Plant Anatomy: Workshop - Paniuta O.O. 2019

Topic 2. Tissues
Laboratory Work No. 8. Vascular Tissues

Theoretical Background. Conducting (vascular) Tissues are responsible for transporting Water and mineral nutrients from the roots to the leaves, as well as Organic compounds from the leaves to all other plant Organs.

The Movement of water with dissolved mineral nutrients is referred to as the ascending stream, whereas The transport of water containing dissolved organic compounds is known as the descending stream.

As a rule, the ascending stream moves through the plant's xylem, and the descending stream through the phloem. In many plants, conducting elements are grouped into vascular bundles which, as the name implies, consist not only of conducting tissues but also of mechanical (supporting) tissues and parenchyma.

A vascular bundle is composed of xylem and phloem. The phloem component comprises sieve tubes with companion Cells, parenchyma, and bast fibers. The xylem component includes vessels (or tracheae), tracheids, parenchyma, and wood fibers (xylary fibers), though the conducting function is performed solely by the sieve tubes, tracheids, and vessels.

In most plants, companion cells lie adjacent to the sieve tubes, having originated from the same mother Cell as the sieve tube members.

In longitudinal section, companion cells may extend along the entire length of the sieve tube members (for instance, in the primary phloem of herbaceous plants) or they may be very short (in the phloem of woody plants). In transverse section, they are smaller than other phloem elements and can be triangular, rectangular, or sometimes rounded in shape.

As previously noted, the ascending stream travels through the xylem portion of the vascular bundle, specifically via tracheids and vessels.

Depending on their origin and potential for Secondary Growth, vascular bundles are classified into open bundles—which retain and actively function with a cambium layer between the phloem and xylem—and closed bundles, which lack cambium.

Based on the relative arrangement of phloem and xylem, vascular bundles are categorized as collateral, bicollateral, radial, and concentric.

Objective: To study the Structural Features of conducting tissues.

Materials and Equipment: Light microscopes, Glass slides and cover slips, dissecting needles, forceps, glass rods, watch glasses, filter paper, distilled water, phloroglucinol with Hydrochloric acid, aniline sulfate, chlor-zinc-iodine, chromic acid, 20% potassium hydroxide solution, 10% sodium carbonate solution, methylene green, gallic carmine, plant material.

Prepared Slide: Vascular bundle in a corn (maize) stem (Zea mays L.)

Plant material for investigation should be harvested in summer and preserved in alcohol or formalin.

Take a piece of the stem, use a scalpel to remove the tough outer epidermal layers, and carefully level the surface so that the bundles are sectioned precisely in cross-section. Make several transverse slices through a segment of the stem. Place the sections in a drop of water on a Microscope slide and cover with a coverslip. White dots are visible to the naked eye amidst the ground tissue of the section; these are the vascular bundles extending longitudinally along the stem like whitish Veins. The bundles are smaller and more densely packed at the periphery of the stem, whereas toward the center they become larger and more widely spaced. Consequently, the best sections are obtained from the central part of the stem. Place the prepared slide on the microscope stage and examine it under low magnification, selecting the thinnest area where an entire vascular bundle is clearly visible.

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Fig. 41. Closed collateral vascular bundle in a corn stem:

1 - parenchyma, 2 - sclerenchymatous sheath, 3 - lacuna, 4 - xylem, 5 - protophloem, 6 - protoxylem

Vascular bundles are oval or rhombic in shape and are embedded within the thin-walled ground parenchyma (Fig. 41).

Even under low magnification, it is readily apparent that the entire bundle is surrounded by a sclerenchymatous sheath. The layer of sclerenchyma cells is thicker above the phloem and below the air cavity (lacuna) within the xylem, while being considerably thinner on the sides of the bundle. The Cells of the sclerenchymatous sheath fit tightly together; their walls are significantly thickened and lignified, their cellular contents have disintegrated, and The Cell lumens are filled with air. Remnants of Cytoplasm and occasionally even a Nucleus can be observed in only a few cells. The cell walls of the sclerenchyma are thoroughly permeated with numerous pits throughout their thickness.

Beneath the upper, coarser part of the sclerenchymatous sheath lies a small area where the cell walls have fused to such an extent that individual cells are no longer distinguishable, leaving only visible cell lumens. These are the remnants of the primary phloem cells, specifically the protophloem. These cells formed early in plant development through The activity of the primary meristem. Underlying the protophloem is the secondary phloem, which appears under high magnification as a translucent circle of cells. By adjusting the fine-focus knob, one can observe fairly large cells within the phloem—either empty or bearing sieve plates—which are the sieve tubes. Small, thin-walled cells filled with granular cytoplasm lie snugly against them; these are the companion cells. On well-prepared slides, it is evident that the companion cells and the sieve tube originated from the same cell. In such cases, the companion cell appears as a corner of the sieve tube, partitioned from it by a delicate membrane.

