Plant anatomy : from the standpoint of the development and functions of the tissues : and handbook of micro-technic

Preface

To one interested in biology the study of plant anatomy affords a rich and alluring field, since it reveals how plants, under conditions the most exacting, have met and solved the problems of their existence by achieving the power and habit of cell differentiation and cell association into tissues adapted to carry on the different physiological functions ; and when the study of plant anatomy is directed to reveal the process of cell differentiation and the steps by which the mature tissues are made fit for their functions, the student cannot fail to see at once its high biological significance.

The ontogeny and physiology of the tissues is in fact so illuminating to their mature form and structure that the student of anatomy works at a distinct disadvantage if he is not constantly reverting for enlightenment to questions of origin and function; and whatever motive may incite him to the study of plant anatomy, whether it be purely intellectual curiosity, or the recognition of the necessity of a knowledge of plant anatomy to the scientific pursuit of pharmacognosy or agriculture, he will find the outcome more worthy of his efforts if he has sought out the physiological and ecological interpretation of his anatomical findings. It is not nature's way to evolve cells and tissues at random, with no problems to be solved by their evolution. The tissues are not an aimless expression of the power of variability. Rather, they represent the means of the triumph of living organisms over the conditions and forces which make up their environment.

This book attempts to point out in a brief and elementary way how plants arrive at this achievement by the evolution of the different physiological tissue systems from a primitive undifferentiated embryonic tissue, and how the tissue systems are adapted by their character and relation to each other to

VI PREFACE.

carry out the plant's vegetative functions. It seeks to answer in some measure questions about what kind of organisms plants are; how they wrest their living from the inorganic world ; and how they are equipped to make satisfactory terms with their environment.

At the close of each chapter are given directions for observations that will afford a good foundation for critical discussion. In carrying out the work as there outlined the student will become familiar with the most important practices in microtechnic, and he will at the same time get training in independent work that will prove a significant part in his education. Chapters dealing in sufficient detail with microtechnic and microchemistry are given to help the student to pursue the subject beyond the limits of this book, and to undertake practical work in pharmacognosy and pure food and drug investigations.

In the illustrations much use has been made throughout the book of generalized diagrams. I have found these very helpful in my teaching, and I offer them here in the hope that they may prove suggestive to students in correlating and interpreting the details of the isolated sections with which the histologist has to deal, and of service in throwing light on the operation of the physiological functions.

I must make acknowledgment of especial indebtedness for substance and point of view to Strasburger's Botanisches Practicum, and Leitungsbahnen, Haberlandt's Physiologische Pflanzenanatomie, Pfeffer's Physiology of Plants, Zimmermann's Microtechnic, Czapek's Biochemie der Pflanzen, Meyer's Grundlagen und Methoden fur die Mikroscopische Untersuchung von Pflanzenpulvern, Chamberlain's Methods in Plant Histology, and Winton's Microscopy of Vegetable Foods. The Bausch and Lomb Optical Company have kindly supplied the cuts for Figs. 127, 133, 135, and 136; and the Spencer Lens Company the cut for Fig. 131.

I am indebted to Miss Eugenie Sterling for preparing the drawings for most of the illustrations, and to Mrs. Marguerite

PREFACE. Vii

Wise Sutton for some of the drawings. Mr. Alban Stewart prepared the camera lucida drawings of tissues of Avicennia, Psidum, and mangrove, from materials collected by him in the Galapagos Islands. Mr. L. M. Peace made the photomicrographs appearing here, prepared many of the sections from which the drawings were made, and gave me many valuable suggestions for the chapters on microtechnic.

My thanks are due my colleagues, Dr. M. A. Barber, Dr. C. E. McClung, Dr. F. H. Billings, and Prof. Chas. M. Sterling for reading and criticizing different parts of the book, and to Ada Pugh Stevens for reading all of the proof sheets.

