ABSTRACT/SUMMARY The experiment was done to study the cell reproduction in mitosis.

During the experiment, students need to prepare a slide on onion root tip. Using the materials and apparatus provided, slide on onion root tip was prepared. Staining method was using during preparing the slide. The slide was then observed under the microscope to identify the stages in cell reproduction. The data was collected by sketching the image under the microscope and by counting the number of mitotic stages. The data obtained from the other group was also important to determine the percentage of the whole class. After that, the time taken for the cell to complete each stage was calculated using a several formula. A pie chart has been done to compare the time taken for the cell to complete in each stage.

INTRODUCTION Cell division is the process by which all cells of a multicellular organism are formed. Cell division is also responsible for repair and replacement of cells and tissues during one's lifetime. There are two type of cell division which is mitosis and meiosis. Mitosis is studied in this experiment. Mitosis is for growth and replacement of damage cell. Mitosis is a part of the cell cycle. The cell cycle start when a cell is formed and continues until it dies. Cell division is a brief part of the life cycle. Most of the life of a cell is spent in normal activities of growth and maintenance.

Just before mitosis the cell is in interphase. In this the part where the cell will have a distinct nucleus and nucleoli which the thin threads of chromatin are duplicated. After duplication the cell is ready to begin mitosis and its starts with a step called prophase. In prophase, the chromatins thicken into distinct chromosomes and the nuclear envelope breaks open releasing them into the cytoplasm. The first signs of the spindle begin to appear. Next the cell begins metaphase, where the spindle attaches to the centromere of each chromosome and moves them to the same level in the middle of the cell. This level position is called the metaphase plate. Anaphase begins when the chromatids are separated and pulled to opposite poles. Then, the final stage is telophase. The nuclear envelope is reformed and the chromosomes gradually uncoil.

OBJECTIVES To study the cell cycle and understand how, when and why cells divide. To study and identify the major stages of cell division. To relate the mitotic process to living organisms. To understand the differences between plant and animal cell division. To graphically illustrate the roles of cell division and cell elongation in root growth. To learn how chromosomes are stained.

THEORY

It was discovered in 1858, by Rudolf Virchow, that new cells can only arise from previously existing cells. This is done in two ways: mitosis and meiosis. Somatic (body) cells divide exclusively by mitosis followed by cytokinesis, while germ cells produce gametes by the process of meiosis. Plant cells grow by enlargement, essentially by taking up water. When they reach a certain size, they divide, forming two identical daughter cells. The various parts of the cell are divided in such a way that the new daughter cell is identical to the parent cell. Strictly speaking, mitosis implies only the division of the nucleus, and is therefore distinct from cell division, in which the cytoplasm is divided. In most organisms, cells divide by ingrowth of the cell wall, if present, and the contraction of the cell membrane, a process that cuts through the spindle fibers. In land plants (bryophytes and vascular plants) and a few algae, cell division takes place by the formation of a cell plate. Small droplets appear across the equatorial plate of the cell and gradually fuse, forming a disc that grows outward until it reaches the wall of the dividing cell, which completes the separation of the two daughter cells. The DNA of prokaryotes is simply replicated before division. In eukaryotes, however, the hereditary material is part of their complex chromosomes. Equal division of this material requires a more complex method by which the chromosomes are replicated, separated, and apportioned precisely between the daughter cells. Mitosis, or nuclear division, ensures the equal division of the nuclear material between the daughter cells in eukaryotic organisms. During mitosis the chromosomes condense, and move to the center of the cell where they fully contract. They then split longitudinally into two identical halves that appear to be pulled to opposite poles of the cell by a series of microtubules. In these two genetically identical groups, the coiling of the chromosomes relaxes again, and they are reconstituted into the nuclei of the two daughter cells. It is a continuous process that can be divided into five major phases: interphase, prophase, metaphase, anaphase, and telophase.