Living, thin-walled cells of the phloem parenchyma are located between the sieve tubes.

The phloem portion of the bundle adjoins the xylem (which is shaped like a triangle pointing toward the stem periphery), yet it remains sharply demarcated from it. Two large reticulate vessels are situated on the sides of this triangle, with a smaller-diameter annular-spiral and an annular vessel positioned slightly lower between them, underneath which lies an air cavity. Occasionally, unbroken rings of annular or spiral vessels can be seen within the air cavity. The vessels are surrounded by sclerenchyma cells. The space between the reticulate vessels is filled with thick-walled sclerenchyma cells, whereas the remainder of the xylem portion consists of wood parenchyma cells. The wood parenchyma cells are living, containing cytoplasm and a nucleus, and their walls may be thin or moderately thickened.

Thus, in the corn vascular bundle, it is clearly observable that the phloem borders the xylem on one side, with no cambium present between them. Consequently, this is a closed collateral bundle.

To better distinguish the individual Components of the vascular bundle, the section must be treated with phloroglucinol and hydrochloric acid or with aniline sulfate. To do this, remove the coverslip from the slide, apply a drop of phloroglucinol and hydrochloric acid to the specimen, and wait a few minutes. Then, transfer the section into a drop of water on a clean glass slide, cover with a coverslip, and examine under the microscope. As a result of this Treatment, all lignified elements of the bundle will turn red; if treated with aniline sulfate instead of phloroglucinol and hydrochloric acid, they will turn yellow.

After examining the stained preparation, make any necessary corrections to your drawing.

Slide. Vascular-fibrous bundle in a corn stem (Zea mays L.) in longitudinal section

Take a small piece of a fresh or preserved corn stem and use a scalpel to peel off the upper hard covering on one side. The vascular bundles in corn are easily visible to the naked eye. One of them should be exposed along its entire length so that it stands out clearly among the other stem tissues. Holding the prepared piece of stem in your left hand, slice through the middle of the bundle along its entire length. Make sure that the cut runs precisely along the bundle, as an angled section will make it impossible to examine The Structure of individual elements of the vascular bundle.

Make several sections and place them on a glass slide in a drop of water in the same order they were cut. This is necessary in order to examine all the elements of the bundle sequentially. Depending on the direction of the cut, it may be either tangential or radial.

To make it easier to understand the studied material, you should have a diagram of a cross-section of the vascular bundle in front of you.

Fig. 42. Tangential section of a corn bundle through the sclerenchyma sheath: 1 - thin-walled parenchyma, 2 - cells of the sclerenchyma sheath

In the section (Fig. 42) made in the tangential direction, the ground parenchyma is clearly visible on the left and right; it consists of fairly large, thin-walled living cells, in which the cytoplasm can be seen as a thin layer along the cell walls. A nucleus and even METABOLISM/14.html">Chloroplasts may be noticeable inside the cell. Beyond the thin-walled parenchyma cells, the cells of the sclerenchyma sheath, composed of sclerenchyma fibers, are visible. They are elongated, tapered at the ends, and have thickened walls penetrated by numerous slit-like pits. The sclerenchyma cells lack cell contents and are filled with air. After sketching the designated areas, shift the slide so that another section comes into view, showing the elements located deeper in the bundle behind the sclerenchyma sheath.

Under high magnification, the transverse partitions of the sieve tubes with their tiny pores clearly look like a sieve, topped with a dense, fine-grained substance. Companion cells are elongated, thin-walled, and filled with granular cytoplasm containing a clearly visible nucleus. Having carefully studied the section, sketch it, then move the glass slide again to find the section that passes through the xylem part of the bundle.

Fig. 43. Tangential section of a corn bundle through the xylem:

1 - ground parenchyma, 2 - sclerenchyma, 3 - reticulate vessels, 4 - annular-spiral vessel, 5 - wood parenchyma

In the section passing through the xylem part of the bundle (i.e., through its middle), the ground parenchyma is located on the sides, followed by the sclerenchyma sheath (Fig. 43). The layer of sclerenchyma cells here is much smaller than above the phloem. This corresponds entirely to what we saw in the cross-section, where the sclerenchyma sheath on the sides of the vascular bundle was much thinner than at the top and bottom. Two reticulate vessels are located on both sides behind the sclerenchyma, with their longitudinal walls showing distinct small thickenings that give the vessel walls a net-like appearance. The transverse cell walls that formed these vessels have disappeared. Between the reticulate vessels lies an annular-spiral vessel, whose inner wall features spiral thickenings. The space between the vessels is filled with wood parenchyma cells. They are living, although they have quite thickened walls.

