Exercise 2 THE PLANT CELL

In beginning to examine in the internal structure of a cell requires a light microscope of sufficient quality to magnify (i.e., increase the apparent size of the object) with adequate resolution (i.e., ability to visually separate two objects that are close to each other), and contrast (i.e., proper lighting conditions so that objects can be seen more clearly and not appear washed out against the brightness or hidden in shadows by the darkness). Proper use of the microscope is necessary to make the most use out of this instrument so that you can maximize its capabilities and usefulness. Your lab instructor will discuss the parts and proper usage of the light microscope before you begin this laboratory exercise.

Compare what you see in lab with the figures given in the photographic atlas (i.e., Figure 1.1).
I. PREPARING A WET MOUNT A. ELODEA Unless you are using a prepared slide, most of your microscopic work will require the use of a wet mount (a slide which you prepare yourself by placing the object in a drop of water on a slide and adding a cover slip). Except for very elementary observations, all objects viewed through the microscope with transmitted light must be mounted in some medium. Obtain a clean, dry slide and cover slip from the supply table. With forceps, remove a young leaf from the actively growing tip of Elodea. Place the leaf in a small drop of water on your slide and carefully add the cover slip. You may find it best to hold the cover slip by the edges, touch it to the slide, and then move it toward the drop of water at an angle of about 45 degrees. As the lower edge of the cover slip contacts the water, gently lower it to the slide. If you have done this carefully, you should have the Elodea leaf mounted in the center of your slide in just enough water to fill the space between the slide and cover slip and there should be no entrapped air bubbles. Your instructor will demonstrate the proper procedure. Study your wet mount under low power and then select an area near the tip about mid-way between the margin and the mid-rib. Using higher magnification, carefully focusing, and changing diaphragm settings. What structures do you see? In some cases you may see movement of organelles and cytoplasm within a living Elodea leaf cell. This movement is known as cyclosis or cytoplasmic streaming.

Compare what you see in lab with the figure given in the photographic atlas (i.e., Figure 1.5).
B. ONION EPIDERMAL CELLS The outermost or surface layer of cells in plants is referred to as the epidermis. In most cases the epidermis is composed of only a single layer of cells and can frequently be removed by the peel technique. Your instructor will demonstrate this process. Following this procedure, prepare a wet mount of a small piece of epidermis from the inner side of an onion bulb scale. Mount the tissue so that it is not folded and so that the surface of the epidermis is toward the top in your slide. Examine your preparation under the microscope and notice the general shape, size, and arrangement of the cells. Switch to high power and regulate the diaphragm setting so that you can see clearly the cell wall and the living protoplast with composed of the cytoplasm and the nucleus ( plural, nuclei). If you look closely you should also be able to see a nucleolus (plural, nucleoli) within the nucleus of some cells. By careful focusing and light adjustment you may be able to discern the central vacuole containing the so-

called cell sap (water and dissolved substances). Thus far you have been observing a living cell and have probably noted that some of its features are difficult to observe In order to enhance details of the cell contest, place a drop of iodine stain at one edge of the cover slip. With a small piece of paper towel gently touch the opposite edge of the cover slip in order to drop the iodine under the cover slip. Now observe your preparation.

Compare what you see in lab with the figure given in the photographic atlas (i.e., Figure 1.3). For a detailed list of the parts of a typical plant cell check out Table 1.1 on page 2 in the photographic atlas.
II. PERMANENT SLIDES

During this semester you will be examining a wide variety of plant materials with your microscope. Much of this material will be in the form of permanent mounts, in which the tissue is cut into very thin slivers, stained to bring out finer details or differentiate tissues, and embedded in a permanent mounting medium. In order to produce a permanent slide of good quality a long tedious process must be followed that may extend over a period of several days or even weeks. The main steps in this process are usually as follows: A small piece of tissue such as a portion of a root, stem, or leaf is placed in a special solution to kill and fix cellular detail as nearly as possible to the living state. The tissue is dehydrated by passing it through a progressively stronger series of alcohols. Alcohol is then replace with a solvent, which in turn is replaced by melted paraffin (embedded). After cooling, the paraffin (which now contains the tissue) is cut into thin sliver by a precision cutting device called a microtome. Sections are affixed to a slide and the paraffin is then removed with solvents. Slides are passed through one or more stain series to enhance details and differentiate tissues within the section. Finally, after another dehydration sequence, a drop of special permanent mounting medium is place on the specimen and cover slip added.