W. C. Stevens.

Contents

CHAPTER I The Plant Cell The Protoplast. — Plasma Membrane or Ectoplasm. — General Cytoplasm. — The Nucleus.— The Plastids. — Cell Division. — Cell Differentiation. — Sizes of Cells. — The Cell Wall. — The Chemical and Physical Nature and Physiological Powers of the Protoplast. — Illustrative Studies 1-24

CHAPTER II Differentiation of the Tissues General Survey. — The Primordial Meristem. — The Protoderm. — The Procambium. — The Ground Meristem. — The Primary Permanent Tissues. — Illustrative Studies 2 5~47

CHAPTER III Secondary Increase in Thickness Dicotyledons and Gymnosperms. — Growth of the Vascular Bundles. — Increase in the Cortex. — Monocotyledons. — Unusual Growth in Thickness. — Illustrative Studies 48-63

Chapter I

The plant body is composed of structural units termed cells. These are minute boxes often barely visible and usually entirely indistinguishable to the naked eye. The walls of the boxes are composed of cellulose, wood, or cork, or a substance allied to cork called cutin. As a rule the walls are without apparent perforations. The term cell has also come to include the living body which is always present in young cells and very frequently in old ones.

The discovery that the plant body is composed of the cell units was made by Robert Hooke, who, about 1660, with a compound microscope improved by himself, saw the cellular structure of cork. The cells of cork have the appearance of the cells of honey comb, and this similarity led to his use of the word " cell " for the structural units of the cork, and ultimately to the extension of the usage to plants in general. The term is not an unfortunate one for cells considered merely as boxes; but its application to the living parts inclosed within the cell walls, or to the living part where no wall is present is inapt. The term protoplast (Gr. protos, first, and plastos, formed, the thing first formed) is now in general use to designate the living part of the cell as a morphological unit; and the term protoplasm or plasma is applied to the substance composing the protoplast, just as a brick would be a morphological unit of a brick house, and burnt clay the substance composing it. Following the general usage the word cell will here be applied to the box and its living content taken collectively, or to the box alone when the living parts have disappeared, and the word protoplast will be used to designate the living part alone.

The box or cell wall is manufactured by the protoplast for its own stability and protection, and the protoplast must, therefore, exist before the wall which encloses it. Since the protoplast is the living structural and physiological unit of the plant body, and since everything that the plant performs is really the work of its individual protoplasts, it necessarily follows that a satisfactory comprehension of plant anatomy and physiology is impossible without a knowledge of the nature of the protoplast itself.

The Protoplast. — If we study under high magnification sections of an onion root tip that has been fixed, imbedded, sectioned, and put up in permanent, stained mounts as directed in the chapter on The Preparation of Sections, we shall find near the root apex young cells that have not yet secreted their cell walls. Each cell in this instance is therefore a protoplast and nothing more. It often happens in the preparation of the sections that some of the protoplasts become separated from the others and so reveal to us just how much constitutes a single protoplast. Fig. I, A, shows us such a protoplast. In the root all of the parts here shown were living, excepting possibly the nucleolus b. The cytoplasm c constitutes the bulk of the protoplast ; the nucleus a is imbedded in the cytoplasm ; the plasma membrane d is a specialized outer portion of the cytoplasm; the plastids e are relatively very minute parts of the protoplast, but have a special work to do, as will be learned later on. In older cells farther back from the root tip we find that the protoplasts have secreted a wall, as shown in Fig. i, B.

Comparing A and B of Fig. i we see that in the younger protoplast the cytoplasm looks something like a sponge with very fine meshes while in the older protoplast the cytoplasm does not so completely fill out the space inclosed by the plasmatic membrane, some of the meshes having widened into

PLASMA MEMBRANE 3

relatively large rifts. It seems that as the protoplast grows older the cytoplasm does not keep pace with the general increase in size and its network in consequence becomes broken in places, allowing the small meshes to coalesce into larger ones known as vacuoles. Both the meshes and the vacuoles are filled with cell sap. The cell sap is largely water containing in solution salts that have come up from the soil, and food substances together with various other compounds manufactured within the plant.

