of absolute alcohol is added to the liquid. After nitration the liquid is ready for use. Carmine readily stains the protoplasm and the nucleus ; Thiersch's preparation is especially useful for bringing out the structure of the nucleus. It can very well be used for sections which have been previously treated with picric, chromic, and osmic acids. The time during which the section is to be exposed to its action varies very much, as is the case with hsematoxy- lin. The rule is in both cases, that the most satisfactory results are obtained by a prolonged immersion in a dilute solution. In case of overstaining, the section may be washed for a moment in water, to which a trace of ammonia has been added. Preparations stained with carmine are best mounted in glycerine. METHODS. 9 3. Picro-carmine (or ammonium picro-carminate) is another useful preparation of carmine. It is prepared by adding a strong ammoniacal solution of carmine to a quantity of concentrated solution of picric acid in water, until a precipitate begins to be formed ; it is then evaporated to about one-fifth of its bulk filtered, and the filtrate is evaporated to dryness. The crystal- line residue is dissolved in water so as to make a 5 per cent solution, and this may be diluted as occasion requires. Another method (Gage) is to dissolve a quantity of picric acid in ] 00 parts of water, and an equal quantity of carmine in 50 parts of solution of ammonia ; these are then mixed, filtered, evaporated to dryness, and the residue dissolved in 100 parts of water. Picro-carmine is used especially for staining nuclei, the staining being more uniform than when carmine alone is used : it has this further advantage, that a prolonged exposure to it does not pro- duce overstaining, as is the case with the other preparations of carmine. The objects should be previously kept for some time in absolute alcohol. If it is desired to retain the double staining which this reagent produces, the sections must be mounted at once in glycerine ; but if the carmine staining only is required, the sections must be washed in water, which will dissolve out the picric acid. When stained sections are mounted in glycerine, a small quantity of picro-carmine must be added to the glycerine in order to preserve the colours. The various preparations of carmine can be used as well for tissues which have been hardened in chromic, picric, or osmic acid, as for fresh tissues, but the former stain less readily. 4. Cochineal. The ordinary preparations of carmine frequently fail to give good results, especially when the tissue has been previously treated with chromic acid. Other preparations of the same colouring matter made directly from the cochineal insect have therefore been employed. 1. Alcoholic Solution. A quantity of finely powdered cochi- neal (best grey) is extracted for several days with 70 per cent, alcohol ; the liquid is filtered off and is ready for use. 2. Solution in water. Seven grammes of cochineal and an equal quantity of burnt alum are rubbed up together in a mortar until the whole is a fine powder : the powder is then added to 700 c.c. of 10 PRACTICAL BOTANY. distilled water ; the whole is then boiled, and evaporated to 400 c.c. : when it is cool a trace of carbolic acid is to be added, and then the liquid is passed two or three times through a filter. A dirty -red substance remains on the filter, and the filtrate is a clear fluid, thin layers of which appear red and thicker layers violet. This fluid will keep well for some months, but every now and again a trace of carbolic acid must be added to it, and it must be filtered. Both these preparations give good results, the differentiation being very marked. In using the alcoholic solution, the sections must be first soaked in 70 per cent, alcohol before they are placed in the staining liquid : it is also necessary, when sections are to be stained, to dilute the solution considerably with 70 per cent, alcohol. The watery solution acts very rapidly, staining fresh or alcohol material in a few minutes (3 5). The solution of cochineal in water stains especially the bast- fibres of vascular bundles. In some cases the whole of the wood stains, but if the section be treated with dilute hydrochloric or sulphuric acid, the colour will be removed from all the cell-walls except those of the bast-fibres. Gold-chloride, in 0'5 per cent, solution in water, has been employed for staining Fungi. They must remain in it from one to six hours, and be mounted in dilute glycerine. Aniline colouring-matters.; A large number of these have been employed, only the more important are mentioned here ; they all stain rapidly. 1. Safranin. This is used in solution in absolute alcohol. It is especially adapted for staining sections which have been previously hardened with chromic or picric acid ; it is not quite so good for those which have been treated with osmic acid. The sections must be well washed in distilled water, and then placed in a small quantity (1 c.c.) of the saturated alcoholic solution mixed with an equal volume of distilled water ; they require to be left for several hours in the staining fluid. They must then METHODS. 11 be removed, and washed for a short time in alcohol ; then they must be placed in absolute alcohol and kept there until they appear transparent. The sections can now be mounted in dis- tilled water in order to see if the results are satisfactory, or, if they are to be preserved, they must be cleared by means of oil of cloves, and mounted in Canada balsam or Dammar. By this means very successful preparations of the structure of nuclei can be obtained. 2. Fuchsin. This is used in alcoholic solution. It is useful for bringing out the structure of thickened cell- walls. The sections must be previously treated with alcohol. It is also a good reagent for corky tissue. When a section is stained and is then washed with absolute alcohol, the coloration is removed from all parts excepting the corky tissue. 3. Methyl-green. A tolerably strong alcoholic solution of this is used. The sections of the object, which must have been previously kept in absolute alcohol, are to be treated with the staining-fluid for from 5 25 minutes, then quickly washed with distilled water, and mounted in glycerine. The nucleus stains of a green or bluish-green colour, the protoplasm remaining un- coloured. It is especially good for staining nuclei which are dividing. It has been used for staining chlorophyll-corpuscles, and is also useful in bringing out the nuclei and protoplasm in the cells of Fungi, which have been previously preserved in absolute alcohol and in glycerine. Strasburger recommends the following method for obtaining preparations of nuclei : A section of the fresh tissue is mounted in 1 per cent, acetic acid solution, to which a little methyl-green has been added ; the nuclei are fixed almost instantaneously. 4. Methyl- violet, This is used in concentrated alcoholic solution. It is especially useful for staining bacteria. A few drops of the solution are added to 15 20 c.c. of distilled water, and a drop or two of the mixture should then be placed on the bacteria- membrane (zooglcea), and be allowed to remain there for a short time until the membrane appears to be coloured : if the solution used is too strong, the substance between the bacteria will become stained. The colouring-matter is then washed off with distilled water, or better with a 10 per cent, solution of acetate of potash. The preparation may then either be allowed to dry in the air and 12 PKACTICAL BOTANY. be then mounted in Canada balsam, or it may be mounted in a 50 per cent, solution of potassium acetate in water. A useful preparation of methyl- violet is the following : Some of that substance is dissolved in strong sulphuric acid, forming a brownish-green solution : on the gradual addition of water the violet colour reappears. This is especially useful for sieve-tubes. If a section be treated with this fluid for a short time, and be then washed with water, it, will be found that the cell- walls have become swollen and transparent, that the protoplasm has become deeply stained, aod that the sieve-plates are very well brought out. Lignified tissues treated with this fluid assume a yellow colour, as they do when treated with aniline sulphate. 5. Hanstein's Aniline-violet. This is prepared by dissolving equal parts of fuchsin and methyl- violet in alcohol. It stains cellulose cell- walls of a faint violet colour, and lignified cell-walls red. It is especially useful for bringing out the different parts of the bast, since the bast-fibres stain red, whereas the sieve-tubes and the parenchyma scarcely stain at all. The protoplasm is stained pink ; amyloid substances, gums, and nuclei stain different shades of red, resins blue, and tannin brick-red. 6. Hoffmann's Blue. Used in solution in dilute alcohol slightly acidified with acetic acid : it is a useful reagent, inas- much as it stains the protoplasmic cell-contents and not the cell- wall : it stains also the callus which closes the perforations of the sieve-plates during the winter in perennial plants. (Water blue is almost as good a reagent.) 7. Methylene blue. Used in solution in water : stains the cell- wall but not the protoplasm. To produce the differentiated staining mentioned in 6 and 7, the preparations must be washed in water after staining, and also before staining if the material has been previously kept in alcohol. 8. Alizarine. Many of these aniline-dyes will not stain the protoplasm of Fungi. Alizarine will do so at least in some cases. 9. Eosin. Used in strong alcoholic solution for demonstrating the structure of sieve-tubes, as it stains the protoplasm deeply : a solution in water may also be used. 10. Corallin (rosolic acid). A solution in 30 per cent, sodium carbonate colours lignified tissue, the callus of sieve-tubes, and starch grains pink. METHODS. 13 To the detailed instructions given above, the follow- ing general remarks may be added. All the above- mentioned staining-fluids may be used for protoplasm and nuclei. The stain produced by aniline-colours is apt to fade, so that they are not to be recommended for preparations which are to be kept for a long time. The staining of haematoxylin also fades, but more slowly. In order to prevent fading, the preparations should be kept in the dark. Clearing the preparations. If it is not desired to observe the details of structure of the protoplasm or of the nucleus, the best clearing agent is a solution of potash, either in water or alcohol. The most generally useful is the 5 per cent, solution made by dissolving five grammes of solid caustic potash in 100 c.c. of dis- tilled water. The alcoholic solution is made by adding strong alcohol (ordinary methylated alcohol will do) to a quantity of a concentrated solution in water until a precipitate begins to be formed. The mixture must then be well shaken, and allowed to stand and settle for twenty-four hours; the clear fluid is then poured off. For use a mixture of equal parts of this solution and of distilled water may be made. The clearing action of potash is due to the swelling of the cells and their contents, so that they become more transparent ; at the same time it dissolves many of the granules in the protoplasm, and saponifies the oil-drops. The swelling caused by the action of the solution in water is often too great, especially when it is desired to see the cell- walls distinctly; this difficulty may be got over by the use of the alcoholic solution. 14 PRACTICAL BOTANY. After treatment with the aqueous solution, the sections should be washed in distilled water, and after treatment with the alcoholic solution in dilute alcohol; the sec- tions, in both cases, should be mounted in glycerine. Another method of clearing, which is especially re- commended for obtaining good preparations of growing points, is to treat sections with calcium chloride. The sections are placed on a slide in a drop of water, and are then covered with some dry powdered calcium chloride ; the slide is then warmed over the flame of a spirit-lamp until the water has nearly all evapo- rated; a drop or two of water is now placed on the sections, and they are to be mounted in glycerine. In the case of tissues, which have been hardened in alcohol, with or without treatment with other hardening agents, another method of clearing may be used. The sections, after staining, if that is desired, should be placed for a few minutes in absolute alcohol ; they should then be transferred to a watch-glass, containing either a mix- ture of turpentine and creosote (four parts of the former to one of the latter), or some oil of cloves ; sections which have been stained with aniline dyes are best cleared by cedar-wood oil ; they should be left in this for a short time, until they appear to be quite trans- parent, and should then be mounted in a drop of Canada balsam or Dammar. Mounting. For the observation of the coarser features of the histology of plants, it suffices to mount the sections in a drop of water, or, in certain cases, in a drop of alcohol. This is the only possible method when micro-chemical observations have to be made. Sections of objects which have been hardened, or otherwise METHODS. 1 5 specially prepared, and which it is desirable to pre- serve, should be mounted in glycerine, or in glycerine- jelly, or in Canada balsam or Dammar. Glycerine and glycerine-jelly may be used for sections which have been prepared by any of the methods described above. Dilute glycerine should be used for this purpose, con- sisting of a mixture of pure glycerine with an equal bulk of water. The cases in which these media are especially suitable have been mentioned. Only those sections which have been treated with absolute alcohol, and either oil of cloves or the mixture of turpentine and creosote can be mounted in Canada balsam or Dammar. When preparations are mounted in glycerine-jelly, a trace of carbolic acid should be added in order to pre- vent the growth of Fungi. The sections should be previously soaked in glycerine so as to remove water or alcohol from them. In order to make the preparations mounted in glycerine quite permanent, the cover-slip should be fixed to the slide by applying a coating of gold-size or Brunswick black round its edge with a brush. Care should be taken that no glycerine is on the slide outside the v cover-slip; if any is there it should be removed by means of blotting-paper before applying the varnish. It is better to varnish Dammar preparations in this way also ; but it is not necessary for those in Canada balsam. Preparations of green parts of plants in glycerine lose their colour. These may be best put up in a drop of a strong solution of potassium acetate, or of alu- minium acetate. The cover-slip must be fastened down as above described. 16 PRACTICAL BOTANY. It is often desirable to observe objects in the living state for a considerable time under the microscope. This must be done in a moist chamber. A moist chamber may be readily constructed as follows : A piece of thick rough cardboard is cut to the size of the glass slide, and a circular hole is punched out of the middle of it of such a size as to be covered by a cover-slip. The piece of cardboard is then soaked in water (or boiled in water when pure cultures of Fungi are to be made), so as to saturate it, and placed on the glass slide. A drop of water (or solution as described below), is placed on the cover-slip, the object is immersed in it, and the cover- slip is then inverted over the hole in the piece of card- board. Thus the object is suspended in a drop of liquid on the under surface of the cover-slip. Any loss from the chamber by evaporation is prevented by occasion- ally wetting the cardboard on the slide. The liquid to be used will of course vary with the nature of the object to be observed. In the case of AlgaB, water may be used ; in the case of Fungi, decoc- tions of various organic substances (fruits, animal tissues, &c.), or a solution of sugar, according to the habit of the Fungus. For observing the germination of the spores of Mosses and Ferns, water will suffice ; but in the case of pollen-grains a solution of sugar is neces- sary (1 20 or even 30 per cent, the concentration being different for different plants) ; for observing the process of cell-division in the hairs on the stamens of Tradescantia, a 1 per cent, sugar solution may be used. REAGENTS. 17 B. Miero-ehemical Reagents. Besides the fluids which are used for hardening and staining the tissues, a considerable number are employed, which, on account of the characteristic effects produced by their action on cell-walls and cell- contents, may be regarded as chemical tests for the various substances which may be present. The follow- ing are the principal reagents which are used in this way : the mode of preparing them is also given, and some indication of their uses; but this latter subject is more fully treated in the next chapter. I. Acids. Sulphuric acid. This is used either concentrated or dilute (1 to 3 of water). It causes, in either case, the swelling-up of cellulose cell-walls, starch-grains, &c. ; when cellulose cell-walls which have been pre- viously saturated with solution of iodine are treated with sulphuric acid, they turn blue. Concentrated sulphuric acid dissolves cellulose and starch, but cuticularised cell-walls and the middle lamella of lignified cells resist its action. It is used with cane-sugar, as a test for proteids, and with aniline sulphate as a test for lignin. Nitric acid. It colours cuticularised cell- walls and proteids yellow ; it also causes swelling-up of cellulose and of lignified cell-walls. It is useful for dissolving the crystals of calcium oxalate which are frequently present in the cells. It is used with ammonia as a test for proteids (xanthoproteic reaction), and with potassium chlorate as a test for suberin, and as a macerating fluid. C 18 PRACTICAL BOTANY. Hydrochloric acid. Used, with aniline chloride phloroglucin, or carbolic acid, as a test for lignin. By itself it turns lignified cell-walls yellow ; when its action is prolonged, the cell-walls become violet, owing to the presence of various substances such as phloro- glucin, coniferin, and pyrocatechin. Chromic acid. A strong aqueous solution of this acid dissolves lignified and cellulose cell- walls ; cuticu- larised cell-walls resist its action ; but they become very transparent, and may be easily overlooked. A dilute solution brings out the stratification of cell-walls very clearly. Acetic acid. This is used as a dilute aqueous solution (1 per cent.). It dissolves crystals of calcium carbonate ; it causes swelling-up of cell- walls, starch- grains, &c. ; it brings out nuclei very clearly ; it is useful as a corrective after treatment of a preparation with potash. II. Alkalies. Potash. This may be used either in a dilute or a concentrated solution in water. The dilute solution is chiefly used for clearing preparations, as already de- scribed. It causes cell-walls, starch-grains, &c., to swell up very much, and it dissolves proteid crystalloids, and most aleurone-grains. It gives a reddish colour to cells in which tannin is present. It may be used as a macerating fluid ; when woody tissues are boiled in potash, the cells of the vascular bundles become more or less isolated, for the lignin of their walls undergoes solution. It dissolves inulin. The concentrated solution is used as a test for suberin. When sections of cork are boiled in strong REAGENTS. 19 potash, the suberin escapes in the form of yellow viscid drops ; when the sections are only slightly warmed in potash solution the cuticularised cell-walls assume a yellow colour. Potash is also used, together with copper sulphate as a test for proteids, and for various kinds of sugar. Ammonia.. The solution in water is often used instead of potash for clearing preparations, as its action is less intense. It is used, together with nitric acid, as a test for proteids, and with copper sulphate as a solvent for some forms of cellulose. III. Non-Metallic Elements. Iodine. This is .one of the most useful micro- chemical reagents. It is used in solution, in water, or alcohol, and in the chloride of zinc mixture. 1. Solution in water. Dissolve a small quantity of potassium iodide in the requisite quantity of water ; then dissolve iodine in it until the liquid has a dark sherry colour. This may also be prepared by diluting the liquor iodi of the pharma- copoeia. 2. Alcoholic solution. Dissolve iodine in alcohol until it has a dark sherry colour. This may also be prepared by diluting with alcohol the tinctura iodi of the pharmacopoeia. Iodine stains proteid substances brown, cellulose faintly yellow (as a rule, see next chapter), cuticularised and lignified cell-walls yellow, gum purple, starch blue (only in the presence of water). Iodine is used as a micro-chemical test for starch and for cellulose. The blue colour which it gives with starch, and the conversion of the faint yellow colour of a cellulose cell- wall stained with iodine into blue when c 2 20 PRACTICAL BOTANY. it is treated with sulphuric acid, are characteristic The cellulose reaction is also given with the chloride of zinc mixture. IV. Inorganic salts. Sodium chloride (common salt). This is used both in dilute (10 per cent.), and in saturated solution in water as a solvent for the proteid crystalloids. The 10 per cent, solution is used for producing plasmolysis. Ferrous sulphate. Used in dilute solution in water, to which a drop of nitric acid has been added, as a test for tannin. Potassium bichromate. Used in dilute solution in water as a test for tannin ; used also (in 1 per cent, aqueous solution) for hardening tissues. Potassium chlorate.