Plant Anatomy - 2013

Chapter 2. MODULE 1. PLANT CELL AND ITS METABOLIC PRODUCTS

Objects of Study: inner epidermis of a succulent onion scale, leaves of Canadian waterweed (Elodea canadensis), rosehips, wandering Jew (Tradescantia) leaves, petioles of garden sorrel (Rumex acetosa), belladonna, bergenia, potato tubers, corn and rice grains, pea seeds, outer scale of onion, aloe leaves, horse sorrel stem.

Equipment and supplies: microscopes, magnifying glasses, Microscope slides and coverslips, razors, dissecting needles, Petri dishes, forceps, dropper bottles, distilled Water, methylene blue solution, chloral hydrate solution, Lugol's solution, 5% hydrochloric and acetic acid solutions, filter paper, litmus paper, test papers, permanent microscope slides, charts: "Microscope", "Plant Cell", "Types of Plastids in Plant Cells",

"Starch and Aleurone Grains"; "Crystalline Inclusions in Plant Cells".

Task 1.

To study The Structure of the Light Microscope.

Examine and study the optical PARTS OF THE microscope (objective lenses and eyepieces), the illumination system (mirror, condenser, light filters), and the mechanical components (stand, stage, body tube, revolving nosepiece, coarse and fine adjustment knobs). Pay attention to the functional purpose of each part of the microscope.

Describe the STRUCTURE OF THE light microscope in your laboratory notebook.

Task 2.

To master the techniques of working with a microscope.

Place the microscope closer to your left shoulder. Lay out your laboratory notebook to the right of the microscope. Lock the low-power objective lens over the aperture of the stage, on which the specimen to be examined is placed. Looking through the eyepiece, use the mirror to illuminate the field of view of the microscope. Lower the body tube using the coarse adjustment knob until it is 0.3 cm away from the stage with the specimen. Turn the knob toward yourself until the image becomes visible in the microscope. Focus the image sharply using the fine adjustment knob, and examine the object first under low magnification and then under high magnification. Switching objectives is performed by rotating the nosepiece clockwise. Before doing so, center the point of interest in the field of view of the microscope; otherwise, the area under study may fall out of the field of view at high magnification.

Describe the rules for working with a microscope in your laboratory notebook.

Task 3.

To prepare a temporary mount of the inner epidermis of a succulent onion scale.

Peel off the epidermis from the inner side of a succulent onion scale and place it into a drop of methylene blue solution applied to a microscope slide. Spread the epidermal tissue out with a needle and lower a coverslip onto it at an angle. Press down slightly. Blot the excess moisture around the coverslip with filter paper.

Task 4.

To study the structure of a plant cell.

Examine the prepared temporary mount of the onion scale epidermis in methylene blue under low magnification of the microscope. Among the many cells, select one with a distinct Nucleus and vacuoles, and move it to the center of the microscope's field of view. Rotate the nosepiece to lock the high-power objective lens over the specimen. Examine the Cell Structure under high magnification. Pay attention to the prosenchymatous shape of The Cell and its straight walls. Locate The Nucleus with its nucleolus, Cytoplasm, and vacuoles. Stained with methylene blue, the nucleus appears blue, the cytoplasm light blue, while the cell walls and vacuoles remain colorless.

Draw the cell with its Organelles. Color them accordingly, label and annotate the drawing. Describe the results of the completed task in the form of Conclusions.

Task 5.

To prepare a surface mount of a leaf, determine the type of plastids, and observe cytoplasmic streaming in cells.

Take an Elodea leaf from the Petri dish and place it on a microscope slide in a drop of water. Cover with a coverslip and examine under low magnification. Numerous green plastids—METABOLISM/14.html">Chloroplasts—are visible within the rectangular cells. Study the shape of the chloroplasts. Small granules within the chloroplasts represent primary starch. Locate a cell in which chloroplasts are in motion, and examine it under high magnification. Focus your attention on a single chloroplast and use its movement to determine the direction of cytoplasmic streaming. If the cell contains one large vacuole, the cytoplasm streams around the periphery along the cell walls (rotational streaming). If the cell contains several small vacuoles, such cytoplasmic strands noticeably converge near the nucleus, allowing rotational-circulatory or streaming movement to be observed. Note that cytoplasmic streaming may occur in different directions in different cells (either clockwise or counterclockwise).