This preparation should also be sketched.

In the longitudinal-radial section (Fig. 44), the elements are arranged in the following order: at the edges of the section on the right and left, thin-walled ground parenchyma is visible. This is followed on both sides by areas of the sclerenchyma sheath. To the left of the sclerenchyma sheath lies an air cavity, followed by the annular and spiral vessels, as well as wood parenchyma cells. Located behind the xylem is the phloem, specifically the sieve tubes and companion cells. If the section passed through the middle of the bundle, it will not show reticulate vessels, because

they are located slightly to the side and higher up.

Fig. 44. Radial section of a corn bundle:

1 - ground parenchyma cells, 2 - sclerenchyma sheath, 3 - air cavity, 4 - annular vessel, 5 - spiral vessel, 6 - wood parenchyma, 7 - sieve tubes, 8 - companion cells

As a rule, sections do not always pass in the exact direction described above. Therefore, one must learn to clearly distinguish all the elements of the vascular bundle in a longitudinal section in order to correctly label them in the drawing later.

It is advisable to pay special attention to the Different types of vessel thickenings and the STRUCTURE OF THE sieve tubes. In addition to the overall drawing, it is recommended to separately sketch, at a larger scale, all types of vessels and the sieve tube with its companion cells, which serve as the actual conducting elements.

It is also recommended to treat this section with phloroglucinol and hydrochloric acid or aniline sulfate to highlight the lignified elements.

Slide. Closed collateral vascular bundle in the stem of German iris (Iris germanica L.)

Take a small piece (a few centimeters) of the iris flower stem, make several thin cross-sections, place them in a drop of water on a glass slide, cover with a coverslip, and examine under low magnification.

Scattered vascular bundles can be seen amidst the ground tissue. Having selected the thinnest one, center it in the field of view and switch the microscope to high magnification. By moving the slide, you can see that the vascular bundle is surrounded on all sides by thin-walled parenchyma. A small area of sclerenchyma cells is located above the phloem. Sieve tubes and companion cells are clearly visible in the phloem (Fig. 45).

Fig. 45. Cross-section of an iris vascular bundle:

1 - thin-walled parenchyma, 2 - sclerenchyma, 3 - sieve tubes, 4 - companion cells, 5 - tracheids, 6 - wood parenchyma

The sieve tubes are quite wide and empty, or show visible sieve plates. The companion cells are significantly narrower than the sieve tubes and filled with dense granular cytoplasm. The xylem part of the bundle is directly adjacent to the phloem. A characteristic feature of the iris stem is the absence of an air cavity and true vessels, or tracheae, in the xylem. The xylem portion of the vascular bundle consists of tracheids and wood parenchyma. The walls of the tracheids are quite thick and heavy, whereas the wood parenchyma cells are thin-walled, living, and filled with protoplasmic contents. To better differentiate the individual elements of the vascular bundle, remove the microscope slide from the stage, take off the cover slip, and treat the sections with phloroglucinol and hydrochloric acid. After a few minutes, transfer the sections into a drop of water on a second microscope slide, cover with a cover slip, and examine the preparation first under low and then under high magnification. Groups of sclerenchyma cells above the phloem, as well as the tracheids in the xylem, stain cherry-red, indicating their lignification. The remaining Tissues of the section remain unchanged.

Make a drawing of the examined preparation and label the main tissues on the diagram.

Note. In the iris stem, the bundle is not referred to as a fibrovascular bundle because it lacks true vessels and has very poorly developed sclerenchyma fibers.

Slide. Closed collateral vascular bundle in the stem of the German iris (Iris germanica L.) in longitudinal section

Cut a small piece of the stem and prepare several thin radial sections so that they pass through the vascular bundle. Ensure that the cutting surface is not skewed. Place the prepared sections in a drop of water on a microscope slide and examine them first under low and then under high magnification.

Flanking the vascular bundle is a thin-walled parenchyma, bordered on the right by a group of sclerenchyma cells (Fig. 46) (in the cross-section, these cells were located above the bast). The sclerenchyma cells are elongated with tapered ends. Their walls are quite thickened and penetrated by A large number of slit-like pits. Beyond the sclerenchyma lies the bast (phloem) portion of the vascular bundle, consisting of sieve tubes and companion cells.

Fig. 46. Longitudinal (radial) section of an iris vascular bundle:

1 - stem thin-walled parenchyma, 2 - sclerenchyma, 3 - bast (phloem), 4 - sieve tubes, 5 - companion cells, 6 - wood (xylem), 7-9 - spiral vessels, 10 - annular vessels, 11 - wood parenchyma

The transverse septa are clearly visible in the sieve tubes. They are perforated in many places, resembling a small sieve, above which a callose plug is sometimes visible. The companion cells are narrow and shorter than the sieve tubes. They are living, thin-walled, and filled with a granular cytoplasm in which an elongated nucleus is occasionally distinct.