^Used, together with nitric acid, as a macerating agent. Copper sulphate. Used in very dilute solution in water ; the blue colour of the solution must be only just perceptible. It is used, with potash, as a test for some kinds of sugar, and for proteids. It is used also in the preparation of ammoniacal solution of cupric hydrate, which dissolves pure cellulose. For the preparation of Fehling's fluid the following directions are given in Foster's Practical Physiology : (a). Dissolve 34'65 grm. of pure crystallised cupric sulphate in about 160 c.c. of distilled water. (&). Dissolve also 173 grm. of pure crystallised potassic-sodic tartrate in 600 to 700 grm. of sodic hydrate sp. gr. 1.12. Add (a) to (b) stirring well to cause a thorough mixture, and dilute with distilled water to a litre. Fehling's fluid should be fresh made whenever it is required, since it decomposes on keeping ; it will keep some little time if REAGENTS. 21 kept in a cool place in the dark, and in completely filled, well- closed bottles (Hoppe-Seyler). The solution (6) may be prepared, and kept for adding to (a ) freshly prepared when required. Before using a kept solution to test for sugar, always boil a little of it by itself to see if any reduction will take place. From 1 c.c. of this solution the copper is completely reduced by '005 grm. of grape-sugar. Y. Organic substances. Alcohol. Used as a solvent for various substances, such as fats, oils, resins, colouring-matters, &c., and as a precipitant for various substances. It has a peculiar action upon some proteid crystalloids. Ether. Used as a solvent for wax, fats, resins, &c. Cane sugar. The concentrated aqueous solution is used, together with strong sulphuric acid, as a test for proteids. A dilute (1 per cent.) solution is useful for mounting living cells for observation under the microscope. Alkanet. The alcoholic extract, or better, an alco- holic solution of alkannin, is used as a test for resin and caoutchouc : a fresh solution must be prepared on each occasion. Phloroglucin. Used in alcoholic or aqueous solu- tion as a test for lignin. The section is first treated with hydrochloric acid and then with solution of phloro- glucin : the lignified cell- walls assume a bright red colour. If phloroglucin cannot be obtained, it may be replaced by an extract of cherry wood. Shavings of young cherry- branches are extracted with alcohol for twenty-four hours to remove chlorophyll and other substances ; then the alcohol is poured off. The shavings, after being pressed, are extracted for several days with alcohol, 22 PKACTICAL BOTANY. the alcohol extract is poured off and filtered and then evaporated nearly to dryness, until a piece of coarse blotting paper moistened with it and treated with hydrochloric acid turns violet. The extract is then ready for use. It gives a violet colour to lignified cell-walls, as it contains other substances ( especially pyrocatechin) besides phloroglucin. Phenol (carbolic acid). Used, together with hydro- chloric acid, as a test for lignin. The best preparation of it is its solution in hydrochloric acid : this is pre- pared by dissolving carbolic acid in warm hydrochloric acid, adding, whilst the mixture is cooling, sufficient hydrochloric acid to dissolve any precipitate that may be formed. Lignified cells, treated with this mixture and exposed to sunlight, assume a bright green colour in consequence of the presence of coniferin. Aniline sulphate and chloride. These salts are also used as tests for lignin in cell-walls ; the chloride is preferable. They may be used in solution either in water or alcohol, but the alcoholic solution gives the best results. The section is first treated with the solu- tion and then with sulphuric or hydrochloric acid re- spectively, or better, the solution may be kept slightly acidulated by one or other ot these acids : the lignified cell- walls assume a bright yellow colour. VI. Mixtures. Schulze's Solution 1 (Chlor. Zinc lod.). Used as a test for cellulose ; cellulose cell-walls turn blue when treated with this mixture ; corky and lignified cell- walls turn yellow, protoplasm brown, and starch blue. 1 Since there has been some uncertainty as to the exact name of the botanist who introduced these reagents, it may be here stated that they were first used by Professor Franz Sclmlze, of Rostock. See Flora, 1850, p. 643. REAGENTS. 23 It is prepared by dissolving zinc in pure hydrochloric acid, and evaporating the solution, on a water bath, in the presence of metallic zinc until it has a syrupy consistence ; it is then saturated with potassium iodide, and then with iodine ; a few grains of iodine should be left in the liquid after it is poured off for use. It may also be prepared by dissolving 25 parts of pure fused zinc chloride and 8 parts of potassium iodide in 8^ parts of water, filtering through asbestos, and saturating with Iodine. On adding Iodine to Schulze's solution till precipitation begins, a fluid is obtained which stains the cell- walls yellow, and the oallus of sieve-plates a deep brown (Russow.) Schulze's Macerating Fluid. 1 One gramme of potassium chlorate is dissolved in 50 c.c. of nitric acid ; the tissue is then placed in a small quantity of it, and the whole is boiled for a short time in a test tube ; the liquid is poured off, and the residue is well washed with water. A filter may be used for washing. The cells become isolated in consequence of the solution of the middle lamella. 1 See Note, p. 22. II. THE STRUCTURE AND PROPERTIES OF THE CELL. A. General Structure. CUT longitudinal sections of a parenchymatous tissue, the young shoot of the Elder, for example, mount in water, examine the parenchymatous cells of the pith with a high power ; Note, 1, the Cell-wall, transparent, colourless, and apparently homogeneous ; 2, the Protoplasm, forming a layer (the pri- mordial utricle), closely lining the cell-wall, and connected by bridles with a more centrally placed mass in which 3, the Nucleus, a well-defined, roundish, highly refractive body, is situated ; 4, the Vacuole, filled with colourless fluid, the Cell-sap. Structure of the Protoplasm and Nucleus, Harden a small piece of a young growing shoot or root of Pinus in picric acid or in absolute alcohol ; stain with ammonia-haematoxylin ; mount in dilute glycerine, or stain with Kleinenberg's hsematoxy- lin, and mount in Canada balsam ; examine with a high power : Observe in the protoplasm 1. The Ectoplasm, a hyaline layer, but little stained, next to the cell- wall. STRUCTURE OF THE CELL. 25 2. The Endoplasm, the more internal, deeply stained proto- plasm ; note that the staining is confined to fibrillse which form a sort of network in the endoplasra, and to numerous minute particles, the Microsomata. Observe in the. nucleus 1. Stained fibrillae forming apparently a reticulum (ehromatin). 2. The unstained matrix (achromatin) in which the fibrillaj are imbedded. 3. Cell-division. In order to study the process thoroughly the hairs on the stamens of Tradescantia may be taken. A stamen is to be removed from a bud, on a warm day, and is to be placed at once in a drop of 1 per cent, sugar- solution on a cover-slip ; the cover-slip is then to be placed over a moist chamber as previously described. A magnifying power of about 500 diameters is to be used. A terminal cell of one of the hairs, with a large and conspicuous nucleus, is to be observed. It will be seen that the nucleus gradually elongates in the direction of the longer axis of the cell ; it becomes more granular, and the protoplasm of the cell aggre- gates at its poles ; then the nucleus presents a striated appearance, the fibrillse gradually arrange themselves parallel to the longer axis of the nucleus, and approach each other at the poles ; thus a characteristic nuclear spindle is produced ; the fibres are then ruptured in the equatorial plane, and gradually collect at each pole, so that two new nuclei are found. A layer of granular protoplasm (the cell-plate) consisting of microsomata, is now found in the equatorial plane, and it extends on each side until it reaches the wall of the cell ; this layer becomes converted into cellulose, and constitutes the dividing wall between the two cells. Good preparations of nuclei may be obtained by making longi- tudinal sections of growing points (e.g. of the young roots of }* and staining with heematoxylin. 4. Structure of Chlorophyll-corpuscles and of Leukop last ids. a. Chlorophyll -corpuscles, or chloroplastids. Mount a thin leaf of a Moss (e.g. Funaria), or the prothallus of a Fern, in water ; note the corpuscles in the cells. 2G PRACTICAL BOTANY. Treat with alcohol ; the green colouring matter (chlorophyll) is gradually dissolved out, and the corpuscle is left colourless. Press out the contents of an internodal cell of Nitella or of Cham on a slide ; put on a cover-slip and examine with a high power. Run in some distilled water : observe that the corpuscles swell up, assuming the form of large hyaline vesicles ; the chlorophyll is con- fined usually to one portion of the vesicle. If chlorophyll-corpuscles, which have been treated with picric acid and decolorised with alcohol, be stained with iodine, Hoffmann's blue, or hsematoxylin, and be examined with a very high power, it will be seen that they have a trabecular structure ; it is from the inter- stices of the trabeculaB that the colouring-matter has been removed. The leaves of Vallisneria afford good material. The same result may be obtained by prolonged treatment with dilute acid (hydrochloric acid mixed with water in the proportion of 1 : 4, is most effectual), or by exposure for one or more hours to steam (Pringsheim). The minute structure of the corpuscles can be very readily made out in cells of the leaves of JEcheveria. If the plants used have been previously exposed to light, it will be observed that the chlorophyll-corpuscles contain granules. If a decolorised corpuscle be treated with iodine, the inclosed granules will turn blue, showing that they are starch -granules (see p. 33). b. Leukoplastids. These are colourless protoplasmic corpuscles of various shape, which are to be found in the cells of those parts of plants which are not exposed to light, and in which starch is deposited. STRUCTURE OF THE CELL. 27 The material must have been previously treated for a short time with picric acid, so as to prevent their swelling up and disappearing when they are mounted in water or in dilute glycerine. The most suitable material is any tissue of which the cells contain but few starch-granules ; the best is the tubers of the orchid Phajus grandifolius, (Bletia Tankervillice). In this starch- grains can be easily seen borne on the leukoplastids. 5. Structure of Thickened Cell-walls and of Starch-grains. a. Cell-walls. Cut a transverse section of an old branch of Clematis Vitalba ; mount in water ; examine with high power. Observe the thick-walled cells of the pith ; the wall appears to consist of a series of concentric layers ; this is described as the stratification of the cell-wall. Strip a piece of the bark from the branch, and remove with a needle some of the fibrous internal layer of the bark ; mount in water, tease out with needles, and examine with a high power. Observe the dark lines running in the wall of the fibre at an acute angle to the longer axis of the fibre ; note that these lines run in different direc- tions in different layers of the wall of the fibre ; this may be seen by carefully focussing first the surface and then the deeper layers of the wall; these lines are described as constituting the striation of the cell-wall. Observe the canals running transversely across the cell-walls. Some of the cells will present their upper walls (those nearest the observer) : on these pits 28 PRACTICAL BOTANY. will be seen, which are the terminations of canals like those seen in the sections of the longitudinal walls. Pits can be readily seen, without making sections, in the leaves of some species of Trichomanes. Cystoliths may be included here, since they are developed from the cell- wall. Cut a transverse section of a leaf of Ficus elastica : mount in water; examine with a high power. Observe the layer of large clear cells underlying the superficial layer of the epidermis of the upper surface of the leaf : here and there one of these cells is seen to contain a botryoidal body suspended by a stalk from the top of the cell ; this is a Cystolith : it consists of a mass of cellulose developed as an outgrowth from the cell-wall, encrusted with calcium carbonate. Run in a drop of acetic acid : observe that the cystolith becomes gradually transparent, and that an evolution of bubbles of gas is taking place from it. When the calcium carbonate is all dissolved, a mass of cellulose will be seen to remain, presenting both striation (from above downwards) and stratification (parallel with its margin). Apply tests for cellulose (p. 29). 1. Starch-grains. Scrape lightly with the blade of a knife the freshly cut surface of a piece of a potato ; mount the scrapings in a drop of water ; examine with a high power. A number of somewhat ovoid bodies of various sizes will be seen ; these are Starch-grains. Near the pointed end of a well-developed grain will be seen a small, round, clear spot, the hilum. MICRO-CHEMISTRY OF THE CELL. 29 On each side of the hilum a number of layers will be seen, constituting the stratification of the grain. The layers near to the hilum are concentric with it, and are complete ; the more external layers are excen- tric, and many of those between the hilum and the broad end of the grain will be seen to be incomplete ; hence the layers are more numerous between the hilum and the broad end than between the hilum and the pointed end. Here and there will be seen a compound grain, consisting of two small grains in contact b} r their broad ends, and invested by several layers common to both. !>. The Micro-Chemistry of the Cell. I. The CELL-WALL. a. Cellulose cell-walls, i. Coloured faintly yellow by iodine. ii. Coloured violet on treatment with Schulze's solu- tion (p. 22). iii. Coloured blue on treatment with iodine and sulphuric acid. In some cases the cell- wall turns blue when it is treated with iodine alone, a substance allied to starch being probably present (amyloid) ; instances of this are, the asci of Lichens, the bast in the stem of Lycopodium and in the root of Ruscus, the endosperm-cells of Pceonia, and the cells of the cotyledons of various Leguminous seeds. In other cases the characteristic reactions are not given on treatment with Schulze's solution, or with iodine and sulphuric acid ; instances of this occur in the tissues of young seedlings, of growing-points, of the cambium, and of Fungi. In the case of young tissues it suffices to treat them previously with hydrochloric acid or with solution of potash for a short time ; they then give 30 PRACTICAL BOTANY. the reactions mentioned above ; the tissues of Fungi require a long treatment (three or four weeks) with potash. It appears that in these cases other substances are present which must be extracted from the cell- walls before the characteristic cellulose-reaction can be obtained. iv. Dissolved by ammoniacal solution of cupric hydrate and by strong sulphuric acid. v. Stained by solutions of carmine and of hsematoxylin which contain a mordant, by methylene blue, and in various degrees by other aniline colours. 1. Lignified cell-walls i. Coloured yellow by iodine and Schulze's solution, ii. Coloured deep brown by iodine and sulphuric acid. iii. Coloured bright yellow when treated with solution of aniline chloride or sulphate, the colour being intensi- fied by subsequent treatment with hydrochloric or sulphuric acid. iv. Coloured red when treated with solution of phloroglucin (p. 21), and with strong hydrochloric acid. v. Coloured green when exposed to light (\ 1 min.), after treatment with carbolic and hydrochloric acids (p. 22). vi. Swollen and slowly dissolved in strong sulphuric acid ; dissolved slowly in concentrated chromic acid ; soluble in Schulze's macerating fluid (p. 23). When the lignification is not complete the cell-wall becomes disorganised and dissolves partially in strong sulphuric acid ; this is due to the presence of a considerable proportion of cellulose. Lignified cell- walls give the characteristic cellulose-reactions after maceration in Schulze's fluid. The solubility of lignin in this fluid affords a means of isolating the cells of a woody tissue. vii. Stained slightly or not at all by solutions of carmine and hsematoxylin, but readily by aniline colours MICRO-CHEMISTRY OF THE CELL. 31 c. Cuticularised cell- walls (including cork) i. Coloured yellow by iodine, by Schulze's solution, and by iodine and sulphuric acid. ii. Coloured yellowish by concentrated solution of potash ; on gradually warming (without boiling), it becomes bright yellow ; on boiling, yellow drops of suberin escape. iiii. On treatment with Schulze's macerating fluid, the cuticularised cell-walls become conspicuous ; on boiling, viscous drops (impure suberic acid) escape, which are soluble in hot alcohol, ether, benzol, chloroform, and dilute potash solution. Traces of cuticularisation may be detected by treating the tissue for a short time with Schulze's fluid without heating, and then with potash ; the cuticularised cell-walls become conspicuous and turn yellow ; the colour may be intensified by gently warm- ing in potash. iv. Dissolved very slowly in concentrated chromic acid ; hence on treatment of a section with this reagent the cuticularised cells are the last to disappear. v. Not stained by solutions of carmine or hsematoxy- lin ; stained by aniline solutions. The cuticle may be isolated, from the surface of a leaf for instance, by boiling for a few minutes in hydro- chloric acid, and then washing with water. d. Callus. To be found on the plates of the sieve- tubes. i. Soluble in sulphuric acid. ii. Stained by Hoffmann's blue, and by hsematoxylin. The most delicate reagent for callus is the following : to a quantity of chlor. zinc, iod., add an equal volume of the ordinary solution of iodine in potassium iodide ; to 32 PRACTICAL BOTANY. the mixture add a saturated solution of iodine potassium iodide drop by drop, until precipitation begins, This mixture stains the callus a deep brown. e. Mucilaginous cell- walls. Resemble cellulose cell-walls in their reactions. On treatment with iodine and sulphuric acid they some- times assume a brownish colour in addition to the blue. Cell- walls which have become converted into gum do not turn blue on treatment with iodine and sulphuric acid : Hanstein's aniline- violet colours them red. Both mucilaginous and gummy cell-walls are stained by inethylene blue. Mucilages stain pink with corallin solution ; certain kinds stain with Hoffmann's blue. Gums stain with neither of these reagents. f. Mineral deposits in cell-walls. i. Silica. On heating a section of tissue containing silica on platinum foil with nitric acid, a complete skeleton of the silicified cell-walls remains. (ii.) Calcium oxalate. Occurs in the form of crystals: insoluble in acetic acid ; soluble, without evolution of gas, in nitric acid. (iii.) Calcium carbonate. Occurs either in distinct crystals, or, apparently, as granules : soluble in acetic acid with evolution of bubbles of gas The most characteristic form in which it appears is in special outgrowths of the cell- wall which are incrusted with it ; these are termed cystoliths (see p. 28). MICRO-CHEMISTRY OF THE CELL. 33 II. The CELL CONTENTS. a. The Protoplasm. i. Coloured yellow by iodine, and by Schulze's solution. ii. Coloured yellow by nitric acid, the colour becoming more intense on warming ; on the addition of potash or ammonia a bright yellow colour is produced (xantho- proteic reaction). iii. Coloured violet after treatment with dilute solu- tion of copper sulphate on the addition of potash. Fehling's solution may be used. See page 20. iv. Coloured pink after treatment with syrup on the addition of dilute sulphuric acid. v. Stains readily with solutions of carmine, hgema- toxylin, and Hoffmann's blue ; bright red with Hanstein's aniline violet. These reactions are given by all bodies consisting of proteids. I. The Chlorophyll-corpuscles. On treatment with alcohol the green colouring- matter (chlorophyll) is dissolved, and the substance of the corpuscle is left: this gives the reactions enu- merated above as being characteristic of proteids. The orange colour of many fruits and flowers is due to the presence of coloured granules which appear to be modified chlorophyll-corpuscles (chromoplastids). These may be well observed in the petals of Tropceolum. c. The Starch-grains. Coloured blue on treatment with iodine. Coloured pink with corallin solution (p. 12). In order to detect the presence of minute starch-grains in chlorophyll-corpuscles, the tissue must be kept in alcohol exposed D 34 PRACTICAL BOTANY. to light until the whole of the chlorophyll is dissolved out ; it must then be treated for several hours in strong solution of potash ; after neutralisation with acetic acid the tissue may be treated with iodine. d. Oil-drops. i. Coloured black on treatment with osmic acid, ii. Soluble in alcohol, in ether, and in potash (sapo- nified). e. Mineral substances. i. Calcium oxalate : occurs with two molecules of water of crystallisation in crystals belonging to the clinorhombic system (including raphides), or with six molecules in crystals belonging to the quadratic system. Clusters of crystals and sphsero-crystals may consist of crystals belonging to either system. Insoluble in acetic acid ; soluble in nitric acid, without evolution of gas. ii. Calcium carbonate : occurs usually in small crystals, the crystalline nature of which can only be ascertained by means of the polariscope. Soluble in acetic acid, with evolution of bubbles of gas (CO 2 ). iii. Calcium phosphate (also magnesium phosphate) : occurs in the form of granules (e.g. the globoids). Soluble in acetic acid without evolution of gas. iv. Calcium sulphate : occurs in the crystalline form. Soluble with difficulty in acetic or nitric acid. /. Crystalloids : may be seen in the more external cells of potato-tubers, in the form of cubes. i. They give the reactions characteristic of proteids. ii. Soluble in potash. iii. Soluble in saturated solution of common salt. g. Aleurone-grains : occur most prominently in oily seeds. MICRO-CHEMISTRY OF THE CELL. 35 i. Give the reactions characteristic of proteids. ii. Soluble, usually, in potash. The reactions of these bodies are very different in different seeds ; the following will serve as types : 1. Grains without crystalloids. (a). Soluble in water : peony, almond, cherry, apple. (6). Partially soluble in water ; more or less readily soluble in 10 per cent, solution of common salt. a. Soluble in saturated solution of common salt : lupine, pea, bean, scarlet runner. j8. Soluble in saturated solution of common salt only after treatment with alcohol : sunflower, turnip, cress, 2. Grains containing crystalloids. (a). Partially soluble in water ; more or less readily soluble in 10 per cent, solution of common salt. a. Soluble in saturated solution of common salt : Brazil nut, pumpkin. . Soluble in saturated solution of common salt only after treatment with alcohol : castor-oil plant, walnut. In all cases a mass (globoid) of mineral matter remains behind after the solution of the grain ; this is soluble in acetic acid. The sections should be examined in alcohol. h. Tannin : gives the cells in which it is present a brownish colour. i. Coloured deep brown by potassium bichromate, or chromic acid. ii. Coloured greenish-blue by dilute solution of iron sulphate. iii. On treatment with a solution of ammonium molybdate in a strong solution of ammonium chloride, either a voluminous yellow precipitate is formed (showing presence of tannin), or a red colour is produced (show- ing presence of tannic, i.e., digallic acid). i. Resin : occurs in drops in the cells bounding resin-passages as well as in the passages themselves. D 2 36 PRACTICAL BOTANY. i. Coloured red by tincture of alkanet. ii. Coloured blue by Hanstein's aniline violet iii. Decomposed by potash. iv. Soluble in alcohol and ether. 