After this general view of the several parts of the protoplast we are ready to examine into the character and uses of each more thoroughly. In doing this let us not lose sight of the fact that the protoplasts taken collectively constitute the. living body of the plant and whatever the plant 'does as a living organism is accomplished by them. They put to use various forces of the external world; they compound the plant's food and a multitude of other products, many of which are employed by the pharmacist; they make the cell-wall framework of the plant; they multiply and increase in size so that the plant as a whole is made to grow ; they differentiate the various tissues, each suited to perform a particular service; they are sentient to gravity and light, moisture and temperature, and to the general state of the whole organism of which each protoplast forms a part, and they are capable of responding to these things in a definite and useful way. With this knowledge we might anticipate, and it is not surprising to find, that each protoplast is a complex thing with visibly distinct parts, and that each part has its own physiological significance. We shall now take up the parts of the protoplast in the following order: plasma membrane, general cytoplasm, nucleus, plastids.

Plasma Membrane or Ectoplasm. — At the exterior of every protoplast is a very thin, hyaline membrane, which, as has been said, is morphologically a part of the cytoplasm, for when a protoplast is torn or cut in two a membrane is produced from the cytoplasm over the wounded surface. This membrane is known as the plasma membrane or ectoplasm

(Fig", i, A, a). It is approximately .0003 mm. thick, or about 1/400 of the thickness of this page. After the cell wall has

been formed it is very difficult to distinguish the plasma membrane because of its extreme thinness and close contact with the wall; but the greater part of this difficulty vanishes when the protoplast is made to shrink away from the wall. This we can do to advantage with the epidermal cells of Tradescantia zebrina containing a colored cell-sap. We strip a bit of epidermis from the under side of a midrib of a leaf and mount it under a coverglass in a drop of water. We bring some of the colored cells under the objective and run a 5 per cent. NaCl solution under the coverglass. The salt solution draws water out of the cells by osmosis and the protoplast soon shrinks away from the walls because it is elastic and had been stretched by the water within the cell. We can now make out the thin plasma membrane at the surface of the shrunken protoplast (Fig. 1, C). If we replace the salt solution under the coverglass with fresh water the protoplast quickly swells up and presses against the cell wall all around as before.

Fig. root-tip ; plasm;

t. A, embryonic cells from onion d, plasmatic membrane; c, cyto- , nuclear membrane enclosing the threadlike nuclear reticulum; b, nucleolus;

e, plastids (black dots scattered about). B, older cells farther back from the root-tip. The cytoplasm is becoming vacuolate;

f, vacuole. C, a cell from the epidermis of the midrib of Tradescantia zebrina, in its natural condition on the right, and plasmolyzed by a salt solution on the left;

g, space left by the recedence of the cytoplasm from the wall; the plasma membrane can now be seen as a delicate membrane bounding the shrunken protoplast. All highly magnified.

GENERAL CYTOPLASM 5

In this experiment we can see that while the plasma membrane allows the water to be drawn from the protoplast by the salt solution it does not permit the coloring matter or the osmotic substances in solution in the cell sap to escape, for the color does not at all diminish in the protoplast as it shrinks, and the protoplast could not again swell up on the replacement of the salt solution by water if its osmotic substances had been lost. This guardianship of the exchange of materials between the cells or between the cells and the external world is one of the well-recognized functions of the plasma membrane. Since the plasma membrane lies at the surface of the protoplast it must receive and transmit to the other parts the stimuli that come from without. Until the cell wall is built the plasma membrane doubtless affords some rigidity and protection to the parts within; and when the time for the building of the cell wall arrives it seems that the plasma membrane constructs it by the chemical transformation of its own substance into the substance of the wall.

General Cytoplasm. — The cytoplasm is the living matrix in which the nucleus and plastids are imbedded. In very young cells it fills out all of the space not occupied by the nucleus and plastids (Fig. i, A), but in old cells it becomes a very thin film, hardly greater than .0006 mm. in thickness lining the cell wall. In cells that have been killed, fixed and stained in the usual ways (see Chapter on The Preparation of Sections) the cytoplasm has a spongy or netted appearance (Fig.l,A).

It is uncertain whether the cytoplasm is really spongelike with irregular and intercommunicating canals, or alveolar with each cavity a closed sac. Whatever the exact character of the cavities may be they are filled with cell sap, or, in many instances, with insoluble reserve food, such as starch, proteids, and oils, and excretions, such as crystals of calcium oxalate.

