An Investigation into the Mitotic Nuclear Division of Allium Sativum Root Tip Cells, and the Relative Duration of Each Phase of this Cellular Cycle

---

Results

The Raw Results

A Table of Results to Demonstrate the Relative Quantities of Cells Undergoing each Relevant Stage in the Process of Mitotic Nuclear Division

Calculating an Average Number of Cells that were Observed at Each Phase of Mitosis

Please Note: Unless explicitly stated, the analysis of results and the evaluation of the investigation will refer to the mean average of data calculated. It should additionally be noted that the values contained in the above table (especially the average results) have been rounded to whole numbers of cells to preserve the same level of accuracy and hence precision present throughout the results presented.

---

Explanations of Raw and Average Results and their Biological Reasoning and Significance

The most distinct analysis that can be made concerning the series of results obtained is that the cell counts for the varying stages of mitosis differed; hence signifying an agreement with the suggested hypothesis, and indicating that as a result of the differing nature of the biological reactions and events occurring in each phase of mitosis, the relative lengths of these aforementioned phases also differs. The difference in the lengths of these phases can be explained by the cellular and cytological events occurring in the cell, and namely their relative energetic requirements. It is this range of cellular phase lengths that will be considered and analysed; providing a variety of detailed scientific reasoning to reinforce and justify the results gained through the experimental procedure.

From the average calculated, it is clear that when considering the process of mitotic nuclear division, the most commonly appearing phase in the provided Allium Sativum meristematic root tip cells was prophase. This is evident from the average number of cells observed; at prophase 21 cells are seen, compared to 7, 7 and 15 corresponding to the additional stages .This suggests that this phase also has greatest duration; taking the greatest amount of time to complete. One possible suggestion of an explanation of this occurrence is that the wide range of intercellular transport relies heavily on the concept of the provision of ATP to the cell; a theory that is linked closely with the next point. The synthesis and activation of the required mitotic cellular components (namely the microtubule spindle fibres of the cytoskeleton) and the condensation of chromosomes also requires a great deal of factors to be present within the cell. In addition, the disassembly (or “breakdown”) of the nuclear membrane into vesicles in the cytoplasm also has energetic and enzymatic requirements. This wide range of biochemical and cytological events hence indicates that the process of prophase will have a long duration due to the length of the occurring reactions and the requirements that must be fulfilled for successful completion of the phase. The concepts identified correspond well to the gained results (and to the background research ratio), which shown that this trend in prophase is present throughout the mitotic cycle.

Unlike prophase, at metaphase the average of the observed results were relatively small in comparison to the other stages (7 cells were observed, compared to the 21 at prophase, 7 at anaphase and 15 at Telophase). As it was previously explained in the hypothesis, this is due to the scale of mitotic events occurring; there is no synthesis of new cellular components, and the movement that occurs is due to the constrictive forces of the microtubule spindle fibres; a process that has a low energetic requirement. Hence, the cell counts observed are in accordance with those predicted in the hypotheses, and those obtained during research, demonstrating the regularity of the metaphase duration throughout numerous sets of data and therefore experimental investigations.

At anaphase, the cell counts observed are relatively lower in direct comparison to those observed during the other phases of this mitotic cell division. This is due to the few biological processes that occur at this stage. A number of physical, force-related cellular changes occur during anaphase, including the division of chromosomes at the centromere to form individual chromatids, and their assisted movement to the poles of the cell. These processes are more electrochemical in nature than biological, as this movement occurs due to the interactions of a “motor protein”, kinesin, which utilises the charged nature of the microtubule to stimulate coordinated locomotion. This process requires a relatively small amount of extranuclear/extracellular substances, (including the cellular energy currency, Adenosine Triphosphate), and hence it has a short duration compared to those of the other stages; a fact that is evident from both the observed cells throughout the investigation, and from the background research collected on the mitotic phase lengths in the embryonic grasshopper cells.

