Learning Objectives

Tobacco Growing Made Easy

How To Grow Tobacco At Home

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After completing this laboratory topic, students should be able to:

  1. Describe the events that occur during all the stages of cell division.
  2. Rccognize the stages of cell division under the microscope.
  3. Describe the process of tissue culturing.
  4. Understand the need for sterile technique in tissue culturing.
  5. Understand how cell division contributes to asexual reproduction.
  6. Describe how to make cuttings for plant propagation. EXERCISE A: Cell Division

Cell division is just a small part of the cell cycle, but it is the only time when chromosomes can be seen under the microscope. The rest of the ccll cycle, when the cell is not dividing, is called interphase (fig. 2.1/? . During interphase, the genetic material exists as long strands of DNA and protein known as chromatin; during ccll division, the chromatin becomes organized into chromosomes.

The cell is mctabolicallv active during interphase and is also making all the preparations for the next round of cell division. Three metabolic stages characterize interphase: Gt, S, and G:. During G (first gap), the cell is actively growing and rapidly synthesizing proteins and other metabolites. The S (synthesis) stage is when DNA and other components of the chromosomes are duplicated. The final preparations for ccll division take place during die Cj (second gap) stage, and as this stage ends, mitosis begins. The process of mitosis occurs in four continuous phases: prophase, metaphase, anaphase, and telophase.

Prophase

Numerous events occur during prophase. The threadlike chromatin condenses and coils into recognizable chromosomes. When the chromosomes become visible, they arc seen to consist of two identical chromatids, which are joined at a constriction known as the centromere (fig. 2.1b). As prophase continues, the nucleoli and nuclear membrane disperse into the cytoplasm, leaving the chromosomes free in the cytoplasm, and spindle fibers begin appearing (fig. 2.2).

Metaphase

During metaphase, the cliromosomcs rearrange themselves so that the centromeres lie across the center of the cell «metaphase plate). 'Die spindle, which consists of numerous spindle fibers, becomes fully developed. Some spindle

Botany Drawings Broadbean

Phase

Main Events

VIci3 {aba

gi

Cells rr.etabclioaliy active; organelles begin to increase in number

4.9 hr

s

Replicat:on of DNA

7.5 hr

g2

Synthesis of proter-s; final preparatory for coll division

49

M

Mitosis

2.0

Total

19.3 hr

Tissue Culture Vicia Faba
Si«*-ctvorvatidft

FIGURE 2.1 (A) THE CELL CYCLE CONSISTS OF FOUR STAGES (G . Sf G2, AND M). THE EVENTS THAT OCCUR IN EACH STAGE ARE DESCRIBED USING THE BROAD BEAN (VICIA FABA) AS AN EXAMPLE. (8) MICROGRAPH AND DRAWING SHOWING THAT A DUPLICATED CHROMOSOME CONSISTS OF TWO SIS-TER CHROMATIDS HELD TOGETHER AT THE CENTROMERE ( x 9.600).

fibers extend from the centromeres to the poles of the cells, while other libers extend from pole to pole (tig. 2.2).

Anaphase

The two chromatids that make up each chromosome are separated during anaphase. Spindle fibers pull the chromatids to the opposite poles of the cell. Once thev separate, each former chromatid is called a chromosome. The end result of anaphase is that the genetic material is divided into two identical sets, each with the same num ber of chromosomes. By the end of anaphase, the spindle is no longer apparent fig. 2.2).

Telophase

At each pole of the cell, the chromosomes unwind and lengthen during telophase. Also, nuclear membranes and nucleoli reappear, so that at the end of telophase, two distinct daughter nuclei are visible (fig. 2.2).

During the later part of anaphase and continuing through telophase,' cytokinesis takes place. The

Cell Wall Late Telophase

Nucleolus

Centromere

Centromere

FIGURE 2.2 MITOSIS IN A PLANT CELL.

Nuclear membrane

Chromatin interphase

Spindle fibers beginning to asserrole

Dojb'e chromosome

Prophase

Late telophase

Spinae

Plant

Mitosis

Early telophase and cytokinesis early rretasnase

Anaphase

Late rr-etapnase

Single chromosome phragmoplast. which consists of vcsiclcs and portions of die endoplasmic reticulum, forms between the daughter nuclei. The phragmoplast participates in the formation of a disc-shaped structure called the cell plate, which expands outward and becomes the cell walls separating the newly formed daughter cells (tig. 2.2).

In this exercise, you will first study mitosis in a prepared slide of the onion root tip. In the sectioned material on the prepared slides, all the srages of mitosis can be found quite easily; however, it is nor always easy to distinguish individual chromosomes. You will then make your own squash preparation of a fresh root tip from Vic in fab a (broad bean). In a squash preparation, the cells are flattened, and the large chromosomes are more dispersed and usually seen in the same focal plane. This allows for better examination of chromosomes.