As has been stated, as the cell grows older some of the cavities in the cytoplasm enlarge and coalesce, and are then known as vacuoles. A plasma membrane is formed about the vacu-

O THE PLANT CELL

oles similar to the exterior plasma membrane already described, and it exercises a selective function over the passage of materials to and from the vacuole just as does the exterior membrane to and from the protoplast as a whole. The spongy, or alveolar, condition of the cytoplasm persists until the division of the nucleus preparatory to cell division sets in, when a part becomes threadlike and seems to assist in the translocation of the chromosomes (the definite parts into which the nuclear substance becomes segmented during nuclear and cell division) to the opposite poles of the cell; this process may, therefore, be classed as one of the functions of the cytoplasm (see Fig. 3).

Throughout the life of the cell the cytoplasm has many things to do of a chemical nature, but it is very improbable that it often works independently of the nucleus. Where storage of proteids and oils is taking place we find them stowed away in the meshes of the cytoplasm, and this is good circumstantial evidence that the cytoplasm has manufactured them where we find them. This is particularly true of the insoluble proteids. The cytoplasm probably secretes the ferments by means of which the stored materials are digested when they are wanted for food. Many other of the ceaseless activities of the cell are doubtless accomplished with its assistance.

The Nucleus. — In young cells the nucleus is spherical in form and lies at the center imbedded in the cytoplasm and occupies from .5 to .8 the diameter of the cell. It consists of the nuclear membrane, nuclear reticulum, and nuclear sap, and usually contains one or more nucleoli (Fig. 1, A, b). The nuclear membrane appears to be really a part of the cytoplasm, similar to the plasmatic membranes lining the exterior of the cell and of the vacuoles. The reticulum is the essential living part of the nucleus. The nuclear sap appears to be a fluid which furnishes to the reticulum water and food, and in other ways serves it; while the nucleolus seems to be reserve food of a peculiar kind needed to help in the processes of nuclear and cell division.

The nucleus is often spoken of as the center of life of the

THE NUCLEUS 7

cell. While the statement is vague it conveys a meaning not entirely misleading-. Some of the facts at the foundation of this conception are these: (a) When a protoplast is segmented into two parts by plasmolysis or other artificial means the part containing the nucleus has the power to construct a wall about itself, while the enucleated part has not. Although the plasmatic membrane is the immediate agent in the construction of the wall it can not do its work without the influence of the nucleus, (b) We have good reasons for the belief that the nucleus is the cause of oxidation in the living cell, and, without it, the tearing down and building up processes of the cell that depend upon oxidations can not go on. (c) After the nucleus has been removed the remainder of the protoplast soon dies. While, on these grounds, the nucleus may be spoken of as the center of life of the cell, the fact must not be left obscured that a nucleus dissociated from the rest of the protoplast can not long maintain its existence, and the cooperation of the other parts is necessary to the success of the functions of the nucleus.

The nucleus is also frequently spoken of as the bearer of the inheritable characters and qualities. This, too, is not without foundation, and if true the nucleus stands forth as the architect and master of the form, character, and activities of the cell and entire plant body. The grounds for the assumption are: (a) In the embryonic cells of the growing apex or cambium the nucleus is relatively large, having an average diameter 0.6 that of the entire cell. These cells are to undergo profound changes in size, form, and general character as the differentiation of tissues proceeds, and why should the nucleus be allotted so much space within the cells unless it plays a dominant part in the hereditary differentiations that are to follow ? The evidence here is enough to raise the question, but it does not go far towards solving it. (b) During nuclear and cell division the nuclear reticulum, which is the essential part of the nucleus, divides into many pieces with great precision, and these pieces are equally distributed between the nuclei of the two resulting cells. What but the vehicle of hereditary transmission should require such exactness of distribution? Here again the facts afford us hardly more than the suggestion of the question, (c) In fertilization, when the sperm cell from the pollen grain is fusing with the egg cell within the ovule, the sperm cell consists almost entirely of nucleus, and the egg cell is relatively rich in cytoplasm ; yet the offspring may partake as much of the peculiarities of the male as of the female parent.