The xylem of the vascular bundle is located behind the phloem. In longitudinal section, it is clearly visible that it is composed of tracheids rather than vessels. These are elongated cells with tapered edges that fit tightly against one another. Spiral or annular thickenings are visible on the inner walls of the tracheids. Wood parenchyma cells are located between the annular tracheids and the ground parenchyma.

After examining the preparation, remove it from the microscope stage and treat it with phloroglucinol and hydrochloric acid, or aniline sulfate. The sclerenchyma cells, tracheid walls, and thickenings on the inner walls of the tracheids will stain cherry-red (with phloroglucinol and hydrochloric acid) or yellow

(with aniline sulfate).

Make a drawing of the preparation.

Slide. Open bicollateral fibrovascular bundle in the stem of the pumpkin (Cucurbita pepo L.)

Before proceeding with the preparation of the anatomical slide, make a cross-section through the entire stem and place it in a drop of water on a microscope slide. Examining the section against the light reveals that the pumpkin stem is ridged both internally and externally. Fibrovascular bundles are located in the angles of the ridges—smaller ones on the outer ridges and larger ones on the inner ridges. In both cases, the lumens of the vessels are clearly visible. The bundles are arranged in regular rows in a circle at certain distances from one another. In the center of the stem lies a central cavity with large depressions extending into the parenchyma between the bundles.

Fig. 47. Diagram of the pumpkin stem structure:

1 - epidermis, 2 - collenchyma, 3 - parenchyma, 4 - sclerenchyma, 5 - xylem, 6 - cambium, 7 - phloem, 8 - cambium, 9 - air cavity

Next, prepare a thin section, examine it under low magnification, and sketch the general layout (Fig. 47). The stem is externally covered by an epidermis, which bears a large number of straight multicellular trichomes (hairs). The Hair cells are quite wide at their base and narrower toward the apex, terminating in small, sharp, conical cells. The hair cells are living, filled with cytoplasm containing a nucleus, numerous chloroplasts, and many vacuoles.

Beneath the epidermis in the angles lie small patches of mechanical tissue—collenchyma. The cell walls of the collenchyma are thickened at the corners, and under a microscope, these thickenings appear as shiny triangles or quadrangles.

Several layers of parenchymal cells lie beneath the collenchyma, followed by a continuous ring of mechanical tissue—sclerenchyma—several rows thick, whose cell walls are thickened and lignified.

Further inward lies the ground parenchyma, which contains the fibrovascular bundles. An air cavity is located in the center.

To better differentiate all PARTS OF THE pumpkin stem, the section should be treated with phloroglucinol and hydrochloric acid or aniline sulfate. The sclerenchymatous ring and vessels in the vascular bundle stain cherry-red (with phloroglucinol) or yellow (with aniline sulfate). All lignified elements in the section stain in the same manner, which helps to clearly distinguish and illustrate the phloem and xylem parts of the bundle on a diagram.

After sketching the general layout of the individual tissues in the pumpkin stem, make several new thin sections to capture the part of the stem where the large fibrovascular bundles are located. Place some of the sections in a drop of water on a microscope slide, and others in a watch glass to be treated with phloroglucinol and hydrochloric acid or aniline sulfate, after which they are transferred to a drop of water on a second microscope slide. Having covered the prepared specimens with coverslips, examine the unstained specimen first.

Select the thinnest, non-skewed section of the fibrovascular bundle, in which all its constituent elements can be clearly distinguished even under low magnification. Outline the contours of the fibrovascular bundle in your laboratory notebook and use dashed lines to mark the positions of the phloem, xylem, cambium, and ground tissue, ensuring an accurate representation of the arrangement and scale of all elements.

Having examined the general structure of the fibrovascular bundle, switch the microscope to high magnification and, starting from the peripheral part of the section, sketch the corresponding elements of the bundle. The fibrovascular bundle lies among the cells of the stem ground parenchyma. The phloem is located at the periphery of the bundle, comprising sieve tubes with companion cells and bast parenchyma (Fig. 48).

