Examining mitosis at the tip of the plant

Cells which are actively dividing mitotically can be found at the root tip region of a plant. This experiment is conducted to observe the mitosis process occur at the root tip of the plant, comparing the sizes of cell undergoing division with the one that is not, and to calculate the mitotic index of the tissue region. The mitosis occurs at the root tip is observed under the microscope. The root tip slide is prepared by using a squashing method. The root tip is put into carnoy fixative to denature the proteins and extract the lipids. The root tip is then put into hydrochloric acid to partially break down the cell and their components. After that, the root tip is stained using the orcein ethanoic stain to make the chromosome more visible. As for the result, mitosis consists four stages which are prophase, metaphase, anaphase and telophase. The size of cell undergoing division, 28µm, greater than the size of undividing cell, 20µm. The expected result for mitotic index at the root tip is high but due to some errors and limitation in conducting the experiment, the mitotic index is quite low for the root tip.

Introduction

The best characteristic of organisms is the ability to reproduce itself that best differentiate the living things from the nonliving matter. This continuity of life reproduction is based on the cell cycle. Cell cycle occurs in three stages or periods which are the interphase, karyokinesis and cytokinesis. Interphase is a long period which the cell grows and it is a period in which the cell is carrying an intense activity. During this phase, the proteins and the cytoplasmic organelles such as mitochondria, centrosome and endoplasmic reticulum are synthesized and the chromosomes are replicated and duplicated in preparation for mitosis. The chromosomes are in thin, long thread-like form which is called as chromatin. The chromatin is not so visible as they are not in a condensed form yet.

Cell cycle continues with cell division after going through interphase. Cell division consists of two stages which are karyokinesis and cytokinesis. Karyokinesis is a process where the nucleus divides either through mitosis or meiosis. If the cell undergoes mitosis, the each daughter cell will have the same number of chromosome as to their parent cell and both of the cell are genetically identical to one another. If the cell undergoes meiosis, four daughter cells are produced at the end of the process. Each of them have half number of chromosome of their parent cell and all of them are genetically varied from each other. Cytokinesis is the continuation process after the karyokinesis where during this process, the cytoplasm divides.

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In karyokinesis, the mitosis is conventionally broken down into several distinct phases. There are four main phases which are prophase, metaphase, anaphase and telophase. During the first mitotic phase, the prophase, the chromatin has become more tightly coiled and condensed which is now visible under the microscope. The nucleoli disappear and the nuclear membrane disintegrate. Each duplicated chromosome are now in pair of sister chromatids, joined together at the centromere. The spindle fibre starts to form and extend from the centrosome. The centrosome move away from each other towards the opposite poles.

Prophase is followed by metaphase. During metaphase, the centrosome are now at opposite ends of the cell. Each pair of sister chromatids arranged and aligned themselves at the metaphase plate, which is at the equator of the cell. For each chromosome, the kinetochores at the centromere of the sister chromatids is attached with the spindle fibre coming from the opposite poles.

The next stage is anaphase. Anaphase begins when the centromere divide, separating the sister chromatids. Each sister chromatid is now has completely become a fully-fledged daughter chromosome. As the spindle fibre shorten itself, the two separated chromosomes begin moving toward opposite poles of the cell as their kinetochore at the centromere attached to the spindle fibres.

Telophase is the final stage of mitosis. During telophase, the daughter chromosomes are now arrived at the corresponding opposite poles. The nuclear membrane begins to reform and the nucleoli now reappear in the two new nuclei. The chromosomes start to uncoil, returning to thin, long thread-like chromatin and less condensed then before.

After karyokinesis complete, the cell cycle continue and entering into cytokinesis. Although mitosis is the same in both animal and plant cells, cytokinesis is different. In animal cells, the cytoplasm divides by constricting inward in a process called furrowing. In the furrow region, the protein actin encircles the cell. This contractile ring gradually pinches the cell in half, forming two daughter cells. In plant cells, there is no constriction process. Instead, membrane vesicles containing cell wall components and derived from the golgi apparatus migrate to the center of the cell. These vesicle fuse with each other and the plasma membrane to forn the end membranes of two new daughter cells. Hence, the phrase cytokinesis by cell plate formation is used.(1)

Mitosis is best to be observed in region of cells that are growing at a rapid pace, such as the lateral and the apical meristimatic tissue. Mitosis occurs most frequently at the apical meristems such as the root tip. The root of a plant continually grow in order to search for nutrients and supply the plant to keep them growing and survive. The tissue region at the root needs to have very active dividing cells, especially at the very root tip, as to enable the root to grow in length. Therefore, various stages of mitosis can be observed at this region.

