Plant Anatomy: Workshop - Panyuta O.O. 2019

Topic 1. The Cell
Laboratory Work No. 4. Inclusions in the Plant Cell

Theoretical Background. Inclusions are temporary Cell components that can be formed and enzymatically degraded at various stages of cellular activity. They are primarily localized in the Cytoplasm, though they can occasionally be found in The Nucleus. Inclusions include reserve nutrients (CARBOHYDRATES, Proteins, Lipids) and various crystals.

Carbohydrates accumulate in plant Cells in the form of starch, sugars, inulin, and Other Compounds.

Starch. Starch is one of the primary reserve carbohydrates, consisting of two Polysaccharides—amylose and amylopectin—which are composed of glucose residues. It is among the most widespread polysaccharides, deposited in all plants except Fungi and blue-green Algae. Based on its physiological role and cellular Location, starch is classified into three types: assimilation (autochthonous), transitory, and reserve.

Assimilation starch forms as small granules within the METABOLISM/14.html">Chloroplasts of the leaf mesophyll, the cortex of young stems, and other chlorophyll-bearing cells undergoing active Photosynthesis. This primary starch does not linger in the chloroplasts; it is rapidly hydrolyzed into soluble sugars, which are then transported via the phloem to consumption sites. Assimilation starch is termed primary because it is synthesized directly within the chloroplasts as a product of photosynthesis.

Transitory starch forms temporarily along the pathways of sugar translocation to storage Organs.

Large amounts of these two starch types are formed in the leaves of potatoes, peas, and beans; lesser amounts in buckwheat and carnation families; and the least in goosefoots. Among monocots, cereal leaves produce little starch, while it is virtually absent in lilies and orchids.

Reserve, or secondary, starch accumulates in large quantities in specialized storage Tissues and organs (endosperm and cotyledons, stem and ROOT wood parenchyma cells, tubers, bulbs, rhizomes), from which it is gradually mobilized to meet the metabolic needs of the plant Organism. However, some starch is not consumed even during plant starvation (for example, in root cap cells).

Starch is easily detected by treating a specimen with an iodine-potassium iodide solution. Iodine turns starch blue. If a large amount of iodine is added, the starch grains turn black. Upon heating, the blue or black color disappears, only to reappear upon cooling. The absence of starch in the leaves of certain plants does not necessarily indicate that the plant is incapable of producing it. For instance, if a leaf of a common iris, which is low in starch, is placed in a 20% sugar solution, starch deposition can subsequently be detected in the living cells of this plant.

Starch is deposited in the form of round or ellipsoidal grains, and even within a single cell, starch grains vary in size. Furthermore, individual plant species possess starch grains of a characteristic shape specific to that particular plant. The morphological appearance of starch grains makes it easy to determine their plant origin, which can be utilized to identify the type of grain used in flour production.

Starch grains may be elliptical (in potatoes), flattened (in cannas), lenticular (in wheat), among others. A starch grain grows unevenly, forming layers that are readily visible under a Microscope. As a rule, starch layers are deposited around a center known as the hilum of the starch grain. Depending on THE POSITION OF the hilum, starch grains are classified as concentric or eccentric. In concentric starch grains, the hilum is located in the center of the grain (e.g., in wheat and rye), whereas in eccentric grains, it is situated near the periphery. The stratification of starch grains results from their uneven growth throughout the day. Moreover, eccentric starch grains are characterized by stratification that forms and develops near The surface of leucoplasts, whereas starch grains with concentric stratification originate and develop in the center of the leucoplast. Notably, the size of a starch grain almost always exceeds the size of the leucoplast by several times.

Starch grains are also categorized into simple, compound, and semi-compound grains. In potatoes, simple, compound, and semi-compound starch grains can frequently be observed within a single cell.

Starch grains from different plants vary in size. For example, they reach 70–100 mkm in potatoes, 60 mkm in beans, 30–45 mkm in wheat, 15–35 mkm in corn, and 3–10 mkm in rice. The largest starch grains are found in toothwort (Lathraea squamaria), reaching up to 275 mkm. The size of starch grains is of practical importance in the baking industry.

Starch is a highly versatile substance that readily breaks down into simpler compounds under various environmental influences, which the plant then utilizes in its vital metabolic processes.