The inference is that, since the male contributed little more than a nucleus and the female a relatively large amount of cytoplasm in addition to the nucleus, it must be that to the nucleus have been given the inheritable qualities to transmit from generation to generation; for if the cytoplasm bears these qualities equally with the nucleus the impress of the female might be expected to preponderate in the offspring, {d) In the nuclear and cell divisions resulting in the production of pollen spores and embryosac spores the number of chromosomes (see Fig. 3) in each nucleus is reduced one half. The sperm cell and egg cell arising from the germination of pollen spore and embryosac spore each contain the reduced number of chromosomes, but when they fuse in the act of fertilization the original number is restored in the fertilized egg and so continues throughout the body of the resulting offspring until pollen spores and embryosac spores are again produced by it with the reduced number. What the significance of this halving and doubling may be is still open to debate, but the meaning evidently will be found in relation to the transmission and blending of the male and female qualities. Since the chromosomes are segments of the nuclear reticulum the phenomenon here described may be classed as a part of the evidence that the nucleus is the bearer of the inheritable qualities, (e) A new line of evidence is at present being opened up in the study of hybrids, where the chromosomes from each parent are found to retain their individuality in the offspring. This fact is being used to illuminate some problems in heredity, and if future investigations

THE PLASTIDS 9

confirm the conclusions that are being drawn from present evidence the hereditary function of the nucleus will be firmly established.

The Plastids. — The plastids are distinct and usually relatively small parts of the protoplast. They vary in size from barely discernible points with the highest magnifications to discs and bands that traverse the entire length of the cell. In form they may be orbicular, ellipsoidal, disc, or ribband shaped. They are classed under three names according to their color: the leucoplasts are colorless ; the chloroplasts contain the green coloring matter chlorophyll; the chromoplasts are yellow, orange, or red. They are, however, really the same thing under different guises and performing different functions; for the leucoplasts may become chloroplasts, and the chloroplasts chromoplasts. This fact is well made out in the tomato, for instance, where the very young pistil in the bud contains no other plastids than the leucoplasts and is colorless in consequence; later when the corolla drops away and the pistil emerges into the light the leucoplasts produce chlorophyll and the young fruit is green; but when the fruit begins to ripen the chlorophyll is gradually replaced by red and orange coloring matters, and the chloroplasts become chromoplasts. The leucoplasts are therefore the progenitors of the other plastids, but they have their own functions to perform as leucoplasts : they take carbohydrates out of solution in the cell sap and store these within themselves in the form of insoluble starch grains. This is well seen in those cells and tissues, as in the potato, and the endosperm of seeds, where reserve food is stored away. The chloroplasts secrete two coloring matters known as chlorophyll-green and carotin or chlorophyll-yellow. The chloroplasts employ these pigments in arresting the sun's energy, by means of which they make the food of the plant. In Chapter 9 the chloroplasts will be more fully discussed in connection with the photosynthetic system. The chromoplasts impart red, orange, and yellow colors to flowers and fruits, where, within the chromoplast body, the reds occur as crystalline carotin and the yellows as amorphous xanthin. These may be found separately or together in the same chromoplast. The presence of both produces an orange color (Fig. 2). The reddish and bluish pigments occurring in solution in the cell sap are known as anthocyanin, and it does not appear that the chromoplasts are necessary to their production.

Fig. 2. A, cell from the epidermis of the upper side of the calyx of Tropaeolum majus with crystalline chromoplasts; B, cells from the petal of Lupinus luteus with yellow chromoplasts; C, cell showing numerous chloroplasts scattered through the cytoplasm. (A, after Strasburger; B, after Frank.)

Cell Division. — In the growing apices of root and shoot and in the cambium cell division may go on indefinitely. The process of cell division begins in the nucleus and terminates by the formation of a dividing cell wall, as will be seen in Fig. 3, where the different stages may be followed. The nuclear reticulum (in a) becomes transformed into a thick winding thread (in b), which in successfully stained sections is seen to consist of colored discs or granules termed chromatin, imbedded in a colorless matrix called linin. The thread splits longitudinally throughout its length (in c), and then breaks into rod-shaped pieces, each of which consists of two longitudinal halves arising from the longitudinal division of the thread (in d). These rods are known as chromosomes, and their number varies with the species. Next the nuclear wall

CELL DIVISION

I I disappears and threads arise in the cytoplasm and converge to a point at two opposite poles forming what is known as the nuclear spindle (in e). Some of the threads extend uninterruptedly from pole to pole while others become fastened to the wm f