Fig. 48. Bicolateral fibrovascular bundle of a pumpkin in cross-section:

1 - ground parenchyma, 2 - outer phloem, 3 - cambium, 4 - secondary xylem, 5 - primary xylem, 6 - inner phloem

The sieve tubes are large, with slightly thickened, cellulosic walls, and differ from all other cells by a distinct matte tint. During sectioning, most sieve tubes are cut into equal hollow segments, but a significant number are cut at the level of the transverse septum; consequently, in cross-sections, perforated septa are visible in the sieve tubes, where large pores are separated by a relatively thin Cellulose mesh. The pores are often filled with the Contents of the sieve tubes, which are rich in plastic substances, especially Proteins. In fixed material, the contents of the sieve tubes coagulate. If the material was collected in autumn, so-called callus pads—deposits of callose that hinder the movement of nutrients through the sieve tubes—can often be observed on the sieve plates. The formation of such pads can completely halt the flow of sap. This phenomenon is observed in many plants during winter; in spring, however, these pads can completely dissolve, and the sieve tubes resume their function.

A companion cell is closely appressed to each sieve tube. Companion cells develop together with the sieve tube from a single mother cell, which divides via a longitudinal partition into two unequal parts: the larger one, which forms the sieve tube, and the smaller one, which forms the companion cell. The companion cells are thin-walled and filled with dense granular cytoplasm in which The Nucleus is clearly visible. Companion cells regulate the movement of sap through the sieve tubes. A layer of meristematic cambium tissue, 5-6 rows thick, lies between the phloem and the xylem. Cambium cells have the shape of elongated tetrahedral prisms with pointed ends, arranged in regular rows one below the other, and originate from the primary meristematic tissue, the procambium. In cross-section, cambium cells are rectangular.

The soft, elastic walls of the cambium cells consist of cellulose and a small amount of pectic substances. The tangential walls are thinner than the radial ones. The lumen of the cambium cells contains cytoplasm housing a spindle-shaped or elongated nucleus, leukoplasts, and Mitochondria.

The xylem lies beyond the cambium in the pumpkin fibrovascular bundle. In this part of the bundle, attention is first drawn to the vessels, which, based on The Nature of their inner wall thickening, can be pitted (larger vessels), annular, or spiral.

The spaces between the vessels are filled with living, thin-walled wood parenchyma cells and individual tracheids whose walls feature reticulate thickening and are pierced by large pits. Underlying the xylem region formed by cambial activity is the primary xylem, which consists of isolated or clustered annular and spiral tracheids with significantly thickened walls, alongside thin-walled living parenchymal cells.

Beyond the primary xylem, deeper toward the center of the stem, lies the inner phloem layer, which has a structure identical to that of the outer phloem. A narrow strip of thick-walled parenchyma—whose cells are incapable of division—is situated between the primary xylem and the inner phloem. There is no cambium between the xylem and the inner phloem.

After carefully studying and sketching the specimen, replace it with another treated with phloroglucinol and hydrochloric acid, in which the lignified elements stained cherry-red are clearly visible even under low magnification. Next, verify the accuracy of the individual bundle elements shown in the drawing and make any necessary corrections.

The pumpkin fibrovascular bundle is called bicolateral because the phloem adjoins the xylem on both sides.

Slide. Open fibrovascular bundle of common pumpkin (Cucurbita pepo L.) in longitudinal section

Take a small piece of the pumpkin stem and cut it so that one of the large fibrovascular bundles is exposed On the surface.

The section must be radial, capturing the central part of the bundle and the adjacent tissues. Ensure that the section is not skewed and runs parallel to the length of the stem. Make several sections so that the thinnest one can be selected and all components of the fibrovascular bundle can be examined. It is advisable to place the sections on a microscope slide in a drop of water in the exact order they were cut.

Place a similar series of sections on a second microscope slide and treat them with phloroglucinol and hydrochloric acid or aniline sulfate.

The prepared specimen is examined first under low magnification to outline the general layout of the individual elements, and then under high magnification to study in detail the structure of all tissues comprising the fibrovascular bundle.

Fig. 49. Cytology/practical/54.html">Longitudinal section of a pumpkin fibrovascular bundle:

1 - ground parenchyma, 2 - sieve tubes, 3 - companion cell, 4 - cambium, 5 - pitted vessel, 6 - wood parenchyma, 7 - spiral vessel

If the section is radial and passes through the entire width of the fibrovascular bundle and its adjacent tissues (Fig. 49), the ground parenchyma, consisting of large thin-walled cells, will be visible on the left and right sides of the specimen. Sieve tubes are visible beyond the ground parenchyma. Individual segments of the sieve tubes are cylindrical and slightly expanded at the points where transverse partitions—sieve plates—occur. The lateral walls are thickened and perforated by numerous pores, giving them a bead-like appearance.

Transverse sieve plates may lie horizontally or obliquely. They may resemble the longitudinal walls or become significantly thickened due to the deposition of callose on both sides of the primary partition.

If the specimen is prepared from living material without The Use of any Reagents, the proteinaceous content can be observed within the sieve tubes. In fixed material, this protein content contracts, pulls away from the walls, and appears as a thick cord running along the tube, expanding at the level of the transverse partitions, to which it is anchored by strands of the same protein substance passing through the pores.