Within every plant cell, starch grains are not isolated; rather, they are closely associated with other cellular inclusions. The number of starch grains in a storage cell can be remarkably high. For instance, potatoes may contain up to 23% starch relative to the total fresh weight of the tuber. In such cases, the cytoplasm is reduced to a delicate net-like matrix wedged between densely packed starch grains that fill the entire cell.

Sugars. In certain plants, the primary reserve carbohydrates are Water-soluble sugars such as sucrose, glucose, and fructose. These sugars serve as building blocks for more complex polysaccharides, including starch, inulin, hemicellulose, and Cellulose. Additionally, they are Key Components of various Glycosides, Tannins, and protein compounds.

Glucose and fructose belong to the Monosaccharides known as hexoses. Every living plant cell contains a certain quantity of at least one type of hexose, which serves as the primary energy source utilized by the plant in its metabolic processes.

The concentration of hexoses varies significantly among different plant species. For example, in ripe apples, hexoses can account for up to 10% of the fresh mass, whereas in potato leaves, they constitute a mere 0.15–1.27%.

Glucose is frequently accompanied in plants by fructose, often referred to as fruit sugar. Fructose differs from glucose in its optical properties: it rotates the plane of polarization to the left, whereas glucose rotates it to the right. Furthermore, unlike glucose, which readily crystallizes, fructose does not form crystals and remains in a syrupy state. Fructose also tastes sweeter than glucose.

Sucrose is widely distributed in PLANT CELLS AND is formed by the Condensation of two hexose molecules—specifically, one molecule of glucose and one molecule of fructose.

Sucrose has a sweet taste and readily crystallizes. In some plants, it can accumulate in substantial quantities; for instance, sugar beets contain up to 20–23% sucrose. High concentrations of sucrose are also found in sugarcane, sugar maple, sweet sorghum, corn, and various fruits such as wild strawberries, garden strawberries, apricots, oranges, apples, melons, and dates, among others.

Under The Influence of acids and Enzymes, sucrose undergoes Hydrolysis, breaking down into glucose and fructose. This process is known as inversion. Sucrose is widely consumed as a food product and holds significant economic importance.

The various sugars accumulating within cells can be quantified using standard Analytical Methods applied in diverse research contexts. In practical plant anatomy classes, qualitative methods known as Microchemical Reactions are employed.

Sugars cannot be observed directly under a microscope because they are dissolved within The Cell sap. Instead, their presence is detected through microchemical reactions using Fehling's solution.

Inulin. In many plants, reserve substances are stored not as starch, but as inulin, which remains dissolved in the cell sap. Inulin is a polysaccharide composed of multiple fructose units and shares the same chemical formula as starch. It is particularly characteristic of plants belonging to the Asteraceae family, accumulating in large amounts in the tubers of dahlias, as well as in the roots of kok-saghyz, dandelion, chicory, burdock, wormwood, and other species.

When heated with acids, inulin breaks down into fructose. Normally, inulin does not crystallize; however, if dahlia tubers are immersed in alcohol, inulin eventually precipitates out of solution in the form of sharp, radially arranged needle-like clusters known as spherocrystals. The size of these spherocrystals can vary.

A single spherocrystal can occupy several cells, and conversely, a single cell may contain several small spherocrystals.

Inulin spherocrystals differ from other crystals in their ability to swell slightly, resembling starch grains. Inulin spherocrystals are nearly insoluble in cold water, which causes them to be retained within living cells, whereas they dissolve very readily in hot water. Inulin can be hydrolyzed by chemical Reagents or enzymes, particularly inulinase. This enzyme has been detected in sprouting Jerusalem artichoke tubers and in the mycelia of Molds. Under the action of inulinase, inulin is converted into sugar, specifically fructose.

Inulin was first discovered in the underground Organs of the elecampane plant (Inula), from which it derives its name. The pathways of inulin formation in the underground organs of plants remain unclear.

If a thin section from a fresh dahlia tuber is placed in a drop of alcohol, a turbid precipitate initially forms; after some time, the turbidity disappears and inulin spherocrystals develop. This is why Practical Classes always require The Use of preserved plant material stored in alcohol.

Proteins. Among the cytoplasmic substances that directly participate in metabolism, proteins are of particular significance.