7c. Caoutchouc : occurs in the laticiferous vessels in the form of granules of different size in different plants : stains red with alkannin solution. By means of this reaction good preparations of laticiferous vessels can be made. I. The Cell-sap may contain in solution : 1. Colouring matters. 2. Cane-sugar (as in the Beet-root) (C 12 H 22 O n ), which does not give a reaction with Fehling's solution. See p. 20. If much is present it may be made to crystallise out by treatment with absolute alcohol. 3. Grape-sugar (Glucose) (C 6 H 12 O 6 ). If a section be boiled in dilute Fehling's solution, it will, if the cells contain glucose, turn yellow, owing to the reduction of the copper. See p. 20. The precipitate (cuprous oxide) appears in the cells under the microscope as small black granules. 4. Inulin (C 6 H 10 O 5 ). When the material or the section has been treated with alcohol, the inulin is precipitated in the form of sphsero-crystals, which may be readily observed. These crystals are insoluble in cold, but readily soluble in warm water, and in dilute acids and alkalies. Coloured slightly brown by iodine. 5. Asparagin (C 4 H 8 N 2 O 3 ). When a section of a tissue containing asparagin is treated with absolute alcohol for some time, the aspara- gin is precipitated in the form of prismatic crystals, MICRO-PHYSICS OF THE CELL. 37 either in the cells or at their edge, which are readily soluble in water. The best method is to maintain a stream of alcohol under the cover-slip by means of blotting-paper. A saturated solution of asparagin may be used as a further test ; the precipitated crystals will not dissolve in it, but will be dissolved on the addition of water. In performing these tests it is better to use longitudinal than transverse sections. C. The Micro-physics of the Cell. I. Imbibition. This term is used to express the fact that the cell-wall and certain of the cell-contents (proto- plasm, starch-grains, aleurone-grains, crystalloids) usually contain a certain amount of water, termed the water of imbibition. The amount of water of imbi- bition may be made to vary by appropriate re-agents, and this involves variation in size of the body observed. These phenomena are best seen in cell-walls and in starch-grains ; the cell-walls should be such as are thickened, and consist of cellulose; those which are chemically altered (either cuticularised or lignified) cannot be made to vary to any considerable extent. Cut a transverse section of the petiole of the Sun- flower (Elder or Mallow will do as well) ; mount in water ; examine with high power. Observe just beneath the epidermis, several layers of cortical cells, the walls of which are thickened at their point of junction (collenchyma). Bun in some potash solution, or some moderately 38 PRACTICAL BOTANY. strong sulphuric acid ; notice the swelling-up of the thickened cell-walls. The swelling-up of starch-grains may be observed in the same way. The amount of the swelling-up may be estimated by using a micrometer-eye-piece. The thickened cell-walls of pith-cells of Clematis Vitalba, or those of seeds (Lupine, Date) are also suitable material for this purpose. II. Osmotic Properties. These can be most easily studied in cells which have coloured cell-sap. Cut a rather thick section of a piece of fresh beet- root, and mount in water ; Observe, the thin cell-wall ; the layer of protoplasm (primordial utricle) which lines the cell-wall ; the red cell-sap filling the cavity of the cell (vacuole). Note that the red sap does not escape from unin- jured cells. Examine a similar section which has been dipped for a moment into alcohol ; the red sap diffuses out of the cells. Hence it is evident that the colouring-matter cannot diffuse out of a living cell, but diffuses readily out of a dead cell. Mount another section in water, and run some 10 per cent. Na Cl solution under the cover-slip ; it will be seen that the red sap collects as rounded deeply- coloured bodies in the centre of the cells. This is due to the contraction of the primordial utricle. MICRO-PHYSICS OF THE CELL. 39 A cell in this state is said to be plasmolytic. The contraction is due to the withdrawal of water from the cell-sap by the strong salt solution, this withdrawal not being compensated for by the entrance of salt solution into the vacuole. The salt solution diffuses through the cell- wall, and occupies the space between the cell- wall and the contracted primordial utricle, but it cannot pass through the primordial utricle to any considerable extent. On washing the section with water, the plasmolytic cells gradually reassume their normal appearance. From these observations it is evident that the passage of substances in solution into or out of the vacuole is controlled by the primordial utricle so long as the cell is living. Plasmolysis can also be well demonstrated on a Fern- prothallus by treating it as above with salt solution ; it will be seen that the contracted primordial utricle is connected to the cell-wall by a great number of delicate protoplasmic filaments. III. Optical Properties. 1. Double Refraction. In order to study this subject, apparatus for polarising light must be adapted to the microscope. This consists of two Nicol's prisms, one of which is fitted into an eye-piece, the other being fixed below the stage of the microscope, so that the light which is reflected from the mirror must pass through it : the former prism is termed the analyser, the latter the polariser. The sections to be examined may be mounted in water or in glycerine, but the best results are obtained with sections mounted in Canada Balsam. A twig of a tree affords good material for observation. A thin, nearly median, longitudinal section is to be made and mounted : a high power must be used. 40 .., PRACTICAL BOTANY. The examination is to be commenced by rotating the analyser, so that the field of the microscope is bright : the section will then appear much as it does when examined with an ordinary microscope. The analyser is now to be rotated until the field is quite dark : it is then seen that the outlines of the cells appear bright, the thick, dense cell- walls (those of the fibres and vessels, for instance), being brighter than the thin cell-walls (those of parenchymatous cells). This observation teaches that the cell-walls, but not the protoplasmic cell-contents or the cell-sap, are doubly refractive, and that the denser the cell-wall the more highly refractive it is. A thin transverse section examined in the same way is seen to present similar appearances. It will be observed, in addition, that the transverse sec- tion of a much thickened cell-wall (that of a bast-fibre, for instance), presents, when the field is dark, a dark cross : when the analyser is rotated through an angle of 90, the dark cross is replaced by a bright one the field being also bright. For the explanation of this phenome- non reference should be made to textbooks of Physics. Mount some 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. be prepared. A 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 the left of the line E 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- trum to the right of the line F 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 root- hairs (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, petiolate, 1 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 sul- phuric 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 longi- tudinally, 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 leaf- trace 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 t leaves opposite, leaf- trace with 1 bundle. Clematis, leaves opposite, leaf- trace 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 and their relative position must be accurately marked. A diligent comparison of these (with drawings) will supply the data for de- ducing 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 sur- rounding 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 2/ A 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 inter- cellular 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 excep- tion 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. E 2 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 refrac- tive, 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 cell- walls. 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 diffi- culty. 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 char- acters 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 bundle- sheath 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 from it. We have thus four portions of the bundle which, taking them in succession from the periphery to the centre, are named as follows : f (i.) Sclerenchyma. A. Phloem. ^ ]'/. f { (n.) Soft Bast. B. (iii.) Cambium. C. (iv.) Xylem. Taking first (A) the Phloem examine (i.) The Sclerenchyma. This appears as a half- moon 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 appear- ance. 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 appear- ing 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 cam- bium 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 develop- ment 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 (yellow with Schulze's solution, or with H 2 S0 4 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 suc- cessively 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 satis- factorily represented in a single radial section, therefore the study of the tissues and their relative positions should be conducted by com- parison 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 prosenchyma- tous 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 develop- ment : 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 proto- plasmic 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. Note on Interfascicular Canibium. We 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 cam- bium 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 longi- tudinal 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 layers. We 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 protuber- ances of the dermatogen and the subjacent cells. As they increase in size their internal tissues become differentiated into (1) procambium, which is subse- quently 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 multi- cellular 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. Ob- serve also the various stages of the process of vacuoli- sation 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 bundle- system 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). F 2 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 parenchy- matous 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 posi- tion 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 gradu- ally 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 ex- tent 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 ex- tended, 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 con- necting two consecutive medullary rays laterally. The cells of the Medullary rays are in the xylem thick- walled (lignified) and pitted ; they have proto- plasmic 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. Muci- lage 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 thy loses. (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. (iii). Vessels 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 prosenchyma- tous, 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 connec- tion 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. (a). Sieve-tubes, by lateral disten- sion 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. (ii). Xylem. (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 inter- weaving 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 : 1. A 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. 3. A 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 vege- tationis). 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. 3. A 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 at a higher point on the axis than that of any of the leaves. We 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. G 2 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 Schulze's solution, or aniline sulphate and H 2 S0 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 granu- lar 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 sections with iodine, then dry off the superfluous fluid with blotting-paper, and mount in a single drop of strong sulphuric acid. The cellulose walls and callus will swell; the protoplasm will contract; look carefully over the protoplasmic contents of the sieve-tubes for the points where sieve- plates have been ; here it will be found that fine strings of protoplasm, which passed through the sieve -plate, connect the protoplasmic masses on opposite sides of the sieve with one another (cf. Sachs' Textbook, Fig. 47.) By this reaction the continuity of protoplasm through the sieve is demonstrated. It will be noted that the sieve-tubes of Cucurbita, closely resemble those of Helianthus, the sieve-plates being transverse and simple. This is the usual type of sieve-tube to be found in primary phloem of Angio- sperms, and generally in herbaceous stems of the same group. In the secondary phloem of ligneous stems a more complicated type of sieve-tube is found. This will be studied below in the stem of the Lime. ii. Tilia (Lime). I. Cut radial sections of the phloem of a stem of Lime more than three years old. Mount in glycerine and examine with a high power for sieve-tubes. The general arrangement of the phloem is similar to that in STEM. LATICIFEROUS TISSUES. 87 the Elm. The sieves occur on oblique walls facing the radial plane, and are therefore here seen in surface view. Note that they have a similar appearance to those above described, but here three or more sieve- plates occur on each oblique wall. II. Cut tangential sections of the same ; mount as before. The oblique walls are here cut longitudinally ; the sieve-plates are often callous, especially in autumn. Note the form of the segments of sieve-tubes ; it is fundamentally the same as that of the cambium cell, as seen in tangential section. LATICIFEROUS TISSUES. The material for the study of these tissues should be prepared by treatment with alcohol to coagulate the latex. Care should be taken to place the material in alcohol directly it is cut, or at least the cut surfaces should be wetted with alcohol so as to check the flow of latex from them. If the latex be allowed to escape, the laticiferous tissues are emptied, and are then much less easily traced than when they are full. The best method is perhaps to preserve the -whole plant without injury in alcohol, in which case the latex will not be lost at all. i. Laticiferous Vessels. I. Cut tangential sections from the phloem of the root of the Dandelion (Leontodon Taraxacum}, mount in potash and glycerine, and warm ; examine under a low power. 88 PEACTICAL BOTANY. The main constituents of the tissues are parenchy- matous cells, with thin walls (phloem-parenchyma) : sieve-tubes are to be met with here and there. The whole mass of tissue is permeated by a ramifying, and profusely anastomosing network of laticiferous vessels. The communication of these tubes with one another is demonstrated by the continuity of their contents (latex), which appear brown and granular. The course of the vessels is mainly longitudinal, while lateral, horizontal branches frequently connect the parallel tubes. With a high power make out more accurately the course of a group of the vessels. II. Cut transverse sections of the same ; mount in glycerine, and examine with a low power. The laticiferous vessels appear circular in transverse section, and have brown contents ; they are distributed in groups, which form more or less regular concentric rings round the central xylem. Note in these sections the presence of sphere crystals of Inulin. In the former section they will have been dissolved by the treatment with potash. Observe that they are formed quite irrespective of the cell-walls, which are often included in them. Treat the sections with iodine solution. They are not definitely stained. Run some potash under the cover slip. They will be gradually dissolved without swelling. The development of tlie laticiferous vessels may be traced by cutting thin longitudinal sections through the cambium of the root of the Dandelion. By careful comparison of such sections it STEM. LATICIFEROUS TISSUES. 89 will be found that they originate from a number of originally separate cells of the cambium, the cavities of which are thrown together by the partial or complete absorption of the walls. Such fusions may appear in the terminal or the lateral walls. ii. Laticiferovs Cells, I. Cut tangential sections of the cortex of Euphorbia splendens (other species will do) just outside the vascular ring, and mount in water, or dilute glycerine. Examine with a low power. Running through the cortical parenchyma will be seen long tubes, with thick cellulose walls and granular contents. These are the laticiferous cells, which differ from the preceding in being developed, not by fusion of originally distinct cells, but by continued apical growth of single cells. Note cases of branching of these cells. Included in the granular contents are starch-grains of peculiar dumb-bell form. Treat sections with iodine solution, and observe the effect on these bodies. II. Cut transverse sections of the same stem, and note the distribution of the laticiferous cells ; they may be recognised by their walls, which are thicker than those of the surrounding tissues, and appear circular in section. III. Separate the whole cortex from a piece of the stem ; boil it in potash for about five minutes, and tease out the long laticiferous cells with needles ; mount, and observe with a low power. They appear as long cylin- drical structures, with thick walls (note striation). Observe occasional branching. They are usually broken at the ends. 90 PRACTICAL BOTANY. LEAF. A. PETIOLE. External Characters. Note in the leaf of the Sun- flower the channelled upper surface, and the insertion on the stem by a broad Pulvinus; in the axil may usually be observed an axillary bud. I. Cut transverse sections of the petiole and mount in glycerine. The details of structure resemble in many respects those of the young stem, from which the petiole differs in the following points : 1. The general outline of the section is semilunar, the concave being the superior (ventral), while the convex is the inferior (dorsal) surface : thus the petiole is dorsi- ventral whilst the stem is polysymmetrical. (This pro- perty extends also to the vascular bundles, of which the xylem is as a rule directed towards the upper surface.) 