Materials Needed for Exercise A

Runscn burner

Compound microscopc

Dissccring needles

Dropper botdes with 1 M HCI

Dropper bottles with toluidine blue stain

Forceps

Germinating seeds of I Ida faba Paper towels

Prepared slide of Allium (onion) root tip Razor blades, single edged Slides and coverslips Procedure for Exercise A

1 Allium (onion) root tip prepared slide This is a slide with stained longitudinal sections of the root apical meristem. First scan the slide with the 10 x objective lens. The end of the root is protected by a root cap. What is the shape of the root cap?

Right behind die root cap is the meristem, where there is active cell division. Now shift to the high-power (40 x ) objective and find cells in each stage of mitosis. Study the section carefully. Arc most of the cells dividing, or are the majority in interphase?

After you have studied the section, your instructor will test your ability to recognize the stages of mitosis. When you can recognize all the stages on the prepared slide, you can move on to prepare die root tip squash.

  1. Broad bean root tip squash Using a razor blade, cur off a secondary root from a germinating seed of broad bean, and placc the root on a glass slide.
  2. Remove 0.5-1 cm of growth from the root tip and discard the remainder of the root. (Note: Your instructor may have already done this for you and placed the roots in (.amoves solution to kill and fix the cells. If so, rinse the roots in distilled water before the next step.)
  3. Cover the root tip with two or three drops of 1 M HCI. The HCI helps dissolve die middle lamella that holds the cells together. Using a slide holder, forceps, or a clothespin to hold the slide, pass the slide over the flame or a Bunscn burner for 5 seconds (or pass it through the flame five rimesj. Put the slide down on a paper towel, and let it sit in the HCI for about 4 minutes.
  4. Use the corner of a paper towel to j>jot up the HCI. Cover the root tip with two drops of toluidine blue stain. Pass the slide above the Bunscn burner two times. Placc the slide on a paper towel for 1 to 2 minutes.
  5. Carefully Wot up the excess stain. Using a single-edge razor blade and a dissecting needle, macerate (chop up) the root Up. Make sure all the pieces of the root tip are on the slide. .Add a drop offrgh stain and then apply a coversljp.
  6. Place the slide on a paper towel (covcrslip up). Cover with a second paper towel. Carefully apply pressure to the covcrslip area with your thumb in order to squash and spreadjtlic root tipjjsaic.
  7. Scan under low power (10 objective I to look tor areas with cells undergoing mitosis, and then examine under high power (40 x objective). Try to lind all the stages of mitosis.
  8. If your first rooi did not yield any dividing cells, try a second n>oi tip.

EXERCISE B: Clones from Tissue Culture

Under suitable Conditions, certain cells from a mature plant can resume cell division or even differentiate into another cell type. This is what happens when plants repair wounds caused by injury. The result is the development of a callus, an irregular mass of undifferentiated plant cells, at the injured site.

A callus can also develop from small pieces of plant tissue placed in an appropriate culture medium in the laboratory (tig. 2.3/?). The tissue removed from a plant and placed into culture is called an explant. The tissue culture medium contains both mineral nutrients and organic com pounds, including sugar, vitamins, and plant hormones. The plant hormone auxin controls cell enlargement as well as cell division in culture. Kinctin, another hormone, also induces cell division. Roth arc included in the culture medium for healthy callus growth. Callus can also be induced to differentiate and develop roots and shoots if the relative proportions of auxin and kinctin are changed. The result is the production of small plandets in the tissue cul turc medium (fig. 2.3/». These plandets, which can then

Callus Culture Picture
FIGURE 2.3 PLANT TISSUE CULTURE. (A) CALLUS GROWTH FROM EXPLANT. (8) ADDITION OF HORMONES ALLOWS THE CALLUS TO DIFFERENTIATE INTO A PLANTLET.

be transferred to soil, arc genetically identical to the small piece of plant tissue originally placed in the culture medium. Tissue culluring allows botanists and horticul-turalists to clone plants with desirable traits, and it is widely used in die nursery business and in plant biotechnology. In this lab, we will use tissue culturing to done tobacco plants.

Tissue culturing requires the use of sterile techniques (as described in steps 2 and 3 in the procedures) because die culture medium we are using is rich in nutrients and can easily become contaminated with bacteria and/or fungi. Fungal spores and bacteria arc abundant in our surroundings, both in the air and on die surface of objects. On the tissue culture medium, these microorganisms would quickly overgrow the plant tissue or developing callus, so every possible precaution should be taken to minimize contamination.