Companion cells closely adjoin the sieve tubes. They are elongated, tapered at the ends, thin-walled, and filled with a granular cytoplasm in which the nucleus is distinctly visible.

Depending on the plane of the section, a row of phloem parenchyma cells may lie adjacent to the sieve tubes. Phloem parenchyma cells are elongated and thin-walled. Their transverse walls are typically oriented horizontally. The cells contain a layer of cytoplasm and a nucleus; these are living cells.

The cambium is located beyond the phloem parenchyma. In a longitudinal section, the cambial cells are elongated and filled with dense granular cytoplasm containing a clearly visible nucleus.

As already noted, the cambium lies between the phloem and the xylem. Consequently, the xylem part of the fibrovascular bundle follows the cambium. Here, attention is drawn to the wide pitted vessel. The inner longitudinal walls of the pitted vessel feature a dense network of thickenings, interspersed with regularly spaced transverse rows of pits.

The xylem parenchyma is located beyond the pitted vessel. Xylem parenchyma cells are short, with slightly thickened, lignified walls, yet their cellular contents do not disintegrate, and they remain alive. The xylem parenchyma cells fill the space between the vessels.

Located even closer to the center are the annular or spiral vessels. They are significantly narrower than the pitted vessels, and their walls feature internal thickenings in the form of rings or spirals.

Interspersed between the reticulate, annular, and spiral vessels, alongside the parenchyma, are tracheids and sclerenchyma fibers. Further past the xylem portion of the bundle, elements of the phloem are once again situated.

After examining the preparation and sketching individual parts of the fibrovascular bundle, it is treated with phloroglucinol and hydrochloric acid, or with aniline sulfate. All lignified elements stain cherry-red (with phloroglucinol) or yellow (with aniline sulfate). On this preparation, one should carefully examine the Nature of the vessel thickenings, the structure of the pits, the arrangement of mechanical elements, and make the necessary corrections to the drawing.

On the preparation treated with phloroglucinol and hydrochloric acid, it is clearly visible that the longitudinal walls of the vessels remain cellulosic, while only thickenings of various shapes undergo lignification.

It is not always possible to obtain a section that captures all the aforementioned elements. Therefore, they must be examined progressively across several sections and sketched in the correct sequence, using the cross-section diagram made at the very beginning of The Study of the pumpkin fibrovascular bundles as a reference.

Preparation. Concentric fibrovascular bundle of bracken fern (Pteridium aquilinum L.)

Take a small piece of the bracken rhizome and make an initial cross-section through the entire rhizome, followed by several small, thin sections designed specifically to capture the fibrovascular bundles, which are clearly visible to the naked eye.

The prepared sections are placed on a microscope slide in a drop of water and, without a coverslip, examined against the light. Oval or circular fibrovascular bundles, embedded within the ground parenchyma tissue, are distinctly visible in the section. Having studied the general arrangement of the concentric bundles, one takes the prepared thin sections and examines one of them under low magnification. Under these conditions, the entire bundle fits within the microscope field of view. On the outside of the bundle lies the primary thin-walled parenchyma, followed by a row of dark brown, thick-walled cells that encircle the bundle in a continuous ring. This layer of cells is called the endodermis. The endodermis is tightly flanked on the inner side by a starch sheath consisting of large, living, thin-walled cells, beyond which lie the phloem elements arranged in a ring one or several layers thick. Among them, one can easily distinguish the relatively large, almost empty sieve tubes, interspersed with much smaller phloem parenchyma cells. Extending inward from the phloem toward the center is a ring of xylem parenchyma, followed by the thick-walled water-conducting elements of the xylem (Fig. 50).

Sketch the overall outlines of the bundle in the lab manual and map out the placement of all its components to maintain the proper proportional relationships among the individual tissues of the bundle. To study the structure of the individual elements of the bundle and draw them accurately, switch the microscope to high magnification and, moving the preparation from the periphery of the section toward the center, examine the structure of the individual tissues step by step.

Fig. 50. Cross-section of a concentric bundle from the rhizome of bracken fern:

1 - stem parenchyma, 2 - endodermis, 3 - pericycle, 4 - parenchyma surrounding the vascular bundle, 5 - phloem ring, 6 - parenchyma, 7 - xylem

The ground parenchyma cells are living and thin-walled; they contain cytoplasm, a nucleus, and rod-shaped starch grains.

The endodermal cells are thick-walled, narrow, and yellowish-brown. They lack cytoplasm and are therefore dead cells. The thin-walled parenchyma cells adjoining the endodermis on the inside are living; they possess cytoplasm and a nucleus, and are packed with rod-shaped starch grains, which is why this cell layer is termed the starch sheath.