2. In the presence of numerous Stomata (two guard cells, cf. infra) ; beneath each stoma the collenchyma is replaced by chlorophyll-containing parenchyma with intercellular spaces. Note beneath each stoma the large respiratory cavity. 3. In the number and arrangement of the vascular bundles. In the petiole there are three main bundles, besides several smaller ones (cf. observation of stem with the naked eye, p. 47). 4. In absence of interfascicular cambium, the larger bundles are, for a time at least, open bundles, [i.e. have an active cambium,] while the smaller ones are closed [i.e. have no secondary thickening by cambium.]. SUNFLOWER. LEAF. 91 5. No general bundle-sheath is present, though each bundle is surrounded by a layer of colourless cells without intercellular spaces, which may be regarded as representing the bundle-sheath. B. LAMINA. Bifadal Type. I. Take a piece of the lamina of the leaf of the Sunflower, including the apex : it is important that it should be previously bleached by treatment with alcohol; warm it gently in a mixture of dilute glycerine and potash, and mount in glycerine : examine with a low power, and observe 1. The midrib, with its strongly marked vascular bundle, running up to the apex of the leaf, where it terminates abruptly in a mass of glandular parenchy- matous tissue. 2. Lateral branch-bundles passing off from it, and forming a network by frequent anastomoses, while some branches run up into and terminate in the serrate projections of the margin of the lamina in a manner similar to the midrib as above described. 3. Smaller branch-bundles, which sometimes end blindly in the parenchyma filling the meshes of the network. II. Cut off a small square piece of the lamina of a leaf of Heliantlius, including one of the main ribs or nerves, and imbed in cocoa-butter or paraffin (cf. directions, p. 4), so that the rib shall be perpen- dicular. Cut transverse sections, and mount in glycerine. If cocoa-butter has been used, it may be dissolved off the sections with ether or chloroform. 92 PEACTICAL BOTANY. Good sections may be obtained by holding the piece of lamina between slices of carrot, or pith ; or by folding the whole lamina repeatedly, and cutting sections from the whole mass. In these cases, though the chlorophyll appears of a better colour, the sections not having been treated with a solvent (alcohol), still the sections are infested with air bubbles, which may be partially removed by leaving the sections for some minutes in water ; they may be completely removed (though the chlorophyll would be dissolved) by treatment with alcohol. Difficulty will often be found in obtaining good preparations of the above ; all the important points may be more easily observed in the Cherry Laurel. Note with a low power 1. The general outline of the section, which is irregular and undulating, though it is in the main of uniform breadth. At the point corresponding to the main nerve the section widens out, the nerve appear- ing semilunar, as in the petiole. The convex side is the inferior (dorsal), and the concave the superior (ventral) surface. 2. That the margins of the sections (i.e. the superior and inferior surfaces of the leaf), are studded with projecting multicellular hairs. 3. That the arrangement of the tissues in the large nerve resembles that in the petiole, though less com- plicated. Thus it often has but one large central bundle, with smaller lateral ones. The position of the xylem and phloem relatively to the whole leaf corresponds to that in the petiole, i.e. xylem towards the upper surface, phloem towards the lower. Occasionally some of the smaller bundles in the vein are in- verted, showing an approach to the arrangement of bundles in the polysymmetrical stem. 4. Smaller veins, with correspondingly reduced SUNFLOWER. LEAF. 93 vascular bundles, are found scattered through the thinner part of the section. Next examine the thinner part of the section, or the Lamina proper with a high power, and, starting the study of the several tissues from the upper surface. Note successively the following tissues : 1. Upper layer of Epidermis, continuous with that covering the nerve ; it is a single layer of cells, covered externally by Cuticle, and with the same characters as that of the stem (cf. p. 51). It bears numerous multicellular hairs (already studied in con- nection with the apical bud). Stomata occur in considerable numbers (cf. infra). Beneath this layer lie- 2. Thin- walled, oblong cells, with copious protoplasm, and chlorophyll grains ; they are arranged with the longer axis perpendicular to the outer surface, and form two layers; this tissue, from the form and arrangement of the cells, is called the Palisade parenchyma ; below it is 3. A mass of parenchymatous cells of irregular form, with large intercellular spaces ; in general characters they resemble (2) ; this is the Spongy parenchyma. (2) and (3) are together included under the general term Mesophyll. Embedded between (2) and (3) are 4. Numerous smaller vascular bundles (nerves) of various size, often reduced to a single pitted or spiral tracheide, surrounded by a colourless sheath of paren- chyma similar to those in the petiole. The course of these bundles is diverse, since they form the reticulate system of veins ; they may thus be seen in the sections 94 PKACTICAL BOTANY. to have been cut transversely, obliquely, or longitu- dinally. 5. A second layer of epidermis bounds the section on the lower side ; it has the same characters as the upper layer, but stomata are more frequent. Note the large respiratory cavity, and two small guard cells. Hairs as before seen on the upper surface. Note the mucilaginous walls of these hairs. Since the leaf of Heliantlius is not a universal type, it would be well to study also the structure of other types, for instance the coriaceous leaves of the Cherry Laurel (Prunus Lauro-Cerasus), and the cylin- drical leaves of the Stonecrop (Sedum acre). Special structural peculiarities are to be observed in the leaves of other plants ; for instance, an epidermis consisting of more than a single layer of cells, e.g. in leaves of Ficus, Piperacece, Begoniacem, &c. ; Cystoliths in the cells of the epidermis, e.g. FicuSj Urtica, &c. ; glandular structures, e.g. in Ruta, Psoralea, &c., &c. III. Taking that of the Cherry Laurel sections maybe prepared as above directed for the Sunflower, and be mounted in dilute glycerine. Starting from the upper surface, observe successively the following tissues 1. Epidermis, a single even layer of cells, with thick walls, and colourless protoplasmic contents ; no hairs or stomata are to be seen ; the lateral walls are pitted ; the outer wall is differentiated into a. Cuticle, a continuous, well-defined layer, covering the whole epidermis externally. CHERRY LAUREL. LEAF. 95 1. Cuticularised layers, of granular appearance; they are intermediate in properties between cuticle and true cellulose. c. The cellulose layer, which abuts on the cavity of the cell. i. These several layers may be readily distinguished in sections treated with fuchsin. a and b stain much more deeply than c. ii. Treat sections with concentrated sulphuric acid, a retains a sharp contour ; the rest of the wall swells, and loses distinct- ness of outline. iii. Boil some sections for a long time with strong potash, a and the cuticular granules of b will he dissolved, while c and the cellulose matrix of b will remain. 2. The Palisade parenchyma, composed of thin- walled, oblong, closely-packed cells, with their longer axes perpendicular to the surface of the leaf; the cells are somewhat irregularly arranged in three layers; observe nuclei and chlorophyll grains ; here and there are cells with but little protoplasm (Idioblasts) in which is inclosed a large crystal. Passing towards the lower surface of the leaf, this tissue merges gradu- ally into 3. The Spongy parenchyma, the cells of which resemble those of (2) in general characters ; but their shape is various, and large intercellular spaces occur. Idioblasts with crystals are scattered here and there. Imbedded between (2) and (3) are 4. Vascular bundles of various size ; the direction in which these run is not uniform (cf. reticulate venation of leaf) ; the positions of xylem and phloem with regard to the whole leaf are the same as in the Sunflower ; the bundles are surrounded by a continuous 96 PRACTICAL BOTANY. colourless sheath of cells without intercellular spaces. At the lower limit of the section lies 5. The lower Epidermis, which resembles (1) in general character; but differs in having numerous Stomata. Note the appearance presented where the two Guard cells of a stoma have been cut transversely, and observe carefully a. The form and position of the two guard cells. I. The cavity or intercellular space between them (the Pore) ; this leads into c. The large, intercellular space (Respiratory cavity) in the tissue beneath the stoma. d. In the sections stained with Schulze's solution or with fuchsin, note the continuity of the cuticle round the guard cells, into the pore of the stoma. IV. Cut tangential sections from the upper and under surfaces of the leaf, and mount separately with the external surface in both cases uppermost. The cells of the upper epidermis are tabular, with sinuous outline ; the surface has a granular appear- ance (explained by the granular cuticularised layers observed in transverse sections) ; the lateral walls are pitted; contents colourless; no stomata. The cells of the lower epidermis are similar to the above ; but stomata are numerous ; they have no definite arrangement. Note the two sausage-shaped nucleated guard cells, inclosing the pore ; they contain chlorophyll. (For development of stomata, cf. Hyacinth, p. 117.) V. No Subsidiary cells are found in the Cherry Laurel. The leaves hitherto studied are of the bifa- cial type, i.e. the difference of the upper and lower STONECROP. LEAF. 97 surfaces is recognisable by a different arrangement of the tissues at or beneath those surfaces. It may be noted that the leaf of the Cherry Laurel is of a more pro- nounced bifacial type than that of the Sunflower, since in the latter case stomata arc found on both surfaces, while in the former they occur only on the lower surface. Centric Type. We have now to study leaves of the centric type, i.e. such as have their tissues arranged symmetrically. It is usually in succulent leaves that this arrangement is found, and they are of an approximately cylindrical form. As an example we may take the leaf of Sedum acre (the common Stonecrop). VI. Cut transverse sections of the leaf of the Stone- crop ; mount in water, or dilute glycerine, and observe that the outline of the section is even and oval; the arrangement of tissues is concentric, and is uniform all round, so that beginning at any point of the periphery and passing inwards we encounter 1. The Epidermis, a single layer of cells of variable size and shape, with well-defined Cuticle, and Stomata, the guard cells of which are much smaller than the epidermal cells. 2. Chlorophyll-containing mesophyll, which is not differentiated into palisade, and spongy parenchyma ; this tissue forms the great mass of the leaf; intercellular spaces occur; the cells are thin-walled, with a proto- plasmic sac, in which are imbedded chlorophyll grains, and there is large central vacuole. Observe the chlorophyll grains undergoing division. Embedded in this tissue lie centrally H 98 PKACTICAL BOTANY. 3. Vascular bundles of small size : their number varies from 3 to 5. Strip off a piece of epidermis from the leaf of Sedum acre, and mount in water. Note : 1. The Epidermal cells with sinuous outline, nucleated : with no chlorophyll. 2. The Stomata with two guard cells surrounding the pore as in the Cherry Laurel. Surrounding these are : 3. Three Subsidiary cells, which differ in size and shape from the ordinary epidermal cells, and are arranged in definite order round each stoma. Beneath the epidermis will usually be found cells of the mesophyll, with thin walls, large vacuole and protoplasmic sac, in which are embedded chlorophyll grains. By making similar preparations from successively younger leaves the development of the stoma and subsidiary cells may be traced as follows. From, one of the similar epidermal cells a smaller cell is cut off, from this are successively cut off the three subsidiary cells, the remaining cell is the mother-cell of the stoma, which divides to form the two guard cells. On the leaves of many plants, stomata of large size are to be found situated above the free endings of the vascular bundles of the lamina, and especially at the tips of the teeth : these are often incapable of closing, and are concerned in the secretion of water : hence they are called water-stomata. In certain cases (Saxifragacece and Crassulacece) a mass of cells of the mesophyll is specially differentiated as glandular tissue (the water-gland) ; it is connected with the termination of a vascular bundle. SCAELET EUNNER. ROOT. 99. ROOT. Observations with the Naked Eye. Germinate seeds of Phaseolus multiflorus (the Scarlet- Runner) in wet sawdust, or pure vegetable mould, till the primary root has attained a length of six to eight inches. Note with the naked eye 1. The Seed, from which the testa can easily be removed, disclosing 2. The two fleshy Cotyledons (no endosperm is present) : between these 3. The Plumule, which develops early as a stem, bearing foliage leaves. 4. Below the cotyledons a short Hypocotyledonary Stem, not clearly marked off externally, except by colour, from 5. The Primary root, on the upper part of which are 6. Numerous secondary, or Lateral roots. These are formed in acropetal order, and are arranged in regular longitudinal rows, usually four in number. On the youngest part of the primary root (i.e. within three inches or more of the apex) no lateral roots are to be seen. Observe that particles of the sawdust, &c., adhere to the older parts of the roots, while the younger apical parts come out of the soil quite clean. Microscopic Observations. Harden the roots in alcohol for two or three days or more. I. Cut transverse sections of the primary root at a point nearer the apex than the youngest lateral roots, H2 100 PRACTICAL BOTANY. i.e. about two inches from the end. Treat with dilute potash for about ten minutes, and mount in glycerine. N. B. It will be found convenient to hold the roots in pith, or otherwise to imbed, while cutting the sections. Observe the following tissues : 1. At the centre of the circular section is a mass of Parenchymatous Pith. At the periphery of this are 2. Four radiating groups of elements of the Primary Xylem, which are the most strongly marked tissues of the young root. They have dark lignified walls (test with Schulze's solution or aniline sulphate), and resemble the primary xylem of the stem. Note fresh elements in course of formation at their central limit. The development is thus centripetal. Alternating with these may be seen 3. Four groups of Primary Phloem, which are not as yet very well marked. These several groups of elements are separated laterally from one another by bands of parenchyma. At the periphery of the central cylinder thus built up is 4. The Pericambium or phloem-sheath, consisting of thin-walled cells, arranged in an undulating band, which is a single layer of cells in thickness, peripherally to the phloem, but opposite the xylem it consists of two to three layers of cells. 5. Immediately outside this is the Bundle-sheath, consisting of a single layer of cells, having the character- istic dark dot on their radial walls. Then follows 6. The parenchymatous Cortex, a thick band of tissue, with intercellular spaces, and 7. The Epidermis, a single layer, not well marked. SCARLET RUNNER. Single cells will be seen to have grown out perpendicu- larly to the surface as root-hairs. II. Cut sections successively at older points in the same root,- and observe the mode of origin of the lateral roots, noting more especially the following facts : a. The lateral roots arise opposite the groups of pri- mary xylem : this explains their arrangement in four rows as above observed with the naked eye. Z>. The pericambium, bundle-sheath, and a small portion of the cortex, all take part in their formation. c. In the older lateral roots it may be seen that their vascular system is continuous with that of the main root. This mode of origin of the lateral roots is the rule in the plants with apical meristem, arranged according to Type II. (cf. infra, p. 104). In the plants whose root-apex follows Type I. the lateral roots are mainly, or even entirely, derived from the pericambium. III. Cut transverse sections of the root, six inches or more from the apex, taking care to avoid the lateral roots : treat as before. The general arrangement of tissues is the same as has been above described, though there has been increase in bulk, and the xylem and phloem, being now more fully developed, are more easily recognised. Observe espe- cially that the parenchyma, lying centrally to the phloem, has begun to divide repeatedly by tangential walls : in fact, four cambium bands are thus formed, from which is derived the secondary thickening of the root. IV. Cat transverse sections of an old root of the Scarlet-Runner, and treat as before. Observe 1. Centrally a parenchymatous Pith, relatively to which PRACTICAL BOTANY. 2. The four Primary Xylem groups retain their original position. 3. Four large wedges of Secondary Xylem have originated internally from the four cambium zones. These are separated from one another laterally by 4. Four broad Parenchymatous rays, which lie on the same radii as the primary xylem. Outside the xylem is 5. The Cambium, having similar characters to that of the stem, and giving rise peripherally to 6. Secondary Phloem. Note if possible 7. The four groups of Primary Phloem now sepa- rated from the primary xylem, but still on radii alter- nating with the latter. The section is bounded by 8. Cork with a Cork-cambium. Apex of the Root. Type I. Cut thin median longitudinal sections of the apex of the radicle of the straight embryo of Helianthus. [The arrangement of the meristem at the apex of the radicle of the embryo is similar to that of the apex of the growing root, and the former is chosen in this case as it is much easier to make preparations from it than from the growing root. The sections are of little use unless they are accurately median.] Treat the sections with potash for ten minutes or more : wash with water, and mount in glycerine : examine with a low power, and observe that 1. The mass of tissue is composed of thin-walled cells, arranged regularly in longitudinal rows. APEX OF THE ROOT. 103 2. That these rows of cells converge towards a point at some distance below the external apex of the root. This is the punctum vegetationis. 3. Note the Procambium-cylinder, or formative tissue of the vascular bundles, which pursues a longi- tudinal course up the centre of the root. Examine with' a high power : and observe that 1. At some distance from the apex a definite layer of Epidermis covers the root externally. Follow this to- wards the apex : at some short distance from it this single layer splits into two : the inner is the Dermatogen, or formative layer of the epidermis : the outer is the outer- most layer of the Calyptra, or root-cap. Following the dermatogen further inwards, it will be seen to split again several times in succession : the dermatogen may be traced as a continuous layer covering the inner tissues. The layers thus thrown off externally from the derma- togen form collectively the Root-cap, or Calyptra. We have in this case a common formative layer for epidermis and root-cap (cf. root of Maize, p. 120.). 2. Between the procambium and epidermis lies a broad band of formative tissue of the cortex, or Periblem : follow this to the punctum vegetationis : it is also a distinct continuous band, though reduced to a single layer of cells at the apex. 3. The Plerome, or central procambium cylinder, may also be traced as distinct up to the apical point. This type of arrangement of tissues of the meristem may then be expressed thus : Calyptrogen \ a single layer of cells, i.e. epidermis and Dermatogen j root-cap have a common origin. 104 PEACTICAL BOTANY. Periblem, distinct from the rest. Plerome, distinct. To this type belong most of the Dicotyledons. The work may be equally well done on Linum usitatissimum, or Polygonum Fagopyrum. Type II. Prepare median longitudinal sections of the apex of the radicle of Phaseolus multiflorus (the Scarlet-Runner), and treat as the above. Examine with a. low power and make out 1. Calyptra (Eoot-cap). 2. Epidermis. 3. Periblem. 