As you can see, a great deal of time, effort, and skill is required to produce a permanent slide. Therefore, these slides are often quite expensive and should be handle with extreme care. So far, you have gained experience in using the compound microscope and examined, among other things, the living cells in a leaf of Elodea. The leaves of plants, as well as all other parts of plants, are composed of cells or cell products. Although we often study the plant as an individual organism or as an intact unit, it is very important to understand as much as possible about individual cells since they are the fundamental structural and functional components of the plant as a whole. As you might suppose, plant cells vary widely in size, shape, origin chemical composition, function, and in almost every other conceivable way. As a matter of fact, it is probably safe to say that no two cells are exactly alike. During this exercise we will study some of this great variation and diversity among plant cells. III. OTHER PLANT CELL STRUCTURES A. CHROMOPLASTS Prepare a wet mount of a small amount of macerated red or yellow pepper fruit pulp and examine it under your

microscope. Locate an intact cell. How do these cells compare in size and shape to the onion epidermal cells? Notice the large number of chromplasts located in the cell. In most preparations these plastids will be seen all over the slide since the thin-walled cells are easily rupture thus releasing the cell contents. The primary pigments present in chromplasts are fat-soluble carotenoids? The presence of chromoplasts in the cytoplasm and the pigments these possess impart the color seen in many fruits and flowers. B. LEUCOPLASTS Another type of plastid found in many plant cells is the leucoplast (a food-storage plastid containing starch or oil). Leucoplasts that contain starch are called amyloplasts. Those that contain oil are known as elaioplasts. Prepare a wet mount of a small amount of tissue scraped from the cut surface of a potato. Examine your preparation under low and then medium power. In this slide most of your potato cells will have ruptured and released the amyloplasts but you may be able to locate a few intact cells. Now add a drop of iodine to the edge of the cover slip and draw it under as you did earlier. What color are the leucoplasts (amyloplasts with starch grains) now? _____________________

Compare what you see in lab with the figure given in the photographic atlas (i.e., Figure 1.6).
Under high power focus on a single starch grain and manipulate the light and fine adjustment until you observe maximum clarity and detail. Are concentric rings or layers evident in the starch grain? If so, these rings represent the daily deposition of starch from one day to the next that were stored between extended periods of photosynthesis (day time) and periods when food could not be manufactured (night time). C. CRYSTALS Prepare a hand cross section of the stem of Zebrina or Transcantia, or some available herbaceous plant. In making hand sections it is very important that you use a sharp razor blade and cut the section as thin as possible. Your instructor will demonstrate the technique of making hand sections but do not be discouraged if your first few attempts are not successful. After you have cut what appears to be a good section, prepare a wet mount and examine under low power. In your preparation you will see (in cross section) a great variety of cell types and tissues, which you will study in more detail in a later exercise. For now, you are mainly concerned with locating crystals. Are you able to locate any long needle-like crystals either individually or in clusters? The needle-like crystals are raphide crystals, while the more faceted diamond-like ones are druse crystals. Most crystals are found in the cell sap and are formed from the precipitation of calcium oxalate. Plants with large stores of calcium oxalate are poisonous to animals and humans. D. PLASMODESMATA Carefully observe the demonstration microscope showing plasmodesmata (plasmodesma, singular) in the endosperm of the persimmon, Diospyros virginianta. What are plasmodesmata and what is their function?

Compare what you see in lab with the figure given in the photographic atlas (i.e., Figure 1.22).

IV. MITOSIS AND CYTOKINESIS

A. OVERVIEW OF THE CELL CYCLE Now that you have examined the plant cell and some of its organelles you are ready to study one of the more important processes which plant cells undergo, that of cell division. Growth in higher plants generally involves an increase in number of cells resulting from the formation of new (daughter) cells by the division of parent cells. Such division usually takes place only in special meristematic tissues (tissue in which cells retain their capacity for repeated division). Meristematic tissues is found in the root and stem tips, which allows them to increase in length, and in the vascular cambium and cork cambium which allows roots and stems to increase in diameter during secondary growth. The type of cell division we are considering here is generally referred to as mitosis, although in the strict sense mitosis refers to division of the nucleus (karyokinesis) while cytokinesis refers to division of the cytoplasm. However, as you will see, in most plants the process of cell division is a continuous and dynamic process in which nuclear division and cytoplasmic division blend together smoothly and seem o occur as part of the same overall process. After cell division, some of the daughter cells mature and as a result of differentiation develop into special tissues and organs in the growing plant. Other daughter cells remain as meristematic cells and after a period of time these in turn undergo cell division. Cytokinesis results in an approximately equal division of the cytoplasm and its components. Nuclear division, on the other hand, is an extremely precise process in which each daughter cell receives the same genetic information as that contained in the parent cell. For sake of convenience, mitosis has been divided into distinct stages, although the actual process is a continuous even in which on stage merges smoothly into the next. STAGES OF MITOSIS i. INTERPHASE. Strictly speaking, interphase is not a part of mitosis, but events that occur during interphase make the process of mitosis possible. The nucleus, as well as the entire cell, is extremely active during the period prior to active division. Many materials such as proteins, carbohydrates, and lipids are synthesized or accumulated during this period. Perhaps the most important synthesis that occurs is that of DNA (deoxyribonucleic acid). The DNA with the nucleus must be precisely replicated at this time so that an ext replication of genetic material may be transmitted to each daughter nucleus. The nuclear cont (chromatin) when viewed with a light microscope during interphase appears as a dispersed network of faintly staining material. In Allium, one or more nucleoli may be observed within the nucleus during interphase. The nucleus is relatively large in those cells, which are in interphase immediately prior to division. The nucleus at this stage is sharply defined, indicating that the nuclear envelope is intact. Also, the tin cell wall and the presence of small vacuoles can be seen. PROPHASE. The initiation of prophase is indicated by the appearance of long, thin, thread-like structures (chromosomes) in the nucleus. A gradual shortening and thickening of these chromosomes occurs as the condensation of chromatin continues and by late prophase they appear as rod or bar-shaped bodies. Each species has a characteristic number of chromosomes in its cells. In onion, the characteristic diploid (2n) number is 16, while lily has 24 and humans have 46. In slides prepared by section, the chromosomes in many cells will be cut away or distorted and because of this it is usually difficult to determine the number in a given cell. At the end of prophase, the nuclear envelope and nucleoli begin to disappear, a spindle begins to form, and chromosomes begin to move toward a median position in the cell. Some authors prefer to call this prometaphase while others use the term early metaphase for this stage. iii. METAPHASE. At metaphase, the chromosomes become arrange so that their centromeres are in