Sketch several Elodea leaf cells containing chloroplasts. Indicate the direction of cytoplasmic streaming in the cells using arrows. Label and annotate the drawing. Draw conclusions from the conducted research.

Task 6.

Prepare a temporary mount of fruit pulp and determine the type of plastids.

Slice the Skin of a rose hip and use a needle to gently scrape a small amount of pulp from underneath. Place it in a drop of water on a microscope slide, mix well, and cover with a coverslip. Using low magnification, locate individual, freely distributed cells, then switch to high magnification to examine their shape and chloroplasts. Pay attention to the coloration and Morphology of the chloroplasts.

Draw several cells containing chloroplasts. Label and caption your drawings. Draw conclusions based on the conducted observations.

Task 7.

Prepare a microscopic mount of the leaf epidermis. Identify and study the type of plastids within the cells.

Using a needle, peel off the epidermis from the lower surface of a Tradescantia leaf and place it in a drop of water on a microscope slide. Flatten the tissue with the needle,

cover with a coverslip, and examine under low magnification. Locate the cells with a nucleus surrounded by tiny, colorless spherical bodies—leucoplasts. Examine the leucoplasts under high magnification.

Draw a cell showing the nucleus and leucoplasts. Label and caption the drawing. Draw conclusions from the study.

Task 8.

Determine the pH of plant cell sap.

Cut the petiole of a garden sorrel (Rumex acetosa) and press litmus paper against the fresh cut. Within a minute, a pink spot will appear on the paper, indicating the acidic pH of the cell sap.

Sketch the litmus paper with the pink spot and write the appropriate conclusions.

Task 9.

Determine the presence of Alkaloids in plant cell sap.

Using a razor blade, cut the leaf petiole of a deadly nightshade and apply a reagent test paper for alkaloids to the fresh cut. A light spot with a dark rim will form on the paper, indicating the presence of alkaloids in the cell sap. Sketch the test paper with the spot and write a Conclusion based on the study.

Task 10.

Determine the presence of Tannins in plant cell sap.

Cut the leaf petiole of a bergenia with a razor blade and press a tannin test paper against the fresh cut. A dark purple spot will form on the paper, indicating the presence of tannins in the cell sap.

Sketch the test paper with the spot. Draw a conclusion from the conducted study.

Task 11.

Determine the shape and structure of starch grains in various plants.

Prepare a temporary mount from the parenchyma of a potato tuber. To do this, scrape a small amount of tuber pulp into a drop of water on a microscope slide, mix with a needle, and cover with a coverslip. In a second drop of water, place a small amount of corn endosperm from a sliced grain, mix thoroughly in the water, and cover with a coverslip. Prepare a third mount in Lugol's iodine solution, adding a small amount of rice grain endosperm into the drop. Examine the structure of starch grains in all prepared mounts, first under low and then under high magnification. Pay attention to the size of the starch grains (the largest are in potatoes, the smallest in rice). In potato starch grains, locate the hilum or center of stratification (at the narrower end of the grain), and in corn starch grains, find the central star-shaped (or cross-like) cleft. Note that the rice starch grains stain blue when treated with Lugol's solution.

Draw potato starch grains: simple, compound, and semi-compound; several corn starch grains with clefts; and simple and compound rice starch grains. Label and caption the drawings. Describe the analysis results along with appropriate conclusions.

Task 12.

Examine the microstructure of simple aleurone grains.

Using a razor blade, make a thin transverse section of a pea cotyledon, place it in a drop of Lugol's solution on a microscope slide, straighten it out with a needle, cover with a coverslip, and examine under low and high magnification of the microscope. Locate starch grains (large) and aleurone grains (very small) within the seed cells. Note that Lugol's solution stains starch grains dark blue, while protein grains turn golden-yellow.

Under high magnification, sketch several pea seed cells containing yellow aleurone grains, against the Background of which large, oval, blue starch grains are located. Label and annotate the drawing, and describe the research findings.

Task 13.

Examine crystalline inclusions in the Cells of the covering tissue of a monocotyledonous plant.