Experimental hypothesis

Mitosis consists prophase, metaphase, anaphase and telophase. Mitosis occurs most frequently in apical meristematic tissue of plant. The size of cell undergoing mitosis is greater than the size of cell that are not undergoing mitosis.

Null hypothesis

There is no distinct phases in mitosis. There is no distinct different between the size of the cell undergoing mitosis and the one that is not. Mitosis occurs averagely throughout the plant tissues.

Materials, apparatus and equipments

Root tip, carnoy fixative, hydrochloric acid (18%), orcein ethanoic stain, distilled water, pair of fine forceps, microscope slide, cover slip, scalpel, filter paper, 2 watch glasses, stopwatch, dropper, cotton cloth, compound microscope.

Variables

Manipulated variables : invalid

Responding variables : invalid

Fixed varaiables : source of root tip,

Procedure

A root tip is obtained from a kit in a holding solution (70% ethanol), prepared in the laboratory.

A few drops of hydrochloric acid (18%) are put onto a watch glass and a few drops of carnoy fixative is put onto another watch glass.

The root tip is placed into the hydrochloric acid (18%) on the watch glass and left for 4 minutes.

Then, the root tip is transferred into the carnoy fixative prepared earlier for another 4 minutes.

After 4 minutes, the root tip is placed onto the microscope slide and 2 mm of the root tip is cut off using the scalpel.

The root tip is covered with a drop of the orcein ethanoic stain for 2 minutes. Then, the stain is blotted away using the filter paper.

When the stain has been blotted away, a few drops of distilled water are put onto the root tip.

A coverslip is gently lowered over the root tip and firmly pressed directly downward on the coverslip with a thumb. The amount of liquid on the slide should be just enough to flow out the edges of the coverslip. Any excess liquid is absorbed using the filter paper.

The slide with the dyed root tip is then placed on the stage of the microscope.

The slide is observed by using a low power of the lens first to locate the cells position.

Once the location is determined, the lens is changed onto the higher one with x400 magnification and various stage of cell division is observed.

One cell for each stage is drawn to illustrate the cell viewed under the microscope.

Number of cells undergoing a particular stage, interphase, prophase, metaphase and telophase, are counted.

The percentage of the cells in each stage of mitosis is calculated and ranked from the highest to the lowest.

Using the formula below, the mitotic index of the root tip observed is calculated.

Using a stage micrometer and eyepiece graticule, appropriate measurements and a comparison is made between the size of the cells undergoing interphase and those that are undergoing M phase.

All the data obtained is recorded in a table with a suitable heading corresponds to its contents.

Safety measure and risk assessment

There is some safety measures that are should be taken when conducting this experiment.

When using the microscope, the lens and the mirror have to be free from dust to avoid from getting blurry image of the slide. If using a microscope with separate illumination, direct sunlight should not be used as the light source for the microscope and avoid it from being reflected through the optics as it may cause eye damage to the person. When changing the lens, make sure that the lens does not touch the slide as it may scratch the lens and spoil your slide.

When handling the orcein ethanoic stain, carnoy fixative and the hydrochloric acid, wear safety goggles, lab coat and protective gloves to avoid any direct contact with it. Orcein ethanoic stain will give rise to pink colour on your skin if there is direct contact with it. The carnoy and orcein ethanoic stain can be harmful if swallowed, inhaled or absorbed through skin as it may cause irritation.

When preparing the slide, start lowering the coverslip at 45o with care and gentle. This is to avoid from having bubbles form on the slide. Use a tissue as a cover when pressing the cover slip directly using a thumb. This is to ensure in getting a clearer image of the slide as you thumb might leave a fingerprint on the cover slip. Press the cover slip gently and firmly downward to avoid any lateral movement of the cover slip as this will roll cells on top of one another and destroy the material.

Results

Figure

Area !

Figure

Area 2

Table

Stage of cell division

Number of cells

Percentage of cells (%) (number of cells in phase / total number of cells counted)

Area 1

Area 2

Interphase

30

29

76.6%

Prophase

4

3

9.1%

Metaphase

1

1

2.6%

Anaphase

1

1

2.6%

Telophase

4

3

9.1%

Total

40

37

100%

At x400 magnification, 0.004mm is equal to 1 division on the graticular eyepiece.

Table

Cell

Size of cells (µm)

Dividing cell

28

Undividing cell

20

The illustration of cell for each stage of mitosis

Table

Stages of cell

Events description

Illustration sketch of cell

Interphase

The chromosomes are not so visible as it is in chromatin, a long thread-like form. The cell has dark stained nucleolus and filled with network of threads, the chromatin.