APPARATUS AND MATERIALS 1 M HCl 1% toluidine blue Blotting paper Microscope Onion root tip (Allium sp.) Slide

EXPERIMENTAL PROCEDURE 1. The roots near the base of the onion bulb that have been rooted in water were cut. 2. 1 or 2 mm of the root tip was cut and placed in a slide provided. 3. A drop of 1M HCL was added to the slide. 4. The root tip was removed from the HCL to the clean slide after 4 minutes. 5. 2 drops of 1% toluidine blue was added to the root tip and let it sit for 2 minutes. 6. The root was blotted around to remove excess stain. The root was rinsed with water and blotted again until the water cleared. 7. A drop of water was added and cover slip was applied. 8. A small pressure was applied to the cover slip to get rid of bubble. 9. The slide was observed under the microscope. 10. The chromosome at the various stage of mitosis was identified. 11. The image seen under the microscope was sketched and the number of the cell during the mitotic stages was count.

RESULTS

Mitotic stages Interphase Prophase Metaphase Anaphase Telophase

Number of cells Group 1 7 14 3 21 13 Group 2 18 8 4 21 9 Group 3 8 13 2 21 8 Total Group 4 31 11 3 2 3 Group 5 20 15 10 2 5 Group 6 11 25 3 8 3 Group 7 7 10 7 20 6 Total

Percentage (%) 27 26 9 25 13 100

Times (min)

102 96 32 95 47 372

388.80 374.40 129.60 360.00 187.20 1440.0

Cell Cycle
Metaphase Prophase Anaphase Telophase

Interphase

SAMPLE CALCULATION

The number of the cells percentage (%) =

Consider that it takes, on average, 24 hours (or 1,440 minutes) for onion root-tip cells to complete the cell cycle. The amount of time spent in each phase of the cell cycle from the percent of cells in that stage can be calculated by:

Minutes of cell cycle spent in stage =

DISCUSSIONS

The stages of mitosis were observed and timed in the experiment. These stages are prophase, metaphase, anaphase, and telophase. Prophase is the most time-consuming phase, while anaphase is the least time-consuming. Mitosis is just one portion of a cells life. The longest time of a cells life (27% to be exact) is spent in interphase, a phase just prior to prophase. During this phase, DNA replication takes place. Interphase

A normal resting cell exists in a state called interphase in which the chromatin is undifferentiated in the heavily-stained nucleus, as illustrated above. Before the cell enters the mitosis phase, it first undergoes a synthesis or S phase where each chromosome is duplicated and consists of two sister chromatids joined together by a specific DNA sequence known as acentromere. Centromeres are crucial to segregation of the daughter chromatids during mitosis.

Early prophase

Late Prophase

The first phase of mitosis is known as the prophase, where the nuclear chromatin starts to become organized and condenses into thick strands that eventually become chromosomes. During prophase, the cytoskeleton (composed of cytoplasmic microtubules) begins to disassemble and the main component of the mitotic apparatus, the mitotic spindle begins to form outside the nucleus at opposite ends of the cell. The photomicrograph above depicts the initial chromosome condensation at the beginning of prophase (early prophase) when the nucleolus is still intact. Late prophase, or prometaphase, begins with the disruption of the nuclear envelope, which is broken down into small membrane vesicles that closely resemble the endoplasmic reticulum and tend to remain visible around the mitotic spindle. During this period the chromosomes continue to condense and gradually shorten and thicken until they have completely formed the units that will undergo mitosis. The nucleolus also disappears during this period. The mitotic spindle microtubules are now free to enter the nuclear region, and formation of specialized protein complexes called kinetochores begins on each centromere. These complexes become attached to some of the spindle microtubules, which are then termed kinetochore microtubules. Other microtubules in the spindle (not attached to centromeres) are termedpolar microtubules and these help form and maintain the spindle structure along with astralmicrotubules, which remain outside the spindle. Metaphase

During metaphase, the chromosomes, attached to the kinetochore microtubules, begin to align in one plane (the metaphase plate) middle between the spindle poles. The kinetochore microtubules exert tension on the chromosomes and the entire spindle-chromosome complex is now ready for

the next event. The photomicrograph above depicts onion root tip cell chromosomes in metaphase, ready for taking apart. The kinetochore and polar microtubules are clearly visible and radiate out the ends of the cell leaving the chromosomes in the middle of the complex. Early Anaphase Late Anaphase