Proteins are divided into two groups:

1. Constitutional or structural proteins, which form an integral part of living cytoplasm and its structural components.

2. Reserve Proteins, which are deposited in the cytoplasm as solid structures known as protein bodies or aleurone grains. Most commonly, they occur in cells as amorphous masses, small granules, or crystal-like formations. Crystallized proteins exhibit The properties of both crystals and colloids simultaneously, which is why they are commonly referred to as crystalloids.

Crystalloids have a regular or hexagonal shape, although in some plants this shape is not entirely distinct.

Protein crystals are located directly within the Cell Cytoplasm and cell sap, and they can also be observed in the Cell Nucleus. Protein crystals are likewise found in Plastids, such as leucoplasts. Most frequently, reserve proteins occur in The Cell as specific formations known as protein bodies or aleurone (protein) grains.

Aleurone grains consist of an outer membrane and an amorphous protein matrix containing Three types of inclusions: globoids, crystalloids, and calcium oxalate crystals. Globoids are composed of phytin, which is the calcium-magnesium salt of Inositol hexaphosphoric acid. Typical globoids are spherical in shape. A single aleurone grain may contain one or more globoids.

The majority of aleurone grains contain either globoids, crystalloids, or both. The most complexly structured aleurone grains are found in castor bean seeds, which contain both globoids and crystalloids.

The formation of aleurone grains can be traced during seed development. Initially, small vacuoles arise in the cytoplasm, containing dissolved reserve protein substances. Subsequently, as the seeds mature, the protein concentration within the vacuoles increases, and the proteins precipitate out in the form of protein grains.

Thus, aleurone grains originate from vacuoles. During germination, seeds absorb large amounts of water, causing the aleurone grains to undergo changes. First, the main amorphous matrix dissolves, followed by the crystalloids, and ultimately vacuoles containing the protein are formed anew. These transformations are driven by the complex metabolic processes that arise from the intense physiological activity associated with seed germination and embryo development.

Lipids. Lipids are found in significant quantities within the cytoplasm of plant cells. In plants, they are predominantly deposited in a liquid state and are referred to as oils. For instance, the oil content in coconut palm seeds reaches up to 67%. High oil concentrations are also characteristic of sunflower, castor bean, and hemp seeds, among others. Oils dissolve in certain organic Solvents and ether, but are insoluble in absolute alcohol, acetic acid, and water.

Oils play a crucial role in metabolic processes, particularly during plant germination.

Under the influence of acids, alkalis, and enzymes—especially lipase—oils undergo hydrolysis, breaking down into glycerol and Fatty acids.

Oils can be deposited in various plant organs. Most commonly, they accumulate in seeds, but they are also present in stems, roots, tubers, and bulbs. Oils are likewise characteristic of various lower plants and microorganisms. The presence of oils in plant Cells and Tissues can be detected using microchemical tests.

Crystalline inclusions. The cells of many plants contain solid inclusions, among which crystals of calcium oxalate and calcium carbonate are the most widespread. Calcium oxalate crystals are formed during metabolic processes marked by the release of a significant amount of oxalic acid, which subsequently combines with calcium.

Calcium oxalate is insoluble in water, acetic acid, and oxalic acid. It dissolves in mineral acids without the evolution of gas bubbles. Calcium oxalate is absent in blue-green and diatomaceous algae; it has likewise not been detected in the cells of mosses and certain seed plants, such as horse chestnut cells.

Objective of the work: to examine various types of inclusions in plant cells.

Materials and equipment: light microscopes, Glass slides and coverslips, dissecting needles, forceps, glass rods, filter paper, distilled water, iodine-potassium iodide solution, Fehling's solution, 1% osmic acid solution, 95% ethyl alcohol, alcohol-ether mixture, Sudan III, iodine, Hydrochloric acid, sulfuric acid, plant material.

Slide. Reserve starch in tubers of the potato plant (Solanum tuberosum L.)

Class="center">

Fig. 9. Starch grains in potato tubers:

1 - simple starch grains, 2 - compound starch grains, 3 - semi-compound starch grains, 4 - parenchyma cells filled with starch grains

To prepare the slide, cut a potato tuber with a scalpel and gently scrape the cut surface so that a small amount of liquid remains on the scalpel. Transfer a drop of this liquid into a drop of water on a microscope slide, mix thoroughly, and cover with a coverslip. First, examine the slide under low magnification to find an area where the starch grains are not too densely crowded, and then switch the microscope to high magnification. The field of view reveals A large number of starch grains with quite diverse shapes: oval, ellipsoidal, and rounded. By adjusting the distance between the objective lens and the slide using the fine adjustment screw, you can see that the narrower end of the starch grain contains its hilum (the center of starch formation), around which starch layers are deposited—thinner near the hilum and thicker toward the edges. Because the starch layers are formed periodically, they vary in density and water content, causing them to refract light differently (Fig. 9).