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a line parallel to the equatorial plane in a median position in the spindle. In order for normal division to occur the centromere of each chromosome must be associated with the fibers (microtubules) of the spindle. These chromosomal fibers along with eh continuous fibers make up the spindle mechanism. In three-dimension, the spindle would be similar to two cons placed base to base. In onion it is relatively easy to see the doubled nature of chromosomes at metaphase. Each chromosome consists of two identical units (chromatids), which are tightly appressed and lie parallel to each other. iv. ANAPHASE. Anaphase begins when the centromere of each chromosome divides allowing the two chromatids to move toward opposite poles of the spindle. In onion 16 chromatids (now called daughter chromosomes) move toward one pole while 16 others move toward the opposite pole. Anaphase ends when the daughter chromosomes reach the poles of the spindle. TELOPHASE. Telophase begins with the daughter chromosomes aggregating or grouping at the poles and becoming less distinct. In terms of appearance, the events of telophase are similar to those of prophase in reverse. Chromosomes become diffuse, a nuclear membrane begins to form, nucleoli reappear, and the spindle begins to disintegrate. As the daughter nuclei are formed, they assume an interphase appearance. In most plant cells the process of cytokinesis begins as the daughter nuclei are reforming. A cell plate appears within the spindle along the equatorial plane of the cell and extends laterally until it reaches the surrounding cell walls, thereby separating the cytoplasm and forming tow daughter cells. As the cell plate forms, new cell wall material is deposited on each side to complete the newly formed cells.

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B. MITOSIS IN THE ONION ROOT TIP Obtain a prepared slide of a longitudinal section of onion (Allium) root tip and examine it under low power of your microscope. You will study root anatomy and function in detail at a later time. For now, a simple orientation to the toot tip will suffice. The root cap forms a layer of loosely packed, thin-walled cells covering the tip of the root. The region of cell division (i.e., meristematic region) is located just back of the root cap and beyond this lies the region of elongation. Cells farthest from the root cap are in the region of maturation may be in various stages of differentiation. Notice that some of the epidermal cells found on the outer surface of the root in this region of the root have hair-like extensions (root hairs). It should be obvious to you by now that these regions of the root are not sharply defined but rather there is a gradation of one into the other. Now return to the meristematic region and switch to higher power.

Compare what you see in lab with the figures given in the photographic atlas (i.e., Figure 1.27, 1.28, 1.29). Refer to these figures to help you locate and study cells, which are in various stages of mitosis.
After you have located a certain stage and studied it carefully under high power, notify your instructor so that she can verify your identification. Be prepared to give reasons for you answer or otherwise justify your identification. Place the major characteristics for each of the following stages in the space provided. Interphase Prophase

Metaphase Anaphase Telophase Daughter Cells C. THE DURATION OF MITOTIC STAGES You have probably suspected by now that the various stages of mitosis are not equal in duration. You should also realize that the total process of mitosis might proceed at a slower or faster rate depending on the species, the tissue, and the time of day, temperature, and a number of other factors. Now that you are familiar with appearance of differentiation mitotic stages, carry out the following calculation, which should give a rough approximation of the relative length of time required for completion of each stage. For this activity, examine the lowest part of the meristematic region of your onion root tip slide. Proceeding upward, determine the stage of all mitotic cells until you have recorded a total of 100 cells. Ignore cells that have nuclei that are in interphase. Record your results in the table below. Remember, not overlook any cell in any stage of division (including early prophase and late telophase). After you have completed your calculations, you may wish to compare your results with those of your neighbor. NOTE: In the event that your slide does not contain a total of 100 mitotic cells, you may finish your count on another onion root tip slide. Number of Cells Prophase Metaphase Anaphase Telophase Totals % Of Total

Which stages of mitosis are most prevalent? If you think of the root tip mounted on the prepared slide as a snap shot of the cellular events that were occurring in this region of the plant when the specimen was collected and preserved, what do these data suggest as to the length of time spent by cells as they go through these stages of mitosis?