Place the outer brown scale of an onion onto a microscope slide in a drop of chloral hydrate solution, straighten it out with a needle, and cover with a coverslip. Warm over a flame to remove air bubbles and improve visibility (bring to a boil, but do not boil). Examine the cleared micropreparation under low and high magnification of the microscope. Locate individual prismatic crystals within the cells.

Sketch a few cells containing individual crystals and describe the results of the study.

Task 14.

Examine crystalline inclusions in the leaf mesophyll cells of a monocotyledonous plant.

Make a transverse section of an aloe leaf, discard the outer green portion, and place the white transparent part into a drop of chloral hydrate, then cover with a coverslip. Clear over a flame and examine under low and high magnification of the microscope. Pay attention to the large parenchyma-like cells. Locate individual needle-like crystals (raphides) and their parallel clusters (so-called "bundles") within the cells.

Sketch several cells with raphides, label, and annotate the drawing. Describe the results of the microscopic analysis along with appropriate conclusions.

Task 15.

Examine crystalline inclusions in the stem cells of a dicotyledonous plant.

Prepare several transverse sections of a horse sorrel stem. Select the thinnest one and place it on a microscope slide in a drop of chloral hydrate. Cover with a coverslip and clear over a flame. Examine under low and high magnification of the microscope. Locate cells containing druses—star-shaped crystals. Note that there is always only a single druse per cell.

Sketch parenchyma-like cells with individual druses. In the drawing, depict the loose arrangement of parenchyma-like cells, interspersed with numerous

intercellular air spaces. Label and annotate the drawing. Draw conclusions regarding the obtained research results.

Task 16.

Identify crystalline inclusions in the cells of the petiole (or stem) of a dicotyledonous plant.

Make several transverse sections of a belladonna leaf petiole or its stem. Place the thinnest section into a drop of chloral hydrate applied to a microscope slide. Cover with a coverslip and clear over a flame until air is completely removed. Examine the section under low and then high magnification of the microscope. Locate cells containing crystal sand. Examine individual grains of sand and note their pointed shape. Pay special attention to dark cells: they may appear packed with crystal sand.

Sketch several loosely arranged parenchymal cells containing crystal sand. Describe the results of the microscopic examinations.

Task 17.

Perform a microchemical reaction to identify a crystalline inclusion.

On a single microscope slide, prepare two identical micropreparations of an onion scale in water. Clear over a flame, examine under low magnification of the microscope to ensure that cells in both preparations contain crystals. Afterward, draw off the water using filter paper and apply a 5% acetic acid solution to one micropreparation, and a 5% Hydrochloric acid solution to the other. After 2–3 minutes, examine both micropreparations under low magnification of the microscope and verify that the crystals did not dissolve in acetic acid. In hydrochloric acid, the crystals undergo gradual dissolution without gas evolution, which distinguishes calcium oxalate from limestone.

In your conclusions, describe the results of the microchemical studies and provide the reaction equations:

1. СаС₂0₄ + СН₃СООН - no reaction takes place.

2. СаС20₄ + 2НС1 = СаСІ2 + Н2С2О4

3. СаСОЗ + 2НС1 = СаСІ2 + Н2О + СО2

Cell Sap

(experiment with anthocyanin)

The pigment anthocyanin is very common in plants. It changes its color depending on the pH reaction of the cell sap (ranging from blue to bright red). This color change can be induced using acetic acid and an ammonia solution.

Equal amounts of red cabbage decoction are poured into two test tubes placed side by side in a test tube rack. The red cabbage decoction has a purple color due to the presence of anthocyanin. A drop of highly diluted acetic acid is added to the first test tube using a Glass rod, and the mixture is shaken. The cabbage decoction turns red. A drop of highly diluted

ammonia solution is added to the second test tube. The decoction remains blue. If too much ammonia solution is added, the anthocyanin is destroyed, and the cabbage decoction turns green.

Another memorable experiment involving anthocyanin is as follows. Red primrose flowers are placed under a glass bell jar containing ammonia vapors. Before your eyes, the red color changes to blue-green, then to green, and finally becomes greenish-yellow.