Alongside simple starch grains, compound starch grains are also visible in the field of view; these have two or even three hila, with each center surrounded concentrically by several layers specific only to that center. However, There are also cases where several additional layers enclosing all the centers are formed around these grains. Such starch grains are called semi-compound. If you use a withered potato tuber that has lost a significant amount of water, or dry starch, for your study, you can observe star- or ray-like clefts or vacuoles at the center of the starch grain.

After examining individual starch grains, you should make a drawing of them. In addition, you need to prepare a slide that shows intact cells filled with starch grains. To do this, make a very thin slice from the center of the tuber using a razor blade and examine it under the microscope. At high magnification, you will see the parenchymal tissue, which consists of thin-walled parenchymal cells packed with a large number of starch grains. Cells in the peripheral part of the tuber contain fewer starch grains than those in the central part. Draw one cell containing starch grains.

On this slide, you should examine the iodine test for starch. To do this, use filter paper to draw water from one edge of the coverslip while adding a very weak solution of iodine in potassium iodide from the opposite side. Starch reacts with iodine to form a blue-colored compound known as starch iodide. Depending on the starch content in the grains and The amount of iodine, the starch grains may turn blue, violet, or blue-black.

When a starch grain is heated in water, it gelatinizes. To observe gelatinization, very carefully heat the prepared slide over a spirit lamp and then examine it under the microscope. Upon heating, a cavity forms at the site of the hilum, from which a network of channels then extends toward the broad edge of the grain. With further heating, the central part of the starch grain dissolves, while a sharply defined ring appears at the periphery. Finally, the starch grains swell into large sacs filled with fluid contents that merge into a single continuous mass before eventually disappearing altogether. This described process is known as starch gelatinization.

Major changes occur in potato starch grains during germination. Starch grains from such tubers have an eroded surface with channels extending deep into the grain. These changes are driven by the enzyme amylase, which is always present in plants. Amylase converts starch into osmotically active compounds, particularly sugars.

Slide. Compound starch grains of oats (Avena sativa L.)

Cut a dry or water-soaked oat grain crosswise, use a scalpel to scrape some floury mass from the center of the seed, mix it in a drop of water, and cover with a coverslip. Under the microscope, you will see a large number of small simple starch grains along with large compound grains of oval or rounded shape (Fig. 10). By turning the fine adjustment screw of the microscope and carefully studying The Structure of the large starch grains, you can see that they consist of individual small grains whose walls are pressed tightly together, forming a net-like structure. Semi-disintegrated compound starch grains can also be observed on this same slide (Fig. 10).

Fig. 10. Starch grains of oats:

1 - simple starch grain,

2 - compound starch grain, 3 - partially disintegrated starch grain

A large compound grain can be separated into individual small simple grains by applying light pressure to the coverslip. The size of a compound starch grain depends on the number of small grainlets it comprises.

Iodine stains oat starch grains blue.

Draw the simple and compound starch grains of oats.

Slide. Starch grains of wheat (Triticum aestivum L.)

Fig. 11. Wheat starch grains: small ones are underdeveloped, large ones are typical starch grains

Cut a dry or soaked wheat grain in half, take a small amount of floury mass from the center of the grain with a scalpel, mix it in a drop of water on a microscope slide, and cover with a coverslip.

Under high magnification, starch grains of various sizes are clearly visible on the slide. The large grains are rounded and concentric, and their stratification is very poorly defined. Stratification is completely invisible in the small starch grains (Fig. 11).

If you draw the water out from under the coverslip using filter paper and add a drop of iodine solution in potassium iodide, you can observe under the microscope that as the iodine solution flows under the coverslip, the starch grains stain blue.

Slide. Starch grains of rye (Secale cereale L.)

Fig. 12. Rye starch grains: small ones are underdeveloped, large ones are typical, well-developed starch grains

The slide is prepared in the same way as those of wheat and oats. Rye starch grains closely resemble those of wheat, differing only in that they often feature a triangular or star-shaped cleft in the center (Fig. 12). Like wheat starch grains, rye starch grains turn blue when treated with iodine.