The sieve tubes are arranged in two rows immediately adjacent to the starch sheath and are closely appressed to one another. Their cytoplasmic contents disappear, and companion cells are absent. Phloem parenchyma cells lie between the sieve tubes; they are small, thin-walled, and filled with cytoplasm containing a visible nucleus.

The xylem parenchyma cells lying inward from the phloem toward the center are living, and their walls are non-lignified. The cells contain numerous rod-shaped starch grains. Similar cells are also found in the center of the bundle among the tracheids.

Occupying the center of the bundle is the primary xylem—specifically, the protoxylem, which consists of tracheids with spiral wall thickenings and large, thick-walled scalariform vessels. Vessels with any other type of thickening are absent from the bundle.

After carefully examining the preparation, one should sketch it, then remove the coverslip and add a drop of phloroglucinol and hydrochloric acid to the slide. After a few minutes, transfer the sections to a clean microscope slide in a drop of water, cover with a coverslip, and examine under the microscope.

The thickened walls of the endodermis, as well as the thickenings in the vessel walls, stain cherry-red.

Slide. Concentric vascular bundle in the rhizome of bracken (Pteridium aquilinum L.) in longitudinal section

Make several longitudinal sections, ensuring that the cut passes directly through the vascular bundle. Place the sections on a microscope slide in the exact order they were cut so that all components of the bundle can be properly examined.

The thin-walled, wide cells of the fundamental parenchyma are located along the edges of the section (Fig. 51). In these cells, a parietal layer of cytoplasm, the nucleus, and rod-shaped starch grains are visible.

The endodermal cells are elongated with straight transverse walls; they are empty and dead. The starch-storing cells are thin-walled, rectangular, and filled with cytoplasm and starch grains.

Fig. 51. Longitudinal section of a vascular bundle from the bracken rhizome:

1 - fundamental thin-walled parenchyma, 2 - endodermis, 3 - starch-storing cells, 4 - phloem parenchyma, 5, 6 - sieve tubes, 7 - xylem parenchyma, 8, 9 - scalariform vessels

The phloem parenchyma cells are narrow and oblong, containing cytoplasm and a nucleus; on one side, they abut the starch-storing cells, and on the other, the sieve tubes. In ferns, sieve tubes differ in that their sieve plates are located not on the transverse partitions, but on the longitudinal walls where adjacent sieve tubes are in contact with one another.

The sieve tubes are elongated, wide, and empty, with thin walls. Chlor-zinc-iodine stains the tube walls blue. The sieve tubes are arranged in 2–3 contiguous rows or are separated by the thin-walled phloem parenchyma. The xylem parenchyma lies behind the sieve tubes, separating them from the vessels. The vessel walls feature scalariform thickenings, and their segments are quite wide and long with tapered ends, between which lies a perforated septum. Through the openings in this septum, adjacent segments communicate with one another. Between the vessels lies one or two layers of xylem parenchyma, consisting of living, elongated, thin-walled cells containing cytoplasm, a nucleus, and a considerable number of starch grains.

Occasionally, tracheids with helical thickenings can be observed in the section.

Thus, scalariform vessels differ from all other vessels in that the transverse partitions between individual segments do not disappear entirely, but merely become perforated.

The concentric bundle in bracken is referred to as amphicribral because the phloem surrounds the xylem.

Slide. Concentric vascular bundle in the rhizome of lily of the valley (Convallaria majalis L.)

The rhizome of the lily of the valley is characterized by the presence of Two Types of vascular bundles: collateral and concentric. The collateral bundles form a ring just beneath the endodermis, which separates the central cylinder from the primary cortex, while the concentric bundles are scattered throughout the interior of the central cylinder.

Prepare several thin sections of the lily of the valley rhizome so as to capture the bundles located in the center of the central cylinder. First, examine them under low magnification to sketch the general outlines of the bundle in your workbook and map out its individual components. Then, switch the microscope to high magnification to examine in detail the structure of all the tissues comprising the bundle.

Externally, the vascular bundle is surrounded by a parenchyma consisting of living, thin-walled cells containing cytoplasm, a nucleus, and a substantial number of starch grains (Fig. 52).

Fig. 52. Cross section of the vascular bundle in the lily of the valley rhizome: 1 - thin-walled parenchyma, 2 - xylem, 3 - sieve tubes, 4 - companion cells

At the periphery of the bundle, immediately adjacent to the fundamental parenchyma, the xylem forms a ring consisting of polyhedral cells with significantly thickened walls. These are pitted and spiral vessels and tracheids. The phloem lies in the center of the bundle and consists of sieve tubes bordered by companion cells filled with granular cytoplasm.

Stain the preparation with methylene green and alum carmine. These reagents stain the xylem bright green and the non-lignified cells pinkish-red.