Metaphase sets the stage for chromosome separation in the next stage of mitosis: anaphase. Almost immediately after the metaphase chromosomes are associated at the metaphase plate, the two halves of each chromosome are pulled apart by the spindle apparatus and migrate to the opposite spindle poles. The kinetochore microtubules shorten as the chromosomes are pulled toward the poles, while the polar microtubules elongate to assist in the separation. The photomicrograph above illustrates the early stage of anaphase where the chromosomes are just becoming completely separated. The microtubules are clearly visible in this complex. Anaphase usually is a rapid process that lasts only a few minutes. When the chromosomes have completely migrated to the spindle poles, the kinetochore microtubules begin to disappear, although the polar microtubules continue to elongate. This is the junction between late anaphase and early telophase, the last stage in chromosome division. The photomicrograph above shows the positioning of the chromosomes in late anaphase. The polar microtubules are a clearly formed network and the synthesis of a new cell membrane has been initiated in the cytoplasm between the two spindle poles.

Telophase

In telophase, the daughter chromosomes arrive at the spindle poles and are eventually redistributed into chromatin. The process of cytokinesis, where the cytoplasm is divided by cleavage, also starts sometime in late anaphase and continues through telophase. After complete separation of the chromosomes and their extrusion to the spindle poles, the nuclear membrane begins to reform around each group of chromosomes at the opposite ends of the cell. The nucleoli also reappear in what will eventually become the two new cell nuclei. The photomicrograph below captures a cell in late telophase where the new membrane is beginning to divide the cell but the nuclei have not completely reformed and cytokinesis has not yet finished. During the experiment, onion root tip have been used due to the high percentage of cells going through mitosis where most of the root growth takes place. The root tip is responsible for the downward growth of the root and therefore, is one of the regions in the plant where cells are actively dividing and elongating. The root tips contain an area called the apical meristem that has the highest percentage of cells undergoing mitosis. Because of this, the root tip is an excellent system in which to study the process of cell division and nuclear division.

HCL was applied to the onion tips root during the experiment. The function of HCL is to soften the tissue texture so that it is easy to press the root tips and put it on the slide of microscope. Besides that, HCL was also being used due to its ability to freeze the stage of mitosis. Thus, the observation can be made easily and clearly.

CONCLUSION Cell reproduction contains several staged which are interphase, prophase, metaphase, anaphase and telophase. The longest time for the cell division is interphase. At this stage, the cell will have a distinct nucleus and nucleoli which the thin threads of chromatin are duplicated. After duplication the cell is ready to begin mitosis and its starts with a step called prophase. In prophase, the chromatins thicken into distinct chromosomes and the nuclear envelope breaks open releasing them into the cytoplasm. The first signs of the spindle begin to appear. Next the cell begins metaphase, where the spindle attaches to the centromere of each chromosome and moves them to the same level in the middle of the cell. This level position is called the metaphase plate. Anaphase begins when the chromatids are separated and pulled to opposite poles. Then, the final stage is telophase. The nuclear envelope is reformed and the chromosomes gradually uncoil. From the data that have been collected during the entire of the experiment, it was conclude that the objective of the experiment have been achieves. From the additional reading, it shows that the experiment is successful.

RECOMMENDATIONS During the experiment, the colouring or dye was used for staining which is 1% Toluidine Blue. This dye is harmful if ingested and it also will stain skin and clothing. The acid provided must be handled carefully. Always wear your gloves to avoid your skin from the acid that have been used. A drop of distilled water is enough for smearing. Make sure that there is no air bubble trapped when put cover glass slide at the top of the slide that contains the onion tips root. A little pressure is needed to squash the onion tips root cell. Do not give too much pressure on it. The cell will get damage if too much pressure is added to it.

REFERENCES 1. http://biology.clc.uc.edu/courses/bio104/mitosis.htm 2. http://bio.rutgers.edu/~gb101/lab2_mitosis/index2.html 3. http://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html 4. http://www.biologyjunction.com/lab_3_sample_ap_mitosis__meiosis.htm 5. History of Biology: Cell Theory and Cell Structure - Biology Encyclopedia - cells, plant, body, function, process, animal, different, organisms, chromosomes 6. http://www.biologyreference.com/Gr-Hi/History-of-Biology-Cell-Theory-and-CellStructure.html#b#ixzz1sVQTSA8W