Note. Instead of rye, one can examine barley starch grains, which share the exact same shape.

Slide. Starch grains of common buckwheat (Fagopyrum esculentum Moench)

It is best to prepare the slide using seeds swollen in water, following the same Procedure as for the previous ones. Under high magnification, small, irregularly shaped starch grains will be visible.

Fig. 13. Buckwheat starch grains: a whole compound grain in the center, individual starch granules at the periphery

Buckwheat starch grains may occur singly or in groups (Fig. 13). Packing tightly inside the cells of buckwheat seeds, they press against one another, acquiring a polyhedral shape As a result. Often, starch grains in buckwheat seeds are clustered into large, amorphous groups. No striation is noticeable in these grains. The nucleus around which the starch grain grows is sometimes visible as a tiny dot.

Slide. Starch grains of maize / corn (Zea mays L.)

Fig. 14. Maize starch grains: small ones are underdeveloped, large ones are typical, well-developed starch grains

The slide is prepared in the same manner as the wheat grain slide. Larger fragments should be teased out of the preparation using a dissecting needle and then covered with a coverslip. To distribute the starch grains more evenly, gently press down on the coverslip with the needle and slide it back and forth in different directions. Examining the slide under high magnification reveals that the starch grains vary in size and shape. The smaller grains are rounded, while the larger ones are angular (Fig. 14).

There is a triangular or star-shaped cleft in the center of the starch grain; no striation is observable in maize starch grains.

Slide. Starch grains of garden pea (Pisum sativum (L.) Cov.)

Pea seeds are soaked, and once swollen, the seed coat is peeled off with a scalpel to separate the cotyledons. A small amount of white mass is scraped from the inner side of the cotyledons using a scalpel and thoroughly mixed in a drop of water. It is then covered with a coverslip and examined first under low and then under high magnification. Pea starch grains are quite large.

Fig. 15. Pea starch grains: small ones are underdeveloped, large ones are typical, well-developed starch grains

In the center of a large grain, there is an oblong, multi-rayed star-shaped cleft, surrounded by concentric layers (Fig. 15).

In addition to the large grains, smaller starch grains will also be visible on the slide, sometimes appearing as tiny dots. Neither striations nor central clefts are detected in such starch grains.

To confirm that these are indeed starch grains, the slide must be treated with an iodine-potassium iodide solution. Both the large and the tiniest grains stain blue upon this Treatment.

Slide. Detection of sugars in the pulp cells of watermelon (Citrullus vulgaris Schrad.)

One or two drops of fresh juice from a ripe watermelon are placed on a glass slide, combined with mashed pulp from the ripe watermelon. Next, Fehling's solution is added using a pipette or glass rod, covered with a coverslip, and gently heated over a spirit lamp almost to a boil. At this Temperature, the blue color of Fehling's solution disappears, replaced by a red color caused by the formation of a red copper(I) oxide precipitate. The formation of copper(I) oxide is due to The oxidation of soluble sugars, such as glucose and fructose, when heated with Fehling's solution. This causes a reduction reaction that yields a precipitate of copper(I) oxide. This reaction is used to detect the presence of soluble sugars such as fructose and glucose in plant cells. This precipitate is worth examining under a microscope.

Slide. Detection of sugars in the fruit cells of domestic apple (Malus domestica Borkh.) or common pear (Pyrus communis L.)

Ultra-thin sections are cut from the pulp of an apple or pear fruit and placed on a glass slide in a drop of water, which is changed several times to wash away the cell sap. The preparations are then treated with Fehling's solution, covered with a coverslip, and heated. As a result of the reduction reaction, a copper(I) oxide precipitate forms within the cells, which can be noticed even with the naked eye and examined even better under a microscope.

Slide. Inulin sphaerocrystals in the cells of dahlia root tubers (Dahlia sp.)

A small piece is cut from an alcohol-preserved dahlia tuber, from which several thin longitudinal slices are prepared and carefully placed in a drop of water on a microscope slide, then covered with a coverslip. Examining the preparation under low and subsequently high magnification reveals large, thin-walled cells containing spherical clusters of needle-like crystals radiating from the cell angles—inulin spherulites. The spherulites are located within a single cell, or a single crystal may span two adjacent cells (Fig. 16).