Inulin in Jerusalem Artichoke Tuber Cells (Helianthus tuberosum)

The polysaccharide inulin (CöHioOsX exists in a dissolved state within the cell sap of storage Organs in plants of the Asteraceae family (such as Jerusalem artichoke, chicory, dahlia, elecampane, etc.). To detect inulin, 96% ethyl alcohol is used, which rapidly dehydrates the cells. In this process, inulin forms complex spherical crystals (spherulites/spherocrystals) composed of numerous needle-like crystals. These inulin spherocrystals rapidly expand, encompassing several cells, and can be clearly observed using the micrometric fine-adjustment screw.

Procedure

Sections of the Jerusalem artichoke are prepared, and the thinnest section is placed on a glass slide in a drop of glycerin, then covered with a coverslip. Inulin is insoluble in glycerin, but readily soluble in water.

Therefore, water should not be used for slide preparation.

Leucoplasts in Tradescantia Leaf Epidermis Cells (Tradescantia zebrina)

To prepare the slide, a leaf is plucked from a Tradescantia SHOOT and wrapped around the index finger of the left hand so that the lower (purple-colored) epidermis faces outward. Using a needle in the right hand, the epidermis is torn over or near a vein, and a small piece is peeled off with forceps. Although a portion of the leaf mesophyll may be captured in the process, a region consisting solely of epidermal cells can always be found at the edge of the section. The peeled fragment is placed exterior side up in a drop of water on a microscope slide and carefully covered with a coverslip.

Under low magnification, elongated hexagonal cells are visible, either colorless or tinted pale purple due to the presence of the anthocyanin pigment. By moving the slide, cells with a clearly visible nucleus can be located. Under high magnification, it is easy to see that the cell nuclei are surrounded by small, colorless globules—leucoplasts. The cell protoplasm is barely noticeable, appearing as a continuous granular layer and granular strands extending from the nucleus to the cell walls.

If the epidermis is peeled from areas other than the midrib of the leaf, the cells will be less elongated, and Stomata will be present among them. Each stoma is formed by two guard cells enclosing a stomatal pore for gas exchange and Transpiration. Chloroplasts and slightly granular, colorless nuclei are visible within the guard cells. Adjacent to the guard cells are two subsidiary cells, in which leucoplasts are also discernible.

2.1. Qualitative Tests for Cell Inclusions

• Suberin Staining Reaction with Potassium Hydroxide Reagents: 33% aqueous KOH solution

Performing the Test

1. Place the section in a drop of the reagent on a microscope slide and cover with a coverslip.

2. Gently heat the slide.

3. Observe the coloration of the cell walls.

Results:

Suberized cell walls stain yellow.

• Histochemical Tests for Mucilage Detection of mucilage using India ink solution

1. Place the section in an India ink solution.

2. Observe shiny mucous aggregates against a dark background.

Detection of Mucilage via Swelling in Water

Mucilage swells in water but does not swell in alcohol; mucilage contained in alcoholic material retains its swelling capacity when transferred to water.

1. Place the section in water.

2. Cover it with a coverslip and apply a drop of water to one side of the coverslip.

3. Draw out the alcohol from the opposite side of the coverslip using filter paper, replacing it with water.

4. Observe the swelling of the mucilage in water.

5. Replace the water with alcohol. Observe the reverse process upon dehydration.

Reaction with Copper Sulfate and Alkali Reagents:

1. Concentrated copper sulfate solution.

2. 50% potassium hydroxide solution.

Procedure:

1. Place the section in the copper sulfate solution for 5-10 min.

2. Rinse the section with water.

3. Transfer the section into a drop of potassium hydroxide and cover with a coverslip.

4. Observe the appearance of coloration.

Mucilage stains blue (Mallow family - Malvaceae) or green (Lily family - Liliaceae).

• Histochemical Tests for Fats

In cells, fats typically occur as droplets of various sizes, where smaller droplets coalesce to form larger ones. Fats can be detected using specific stains as well as through saponification reactions.

Saponification Reaction

Based on the property of fats to hydrolyze under the action of alkalis, yielding Fatty acids (soaps) and glycerol.

Reagents:

1. Concentrated KOH solution.

2. 20% ammonia solution.

Performing the reaction:

1. Place a drop of KOH on a microscope slide and add a drop of 20% ammonia solution.

2. Place the section into the reagent and cover with a coverslip.

3. To prevent the slide from drying out, seal the edges of the coverslip with paraffin.

4. Leave the prepared slide for 1-5 days.

Reaction results:

After 1-5 days, needle-like crystals (fatty acid salts) precipitate around the oil droplets.