4. Plerome, forming the procambium and pith. But here all the different tissue-systems will be found to originate from a general meristem, the original formative tissue of none of them being distinct from that of the others. This type may be expressed shortly, thus : Calyptrogen \ Dermatogen ( All united in a general, undifferen- Periblem ( tiated mass of meristem. Plerome / As alternative plants of the same type, may be named Cucurbiia and Pisum. VEGETATIVE ORGANS. (B.) MONOCOTY- LEDONS. EMBEYO AND GERMINATION. Soak fruits of the Maize (Zea Mais) in water for several hours. N.B. The fruit is a caryopsis, and results from the develop- MAIZE. GERMINATION. 105 ment of both ovule and ovary ; its form is compressed conical, the apex of the cone being the basal point of attachment of the fruit. I. Strip off the external coat of the fruit: this represents both the wall of the ovary and the integument of the ovule. If sections be cut, these two layers may be distinguished from one another, under a low power. Distinguish in the body of the fruit which remains 1. A lateral, smaller, white portion: this is the Embryo. 2. A larger yellow part, which forms the greater mass of the fruit : this is the Endosperm. Separate the embryo from the rest, and note its shape. II. Cut longitudinal sections of the fruit, so as to include the axis of the embryo : mount in glycerine, and examine with a low power ; observe i. The coat of the fruit, consisting of two layers. Note at the apex of the fruit the remnant of the Style, and at the base the attachment. ii. The Endosperm, consisting of thin-walled par- enchyma; the cells contain polygonal starch grains, embedded in a matrix of protoplasm : in the peripheral yellower portion of the endosperm the starch grains are more closely packed than in the central whiter portion. iii. The Embryo, which is in close apposition to the endosperm : the part which is in contact with it is the Scutellum (cotyledon); it extends over the whole surface of contact, and almost completely surrounds the 106 PRACTICAL BOTANY. body of the embryo itself. Note (a) the central attach- ment of the scutellum to the body of the embryo ; (fy the vascular bundles, which form a connection through it ; (c) the Epithelium of peculiar structure, which faces the endosperm. The body of the embryo consists of a. An Apical bud, with several sheathing leaves, which surround the apical cone. b. A Radicle, having similar arrangement of the meristem to that of the older root (cf. infra.). Outside the radicle, and continuous with the root-cap, is a root-sheath or Coleorhiza, the existence of which shows the endogenous origin of the radicle. III. Cut sections of the endosperm, and treat with solution of iodine. Note the polygonal starch grains (blue), and the protoplasmic matrix (brown). Germination. I. Compare plants which have been germinated for different periods : the following facts in the history of germination may be observed : 1. The fruit swells. 2. The outer coat ruptures opposite the apex of the radicle, which soon protrudes aod bursts through the coleorhiza also, which appears as an irregular ring round the young root. 3. The rupture of the coat extends upwards to the point opposite the apical bud, which also emerges. 4. The root elongates, and forms lateral roots : other lateral roots (usually two) appear above the insertion of the scutellum : these soon equal the primary root in length. Hence there is no well marked tap root. 5. Leaves of the plumule unfold, and gradually turn green. MAIZE. STEM. 107 IV. From a young plant with leaves about three inches long cut longitudinal sections, as above : mount in water, and irrigate with solution of iodine. Observe 1. That in the neighbourhood of the surface of the scutellum the starch grains are in course of demolition, and that the central part of each is first attacked. 2. That no starch grains are to be seen in the epithelium of the scutellum. STEM. HERBACEOUS TYPE. I. Cut transverse sections of an internode of a well-grown stem of Zea Mais ; mount in water. N.B. Fresh material may be used, but stems preserved in alcohol are preferable. When fresh, the tissues are crowded with air bubbles. The sections should be cut from the upper part of one of the lower internodes, otherwise the vascular bundles may- be found to be imperfectly developed. Examine with a low power, and, beginning the study of the tissues at the periphery of the section, observe a. A single layer of Epidermis, having the usual characters : immediately below this are b. Irregular groups of Sclerenchyma with thick lignified walls : internally lies c. A mass of Parenchyma, which forms the ground- work of the whole section : embedded in this are d. Numerous Vascular bundles : note that they are smaller, but more numerous near the periphery than at the centre ; also that the position of the parts of the bundles relatively to the centre of the section is uniform. 108 PKACTICAL BOTANY. Treat a section with Schulze's solution : put on a high power, and examine in detail the several tissues above-named. a. The Epidermis appears as a definite layer of cells of unequal size, without intercellular spaces. Note a well-marked Cuticle (brown). Here and there may be found Stomata, with two small guard- cells and two subsidiary cells (the structure and development of the stomata will be studied in the leaf; p. 116). b. The Sclerenchyma consists of cells with thick, highly refractive walls, which stain yellowish brown with Schulze's solution (lignified). Note that it does not occur immediately below the stomata, but, as usual, there is there an intercellular space (respiratory cavity). c. The Parenchyma consists of cells with thin cellulose walls (blue with Schulze's solution). At the angles where the cell-walls meet are intercellular spaces. The external layers have abundant protoplasm with chlorophyll-grains. These are less frequent in the inner layers, while in the central parenchyma the protoplasm is hardly appreciable. d. For the minute study of the Vascular bundles select one of the largest central bundles. The section must be thin. The most prominent elements in the bundle are i. Four large Vessels of the Xylem, arranged like a V, with the angle towards the centre of the stem : of these the two smaller are developed first. Compare sections of young stems. In many Monocotyledons the arrangement of the constituents MAIZE. STEM. 109 of the xylem in the form of a V is much more plain than here, e.g., Asparagus. In other cases (e.g. Calamus) this arrangement is not to be seen. The vessel nearest the centre of the stem has annular thickening : in old stems it is partially surrounded by an intercellular space, while the rings often become detached, in "which case the vessel is not easily seen in transverse sections. Next this is a spiral vessel : the remaining two have thinner walls with pitted marking, and large cavity. Surrounding the pitted vessels, and between them, are ii. A number of Tracheides with pitted lignified walls, and no cell-contents. Surrounding the inter- cellular space above described is iii. A group of parenchymatous cells with thin cellulose walls. These may be regarded as Xylem Parenchyma. The Phloem portion of the bundle lies between the limbs of the V-shaped xylem, and is easily recognised by the thin cellulose walls characteristic of Soft bast. It consists of iv. Elements with large cavities, in which transverse septa (sieve-plates) often occur. These are Sieve- tubes. v. Smaller cells (cambiform) between the sieve- tubes. Surrounding the above tissues of the xylem and phloem is a Sheath of sclerenchyma. Transitional forms may be found on its internal side, between sclerenchyma, and certain of the constituents of the bundle. 110 PRACTICAL BOTANY. II. Cut longitudinal sections of the same, treat as before, and observe a. The Epidermis composed of oblong cells. &. The prosenchymatous cells of the Sclerenchyma. c. The ground parenchyma with roundish cells. d. The Vascular bundles pursuing a longitudinal course parallel to one another, without lateral fusion. In the Xylem observe i. The annular, spiral, and pitted vessels, and note, especially in the latter, the clearly-marked joints, pointing to their origin from a succession of cells. ii. The pitted Tracheides. iii. The thin-walled Parenchyma. And in the Phloem (which is easily recognised by its cellulose walls, blue with Schulze's solution) distinguish iv. The Sieve-tubes, which have a wide cavity, intercepted here and there by transverse sieves. !N .B. If it be found difficult to distinguish the sieve- plates, a fresh section may be treated with potash ; the character of the sieve-plate is then more easily seen. v. The Cambiform cells, which are narrow and pa renchymatous. Note the prosenchymatous constituents of the sheath of Sclerenchyma, and observe transitional forms between these and the pitted Tracheides (ii.) with square ends, which belong to the xylem. III. Cut successive, thick transverse sections through a node : treat them with strong potash [or better, soak them for twenty-four hours or more in dilute potash] ; mount in glycerine, and examine with a low power. MAIZE. STEM. Ill Observe that the vascular bundles here form a dense plexus, in which may be recognised 1. Branching, and anastomosis of the bundles of the main axis with one another, at the base of the internode. 2. Entry of the bundle-system of the leaf-trace, and of its axillary bud, into the main axis, in which the bundles at first pursue an irregular horizontal course. 3. Anastomosis of these bundles with those of the main axis. The result is a thorough intercommunication of the several systems of bundles, one with another, at the node. This modification of the type of bundle arrange- ment characteristic of the Monocotyledons is the rule in those of the group which have long internodes. Observe that the structure of the individual bundles at the node differs from that in the internode, the change depending upon 1. The sheath of sclerenchyma being relatively larger. 2. The irregularities of vascular arrangement result- ing from the fusion of bundles. IV. Cut longitudinal sections through a node in planes parallel to the median plane of the leaf and axillary bud : treat as above, and observe 1. The branching and fusion of the longitudinal bundles of the internode at the node. 2. The entry, horizontal course, and fusions of the bundle system of leaf, and axillary bud. Note that the plexus of bundles at the node does not extend far in a perpendicular direction. V. Apex of stem, to show the fundamental arrange- ment of the vascular system. Cut median longitudinal sections through the apex of a young plant of Maize, 112 PKACTICAL BOTANY. or of a foliage branch of an old plant : treat with strong potash [or better, with dilute potash for twenty-four hours] : examine with a low power, and observe, if the section be median 1. The Apical cone (punctum vegetationis). 2. Leaves, in successive stages of development, seated laterally. 3. In the older leaves, Vascular bundles, which enter the stem. On following the course of these vascular bundles it will be seen that on entering the stem they proceed at first towards the centre : before reaching it they curve downwards, and finally turning again outwards they approach the periphery of the stem. We thus see that in young stems of Maize the course of the bundles cor- responds to the Palm-type, though as the stem grows older, and the internodes develop, the correspondence is less obvious, by reason (1) of the almost straight course pursued by the bundles in the internode, and (2) the complications which arise at the node. STEM. ARBOREOUS TYPE. I. Examine preparations of the old stem of Yucca or Dracaena, in which the thin-walled parenchyma has been allowed to rot away, while the vascular bundles remain. On comparing transverse and longi- tudinal sections of such stems, it may be seen, with the naked eye 1. That the central Primary bundles are isolated, and that the course of each bundle may be traced as starting from below at the periphery of the stem, then STEM. ARBOKEOUS TYPE. 113 curving towards the centre as it ascends, and finally turning outwards, and passing into a leaf. These are therefore common bundles. 2. That the peripheral mass of secondary bundles increases in thickness towards the base of the stem, and has no direct connection with the leaves. These bundles are therefore cauline. II. Cut transverse sections of the stem of Draccena at a point one foot or more from the apex, and mount in glycerine. Examine with a low power, and ob- serve 1. A well-marked Epidermis. Beneath this 2. A band of Cork (cf. Elm). 3. A broad belt of Cortical parenchyma, many cells of which contain crystals (Raphides &c.) Here and there a vascular bundle will be seen in the cortex, these are bundles of the leaf-trace, passing inwards from the leaves. 4. At the inner limit of this is an actively dividing Meristematic ring, which gives rise internally to new vascular bundles, and externally to fresh cortical cells. The new bundles thus formed are cauline (i.e. have no direct connection with the leaves), and are embedded in lignified ground tissue. These together form a dense ring. 5. Centrally, an arrangement of thin-walled Paren- chyma and Vascular bundles, similar to that in the internode of Maize. Note the passage of these central bundles outwards to the bases of the leaves. They are common bundles. Note also the mode of formation of the cauline bundles (cf. Hippuris). 114 PRACTICAL BOTANY. Transverse sections should also be cut immediately below the apical tuft of leaves. Here the secondary thickening will not have begun, the arrangement of tissues resembling, in all essential points, that in the internode of the Maize. LEAF. Note the phyllotaxis in the Maize ; the leaf is sessile, and sheathing in its lower half, with a ligule at the apex of the sheath ; lamina, form lanceolate, margin entire, ciliate, midrib well marked ; venation parallel ; upper surface hirsute ; lower glabrous. I. Cut transverse sections of the lamina ; mount in water, or dilute glycerine. Other sections may be treated with alcohol to expel the air bubbles (the chlorophyll will, at the same time, be dissolved out), and be mounted in Schulze's solution, and kept for comparison with the above. Examine with a low power. The section presents a sinuous outline, correspond- ing to a certain extent to the arrangement of the main vascular bundles. At the mid-rib the section widens out. Note the following arrangement of tissues : 1. Covering both surfaces of the leaf is an Epidermis, resembling that of the stem, but bearing hairs of various form, mostly simple, conical. The largest of them are surrounded at the base by an outgrowth of the neighbouring epidermal cells. Note the Stomata on both surfaces, with small guard cells, surrounded by two subsidiary cells (cf. infra). 2. Vascular Bundles of various size, which, in the MAIZE. LEAF. 115 thinner part of the lamina, lie in a median position between the two epidermal layers. The largest of these correspond in structure to those of the internode, the smaller ones are reduced forms of the same type. Note that the spiral and annular vessels (i.e. protoxylem) are nearer the upper surface of the leaf. Between the epidermis on either side, and the larger bundles, are masses of Sclerenchyma, which, together with the bundles, form complete bridges of rigid tissue between the two epidermal layers. 3. The spaces between the tissues, hitherto con- sidered, are filled with parenchyma (Mesophyll), which may either be (a), green (containing chlorophyll) ; or (&), colourless (without chlorophyll). a. The green chlorophyll- containing parenchyma fills up the greater part of the space ; intercellular spaces occur in it. I. The colourless parenchyma occurs (i.), as a sheath, without intercellular spaces, surrounding each bundle (bundle-sheath) ; (ii.) as groups of cells immediately below the epidermis ; these are more common towards the central part of the leaf. At the mid-rib this tissue forms the bulk of the structure. II. Cut transverse sections of the leaf-sheath, and treat as the above. Compare the arrangement of tissues with that of the lamina, and of the stem. Note that colourless parenchyma preponderates. III. Treat a piece of the thin peripheral part of a leaf (which has been previously bleached in alcohol) with potash till it is transparent ; mount in glycerine, and examine under a low power. Observe 1. The parallel course of the Bundles. I 2 116 PBACTICAL BOTANY. 2. Their frequent lateral fusion, by means of small branch bundles. 3. The absence of Stomata above the vascular bundles, and their arrangement in rows in the spaces between them. 4. The various forms of Hair; and especially the conical unicellular hairs, which give the ciliate character to the margin of the leaf. IV. Cut thin tangential sections from the under surface of the lamina, so as to remove, if possible, only the epidermis. Treat with potash, and mount in glycerine. Observe 1. The ordinary cells of the Epidermis of oblong form, and with sinuous outline. 2. Short cells between the ends of these, which often project perpendicularly to the surface as Hairs of various form. 3. The Stomata holding the same position as (2) relatively to the oblong epidermal cells. Observe with a high power the structure of the stomata. They consist of a. Two narrow guard-cells, which inclose the pore. I. Two triangular subsidiary cells, which com- pletely surround the convex side of the guard-cells. Compare this view of the storna with the same structure as seen in transverse sections of the lamina. V. Cut tangential sections of the upper surface of the lamina. (1). Mount some, and examine them under a low power. (2). Treat others with nitric acid; dry them, and ignite on platinum foil over a spirit lamp. Mount the ash in water, and examine under a low power. The structure will resemble that of (1). MAIZE. LEAF. 117 Treat with acetic acid no evolution of gas. Treat with nitric acid it is not dissolved. The residue is a silica-skeleton of the epidermal tissues. VI. Development of Stomata. Take a young leaf from a bulb of Hyacinthus orientalis in which the leaves have not yet protruded more than about one inch from the apex of the bulb. Strip off pieces of the epi- dermis (or cut tangential sections at successive points) starting from the apex, and proceeding to the very base. Mount in glycerine, and examine under a high power. i. Starting at the basal part, cell-division will be found to be proceeding actively in the epidermal tissue ; the walls are thin, and protoplasm copious. The epidermis consists of a. Larger oblong cells. b. Short, nearly square cells. The cells are arranged in regular longitudinal rows. ii. At a short distance from the base, the difference in size of (#) and (5) increases ; some of the square cells may be seen to be divided by a thin longitudinal wall, into two equal halves (guard-cells of the stoma). iii. Further up again, this division wall may be seen to be thicker at its central part, while the whole outline of the pair of guard-cells tends to become circular. iv. Again further up, the division wall will be seen to have split, so that a channel is formed between the guard-cells into the internal tissues of the leaf. This channel is the pore of the stoma. v. Near the apex of the leaf the mature stomata may be seen of circular outline ; their guard-cells are sausage- 118 PKACTICAL BOTANY. shaped, and surround the nearly circular pore. The cells of the epidermis remain ohlong as before. It will be seen that; the stoma of Hyacinthus is of simpler structure than that of the Maize. It is more difficult to trace the development of the latter ; but it may be done in the same way in a foliage bud. The main point of difference is that after the mother-cell of the stoma has divided to form the two guard-cells, two other cells are cut off from the neighbouring epidermal cells (subsidiary cells). These lie parallel to the guard-cells. Further, the epidermis of the Maize is complicated by short cells, which appear in irregular groups among the ordinary epidermal cells. This is a common character among the Grasses. BOOT. 1. Cut transverse sections of the root of Hyacin- thus orientalis. (N.B. An old root must be taken, and the sections should be cut as far as possible from the apex). Treat them with potash, and mount in glycerine. Starting from the outside, note succes- sively ' 1. An Epidermis, not well marked. Note here and there cells, which have grown out perpendicular to the surface as root-hairs. 