Fig. 16. Inulin spherulites precipitated with alcohol in the cells of a dahlia root tuber

Spherulites in The process of formation (incomplete spheres) can also be observed in the preparation. By adjusting the fine-adjustment screw and closely inspecting the preparation, one can see that the spherulites consist of tiny needle-like crystals arranged radially relative to their longitudinal axis.

Note. If individual crystals are packed very tightly against

each other, they become difficult to distinguish. In such cases, it is necessary to add a few drops of nitric acid to the preparation, which makes the crystals much more distinct. Heating the preparation causes the spherulites to break down.

After examining the preparation and the STRUCTURE OF THE spherulites, be sure to make a drawing of what you have observed.

Note. To prepare inulin slides, other plants besides dahlia tubers can be used, such as roots of kok-saghyz, Jerusalem artichoke tubers, dandelion roots, chicory, etc. These plant materials must be pre-soaked in 95% ethanol to crystallize the inulin.

Slide. Simple aleurone grains in the seeds of garden pea (Pisum sativum (L.) Cov.)

Fig. 17. Storage nutrients of a pea seed:

1 - seed coat, 2 - starch grains, 3 - aleurone grains, 4 - Cell wall showing pores

Pea seeds are soaked in water. Once swollen, the seed coat is removed, and the seeds are cut across the cotyledons. Several thin slices are prepared and placed separately on a microscope slide in a drop of water or glycerin. The prepared specimen is covered with a coverslip and examined first under low and then under high magnification. Pea cells have a rounded-polygonal shape, with intercellular spaces filled with air located between the cell angles, making them appear dark (Fig. 17).

The cell wall is quite thick, yet it features unthickened regions—pores. Large starch grains are clearly visible inside the cell, interspersed with numerous tiny granules. Upon treatment with an iodine-potassium iodide solution, the starch grains stain blue, while the cytoplasm and tiny granules stain yellow. These small granules are aleurone grains. Proteins are invariably stained yellow by iodine.

Slide. Aleurone layer in the grain of oat (Avena sativa L.) and wheat (Triticum aestivum L.)

Fig. 18. Aleurone layer in an oat grain:

1 - Cells of the aleurone layer filled with small aleurone grains, 2 - cells containing starch grains

To prepare the slide, the husk should be removed from an oat or wheat grain, the narrower tip of the grain cut off to level the cutting surface, and several thin cross-sections made to include the peripheral layer. The resulting sections are placed on a microscope slide in a drop of glycerin and covered with a coverslip. Under low magnification, scan all sections to select the thinnest section and the thinnest spot within it. Usually, the best area is found at the edge of the section, so it should be centered in the field of view for subsequent high-magnification observation. Microscopic examination reveals that the peripheral part of the section consists of a layer of flattened cells. These are the cells of the seed coat, beneath which lies the aleurone layer composed of large, thin-walled rectangular cells (Fig. 18).

In some places, this layer consists of two rows of cells positioned one below the other. These cells are densely packed with tiny granules—aleurone grains, and the entire layer of cells is referred to as the aleurone layer. A clearly defined nucleus can very often be observed within the cells of the aleurone layer.

Large, elongated cells filled with starch grains lie beneath the aleurone layer. To make the preparation clearer, a drop of iodine is added. Iodine does not mix well with glycerin; therefore, to apply it, one must lift the coverslip, blot away the glycerin, add a drop of iodine, and then lower the coverslip again. Upon iodine treatment, the aleurone grains in the cells of the aleurone layer stain yellow, while the starch grains stain blue.

The preparation should be carefully examined and sketched, indicating its main structural components.

Slide. Complex aleurone grains in castor oil plant seeds (Ricinus communis L.)

Note. Prior to slide preparation, castor seeds must be kept in 95% ethanol or an alcohol-ether mixture for several days to remove the oil, which is abundant in castor seeds. This is necessary because the oil would interfere with the anatomical examination of the complex aleurone grains.

A small piece is cut lengthwise from the seed, from which several thin slices are subsequently prepared. A portion of the slices is first placed on a microscope slide in alcohol, which must be replaced several times to remove residual oil from the section. Then, after blotting away the alcohol with filter paper, 1-2 drops of iodine-potassium iodide are added, and the slide is covered with a coverslip.