Materials Needed for Exercise B

3 or 4 petri dishes containing callus culture medium

Additional culture medium in magenta jars will be needed in 3 weeks for differentiation of roots and shoots. Antibacterial soap

Bunsen burner or spirit alcohol) lamp Cotton Balls

Growth chamber (if available; set at 28°C with 12-hour photoperiod, or bank of cool white fluorescent lights

Jar containing 15% bleach solution

Jar containing soapy water (two drops of dishwashing liquid in 250 ml water)

Jar containing sterile distilled water

Laminar-flow hood or transfer chamber (if available)

Parafilm

Scalpel and forceps

Small glass plate (approximately 15 cm x 15 cm > Spray bottle containing 70% ethanol Tobacco plant Vinyl gloves • if available)

Procedure for Exercise B

  1. If a laminar-flow hood is available in the lab, turn on the blower. This will bring filtered air into the work area.
  2. Wash your hands with antibacterial soap. Ifgloves arc available, they will provide added protection.
  3. Spray 70% ethanol on the work area of the laminar flow hood or your lab bench and use a cotton ball to wipe the entire area. Maintain a clean work area throughout the culturing. Sterilize all instruments b> dipping each instrument in alcohol and then passing it through the flame of a Bunsen burner or spirit lamp. Be careful to keep the jar of alcohol away from the flame.
  4. Cut a 1 to 2 cm segment from the stem of a tobacco plant. Use a segment from the region between leaves (called the intcrnode) near the top of the plant. Note. the tobacco plant should not be close to your sterile work area.
  5. Use forceps to dip the stem segment in soapy water. This will remove any small insects, loose spores, and bacteria.
  6. Transfer the stem segment to the jar of bleach for 3 minutes.
  7. Rinse the stem segment for 2 minutes in sterile distilled water.
  8. While the stem segment is in the bleach and the sterile water, wipe the glass plate widi 70% alcohol and allow it to dry.
  9. Transfer the stem segment to the glass plate.
  10. Using the scalpel and forceps (remember to dip them in alcohol and flame), trim oft"and discard the epidermis (the outermost layer). Also trim off and discard the ends of the segment. Cut the remaining segment into three or four slices.
  11. Place each section on the surface of the culture medium in a pctri dish.
  12. Seal each pctri dish with a strip of parafilm.
  13. Place the dishes in a growth chamber at 25°C under 12-hour photoperiod. If no growth chamber is available, rhc cultures can be grown under a bank of cool white fluorescent bulbs. If a timer is available, set it for a 12- to 16-hour photoperiod, although the cultures will do well even under continuous illumination.
  14. Check the cultures each week. It may take a while before growth is visible. Record your observations on the appearance of die tissue on worksheet 2-1 at the end of this laboratory topic.
  15. Once the callus is growing well (about 3 weeks from now), it will be time to transfer it to the differentiation medium.
  16. Repeat steps 1, 2, and 3 to sterilize the work area.
  17. Obtain three or tour jars containing the differentiation culture medium.
  18. Open the dishes containing the growing callus. Using sterile technique, transfer the callus to the differentiation medium in die jars. Close the jars. If you are using magenta jars, they will have a good seal. If you are using other jars, you may want to seal the lid with parafilm.
  19. Replace the jars in the growth chamber or under the bank oflights.
  20. Continue to evaluate your cultures every week for another 6 weeks. Remember to record vour obser-

vations in worksheet 2-1. At the end of the experiment, you may want to transfer the developing plants to potting soil in a 4-inch pot.

EXERCISE C: Cloning Herbs From Cuttings

In tissue culturing, the addition of an appropriate combination of auxin and kinetin leads to die development of roots and shoots from undifferentiated callus. This allows new plants to develop exclusively through cell division. This type of reproduction is known as asexual reproduction or vegetative propagation. In addition to tissue culture, there arc several other means of asexual reproduction. One technique that is widely applied by both home gardeners and professional nursery owners is the use of stem cuttings. In this method, a section of the stem is cut from the plant and dicn placcd in an environment to promote the development of adventitious roots (roots that develop from a stem or a leaf). Although other parts of the plant can also be used for cuttings, by far the largest number of plant species are propagated by stem cuttings. Some cuttings readily develop roots, while others only do so when hormones arc added. This is usually done by dipping the cutting in a powder or gel that contains a mixture of hormones and a fungicide to prevent infection of die cutting.

In this lab, you will be starting an herb garden by-making (and rooting) cuttings from several herbs. Herbs may be described as aromatic leaves or seeds from plants native to temperate regions. The plants diat provide herbs for cooking and medicine arc usually nonwoodv (herbaceous) plants or small shrubs. For each herb, we will be comparing the success of hormone treatment with the untreated control. Later in this manual, we will use sonic of the leaves when we work with herbs and spices in Laboratory Topic 17.

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