L in

I

Fig. 3. Semi-diagrammatic representation of nuclear and cell division, a, resting cell ready to begin division; b, the nuclear reticulum is assuming the form of a thickened thread, and the cytoplasm at opposite poles is becoming threadlike to form the spindle fibers; c, the nuclear thread has divided longitudinally through the middle, and the spindle fibers have become more definite; d, the nuclear membrane and the nucleolus have disappeared, and the nuclear thread has become segmented into chromosomes which are assembling at the equator of the cell. All of the phases of division thus far are called prophases, e, the metaphase, where the longitudinal halves of the chromosomes are being drawn apart preparatory to their journey towards the opposite poles; f, the anaphase, or movement of the chromosomes towards the poles, is about completed, connecting fibers extend from pole to pole; g, telophase where the chromosomes have begun to spin out in the form of a nuclear reticulum. The connecting fibers have begun to thicken in the equatorial plane; h, the connecting fibers have spread out and come into contact with the wall of the mother-cell in the equatorial plane, and the thickening of the fibers throughout this plane has made a complete cell plate within which the dividing wall will be produced; i, a nuclear membrane has been formed about each daughter nucleus, and the dividing cell wall is completed. The two daughter cells are now ready to grow to the size of the parent cell in a, when the daughter nuclei will appear as does the nucleus there. All highly magnified.

chromosomes. The chromosomes in some unknown way line up in an equatorial plane half way between the poles, and then one half of each chromosome is drawn to one pole and the remaining half to the other where they form at each pole a nuclear thread (e, j, and g). This spins itself out into

a nuclear reticulum around which a nuclear membrane is soon organized (in h and i).

The connecting fibers extending between the poles bulge out at the equator more and more and new ones evidently are formed until the whole equatorial zone is traversed by them (in g and h). The fibers thicken in the equatorial area and the thickenings fuse together, forming a closed membrane known as the cell plate (progressive stages in g, h, and %). This is a plasmatic membrane which organizes in its median plane a cell wall dividing the mother cell into two. The fibers disappear, the cytoplasm becomes reticulate throughout, and the daughter nuclei increase rapidly in size.

About the time when the nuclear membrane disappears the nucleolus as a rule also vanishes from sight and can not again be found until the completion of the daughter nuclei. This has led to the conclusion that the nucleolus is not a living part of the protoplast but is simply a form of reserve food needed at the time of nuclear and cell division. It does not, however, behave uniformly in all subjects and its exact nature is still a subject of debate.

In the nascent endosperm of seeds the division wall between daughter cells may not be formed until the nuclei have many

Fig. 4. Formation of endosperm in the embryo-sac of Agrimonia Eupatorium. Cell walls are being formed between the nuclei. (After Strasburger.)

CELL DIVISION l J

times divided. Finally, when the embryosac containing the endosperm lias completed its growth, new connecting- fibers spring up in the cytoplasm between the nuclei, and cell walls are laid down in the usual way (Fig. 4).

In the formation of spores in the Ascomycetes nuclear division takes place in the usual way, but the method of formation of the wall about the spores is unique. Here nuclear division continues within the mother cell until the number of nuclei equals the number of spores to be produced, when, at the close

Fig. 5. Free-cell formation of spores in the ascus of Erysiphe communis. A, ascus with single nucleus; C, cytoplasm; N, nucleus; NL, nucleolus; B, successive stages in nuclear division within the ascus; at X, early anaphase, nuclear membrane, NM, still persisting; R, kinoplasmic radiations from the poles; at Y telophase, new nuclear membrane not yet formed; Z, a later stage where the nuclear membranes demark the daughter nuclei; C, A, B and C, are successively later stages than Z in B. At A some of the kinoplasmic radiations are bending downward about the nucleus N; at B the nucleus is nearly enclosed; and at C entirely enclosed by the radiations, which now form a complete membrane cutting off a portion of the cytoplasm of the ascus, and thus forming a complete cell. (Arranged after drawings by Harper. B and C are diagrammatic. For the sake of simplicity of description various stages of nuclear division are shown in a single ascus, although at any given time only one stage would actually be present.)