Fig. 19. Complex aleurone grains in castor bean seeds:

1 - cell wall, 2 - intercellular spaces, 3 - cytoplasm, 4 - aleurone grains, 5 - isolated complex aleurone grains showing: g - globoid, k - crystalloid

Examining the slide (Fig. 19) first under low and then under high magnification reveals that the section tissues consist of parenchymatous cells with thin, colorless walls. The cells are loosely connected, with small triangular intercellular spaces between them. When observing the slide, one should find the thinnest section so that the cells are visible as clearly as possible. Such cells exhibit a finely granular cytoplasm stained yellow by iodine. Immersed in the cytoplasm are fairly large, round or oval aleurone grains, which also stain yellow. This indicates their proteinaceous nature. In each aleurone grain, one can distinguish a thin wall, a rounded body known as a globoid, and a crystal known as a crystalloid.

The structure of globoids can be observed particularly well in isolated aleurone grains that have fallen out of the cell. The crystalloid consists of protein material, while the globoid is composed of phytin. Sometimes an aleurone grain may contain not just one, but several globoids.

After careful examination of the slide, a drawing of it should be made.

The formation of crystalloids can be traced during the GROWTH AND DEVELOPMENT of seeds. As they develop, seeds gradually lose water and become drier; subsequently, the formation of small vacuoles containing reserve Proteins can be observed in the cytoplasm of the seed cells. As the seeds ripen, the protein concentration within the vacuoles increases significantly, leading to the formation of crystalloids.

Slide. Lipids in castor bean seeds (Ricinus communis L.)

Thin sections are cut from castor bean seeds and placed on a glass slide, to which one or two drops of a 1% osmic acid solution are applied; under its influence, the oil droplets within the cells turn black. The presence of lipids can be detected using an alcoholic solution of Sudan III, which stains the oil droplets red.

After examining the slide, make a drawing of it.

Slide. Crystals and druses in the petiole cells of begonia leaves (Begonia sp.)

Fig. 20. Calcium oxalate crystals and druses in the petiole cells of a begonia leaf:

1 - cell wall, 2 - cytoplasm, 3 - druses

A superficial longitudinal layer is cut from a piece of a begonia leaf petiole, and several thin longitudinal sections are prepared from deeper tissue layers. These prepared sections are placed in a drop of water on a microscope slide, covered with a coverslip, and examined first under low and then under high magnification of the microscope.

As seen under the microscope (Fig. 20), the petiole tissues containing the crystals consist of large parenchymatous cells with thin, colorless walls. A thin parietal layer of cytoplasm lies beneath the cell wall, in which chloroplasts are clearly visible in fresh material. The central part of the cell is filled with cell sap and single or fused crystals of calcium oxalate, known as druses.

Calcium oxalate crystals are formed directly in the cytoplasm before entering the vacuoles. In the cells of young Tradescantia leaves, crystals are present in the central vacuole in a mobile state. Careful observation under the microscope reveals that the crystal is surrounded by a cytoplasmic sheath, which stains brownish-yellow with iodine. The shape of calcium oxalate crystals can be extremely diverse. One can often observe the onset of druse formation, which occurs as new microcrystals deposit onto the walls of existing crystals until a cluster or aggregate—a druse—is formed.

If a few drops of hydrochloric or sulfuric acid are added to the slide, the calcium oxalate crystals dissolve without the release of gas bubbles.

After examining the slide, a drawing of it should be made.

Slide. Raphides in the petiole cells of hybrid fuchsia leaves (Fuchsia hybrida L.)

A longitudinal section is cut from the tissues located closer to the center of a fuchsia leaf petiole, placed on a microscope slide in a drop of water, and covered with a coverslip.

When examining the slide under low magnification, one should select the thinnest area and then switch the microscope to high magnification. The thin preparation reveals that many needle-shaped crystals—raphides—are clustered within certain cells. Some raphides may lie outside the section because they have fallen out of the cut cells.

Externally, raphides resemble needle-shaped crystals pointed at both ends, gathered into bundles or packets. Initially, each raphide is surrounded by a thin cytoplasmic sheath connected to the parietal layer of cytoplasm. Later, the individual raphide sheaths fuse into a single common cytoplasmic sheath enclosing the entire bundle, and still later, the cytoplasm secretes mucus that surrounds the entire aggregate of raphides.

After examining the slide, a drawing of it should be made.