The concentric bundle in the lily of the valley rhizome is termed amphivasal because the xylem surrounds the phloem.

Slide. Radial vascular bundle in the ROOT of the German iris (Iris germanica L.)

Fig. 53. Radial vascular bundle of the iris root:

1 - primary cortex, 2 - endodermis,

3 - passage cell, 4 - pericycle,

5 - phloem, 6 - xylem, 7 - sclerenchyma

Radial bundles are characteristic of the Introduction/19.html">Primary Structure of roots in all plants. They are distinguished by the fact that phloem strands alternate with xylem strands of the same number, arranged along the radii of the organ.

Xylem strands may coalesce in the center or terminate before reaching it; in the latter case, thin-walled parenchyma occupies the central region of the bundle.

Take an iris root and make several thin cross-sections. Place the sections in a drop of water on a microscope slide, cover with a coverslip, and examine under low magnification to select the thinnest section for sketching the general structural diagram of the root. Even under low magnification, it is clearly visible that the greater part of the root is occupied by the primary cortex, which consists of living thin-walled cells (Fig. 53).

The primary cortex terminates in a layer of endoderm—thick-walled cells forming a ring around the central cylinder, interspersed with occasional thin-walled living cells known as passage cells.

A layer of living cells lies beneath the endoderm, followed radially by areas of xylem and phloem. After examining the preparation under low magnification, sketch the general diagram of the bundle structure and the arrangement of its individual parts in your laboratory notebook; the structural details should then be studied under high magnification.

To better differentiate the individual parts of the bundle, remove the slide from the microscope stage, lift the coverslip, and add a drop of phloroglucinol and hydrochloric acid. Transfer the stained sections to a drop of water on a clean microscope slide, cover with a coverslip, and, after locating the thinnest area of the preparation, switch the microscope to high magnification.

The walls of the endodermal cells are heavily thickened, distinctly layered, and stain a cherry-red color.

Passage cells stand out sharply among the thick-walled endodermal cells. Their walls are thin, and their cytoplasm and nucleus are clearly visible. A layer of thin-walled living cells filled with cytoplasm lies beneath the endoderm. This is the pericycle, from the cells of which lateral roots originate.

The endoderm forms the innermost layer of the primary cortex, whereas the pericycle constitutes the outermost layer of the central cylinder. The xylem strands also stain cherry-red and merge in the center of the bundle. The xylem portion of the bundle consists primarily of vessels with thickened walls. The phloem regions are small, standing out as light patches amidst the xylem. The phloem is composed of sieve tubes and parenchyma. The phloem elements are living, thin-walled, and contain cytoplasm.

Note. The structure of a given tissue can be studied most effectively by isolating it from other tissues via maceration—a technique frequently employed when examining individual conducting elements. Excellent subjects for studying the structure of tracheids and vessels are horseradish root and bracken rhizome.

Preparation. Conducting elements of the horseradish root (Cochlearia armoracia L.)

A piece of horseradish root should be peeled, placed in a 10% sodium carbonate solution or a 20% potassium hydroxide solution, and boiled for 3–6 minutes. The resulting tissues are then thoroughly rinsed with water, freed from the fine white filaments clearly visible to the naked eye, and placed in a drop of water on a microscope slide. Examining the preparation under high magnification readily reveals fairly large tubes composed of several cells. These are vessels in which the transverse partitions between individual segments have not completely disappeared.

Pores are arranged in regular transverse rows along the vessel walls, giving the vessel a reticulate structure. Treatment with phloroglucinol and hydrochloric acid stains the reticulate wall thickenings cherry-red.

Preparation. Conducting elements of the bracken rhizome (Pteridium aquilinum L.)

To isolate individual vessels and tracheids from the bracken rhizome, take a piece of the rhizome, remove the outer soft tissue layer, place the central rigid portion into a test tube, add a 10% sodium carbonate solution, and boil for 3–6 minutes (alternatively, treat with chromic acid and boil for one minute). The rhizome will dissociate into its constituent parts. Rinse the macerated tissue thoroughly with water, place it in a drop of water on a microscope slide, and tease it apart using dissecting needles. Cover the preparation with a coverslip and examine it first under low and then under high magnification. Locate the vessels among the other tissues. They appear as wide tubes whose walls bear distinct transverse thickenings shaped like rods of varying length. These are scalariform vessels. The ends of the vessel segments are tapered, and the transverse partition is heavily perforated, confirming that they are vessels rather than tracheids.

After sketching an individual vessel, examine the preparation by slowly moving it under the microscope until narrow, short tubes—tracheids—are found. The tracheid walls feature spiral thickenings. The transverse partitions between tracheids are solid and inclined.