Sudan III Staining Reaction

The Sudan III staining is based on the dissolution of the dye in oil. The staining is non-specific. Essential Oils, cutin, suberin, Waxes, and resins are simultaneously stained orange. Liquid oils are stained within 5-10 minutes, whereas solid fats and fatty acid crystals stain slowly.

Reagents:

Sudan III solutions of various concentrations:

1. Solution: dissolve 0.01 g of Sudan III in 5 ml of 96% alcohol, then add 5 ml of glycerin.

2. Solution: dissolve 0.1 g of Sudan III in 20 ml of 70-90% alcohol.

Performing the reaction:

1. Place the section in one of the reagents for 10-20 minutes.

2. Transfer the section into glycerin.

3. Observe the stained fat droplets (pinkish-orange).

• Starch Reaction Reagents:

1. Lugol's solution (iodine-potassium iodide solution according to Gram)

Preparation of the solution: dissolve 2.0 g of potassium iodide in 5 ml of distilled water, add 1.0 g of crystalline iodine. Bring the solution volume to 300 ml with water. Store the solution in a dark container.

2. Iodine solution in chloral hydrate according to Meyer.

Preparation of the solution: dissolve 5.0 g of chloral hydrate in 2 ml of water and obtain a saturated solution by dissolving metallic iodine in it.

Performing the reaction with Lugol's reagent.

1. Place the section in a drop of the reagent on a microscope slide and cover with a coverslip.

2. Observe The Development of coloration.

Results:

Starch grains of various sizes are detected in the leaf parenchyma cells. Starch is particularly abundant in the bundle sheath parenchyma. It is absent in the cells of the phloem, xylem, and bundle parenchyma. The endosperm of corn grains stains a deep purple, almost black, and its cells are packed with large starch grains.

• Histochemical Reactions for Inulin

Upon heating with acids, inulin is converted into the sugar levulose, which enables its reaction with α-naphthol or thymol.

Reaction of Inulin Precipitation with Alcohol

Under the action of alcohol, inulin precipitates in the form of spherocrystals, which frequently exhibit a layered structure. Inulin crystals readily dissolve in hot water.

Reagents:

96 % alcohol.

Procedure:

1. Place a section (of a dahlia tuber or another specimen) in alcohol.

2. Observe the precipitation under a microscope.

3. Transfer the section to water and heat it.

4. Observe the dissolution of the precipitate.

• Histochemical Reactions for Pure Cellulose: Reaction with Chlor-Zinc-Iodine

Reagents: chlor-zinc-iodine solution.

Preparation of the chlor-zinc-iodine solution according to Novopokrovsky: dissolve 20.0 g of zinc chloride in 8.5 mL of water. Dissolve 1.5 g of crystalline iodine and 3.0 g of potassium iodide in 60 mL of water. Add the latter solution dropwise to the former while shaking the vessel until a precipitate appears. Store the reagent in a dark container.

Procedure:

1. Place the section in a drop of water.

2. Remove the water with filter paper.

3. Add the chlor-zinc-iodine solution to the section and cover with a coverslip.

4. Observe the development of coloration.

Results:

Cell walls composed of cellulose stain purple.

Reactions with Iodine and Sulfuric Acid

Sulfuric acid converts cellulose into amyloid, which is structurally similar to starch. Amyloid stains blue in the presence of iodine.

Reagents:

1. Lugol's solution.

2. 33% sulfuric acid.

Procedure:

1. Place the section in a drop of Lugol's solution.

2. Transfer the section into a drop of dilute sulfuric acid solution and cover with a coverslip.

Results:

Cell walls containing cellulose stain blue.

• Histochemical reactions for lignified cellulose

Lignified cellulose, i.e., cellulose impregnated with Lignin, stains well.

Phloroglucinol-hydrochloric acid test. Procedure:

1. Place the section in a drop of phloroglucinol.

2. Blot away the reagent using filter paper.

3. Add 1-2 drops of acid and cover with a coverslip.

4. After a cherry-red color develops, transfer the section into glycerin (to prevent structural damage by the acid).

Results:

Lignified cell walls turn cherry-red. The intensity of the staining depends on the degree of lignification.