Slide. Raphides in the rhizome of Solomon's seal (Polygonatum odoratum Miller)

Fig. 21. Calcium oxalate raphides in the cells of Solomon's seal rhizome:

1 - cytoplasm, 2 - nucleus, 3 - bundle of raphides, 4 - individual raphide

Both fresh and preserved (alcohol-fixed) material of Solomon's seal rhizome can be used for slide preparation. Cut a small piece from the rhizome and make several longitudinal sections. Select the thinnest sections for examination, place them side by side in a drop of water on a microscope slide, cover with a coverslip, and observe under low magnification. Under the microscope, colorless, thin-walled cells of varying sizes are visible. Bundles of raphides can be seen within the larger cells. Next, center a cell containing a raphide bundle in the field of view and switch the microscope to high magnification.

Under high magnification (Fig. 21), a layer of cytoplasm can be seen lining the inner cell wall of smaller cells. If preserved material was used, plasmolysis will have occurred, causing the cytoplasm to pull away from the wall and accumulate in the cell lumen. A distinct round nucleus with a nucleolus is visible within the cytoplasm. The larger cells are almost entirely filled with colorless cell sap containing a dense bundle of raphides aligned along the cell axis. By adjusting the fine focus knob to change the distance between the specimen and the objective, the raphide bundle can be examined in detail.

During sectioning, some bundles may break apart, revealing individual raphide needles on the slide.

Raphides vary in size. They can be significantly shorter than the cells that house them, and occasionally even exceed the length of a single cell.

Raphides are composed of calcium oxalate and dissolve in acids. They are most commonly found in monocotyledonous plants.

Note. Raphides are characteristic of many plants and can be observed in duckweed, asparagus, fireweed, as well as in the leaves of tradescantia, aloe, and numerous other species. Many plants also contain druses. These can be observed in mounts prepared from common goosefoot, linden bast, purslane, sorrel leaf petioles, and many other plants.

Slide. Calcium oxalate crystals in the cells of dry onion scales (Allium cepa L.)

Before preparing the slide, soak the dry onion scales in a 15% aqueous solution of glycerin for at least 24 hours to remove trapped air, which is abundant in dead cells. Air can also be removed by placing a piece of scale in pure glycerin (either in a test tube or directly on a microscope slide). Then, cover the preparation with a coverslip and gently heat for 15 minutes. First, examine the slide under low magnification. When viewed under high magnification, solitary or aggregated crystals are easily distinguished within the cells (Fig. 22). These consist of calcium oxalate trihydrate. Similar crystals can also be observed in the withered outer Skin of garlic.

Once the slide has been examined, make a botanical drawing of it.

Fig. 22. Calcium oxalate crystals in dry onion scales:

1 - cell wall, 2 - calcium oxalate crystals, 3 - crystal aggregates

Review Questions

1. What are the main structural parts of a living differentiated plant cell?

2. What plant cell types are distinguished based on their shape?

3. Describe the Structural Features of a plant cell.

4. What is the COMPOSITION OF THE protoplast?

5. Which cells are referred to as parenchymatous, and which as prosenchymatous?

6. What happens to a cell when it dies?

7. Name the substances that make up the cell wall.

8. What is the intercellular substance, and what is its composition?

9. How does the cell wall undergo chemical modification during ontogeny?

10. What are pores?

11. Which Organelles determine the coloration of plants?

12. Describe the structure and shape of chloroplasts, and where are they most commonly located?

13. What are the differences between various types of plastids?

14. The fruits of many plants turn red as they ripen. What causes this?

15. What evidence indicates the evolutionary relationship among Different types of plastids?

16. From which plastids can chromoplasts be formed?

17. What determines the coloration of different types of plastids?

18. What shape do chromoplasts have, and what does this depend on?

19. What factors influence The rate of cytoplasmic streaming?

20. What types of cytoplasmic movement do you know?

21. How can cytoplasmic streaming be stopped in laboratory conditions?

22. What are inclusions?

23. Name the single- and double-membrane organelles of a plant cell.

24. What are the Functions of vacuoles in a plant cell?

25. What determines the color of cell sap?

26. What is the composition and function of the cell wall?

27. Name the secondary chemical modifications of the cell wall.

28. How does the primary cell wall differ from the secondary one?

29. What is tissue maceration, and under what conditions does it occur?

30. Why are reserve nutrients classified as inclusions?