2. A thick band of Cortical parenchyma, consist- ing of rounded cells with intercellular spaces ; in old roots the outer layers of this tissue become disorganised and distorted. The inmost layer of this tissue differs in structure from the rest, and is called 3. The Bundle-sheath : the radial walls of this layer present the characteristic appearance of a black dot, and are cuticularised. Within this is 4. A layer of thin- walled cells (the Phloem-sheath or pericambium), which immediately surrounds HYACINTH. KOOT. 119 5. The central Vascular cylinder. This consists of groups of tissue of two sorts. A. Xylem-tissues, easily recognised by their dark lignified walls. They are arranged in a series of groups of indefinite number, which abut externally on the pericambium, and extend inwards, till they meet in- ternally, and form a central mass. The chief con- stituents are vessels of various form. As may be seen in transverse sections of young roots, the smaller peripheral members of each group are formed first (protoxylem), and have spiral thickening ; then successively the larger vessels towards the centre. Between the peripheral groups of the xylem, and alternating regularly with them may be seen B. The Phloem-tissues, which are groups of ele- ments with small cavity, and bright cellulose walls. II. Cut radial longitudinal sections of the same root : treat in the same way, and observe the several tissues above described. The whole root will be seen to be composed of similar elements to those found in the stem. Transverse sections should also be made of the root of the Maize. The main features of the section are the same, though the structure differs in several minor points from that of the root of Hyacinth. Thus, in the Maize root there is a parenchymatous pith, and the xylem abuts directly on the bundle-sheath. In these sections may be found the point of junction of lateral roots with the main root. It may be seen that the former ori- ginate from the pericambium of the main root, and that they break through the bundle-sheath, cortical tissue, and epidermis ; also that their vascular tissue is continuous with that of the main root ; the activity which produces them begins opposite a phloem-mass of the main root, and not opposite a xylem-mass, 120 PKACTICAL BOTANY. as is usually the case (cf. Dicotyledons). This is to be connected with the fact that the xylem-groups in the Maize (and in most Grasses) abut directly on the bundle-sheath. Apex (punctum vegctationis). It is not easy to cut longitudinal sections of the apex of an ordinary fully developed root without embedding. The arrangement of the meristematic tissues is, how- ever, the same in young as in old roots ; it is therefore more convenient, and quite as successful, to cut longi- tudinal sections of the apex of the young lateral roots, which are to be found growing horizontally out of the nodes of the Maize plant. Or, if fitting material for this be not at hand, longitudinal sections may be made of the radicle of the embryo, in seeds which have been previously soaked for several hours in water. Adopting one of the above methods, cut longitudinal median sections of the apex of the root. Treat them for ten minutes with dilute potash : neutralise with acetic acid, and mount in glycerine. N.B. The section must be accurately longitudinal and median, i.e. the section must include the organic axis of the root, around which the several tissues are symmetrically arranged. In a median section the following arrangement of tissues will be visible. 1. A central mass of tissue, clearly defined laterally, and rounded off at its apex, which is at some distance below the external apex of the root : this is the Plerome cylinder. If this tissue be traced back into the older part of the root it will be found that its central part is continuous with the parenchymatous MAIZE. APEX OF ROOT. 121 pith, while its peripheral part develops into the vascular ring. Note rows of larger cells, which may be traced back as continuous with vessels of the xylem. In the central portion of the plerome are intercellular spaces, which appear black in sections from fresh material, being filled with air. 2. Surrounding the plerome is a broad band of tissue with intercellular spaces, which appear as above. This is the Periblem, which is the formative tissue of the cortex. 3. Outside this is a single layer of cells somewhat elongated radially, and with a thick outer wall : this is the Dermatogen or formative tissue of the epidermis. If the section be accurately median it will be possible to trace (2) and (3) upwards, till, immediately above the apex of the plerome, they merge into a single layer of cells : thus the formative tissue from which the epidermis and cortex are derived, is represented at the apex by a single layer of cells. 4. Outside the dermatogen, at the apex of the root will be found another formative tissue, the cells of which divide parallel to the surface of the dermatogen : this is the Calyptrogen layer, which is formative of the tissues of the Root-cap. The latter appears as a mass of parenchyma, covering the whole apex of the root : the outer cells of it will be seen to be undergoing disorganisation, and mucilaginous degeneration of the cell- walls. 122 PRACTICAL BOTANY. REPRODUCTIVE ORGANS. DEVELOPMENT OF THE FLOWER. I. Examine young Capitula of the Sunflower with the naked eye : they occur in the same positions as the vegetative apical bud, but differ externally from these 1. In their greater bulk, and more especially in their diameter being larger than in these. 2. In their colour, which is usually darker. 3. In being covered externally by a large number of imbricated Bracts (or hypsophyllary leaves), which together form the general involucre. Select a very young capitulum, that is, one in which these characters can be recognised, but are not as yet very pronounced, and, having removed the largest external bracts, cut from it median longitudinal sections : treat with potash for about ten minutes, and mount in glycerine : observe with a low power 1. That in outline and general arrangement of parts the sections resemble those of the vegetative bud, but that the apical cone is broader, and more flat. 2. That the surface of the cone has an irregular outline, owing to the formation of a series of appen- dicular organs, which are developed in acropetal order, i.e. the smallest or youngest are nearest the apex, while on passing towards the periphery the size regularly increases. Put on a higher power, and study these organs in detail. DEVELOPMENT OF THE FLOWER. 123 Beginning at the centre : if the capitulum be young enough, there will be found, as in the vegetative bud, a naked apical cone, with a similar arrangement of tissues to that there observed. Passing from the centre, the external surface assumes an undulating appearance owing to the formation of 1. Bracteoles, leaf-structures, which arise similarly to the leaves as above observed, by outgrowth of the epidermis and subjacent tissue : as they grow older they curve towards the centre. [Note the formation of hairs of various types from single cells of the epider- mis : this being a good opportunity for tracing their origin.] 2. The rudiments of Flowers, which appear in the axils of the bracteoles [i.e. on the side nearer the apex]. These are likewise produced from the epidermis and subjacent tissue, they are morphologically axillary branches. The development of the latter into the complete flower must be carefully studied, by comparison of those nearer the centre with older flowers nearer the periphery of the capitulum, or on capitula of various ages. It is obvious that flowers which have been cut in median section will be best fitted for this study. Note the following successive stages of development (a). Form of papilla, conical. (&). Apex becomes flattened. (c). Periphery of the flattened apex rises into a whorl of five small lobes ; these are the Petals, which are in the mature flower united in a gamopetalous Corolla. (d). Between the corolla, and the now depressed 124 PRACTICAL BOTANY. apex, rises a fresh series or whorl of five lobes, these are the young Stamens. About this stage may be seen externally, below the corolla, a slight protuberance on each side of the flower (as seen in section). This is the first appear- ance of the Calyx, which consists in the mature flower of two scaly sepals. N.B, This order of appearance of the floral whorls is not normal, but is the rule in the order Composite^. In the large majority of plants the calyx is developed first, then the corolla, and then the stamens. (e). Within the whorl of stamens there arise, at the margin of the now much depressed apex, the last series of floral organs, viz., two Carpels, which arch over the apical depression, and thus close in the cavity of the inferior ovary. (/). All the organs increase in size, while from the base of the cavity of the ovary, a papilla arises, which develops into a single anatropous Ovule, with one Integument, and small Nucellus. (For the development of the ovule cf. Helleborus, p. 130.) Cut horizontal (i.e. transverse) sections of a capitu- lum : treat as before : examine with a low power. Note the arrangement of bracteoles, with young flowers in their axils, round the central naked apex. The youngest flowers will appear simply circular in outline (simple papillae of stages a and 5) : older flowers will show successively (i). The five papillae of the Corolla (petals) uniting at an early stage into a gamopetalous corolla-tube. (Stage c.) THE STAMEN. 125 (ii). Five Stamens, alternating with the petals. (Staged.) (iii). Centrally two Carpels. (Stage e.) II. Take a mature flower of Helleborus fcetidus. Observe, and remove successively 1. The five Sepals, polysepalous, regular, inferior, and herbaceous. 2. Petals, number various, polypetalous, tubular, inferior. 3. Stamens, numerous, hypogynous, free. 4. Carpels, number various, apocarpous, superior. Examine a single Stamen, and observe that it con- sists of a. A thin stalk the Filament. 1. A two-lobed head the Anther. In a fully open flower note the lateral, longitudinal dehiscence of the anthers, and the dusty Pollen thus liberated. Examine a single Carpel; it consists of a lower thicker portion, terminated by a thin curved portion (the Style) ; on the inner surface at the top of the style is the Stigma. Slit a carpel open along the dorsal side, turn back the flaps, and observe the numerous ovules, attached, in two rows, to the ventral side of the carpel. STAMEN. III. All the following preparations should be made from materials hardened in alcohol, or better, fixed with saturated solution of picric acid, and then washed and hardened with alcohol. A. Cut transverse sections of a flower bud of Helle- 126 PRACTICAL BOTANY. lorus fcetidus, which was just ready to open, taking care that the anthers shall be cut through transversely. Neglecting the other parts, mount the sections of the anthers in glycerine, and examine with a low power. Note 1. The general outline of the section, and compare it with the form of the bi-lobed anther, as above observed. 2. The two large cavities one in each lobe. 3. The partial Septa, which originally divided each cavity into two Pollen-sacs or Microsporangia ; the anther has thus orginally four pollen-sacs, and these may sometimes be found still distinct even in almost mature anthers (cf. development of anther). 4. A single small Vascular bundle lying sym- metrically between the cavities, in the central part (or Connective) of the anther. 5. Pollen-grains or Microspores, mostly to be found lying free in the glycerine. Put on a high power, and observe that 1. The wall of the anther is composed of (a) An external Epidermis with a well-marked cuticle. Within this (fr) A ]ayer of cells with a fibrous thickening of the walls. (c) Immediately within (6) a narrow ill-defined band, consisting of the remnants of a transitory layer of cells, the Tapetum. 2. A point in the wall of each cavity, opposite the partial septum, where the cells are smaller, and the inner layer not spirally thickened ; this is the point of dehiscence of the anther. THE STAMEN. 127 3. Pollen-grains or Microspores are almost sphe- rical, with smooth walls, and granular protoplasmic contents, in which may be made out, with difficulty, two nuclei. B. Mount in half glycerine half alcohol some almost mature pollen of Fritillaria imperialis, which has been previously preserved in alcohol, and examine with a high power. The grains have a smooth wall, and in the granular protoplasm may usually be seen two nuclei. N.B. If the grains be stained with hsematoxy- lin before mounting in glycerine and alcohol, the nuclei will be more easily made out. Mount and examine, as types of the various forms of the grains, the pollen of HeliantTius, Althcea, Cucurbita, CEnothera, Orchis (pollen-masses or pollinia), Mimosa, Cichorium, &c. C. In order to observe the germination of the pollen-grains, and formation of the pollen-tubes, use may be made of the moist chamber, described on p. 16. Mount some pollen-grains of HeliantJius in one hanging drop of a weak solution of cane-sugar in water (about 5 per cent.). Examine them with a high power, and note their form and the external configuration of their walls. Keep them at an ordinary temperature in the dark for a few hours : on again examining them, many will be found to have put out Pollen-tubes, filled with granular protoplasm, in which one or more nuclei might be detected. The same method may be used for the pollen of other plants, e.g. Orchids, species of Tulipa, Fritillaria, Nymphcea, &c. It 128 PRACTICAL BOTANY. will be found that the time of appearance of the pollen-tube will vary in different cases ; also that to obtain good results solutions of sugar of different strengths will have to be used. In most cases a solution of 10 per cent, or less will be found suitable. Development of Anther and Pollen. If transverse sections be made from very young buds, and suc- cessively from older ones up to the mature flower, the develop- ment of the anther and of the pollen may be traced. The material should be preserved in absolute alcohol (or strong methylated spirit), and the sections should be treated with half glycerine, half alcohol ; this should be left exposed to the air in a watch-glass, so that the alcohol may evaporate ; mount in pure glycerine. (Anhydrous staining reagents may be employed.) By following this method, sections may be prepared illustrating : 1. The formation of the four masses of tissue in the anther (two in each lobe), each of which subsequently becomes differentiated into : (a) A peripheral coat of cells of the tapetum, which take no direct part in the formation of the pollen, and (6) A central mass of pollen-mother-cells. 2. The division of each of the pollen-mother-cells into four special-mother-cells, by the gradual ingrowth of the wall of the mother-cell. 3. The separation of the members of the tetrads thus formed, and their subsequent development as pollen-grains. 4. The gradual disorganisation of the tapetum. 5. The development of the wall of the anther, as above described, and breaking down of the septum between the pairs of pollen-sacs. Compare similar preparations of the young anthers of Trades- cantia, and note the division of the pollen-mother-cells, without any gradual ingrowth cf the wall. Observe, as far as possible, the divisions of the nuclei of the pollen-mother-cells first into two, then into four ; also the two nuclei in the mature pollen-grain. CARPEL AND OVULES. 129 CARPEL AND OVULES. IV. The following preparations must be made from materials hardened in absolute alcohol (or methylated spirit) : Strip off the sepals, petals and stamens from an open bud of Helleborus fcetidus, and cut transverse sections of the Carpels. Treat the sections with one half pure glycerine, one half alcohol, and let the alcohol evaporate gradually. Mount in pure glycerine. Strasburger recommends that tlie transfer to pure glycerine should be made before the sections are cut. Examine first with a low power, and observe 1. The Carpel, having a structure not unlike that of an ordinary leaf. Note the suture or junction of the two margins of the carpel which thus incloses a central cavity. 2. The Ovules (Macrosporangia) seated in this cavity, and attached near the margins of the carpel (it has already been noted that there are two rows of ovules in each carpel, therefore at most only two ovules appear in each section). The form of the ovule is anatropous ; it consists of the following parts : (a) The Funiculus, or stalk, which adheres through the greater part of its course (as the Raphe) to the body of the inverted ovule. A procambium bundle, connected with a bundle at the margin of the carpel, traverses it longitudinally. The body of the inverted ovule consists of K 130 PRACTICAL BOTANY. (b) One Integument several layers of cells thick, united with the funiculus, and covering the body of the ovule completely, excepting a narrow channel (Micro- pyle) near the apex of the ovule. Within this lies (c) The Nucellus, a mass of cellular tissue in which is embedded (d) The Embryo-sac (Macrospore), a large oval cell, situated a short distance below the apex of the nucellus. Examine the embryo-sac with a high power, and observe 1. The granular, vacuolated protoplasm which fills it ; embedded in this are to be found 2. A large central nucleus. 3. At the micropylar end of the embryo-sac, three cells, with clearly denned nuclei. Two of these (the Synergidae) fill the apex of the sac, the third (the Oosphere) being placed laterally, a little below the apex. 4. At the posterior end of the sac are three cells (the Antipodal cells), also with clearly defined nuclei. Note the Tapetum, consisting of cells more or less disorganised, which partially or completely surround the embryo-sac. If similar sections be cut from buds of Helleborus fmtidus of various ages, and be treated in the same way, the development of the ovule, and more especially of the embryo-sac, may be followed, and the various stages of it may be observed. Make similar sections of the ovary of species of Lilium, or Yucca, and compare them with the above. With the exception of a second integument being present in these cases, the structure of the ovule will appear to correspond to that of ffelleboms. FERTILISATION. 131 FERTILISATION. I. Cut median vertical sections through the stigma and upper part of the style of a flower of Datura Stramonium which has just faded. Mount in dilute glycerine, and examine first with a low power. Note 1. The closely-packed tissue covering the Stigmatic surface, the superficial cells of which are slightly elongated perpendicularly to the surface as hairs. 2. The more lax Cortical tissue of the style, with numerous intercellular spaces, which appear dark under the microscope. 3. A central band of more transparent tissue without intercellular spaces (Conducting tissue). 4. Small vascular bundles, two in number, running up the style ; these may or may not be present in the section, according as it has been cut. 5. Pollen-grains adhering to the surface of the stigma; from them pollen-tubes, similar to those grown in sugar solutions (cf. p. 127) may often be traced penetrating the tissue of the stigma. Now gently boil the sections in the dilute glycerine over a spirit lamp, and examine again. Observe 1. The Pollen-grains as before. 2. Pollen-tubes, which may be traced from them through the now more transparent tissues of the style ; they may be recognised by their densely granular contents. Other flowers besides the above may be used e.g. species of (Enothera, &c., or any flower in which the style and stigma are of considerable size. K 2 132 PRACTICAL BOTANY. II. Pick out gently a number of ovules from an ovary of a flower of Datura Stramonium, which has just faded, and mount in dilute glycerine. Observe 1. The Campylotropous Ovules, with curved body. 2. Pollen-tubes, which are often to be found with the end applied closely to the micropyle. Strasburger observed the process of fertilisation itself directly in Torenia asiatica, Gloxinia, and also in Orchids, Monotropa, and Pyrola. His method was to open the ovary of a flower a short time after pollination, and detach and mount the ovules in a 3 per cent, solution of sugar. DEVELOPMENT OF THE EMBRYO. i. Dicotyledon. Pick out the ovules from an ovary of Capsella Bursa- pastoris, which has attained about half the ultimate size of the mature fruit. Treat with dilute potash, and examine with a low power. Observe 1. The form of the ovule (campylotropous, i.e. with a curvature of the body of the ovule). 2. The Funiculus, or stalk. 3. The Integuments. 4. The Micropyle, not very easily seen : a pollen- tube may often be observed entering the micropyle. 5. A large central cavity (the Embryo-sac), which is curved like the whole ovule. In this may be seen, more or less distinctly 6. The Embryo. CAPSELLA. EMBRYO. Io3 To study the structure of the embryo, either longitu- dinal sections of the ovule must be cut, and the embryo be thus laid bare, or the embryo must be removed from the ovule. The former is the more accurate method, though the latter is much the easier : we will therefore adopt the latter. Press gently with a needle upon the cover slip of the above preparation, so as to burst the ovules : the embryo will escape in some cases without injury ; neutralise the potash with dilute acetic acid. The structure of the embryos, which now lie freely suspended in the fluid, may be easily studied. Apply the same method for the preparation of embryos, from ovaries of various ages, both younger and older than that first taken. A series of prepar- ations may thus be obtained illustrating various stages of' development of the embryo, such as are figured in ordinary Text-books. Note more especially the following successive stages of development : 1. The Suspensor, consisting of one or more cells, and terminated by a single Embryonic cell. 2. The embryonic cell divided into octants arranged in two tiers, the terminal cell of the suspensor (Hypophysis) encroaching between the four lower octants. 3. The octants so divided up as to form three layers of cells, which have been distinguished as (a) the external Dermatogen ; (b) the Periblem ; (c) the central Plerome. 