Aniline sulfate (or aniline hydrochloride) test. Reagent preparation:

1. Dissolve 1.0 of dry aniline sulfate in a mixture consisting of 70 ml of purified water, 30 ml of 96% alcohol, and 3 ml of concentrated sulfuric acid.

2. Dissolve 2.0 of aniline sulfate in a mixture consisting of 4 ml of acetic acid and 194 ml of 50% alcohol.

Procedure:

1. Place the section on a microscope slide in a drop of the reagent and cover with a coverslip.

2. Observe the development of color.

Results:

Lignified cell walls turn lemon-yellow.

Reaction with potassium permanganate Reagents:

1. 1% potassium permanganate solution.

2. 10% hydrochloric acid solution.

3. Saturated ammonia solution.

Procedure:

1. Place the section in the potassium permanganate solution on a watch glass for 5 minutes.

2. Rinse the section in water.

3. Rinse the section in 10% hydrochloric acid solution for 2 minutes.

4. Transfer the section to a microscope slide in a drop of ammonia solution and cover with a coverslip.

5. Observe the development of coloration.

Results:

Lignified cell walls stain red.

• Histochemical reactions for suberized and cutinized cell walls

Suberized (i.e., impregnated with suberin) and cutinized (i.e., impregnated with cutin) cell walls do not give reactions characteristic of pure cellulose. Cutin and suberin are well detected by lipid reagents.

Reaction with Sudan III-IV

1. Place the section in the reagent for 10–60 minutes.

2. Transfer the stained section into glycerol and cover with a coverslip.

3. Observe the development of coloration.

Results:

Suberized and cutinized cell walls stain a pinkish-yellow

color.

Reaction with chlorophyll

Alcoholic chlorophyll extract.

Procedure:

1. Place the sections in the reagent for 25 minutes in a dark place.

2. Transfer the sections into a drop of water on a microscope slide and cover with a coverslip.

3. Observe the development of coloration.

Reaction Results:

The cuticle and suberized cell walls stain dark green. This coloration is transient.

• Histochemical Tests for Proteins

Proteins are classified into simple and conjugated. There are virtually no tests specific exclusively to simple or Conjugated Proteins. Some reactions are based on General Properties shared by both simple and conjugated proteins. These include the biuret test and the bromophenol blue test.

Alongside these, specific tests exist for basic proteins and certain conjugated proteins (such as Nucleoproteins).

Biuret Test

The biuret test is used to detect peptide bonds. The peptide bond is the most characteristic feature of proteins, making the biuret test highly specific in this regard. While other bonds may occur in proteins, such as disulfide, ester, ionic, or Hydrogen Bonds, the biuret test is not used for their detection.

The biuret test is based on the principle that in an alkaline environment (in the presence of sodium hydroxide), upon The addition of copper salts (copper sulfate), Polypeptides and proteins form colored complex compounds with copper ions.

The reaction requires at least two peptide bonds to proceed; dipeptides do not react. Polypeptides and low-molecular-weight proteins yield a pink or reddish color, whereas other proteins turn purple.

The biuret reaction derives its name from biuret, a urea derivative that also yields this positive test.

Although individual Amino Acids do not give this reaction, it exceptionally occurs in the presence of asparagine and Histidine.

A drawback of the biuret test is its low sensitivity. The color is particularly pronounced only where proteins are abundant (e.g., in meristematic cells).

Performing the biuret test on fresh material may yield a faint coloration due to poor tissue permeability. Fixed material generally provides better results; however, it should be noted that fixation with alcohol is unsuitable for alcohol-soluble proteins (such as cereal grain proteins).

Reagents:

1. 7% copper sulfate solution.

2. 30–50% sodium (or potassium) hydroxide solution.

Procedure:

1. Place the section in the copper sulfate solution for 5–30 minutes in a watch glass.

2. Thoroughly remove the solution with filter paper and wash the section with water until the blue color is no longer rinsed out.

3. Transfer the section to a microscope slide and treat with sodium (or potassium) hydroxide solution for 10–20 minutes up to 1 hour.

4. Observe the appearance of a purple coloration (sometimes pink in the presence of low-molecular-weight proteins or polypeptides).