Observing Mitosis In Onion Root Tip Biology Essay
All new cells come from previously existing cells. The continuity of life is based on the reproduction of cells, or cell division which involves both replication of cell’s nucleus (karyokinesis) and division of the cytoplasm (cytokinesis) to form two genetically identical daughter cells.
There are two types of nuclear division: mitosis and meiosis. Mitosis typically results in new somatic (body) cells. Mitosis plays several important roles in the life of an organism. Mitosis can produce progeny from some multicellular organisms (for example plants that grow from cuttings). Mitosis also enables an organism to develop from a zygote. After the organism is fully grown, mitosis continues to function in renewal and repair, replacing cells that die from normal wear and tear or accidents. Meiosis, on the other hand, results in the formation of either gametes (in animals) or spores (in plants). These cells have half the chromosome number of the parent cell.
Where does process of mitosis occurs rapidly? Plants and animals differ in this respect. In higher plants the process of forming new cells is restricted to special growth regions called meristems. Meristematic cells are plant cells that are not completely differentiated and are capable of carry out cellular division continuously. These cells are small and have a large nucleus with no intercellular space and vacuole. Meristematic cell are important for the growth of plant as they will be specialized to become xylem, phloem and other tissues in plant. They are found in abundance at the shoot tip and the root tip. Thus, onion root tip is chosen for this experiment as it has a lot of actively dividing meristematic cells.
Mitosis is just one part of the cell cycle. In fact the mitotic (M) phase, which includes mitosis and cytokinesis is the shortest part of cell cycle. The other part of cell cycle is the interphase, which accounts for about 90% of the cell cycle.
Figure - The cell cycle
During interphase, cell grows and copies its chromosomes in preparation for cell division. interphase can be divided into 3 subphases: the G1 phase (first gap), the S phase (synthesis), and the G2 phase (second gap). During G1 phase, a cell undergoes a period of rapid growth and starts to synthesize new organelles in the cytoplasm. It will have a very high metabolic rate. For instance, a particular human cell which has a cell cycle of every 24 hours will spend about 9 hours in this phase. In G1 phase, a cell may enter the S phase to continue the cycle or enter a state of quiescence called G0 phase when it has temporarily or reversibly stopped dividing. Normally, a cell will move on to the S phase so that cell division can occur.
During S (synthesis) phase, the DNA is replicated and chromosomes are duplicated. Each chromosome consists of two sister chromatids, but is still not visible under the light microscope. They are commonly called chromatin at this stage. Histone is built up in a fast rate in this phase. After S phase the cell will proceed to the G2 phase, which is the second growth phase of cell. During this phase, the cell will start to accumulate energy in preparation to the next stage of cell cycle. Organelles such as mitochondria and chloroplasts will start to divide as they have their own genetic materials.
After the cell has gone through interphase, it will go on to M phase. M phase consists of mitosis and cytokinesis. Mitosis, on the other hand, consists of four main phases, which are prophase, metaphase, anaphase and telophase.
Figure -The phases in mitosis
In prophase, thickening of chromatin thread happens and continues until it is obvious that the chromatin has condensed into chromosome. With higher magnification of microsope, it is visible that each chromosome is composed of two chromatids. In late prophase the nuclear envelope are broken down and the chromosomes are free in the cytoplasm. The mitotic spindle begins to form. It is composed of centrosomes and the microtubules that extend from them. The centrosomes move away from each other due to lengthening of microtubules between them.
The next phase, metaphase is the longest stage of mitosis. The centrosomes are now at the opposite poles of the cell. The centromere is attached to spindle. The chromosomes moves and align themselves at the equator of the cells, an imaginary plane that is equidistant between the spindle’s two poles.
In anaphase, the centromere holding the two sister chromatids divides and each chromatid is pulled to the opposite pole of the cell by the contraction of microtubules. Once the two chromatids separate, each is called a chromosome. The daughter chromosomes continue pole ward movement until they form two compact clumps, one at each spindle pole.
In telophase, the nuclear membrane and nucleolus reforms. The chromosomes decondense by uncoiling and become less dense and harder to see. They are converted back into chromatin.
The final stage in the M phase is the cytokinesis. It is followed straight after telophase. In animal cells, cytokinesis involves the formation of cleavage furrow, which pinches the cell into two. In plant cell, cell plate is formed instead. Two daughter cells which are genetically identical to each other and to the parent cells are produced. They will then enter interphase and begin to prepare for the next cell cycle.
Figure - 1% toluidine blue stain http://www.delasco.com/
6M hydrochloric acid
1% toluidine blue stain
Two watch glasses
Microscope slides and coverslips
Pair of fine forceps
Soft tissue papers
1 to 2 cm of onion root tips which have a firm rounded end is cut off by using scalpel from the growing onion roots.
The root tips are then put on the watch glass containing a small puddle of 6M hydrochloric acid. The stopwatch is started and the root tips are left for exactly 4 minutes.
By using forceps, the root tips are transferred to another watch glass containing carnoy fixative for 4 minutes.
The root tip is transferred to a white tile. 1 to 2 mm from the growing tip is cut by using scalpel and the rest is discarded.
The root tip is transferred to a clean microscope slide. The root tip is stained with 2 drops of 1% toluidine blue and let for 2 minutes.
Tissue paper is used to blot around the root tip to remove excess stain.
A drop of distilled water is added to root tip.
It is then covered with a coverslip and blotted firmly with several layers of tissue paper to remove air bubbles.