Prophase

The chromosomes are visible and thick as it is in a condensed form. Each chromosome has duplicated and form a pair of sister chromatids. The nucleolus disappear and the nuclear membrane disintegrate.

Metaphase

All the chromosomes which are now in pair of sister chromatids align themselves at the metaphase plate which is the equator of the cell. The pair of sister chromatids are held in place with its kinetochore at the centromere is attached by the spindle fibre formed from the centrosome.

Anaphase

The centromere divided and the sister chromatids are being pulled towards the opposite corresponding pole due to the shortening of the spindle fibre.

Telophase

The daughter chromosome arrives at the pole and start to uncoil forming the chromatin. The nuclear membrane reform and the nucleolus reappear.

Discussion

There are two methods in observing the mitosis of the root tip which are the squashing method and the cross-sectioning method. Squashing method is chosen for this experiment as it has more advantages than the cross-sectioning method. There are two advantages of using the squashing method. First, the cells slide is easy to observe and identify in squashes under the microscope. Second, the cells may be flatten without bursting when preparing the slide with full care. This allows an easy observation to be made on the chromosome, although it does distort the original three-dimensional of the cells as the cell wall is soften and loses their fixed arrangement.

When preparing the root tip, the root tip is first soaked into the carnoy fixative. This carnoy fixative rapidly kills the cells by denaturing proteins and extracting lipids. As for soaking the root tip into the hydrochloric acid (18%), this is to hydrolyze the cells. In order to stain and squash the cells, it is necessary to hydrolyze it, that is, to partially break down the cell and their components which means to soften the plant cell walls and partially hydrolyze the DNA in the chromosomes.

After then, the root tip is put into the orcein ethanoic stain. The tissue is stained with a coloured dye to make the chromosome visible. This orcein ethanoic stain colour is pink. The chromosome will appear as darker pink and the others will be coloured in much paler pink.

From table 1, it can be seen that most of the time the cell is in the interphase and the calculation showed the mitotic index is qite low for the root tip. Theoretically, the mitotic index for the root tip is expected to be high because as at the root tip, there is the presence of the meristimetic tissue. This tissue actively divide themselves throughout their life. For this experiment, result does not show the positive one because it might be affected by some of the limitation factors in the experiment. Besides, the errors might occur due to the student himself as he cannot truly distinguish between the dividing cells and the undividing cell. Some of the cells that are undergoing division might not be counted in by the student. For example, the prophase and the interphase might look almost the same. Besides, the telophase, the student might actually classified them as already divided into two which means already in the interphase.

From table 2, it shows that the size dividing cell is bigger than the size of the undividing cell. This because when the cell start entering the mitosis, centrioles separate themselves to the opposite poles and the spindle fibres lengthen themselves causing the cell stretch even more. During the anaphase, the single sister chromatid is pulled apart from the homologous causing the cell to stretch even further towards both opposite poles. Later, in the telophase two nuclei are already form at each end of the opposite cell poles. Whereas during interphase, the chromosome is thin and in long thread-like form and only nucleus present with nuclear membrane surrounding them. Thus, that is why the size of dividing cell is bigger than the undividing cell.

Conclusion

Mitosis process consists four process which are prophase, metaphase, anaphase and telophase. The size of the dividing cell is greater than the size of the undividing cell. Mitotic index of the root tip is supposedly high.

Limitation

There are some factors that are beyond our controls that may affect or limit the accuracy of the result. First, the source of root tip. The root tip is already prepared in holding solution obtained from the kit. Thus, we not know where does it come from specifically from the very root tip or along the root. Second, the time constraint affects us to be able to prepare more slides of the root tip. There is not enough time to prepare several slides to be observed and compared between them to get the average which might give a more reliable result.

Suggestion for improvement

Some modification can be made to improve the accuracy and validity of the result. First, when lowering the coverslip over the root tip, lowering it as gently and carefully to avoid trapping any bubbles on the slide. Instead of pressing down the coverslip using your thumb, we can use the back of the pencil. This can ensure the firmness of pressing the slide and let the cells separate into one layer only. Second, orcein ethanoic stain can be replaced by another stain such as toulidine blue stain. This might give us a better view on the slide image and be able to distinguish different stages of mitosis better. Third, more time should be given to the students to conduct this experiment in order to enable them to prepare more slides.

Further work

Observe and compare mitotic index at root tip and shoot tip.

Observe and compare mitotic index at apical meristems and lateral meristems.

Observe and compare mitotic index at root tip and middle part of plant.