4. The two Cotyledons formed by lateral outgrowth from the upper tier of octants, the apex of the Radicle 134 PRACTICAL BOTANY. derived from the hypophysis, the hypocotyledonary stem from the lower tier of octants. 5. Other parts as before. The Apical cone or Plumule formed between the cotyledons. ii. Monocotyledon. Treat ovules of Alisma Plantago in the same way, and observe the following stages of development : 1. Suspensor and Embryo consist of a single short series of cells, produced by transverse divisions. 2. The terminal cell divides longitudinally into four (first tier). 3. The second, third, and fourth cells from the end also divide successively (second, third, and fourth tier). 4. The cells of the body of the embryo divided (as in Capsella) so as to form three layers (a) external Dermatogen, (&) Periblem, (c) central Plerome. 5. A lateral depression of the surface, at the level of the second tier. At the basal lip of this the Apical cone of the plumule is formed. The single Cotyledon is formed from the first tier. The Radicle from the third tier. The Apex of the root from the fourth tier. Compare these results with those obtained in Capsella. For obtaining preparations of the embryo in situ, and of the Endosperm surrounding it, the ovary of species of Potamogeton will be found to be good material : it should be previously hardened in spirit. Cut longitudinal sections of a single carpel, parallel to the flattened sides : they should not be cut too thin. Mount in glycerine, and examine with a low power. ALISMA. EMBRYO. 135 One of the sections will probably be found to include 1. The Embryo-sac, in which are contained 2. The Embryo, with a very short suspensor, the basal cell of which, is greatly enlarged. 3. The Endosperm, a tissue which lines the embryo- sac, and varies in appearance according to the stage of development of the ovary. II. GYMNOSPERMS. VEGETATIVE ORGANS. EXTERNAL CHARACTERS. Take a branch of Pinus Sylvestris, cut in autumn, and which includes at least four years' growth. N.B. The limits of each year's growth may be recognised externally as those points where (false) whorls of strong lateral axes are developed ; and the portion of stem lying between two such whorls may be considered roughly as representing one year's growth. I. Consider first the growth of the year in which the branch was cut, i.e. the part above the youngest whorl of lateral axes. At its apex is a large Bud, surrounded by a variable number of smaller Lateral buds. From a bud, which has been treated with alcohol to remove the resin which covers it, detach some of the brown Scale-leaves, which cover it externally. Note 1. The succulent base of these scales. 2. Buds in their axils. Compare these winter buds with some of the same which have been cut in late spring. The brown scale-leaves will be found to have fallen off, leaving their succulent bases still persistent ; in PINUS. VEGETATIVE ORGANS. 137 the axils of these will be seen the axillary buds above noted. The main axis of the bud has become elongated by extension of the tissues. In studying the growth of the current year, bear in mind that it has been derived from a bud, which had a similar structure to that which is now seated at its apex. Examine the stem of the current year externally, and note 1. The thick Main axis, more or less succulent in appearance; its surface is marked by longitudinal grooves. 2. The brown tooth-like bases of the scale-leaves of the bud, best seen at the lower part of the intern ode. 3. In the axils of these, especially at the upper part of the internodes, are Axillary buds of two kinds. a. Buds with limited growth, each bearing two acicular foliage leaves, surrounded at the base with numerous scale-leaves. These limited foliage shoots occur in the axils of the scales throughout the greater part of the current year's growth. b. Buds with unlimited growth, which are seated close to the apex of the shoot of the current year. They are few in number ; their structure has already been observed ; each may develop into an unlimited axis. It may here be observed that both a and b have a similar origin, both being axillary buds in the axils of the leaves of the main axis of the current year. The apparent difference depends upon the fact that the buds b are more strongly developed than a. 138 PRACTICAL BOTANY. There is great variety in the character of the leaves in the Conifercc. In some cases only foliage leaves are developed (Arau- caria, Juniperus, Thuja). In one case only scale leaves are formed (Phyllocladus), while here in Pinus we have both scale- and foliage-leaves, the former alone being borne on the stronger axes with unlimited growth, while the latter appear only on the foliage shoots with limited growth. Specimens of different members of the group should be examined and compared. II. Passing to the increment of growth of former years, i.e. to the lower and older parts of the branch, in the external appearance and arrangement of parts they resemble that of the current year. The main axis increases in thickness, and is more obviously ligneous, while the limited foliage shoots drop off, leaving scars which mark their former position. HISTOLOGY. THE STEM. It is best to work with material which has been treated for some time with spirit ; by this means the resin, which would otherwise clog the razor, is removed. I. Cut transverse sections of the Axis of a bud, and treat with dilute potash for a few minutes : mount in glycerine. Meanwhile other sections may be mounted in Schulze's solution: examine with medium or low power, and observe at the centre of the section 1. The Pith, composed of cells, with intercellular spaces, and thick cellulose walls (blue with Schulze's solution) ; surrounding this a series of groups of smaller constituents : these are 2. The primary Vascular bundles. Note that they are PINUS. STEM. 139 a. Separated from one another laterally by bands of parenchyma. b. Their form is approximately wedge-shaped. c. That the tissues of which they are composed may be distinguished as i. A Xylem portion, nearer the centre of the stem, the components of which have thick, dark-looking, lignified walls (yellow with Schulze's solution). These first formed xylem elements, since they differ from those formed later, are distinguished as Protoxylem. ii. A Phloem portion, nearer the periphery, with bright-looking cellulose walls (blue with Schulze's solution). The more minute study of these tissues must be deferred for the present. Outside the ring of vascular bundles is 3. The Cortical tissue, a mass of cells similar in structure to the pith. In this occur large intercellular spaces, which are Resin-passages. Since the periphery of the section of the axis of the bud is complicated by great irregularity of outline, the study of the outer tissues will be better carried out in the older stem. II. Cut transverse sections of the stem of the current year. Mount some in glycerine, others in Schulze's solution. The sections have a wavy outline, the in- dentations corresponding to the grooves above observed externally. Starting from the periphery of the section, note the following tissues : 1. Epidermis, a single layer of cells, following the wavy outline of the section : the walls, especially the outer, much thickened : externally a Cuticle. 2. Cortical tissue, consisting of cells with rather 140 PRACTICAL BOTANY. thick cellulose walls (blue with Schulze's solution), and protoplasmic contents with chlorophyll. Many cells have recently divided (this is necessary to keep pace with the growth in thickness of the vascular cylinder). Large intercellular spaces (Resin-passages) occur here and there, and are lined with small-celled epithelium. It must be remembered that in the present case the resin itself has been dissolved out by alcohol. Sections should, there- fore, be made from fresh material in order to see the secretion in situ. It appears amorphous and transparent ; it is soluble in alcohol, leaving a slight residue. N.B. All resins are not so easily soluble. The secretion stains deeply with tincture of alkanet. Near the periphery of the cortex will be found a- layer of Cork and a Cork-cambium (cf. stem of Elm p. 70), derived from cells of the cortex by their division by tangential walls. The cells of the cork have no cell-contents ; their walls are coloured yellowish brown with Schulze's solution. Treat a section with strong sulphuric acid. The walls of the cork retain their sharp contour. At the bases of the indentations of the margin of the section, and immediately below the epidermis note groups of Sclerenchyma, having thick lignified walls (yellow with Schulze's solution). 3. The Vascular system : here a complete ring (cf. the bud) : distinguish as before (a) the external Phloem, (6) the internal Xylem, (c) the misty layer of Cambium. N.B. The vascular bundles were seen to be separated in the bud by intervening parenchyma. Here the ring PINUS. STEM. 141 has been completed by the formation of an Interfasci- cular cambium in the parenchyma between the original bundles. Observe that the internal limit of the vascular ring is sinuous. The convexities mark the position of the primary bundles ; at the apex of these will be found the Protoxylem. 4. The Pith, consisting of parenchyma, having the same characters as in the bud. No resin-passages. Put on a high power, and examine the Cambium. Note i. That the cells are arranged with great regularity in radial rows. ii. That their walls are thinner than those of the surrounding tissues, and are composed of .cellulose (blue with Schulze's solution). iii. That the tangential walls are thinner than the radial. iv. That the cells have copious protoplasm, in which a Nucleus may often be recognised. These facts point to a repeated division of cells by tangential walls. Draw carefully, and compare several of the radial series of cells of the cambium. They will be found to coincide with Sanio's law of cambial division, which was first concluded from obser- vations on Pinus sylvestris. Observe, here and there, radial rows of which the cells are more elongated in a radial direction than the rest. These may be traced outwards towards the cortex and inwards towards the pith. They are the Medullary rays. Some of them may be traced the whole way to the cortex and to the pith (primary medullary rays) f 142 PRACTICAL BOTANY. others only part of that distance (secondary medullary rays). Note that the cells of the medullary rays at the cam- bium zone are less elongated radially than in the xylem or phloem ; the cambium being the formative point of these tissues. The mature cells of the ray usually have cellulose Avails (blue with Schulze's solution), and granular proto- plasmic contents with nucleus. In fact the cells of the medullary rays usually retain their cell-nature. Follow the radial rows of cambium cells outwards, and note the gradual transition to the permanent tissues of the Secondary phloem, the constituents of which are also arranged in radial rows, and have cellulose walls (blue with Schulze's solution). The ring of secondary phloem is cut up into rectangular areas by the Medullary rays, which are easily recognised as above directed. Observe that the tissues filling these areas are of three sorts. i. Elements with cellulose walls, and no very distinct contents ; they are radially compressed. These are the Sieve-tubes, which compose the greater part of the phloem. The walls are differentiated into layers, and have bright globules attached to them (yellow with Schulze's solution). ii. Here and there the radial rows of sieve-tubes are broken by single large cells of the Bast-parenchyma, which resemble in their characters those of the medullary rays. iii. Towards the periphery of the phloem are elements similar in form to the sieve-tubes, whose cell contents are brown, and contain crystals. PINUS. STEM. 143 Note on passing to the periphery of the phloem an increasing irregularity of form of the tissues due to distortion, caused by pressure from without by the cortical tissue upon the vascular system, as it increases in bulk by secondary thickening. Sclerenchymatous elements are absent from the phloem of the stem of P. sylvestris. They are, however, found in the phloem of many of the Coniferce, e.g., Juniperus, in which the different tissues are arranged with great regularity. Follow the radial rows of cambium cells inwards, i.e. towards the centre of the stem. Note the transition from thin-walled cambium to the thick-walled tissue of the Xylem. If the stem was cut in winter the transition will appear sudden, if cut in summer it will appear gradual. The tissue-elements retain the same arrangements in radial rows, as the cells of the cambium. Observe that the xylem ring is cut by the medullary rays into wedge-shaped areas. The chief tissue-elements filling these areas are the Tracheides, which present the following characters : i. They have approximately the same shape as the cells of the cambium from which they are derived. ii. Their walls are thick and lignified (yellow with Schulze's solution), and are differentiated into layers distinguished optically, and by staining. iii. They have no cell-contents. iv. On their radial walls (and rarely on the tan- gential walls) are found irregularities of structure called Bordered pits, which are best seen in the xylem formed at the early part of the year. Note the pit-membrane, 144 PRACTICAL BOTANY. which is always present, traversing the pit-cavity in all cleanly cut sections; the pits are therefore not per- forated. Observe near the centre, and bordering on the pith, the Protoxylem arranged as above observed in the younger stem. No bordered pits occur in the walls of the protoxylem. Note the occurrence of resin-passages in the secondary xylem, lined as before by thin-walled epithe- lium, which may be regarded as xylem-paren- chyma. III. Cut transverse sections of a three years old stem so as to include the whole width of the vascular ring. It is not necessary, however, to have a complete transverse section of the whole stem. Mount in glycerine. Comparing this with what has already been observed in the stem of the current year, note the following differences : 1. The cortical tissue bears evident traces of tangen- tial extension. This is necessary to keep pace with the increase in bulk of the vascular system. 2. The phloem is thicker, and the constituents of the outer part of it are much distorted and displaced. 3. The xylem has increased, in thickness more than any other tissue, so that it is now the chief constituent of the stem. It may be distinguished as being com- posed of three bands (annual rings), in each of which the more central tracheides have large cavity and thinner walls (wood developed in spring) ; passing outwards there may be seen a gradual reduction of the cavity, and increase in thickness of the walls till a certain limit is reached (autumn wood). Outside the latter is a sudden PINUS. STEM. 145 transition to the spring wood. At this point is the limit of each year's growth. IY. Cut radial longitudinal sections of a three years' old stem. Mount some in glycerine, others in Schulze's solution. The sections should be accurately radial and longitudinal, otherwise the difficulty of study of the tissues is greatly increased. Beginning at the centre of the stem and passing out- wards observe successively : 1. The Pith, consisting of two sorts of elements, both of which are of parenchymatous form. a. Cells with cellulose walls (blue with Schulze's solution) pitted, with protoplasm and nucleus. 1. Elements of similar form with pitted lignified walls, and no cell-contents. From the arrangement of these it may be concluded that they had a common origin. 2. The Xylem consisting of a. Tracheides with lignified walls, and no cell- con- tents. Starting from those nearest the pith (i.e. from the protoxylem), and passing outwards, the following forms may be observed, and distinguished mainly by the markings due to unequal thickening of the walls. i.--Tracheides with narrow cavity, and more or less regular annular or spiral marking. ii. Elements wider than these, and with bordered pits scattered between the spirals. iii. Normal Tracheides with bordered pits only. These form by far the greater bulk of the secondary xylem, and must be carefully studied. Their form is prosenchymatous. The greater part of the L 146 PRACTICAL BOTANY. cell-walls is of uniform thickness. On these por- tions of the wall observe with the highest power two intersecting systems of lines of striation. In single longitudinal rows are found the Bordered pits ; each of these appears as two concentric rings, of which the smaller is more strongly marked, and corresponds to the opening of the pit into the cell-cavity. It must be remembered that we are now observing the radial walls in surface view. Compare the bordered pit as seen here with its appearance when seen in transverse section. Note the annual rings recognised here, as in the transverse sections, by difference in width of cavity, and thickness of walls of the tracheides of the .xylem. 5. Here and there the continuity of the mass of tracheides is broken by a longitudinal resin-passage, surrounded by parenchymatous cells (xylem-paren- chyma), which have cellulose walls and retain their cell-contents. The whole mass of xylem is traversed radially by plates of parenchyma (Medullary rays). Note that they extend only a short way longitudinally, but a long way radially ; also that they are composed of cells arranged like bricks in a wall, among which may be be distinguished a. Cells with cellulose walls, and protoplasmic contents. The walls of the tracheides which abut on these are unusually wide. b. Elements, with no protoplasm, and with lignified walls marked with bordered pits. Both tissue-forms may often be found in the same ray, though rays will often bo seen consisting of (b) alone. PINUS. STEM. 147 3. The Cambium-layer consisting of elongated thin- walled cells, the ends of which are difficult to observe (cf. tangential sections). They have copious protoplasm, and an elongated nucleus. Note that the medullary rays are continuous through the cambium, and observe the differentiation from the uniform cambium of the ray to the forms (a) and (ft). In the sections through the cambium of a stem cut in summer, the development of the bordered pits on the walls of the tracheides may be studied. The sections should be treated with Schulze's solution for a long time. 4. The Phloem tissues, which are best studied in sections, which have been treated for some hours with Schulze's solution, consist of a. Sieve-tubes, elongated structures with cellulose walls, those which are radial being marked by numerous circular sieve-plates, here seen in surface view. These sometimes stain a sherry brown with Schulze's solution. The ends of the tubes are difficult to observe (cf. tangential sections). Their protoplasmic contents are transparent and sparing. I. Bast-parenchyma, cells arranged in longitudinal rows, with cellulose walls, and copious protoplasm. c. Occasional elements (prosenchymatous or paren- chymatous) with brown cell-contents, in which crystals are embedded. These are found towards the periphery of the phloem. Medullary rays will be seen with a similar arrange- ment to that seen in the xylem. Their cells, which resemble those of the phloem parenchyma in character, are all alike. 5. Externally to the phloem is the cortical paren- L 2 148 PRACTICAL BOTANY. chyma, which requires no further notice here. Outside this is cork (and sclerenchyma at certain points), and at the periphery of the section ~ 6. The Epidermis. V. Cut tangential sections of a three or four years' old branch, and bear in mind that as a rule the central part of the sections is the most accurately tangential, i.e. that the plane of section is there most accurately per- pendicular to the radius of the stem. Mount as before. A, In sections which pass through the peripheral part of the xylem observe i. The Tracheides of prosenchymatous form. No bordered pits (or very few) are seen in surface view, but they may be seen in large numbers in the radial walls (here cut longitudinally) presenting a similar appearance to that seen in transverse sections. ii. Medullary rays, which resemble a section of a biconvex lens. Note that each ray extends only a short distance in a longitudinal direction : in some cases rays consist of only a single radial series of cells, of which only one lenticular cell appears in this sec- tion. Occasionally a resin-passage is included in a ray. iii. Longitudinal resin-passages (cf. radial sections). B. In sections passing through the cambium will be seen i. The Cambium-cells, resembling the tracheides in form (prosenchymatous) ; cell-walls thin ; protoplasm granular, with elongated nucleus. ii. Cambium of medullary rays, similar in shape to the cells of the rays : thin-walled, with granular protoplasm and nucleus. PINUS. LEAF. 149 If these sections be treated with dilute potash, and mounted in glycerine, their structure may be more easily made out. C. In sections passing through the phloem will be seen i. The Medullary rays as before, but their form is more convex: all the tissues between the medullary rays are derived from cambium-cells of the form above observed. These are ii. Sieve-tubes, which retain the form of the cam- bium-cells : the cellulose walls seen in surface view are smooth : those cut longitudinally appear of wavy out- line (sieves). The structure of the latter is well seen after treatment with Schulze's solution for twenty-four hours. Contents transparent protoplasm. iii. Bast-parenchyma, derived from cambium-cells by their division by transverse walls. iv. Some few cells, especially towards the periphery, containing crystals which give the reactions of calcium oxalate. THE LEAF, Cut transverse sections of a foliage leaf of Pinus syhestris, taken from a stem of the current year. It may be found convenient to embed in paraffin, or to hold the leaf between pieces of pith, or carrot. Mount as before, and examine with a low power. Note the form of the section ; the flat side is the upper, the convex side the lower. Observe successively the following tissues : A. A single layer of Epidermal cells with very thick walls. B. A narrow band of thick-walled Hypoderma. 