The slide, coverslip is placed down on a paper towel. A firm pressure is applied to the coverslip to squash and spread the root tip tissue.
Mount the slide on your microscope.
Use the low power objective on your microscope to look for thin layers of cells and then use the 40X power objective to observe mitotic stages in individual cells.
Step 1 to step 11 is repeated to get a clearer view of each stage of mitosis if the first preparation is not very successful.
Nucleus is small, dense and darker in colour.
Chromosomes are not visible under the light microscope. They are tangled, uncoiled and appear as chromatin thread.
Nucleus is bigger and lighter in colour.
Chromosomes become shorter and thicker and are visible under the light microscope.
the two sister chromatids of chromosome are not seen because the magnification power of light microscope is not as high as electron microscope
The chromosomes are arranging themselves at the metaphase plate of cell.
Spindle fibres formed by centrioles are attached to the centromere of the chromosomes.
Chromosomes separate and each chromatid is pulled towards the opposite pole of the cell through the contraction of spindle fibres.
Most of the chromosomes are seen at the opposite pole of the cell with some of them still moving towards it.
All the chromatids have reached the cell pole and begin to decondense.
The nuclear membranes and the nucleolus reforms.
Cytokinesis follows until two new identical cells are formed.
During interphase, the chromosomes are not visible. During prophase, the chromosomes appear because it is condensed. During metaphase, the chromosomes appear to line up along the equator of the cell. During anaphase, most of the chromosomes are seen at the opposite pole of the cell with some of them still moving towards it. During telophase, all chromatids are seen to have reached opposite poles and the nuclear membrane reforms.
When viewed under microsope, most of the cells are seen to be undergoing interphase. This is followed by the prophase. Least number of cells are seen to be undergoing metaphase, anaphase and telophase. These differences show that the length of time a cell spends on each stage of cell cycle is different. This is because if the length of time for each stage is the same, an equal amount of the cell in each stage will have been seen. Therefore, interphase which has the highest proportion of cells is the longest phase in cell cycle while prophase is the second longest phase. However, prophase is the longest phase among the phases in mitosis. The cell is believed to have spent about the same amount of time in metaphase, anaphase and telophase.
In order to get a clear view on the position of chromosomes, the root tip is put in hydrochloric acid for 5 minutes to break down the middle lamella which holds the plant cell together without affecting its cell wall. This is very helpful in the preparation as the cells will not stick together and can be spread out into a layer when a firm pressure is applied. This will prevent the overlapping of cell. Furthermore, it will be easier for toluidine blue to penetrate into the nucleus of the cell to stain its chromosomes.
Carnoy fixative is used to arrest autolysis and preserve the cell and tissue components in a life stake as possible. The fixative should not cause excessive shrinkage, swelling or hardening of the tissue and should stabilize the tissue against the rigours of processing. This will cause the mitotic activities in the root tips are “frozen”. Finally the fixative should enhance subsequent staining (such as staining using toluidine blue).
Few dividing cells may be found in the root tip when the wrong end of the root tip is taken for observation under microscope. This means that the cells observed are actually located far away from the root tip and are already mature and differentiated. Thus, there will be less dividing cells found. In fact, a denser white and more rounded root tip should be used.
For safety precaution in this experiment, the root tips should be handled gently when they are transferred from one watch glass to another as they are very fragile. Any great force applied to the root tips will break and destroy the structure of the cell because the cell wall in meristematic cell is very thin. Besides that, when blotting the toluidine blue stain from the onion root tip, it is to be ensured that we does not touch the root tip to avoid fingerprints on the cells.
On top of that, lateral movement of the coverslip when squashing the slide should also be avoided so that the clear image is observed under the microscope. This is because some of the cells have already adhered to the coverslip. Furthermore, the lateral movement of coverslip will cause the overlapping and stacking of the cell nucleus.
Another safety precaution is exercising greater care when dealing with toluidine blue stain as it will stain the skin and clothes and is very difficult to remove it. Besides that, concentrated hydrochloric acid should be handled with care as it is very corrosive. Scalpel should also be used with more careful when cutting the root tips because it can injure fingers easily. When dealing with glass wares such as coverslip, suitable precautions should be taken to avoid breaking them.
Source of error
Some errors in the experiment may cause the result to be less accurate. For example, error can occur when the stage of mitosis of a cell is identified wrongly. Some of the stages such as anaphase and telophase might be identified wrongly as their position of chromosomes is about the same if the slide is not prepared clearly. A dividing cell can also be misidentified as the cell in interphase.
Stained microscope slide and coverslip caused by fingerprints may cause blurred sight of the cell. This may lead to misinterpretation of the phases of mitosis.
Limitations of experiment
Existence of unavoidable limitations will affect the accuracy of the result. Limitation occurs when a single layer of cells is not obtained or the middle lamella of the cell is not dissolved completely. Although this can be partly reduced by firmly applying pressure to coverslip, the cell will still be overlapping at some part in the microscope slide. The presence of air bubbles in the slide will also make the image observed unclear. All these will cause the observation of mitosis division of the cells is less visible.
Cell cycle can be divided into interphase and M phase. Interphase can be further divided into three different phases; they are G1 (first gap) phase, S (synthesis) phase and G2 (second gap) phase. M phase consists of mitosis and cytokinesis. Mitosis, on the other hand, consists of four main phases, which are prophase, metaphase, anaphase and telophase. Interphase is the longest phase, followed by prophase. The shortest phase would be the metaphase, anaphase and telophase.
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