150 PEACTICAL BOTANY. C. A broad band of chlorophyll-containing Meso- phyll, with resin-passages. D. A Bundle-sheath, consisting of oval cells. E. A broad band of tissue without chlorophyll, which surrounds F. Two central Vascular bundles. Study these several tissues under a high power. A. The Epidermal cells have their thick walls differentiated into three layers. These may be recognised without staining, or better after treatment with Schulze's solution, as i. A thin external Cuticle, not very deeply stained. It extends as wedge-like processes between the cells. ii. The Cuticularised layers, forming a thick band, which stains a deep brown. Immediately surrounding the cell- cavity is iii. A broad pitted band, not deeply stained. This differentiation may be brought into greater prominence by treating (a) with strong sulphuric acid, or (6) by staining slightly with fuchsine. (c) If sections are boiled for ten minutes or more in strong solution of potash, i. will be dissolved while ii. and iii. remain. Note the larger cells at the angles of the section, with thicker walls. Here and there depressions of the external surface may be observed. These indicate the position of the Stomata. Observe the two guard cells, which are seated some distance below the surface of the leaf. B. The Hypoderma (sclerenchymatous) varies in thickness from a single layer of cells to several layers. It is thickest at the corners of the section ; cells thick- PINUS. ROOT. 151 walled, lignified. Note that it is absent below the stomata. C. The Mesophyll consists of thin-walled, chloro- phyll-containing parenchyma. The cellulose walls (blue with Schulze's solution) show a peculiar infolding. Resin-passages occur in it. Their cavity is lined with thin- walled epithelium, which is immediately surrounded by a layer of thick-walled sclerenchyma. D. The Bundle-sheath, walls stained brown with Schulze's solution. E. The tissue within this consists of two elements : i. Parenchymatous cells, with thin cellulose walls (blue with Schulze's solution), and protoplasmic con- tents. ii. Elements having lignified walls, with bordered pits, and no cell-contents (tracheides, transfusion-tissue. [Mohl.]). F. The two central Vascular bundles, the constituents of which resemble those of the stem. Note that the xylem is directed towards the upper surface. Thick-walled sclerenchyma is scattered irregularly round the bundles. THE ROOT. I. Cut transverse sections of a young primary root of the seedling of Pinus (not necessarily P. sylvestris) ; treat with dilute potash, and mount in glycerine. Observe : 1. A thick band of Cortex, not covered externally by any true epidermal layer (cf. longitudinal sections of apex of root). 2. A Bundle-sheath within the cortex. This is a 152 PRACTICAL BOTANY. single layer of cells, having the characteristic marking on the radial walls. Within this lies 3. The Pericambium, a band three or four layers of cells thick. This immediately surrounds 4. The central Vascular cylinder, in which may be seen a. Y-shaped groups of Xylem elements, the fork of the Y directed outwards ; their number varies (3 6). Be- tween the limbs of the fork of each lies a resin-passage. I. Groups of Phloem elements, equal in number to the xylem groups, and alternating with them. N.B. These tissues of the phloem are not very easily recognised. c. Centrally is a mass of parenchyma, which also extends between the xylem and phloem masses, and separates them from one another. II. Cut other sections from an older part of the root, and treat as before. Observe that : 1. The cortex becomes disorganised and brown. 2. Divisions appear in the outermost cells of the pericambium, forming a layer of cork. 3. Lateral roots may occasionally be found, origi- nating in the pericambium, opposite the xylem. 4. The parenchyma lying centrally to the phloem groups has begun to divide as a Cambium-layer. III. Cut transverse sections of a thin lateral root (about -j\- of an inch in diameter) of a full-grown tree of P. sylvestris ; mount some sections in glycerine, others in Schulze's solution. Observe successively, starting from the periphery of the section : 1. Withered remnants of the Cortex. This may, however, have been already completely thrown off. PINUS. ROOT. 153 2. The Pericambium, with its secondary products arranged thus : a. Externally a thin band of Cork, the cells of which are arranged in radial rows. b. The Cork-cambium, the cells dividing by tangen- tial walls. c. The remainder of the original pericambium in a quiescent state. 3. The Phloem, forming, according to the age of the root, a more or less complete ring. The constituents resemble those of the phloem of the stem, and are often distorted by external pressure. 4. The Cambium, as in the stem. 5. The Xylem, in which may be recognised, near the centre a. The primary xylem groups, arranged in the form of a Y, and each having, as before, a resin-passage in the fork. I. The masses of secondary xylem, more or less fan-shaped, and alternating in position with the groups of primary xylem. The number of the latter, and of the masses of secondary xylem, varies in the lateral root, four being the average number. The constituents of the secondary xylem resemble those of the stem in structure and arrangement. IV. Cut, and mount as before, transverse sections of a root about one-eighth of an inch in diameter. The arrangement of tissues will be as before, but the fan-shaped masses of secondary xylem will have joined laterally, so as to form a complete ring. Annual rings may also be seen in fact, the structure at the periphery of the root now closely resembles that of the stem. 154 PRACTICAL BOTANY. V. Cut median longitudinal sections of the apex of the root of Pinus. This may be easiest done by cutting longitudinal sections of the mature embryo in the seed. Treat with potash till they are transparent, and mount in glycerine. Observe : 1. The central Plerome cylinder, recognised as in the Sunflower and the Maize. It is Bounded off at the apex, and throughout is quite distinct from 2. The Periblem, which surrounds it. This is the formative tissue of the cortex. Outside this no true epidermis is to be found ; but at the apex is 3. A Root-cap, which is formed by the active division of the cells of the periblem at the apex of the root. Compare this arrangement of the apical meristem with those types seen in the roots of Angiosperms. REPRODUCTIVE ORGANS. We have seen at the apex of the ordinary vegetative branch in spring, an apical bud surrounded by a number of lateral buds, all of which normally develop into vegetative axes of the type above described. The reproductive organs of Pinus are produced on buds corresponding in position to these : they are easily recognised, even at an early stage of development, with the naked eye. The following observations should be made upon museum specimens, otherwise they oould only be made at intervals, according to the .period of development of the organs in question. I. Male inflorescence. A. Note that the inflores- cence while young, appears as a bud covered with brown PINUS. REPRODUCTIVE ORGANS. 155 scale-leaves, in the axils of which are lateral axes easily seen on removing the scales. Of these lateral axes (a.) Those nearest the apex of the bud develop as lateral foliage-shoots (cf. ordinary vegetative axis). (b.) Below these, a number bear, in place of the two foliage leaves, numerous Staminal leaves (these axes are Flowers). Comparing the male inflorescence with the ordinary vegetative axis, the main difference lies in the mode of development of the lateral axes. In autumn the male inflorescences of the preceding summer can only be distinguished from the purely vegetative axis, by the absence of the lateral foliage-shoots from the lower parts of them. B. Separate a single male flower, and cut it longitu- dinally in a median plane : it will be found to consist of 1. An Axis, which bears. 2. A number of Staminal leaves. Detach some of these staminal leaves with a needle : each consists of ( concentric circles, the outer being the limit of the two subsidiary cells, the inner that of the two guard-cells. Note also the peculiar radiate marking, which is due to irregularity of thickening of the wall separating the guard-cells from the subsidiary cells. Treat sections similar to the above with Schulze's macerating fluid (KCl 3 + HN0 3 ) for some hours, and then dry them with 220 PEACTICAL BOTANY. blotting-paper, and ignite them in a spirit lamp on platinum foil, or on a cover glass ; then treat the ash with weak acetic acid ; mount the residue, and examine under a high power : a skeleton will then be found to remain, which represents clearly the several details of structure of the epidermis above described. From the treatment which the preparation has undergone it may be concluded that this is a skeleton of silica. VII. Cut radial longitudinal sections of an internode of an underground stem : wash them well with water to remove as much as possible of the starch, and mount some of them in glycerine, others in Schulze's solution. Note successively the following tissues 1. The oblong superficial cells with brown walls, frequently bearing unicellular hairs. 2. The oblong cells of the Cortex with cellulose walls, and containing starch. 3. The Vascular bundles, which may be easily recognised as transparent bands of tissue, in which may be clearly seen a. The elongated Tracheides of the xylem, showing annular, spiral, or irregularly reticulate thickening of the walls : these thickenings stain yellow with Schulze's solution : there are no protoplasmic contents : the lignified rings are often found free in the inter- cellular cavities, owing to the rupture of the thinner parts of the walls : for this reason also the annular vessels, which adjoin the intercellular cavities in the bundles, are frequently not to be found in transverse sections. 1. The Phloem consisting of a. Sieve-tubes, which- are elongated elements, with cellulose walls, and granular protoplasmic contents, EQUISETUM. SPOROPHORE. 221 and are divided into joints by transverse or oblique walls: they correspond in general characters to the sieve-tubes of the higher plants, but the sieve-structure of the terminal walls is not clear. Numerous highly refractive granules are found on both sides of the terminal walls. ft. Cambiform cells of oblong form, with cellulose walls. VIII. From buds which have been hardened in alcohol cut median longitudinal sections : treat them for a short time with a strong solution of caustic potash, then wash them with water, and mount in strong acetic acid. Examine them first with a low power, and observe that the nodes and internodes are easily recognised in the lower, older parts of the sections: the former being the points of insertion of the leaf-sheaths, oppo- site which are various complications of the arrange- ment of the tissues as has already been observed; in the internodes the tissues show greater regularity of arrangement. Note that on passing towards the apex the internodes are successively shorter, and the character of the tissues of both nodes and internodes becomes more uniform ; also that the leaf-sheaths become suc- cessively shorter. Following the axis upwards it may be seen to terminate in a sharp cone, which is the pnnctum vegetationis, consisting of cells undergoing division, which constitute the primary meristem. Here and there it may be seen that lateral buds have been cut through, they are situated at the nodes, and appear to be completely surrounded by the tissues at the bases of the leaves; in their form and structure they resemble the punctum vegetationis of the main 222 PKACTICAL BOTANY. axis, but on a smaller scale. Note also the irregularly annular or spiral tracheides in the internodes, and the way in which their structure is modified at the nodes, where they appear shorter, and are more closely reticulated. Examine the punctum vegetationis under a high power, and observe 1. At the extreme apex a single, large, wedge-shaped cell ; this is the Apical cell. The cells immediately adjoining it are arranged in regular order, and are of definite form, being Segments successively cut off from the apical cell. Observe how the older segments, which are further from the apical cell, have been suc- cessively divided up by walls perpendicular to the outer surface (anticlinal), and parallel to the outer surface (periclinal). The details of arrangement of the suc- cessive walls may with advantage be traced by com- parison of several preparations, and explained by reference to the Text-Books. Since the superficial cells are subject to repeated periclinal divisions it is clear that there is no definite layer of dermatogen : compare this structure of the punctum vegetationis with that of the lateral buds above mentioned. 2. Note the leaf-sheaths, successively smaller towards the extreme apex, and observe how they originate by outgrowth and division of successive zones of cells below the apex. 3. Attention should also be paid to the mode of origin of the lateral buds : a diligent comparison of them in various stages of development will show that they are not of endogenous origin, but are derived from superficial cells lying immediately above the insertions EQUISETUM. SPOROPHORE. 223 of the leaf-sheaths. These cells divide, and form the young buds, which subsequently appear to be completely embedded in the tissue of the leaf-sheath, and ultimately burst through it. 4. It will be useful further to trace the development of the several tissues, and to note their relations to-the apical cell and its segments. IX. Cut a series of transverse sections through a bud : prepare and mount them as above directed (VIII), being careful to keep them in their proper order of succession, and with their upper side uppermost. Some of the sections will only have passed through the upper parts of the leaf-sheaths, which will appear as con- centric rings, with a structure similar to that already ob- served (III.) : note that the leaves of successive whorls alternate one with another. In the centre of these rings there will be found in each of the lower sections of the series a transverse section of the axis, and one of the sections should include the punctum vegetationis, which would thus be seen from above. In this preparation observe that the apical cell appears of triangular outline, while the segments are arranged regularly around it : from this observation, and from its appearance in the longitudinal section, it may be concluded that the apical cell has the form of a three- sided pyramid, and that segments are cut off from three- sides. From the observation of transverse sections cutting the axis below the apical cell, and a comparison of these results with those drawn from a study of longitudinal sections, the mode of subdivision of the segments should be fully made out. X. Cut transverse sections of a well-developed root 224 PRACTICAL BOTANY. of E. arvense, treat them with potash, and mount in glycerine : examine them under a high power, and observe 1. That there is a peripheral band of tissue with dark brown walls : single superficial cells have grown out. as root-hairs. 2. Then follows a broad band of colourless Cortex, with large intercellular spaces ; this is limited internally by- 3. A definite layer of cells having the well-marked characteristics of the Bundle-sheath : 4. Within this is the Phloem-sheath, the cells of which are opposite to those of the bundle-sheath, and are derived with the latter from the inmost layer of the cortex. This surrounds 5. The vascular cylinder, consisting of a. Four Xylem groups, each of which may consist of only one tracheide, while one large element often occupies a central position. I. The spaces between these are occupied by ill- defined groups of Phloem, and Conjunctive paren- chyma. The arrangement of tissues at the apex of the root of Equisetum may be studied in the same way as above described for the root of Aspidium Filix-Mas, and it will be found to be similar to it in all the more important points. Attention should also be paid to the mode of origin of the lateral roots, which here spring from the phloem-sheath, while in Ferns they arise from cells of the bundle-sheath. The Sporangia. XI. Examine one of the fertile stems, which rise above ground in the spring, with the naked eye ; ob- EQUISETUM. SPORANGIA. 225 serve that the internodes and leaf-sheaths of the lower part of it are similar to those of the vegetative axes. Passing upwards, note that the last leaf-sheath below the spike is of smaller size than the rest. The spike itself is covered by closely-arranged peltate scales, of hexagonal outline as seen from without : these are arranged in more or less regular whorls. Remove some of the scales, and examine one of them in detail: it consists of a thin pedicel by which it is attached to the axis ; the pedicel widens out towards its apex into a flattened shield-like structure, from the lower surface of which a number of sacs (Sporangia) are suspended. XII. Cut transverse sections through a spike, so as to include some of the scales : mount in glycerine, and observe under a low power. There will be seen a bulky Pith, a ring of Vascular bundles, and a band of cortex. The Pedicels will appear extending radially from the axis, and widening at the outer limit into the peltate expansion, on the lower surface of which two sac-like Sporangia may be seen. Note that a vascular bundle runs up the pedicel, and ramifies in the peltate expansion. Examine one of the sporangia under a high power, and note a. The Wall which is one layer of cells in thickness : the walls of these cells are strengthened by a spiral or annular thickening : the wall ruptures by a longitudinal slit on the side next the pedicel. b. Many Spores may be found in the sporangia, or scattered through the glycerine : examine them care- fully, and observe the spirally-coiled Elaters, and the Q 226 PRACTICAL BOTANY. smooth inner coats of the spore, which inclose a protoplasmic body with a well-marked nucleus. Scatter fresh spores upon a slide, and breathe upon them gently ; then observe them under the microscope : the elaters will be seen to execute active movements, thus showing that they are hygroscopic. By cutting transverse sections of spikes of various stages of development, which have been hardened in alcohol, or in picric acid and then in alcohol, mounting them in glycerine, and com- paring them, the history of the development of the sporangium may be traced. The chief points to be observed will be (1) that the sporangia appear as multicellular protuberances. (2) A single hypodermal cell, the archesporium, gives rise by division to the spore-mother-cells, while the superficial layer of cells which covers the archesporium divides into three, of which the outermost alone remains as the wall of the mature sporangium. (3) Each of the spore-mother-cells divides into four cells, which develop further into mature spores. THE OOPHORE. The fresh spores may be sown on moist soil, and the first stages of germination, which are rapid, may be easily observed ; the later stages are, however, slow, and to see these the cultures must be carefully kept. The result is the formation of prothalli (oophores) of irregular form, some of which produce antheridia after five to six weeks. Other prothalli of larger size produce archegonia after about two to three months. The antheridia are embedded in the tissue of the prothallus, and produce large antherozoids. The archegonia are borne on the upper surface. The result of fertilisation of the egg- cell of the archegonium is the formation of an embryo, which develops as the spore-bearing plant or sporophore. Endeavours should be made to obtain healthy cultures of the prothalli of Equisetum in which the above and other points described in other Text-books may be observed. END OF PART I. MESSRS. MACMILLAN AND CO.'S MANUALS FOR STUDENTS. THE STUDENT'S FLORA OF THE BRITISH ISLANDS. By Sir J. D. HOOKER, K.C.S.I., C.B., M.D., F.R.S.. D.C.L Globe 8vo 10s. 6d. STRUCTURAL BOTANY, OR ORGANOGRAPHY ON THE BASIS OF MORPHOLOGY. To which are added the principles of Taxonomy and Phytography, and a Glossary of Botanical Terms. By Professor ASA GRAY, LL.D. 8vo. 10. Qd. A COURSE OF INSTRUCTION IN ZOOTOMY (VERTEBRATA). By T. JEFFREY PARKER, B.Sc. With Illustrations. Crown 8vo. 8s. 6d. AGRICULTURAL CHEMICAL ANALYSIS. A Handbook of. By PERCY FARADAY FRANKLAND, Ph.D., B.Sc., F.C.S. 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