The last stage of this process of nuclear cell division, Telophase, also follows the predictions made concerning the observed cellular counts, exhibiting a calculated average of 15 cells, compared to the 21 of prophase, and the 7 of both metaphase and anaphase. Again, in accordance with the cytological and energetic trend shown by the number of cells observed at the other stages of mitotic nuclear division, the number of cells (and hence the phase duration) of Telophase is due to the range and requirements of biological events that occur within the cell at this point in the division process. In respect to the other phases, Telophase has a relatively large energetic requirement, as it is concerned with the synthesis of larger molecules and genetic structures/organelles, from smaller molecules; actively changing the parent cell to 2 daughter cells. An example of one of these energy-requiring processes is the formation of a pair of new nuclear membranes, requiring the stimulated transport of component vesicles from other cellular regions. An analogous way to express the energetic requirements is to refer to Telophase being the reverse process of prophase; reforming the original nuclear and chromosomal structure, and preparing the nucleus through cellular division by cytokinesis. This concept is somewhat reinforced by the background research ratio, which demonstrates Telophase to be the second longest phase, closely following prophase. This was the trend also in the observed experimental results; demonstrating the degree to which the background cellular ratio is corroborated and reinforced by the gained results. This will be analysed further using a statistical method.

---

Notable Observations during the Implementation of Experimental Procedure

(The observations noted here will be considered for further analysis and evaluation later in the investigation)

• The ruler used throughout the investigation is accurate to ±1mm, and so the Allium root sampled had a total length of 10±1mm.
• It was necessary to note down the relative direction at which the root cap was facing on the relevant piece of equipment, using the chinagraph pencil.
• It was essential to carefully regulate the heating process of the root tip tissue when immerged in the acetic orcein/hydrochloric acid solution, to prevent damaging the sensitive tissue.
• The method in which the sample is stained and mounted on a slide has the potential to lead to some minor artefacts that could cause detriment to the analytical process when viewed at a high magnification.
• When counting the cells under the microscope at relatively high magnification, the eyepiece pointer was used to ‘scan’ along the rows of cells, ensuring that each cell counted had not been previously examined and recorded.

Graphical Representations of Results

The Average Results Gathered from Collating Class Data (Angle calculations can be found in appendix)

---

Calculation of the Percentage Averages of Cells Undergoing each Mitotic Phase

Statistical Analysis of Results

The Null Hypothesis

In order to demonstrate that the expected results and the experimental, observed results are concurrent to an appropriate degree, it is necessary to conduct some form of statistical analysis. This is where the concept of a null hypothesis is involved; in statistics, a null hypothesis is a hypothesis set up to be nullified or refuted in order to support an alternative hypothesis. When used, the null hypothesis is presumed true until statistical evidence in the form of a hypothesis test indicates otherwise. It is in this manner that the concordance of both the expected and observed results will be analysed; comparing the difference (and hence, degrees of freedom) between the cell counts. An appropriate biological and genetic model to use when comparing these cellular quantities is the Chi-Squared test; an assessment that can be directly used to evaluate the level of difference between the predicted results (which will be calculated from the suggested time ratio in the background research section), and the observed results (which will be gained through the experimental process). It is hypothesized that the null hypothesis will be accepted; demonstrating the relationship stated in the initial hypothesis; the statement of the expected phase durations.

When considering the validity of the null hypothesis suggested, it is necessary to complete a relevant statistical form of analysis to objectively form a conclusion. The primary statistical analysis that will be utilised will be the Chi-Squared Test, a common test in the field of genetic study to examine the level of difference between the predicted results, and those observed and recorder during experimental procedure.

The Chi-Squared Test: Method of Statistical Analysis

The equation used to determine the validity of the null hypothesis in the Chi-Squared test is:

Where O is the results that were collected through the process of conducting the experiment, and E is the results that should be expected, according to the background research expressed early in the investigation. From this background knowledge, it has been ascertained that the timings are: prophase 100 min, metaphase 15 min, anaphase 10 min and telophase 60 min. These timings form the ratio of 100:15:10:60 respectively, which simplifies to directly form a ratio of 20:3:2:12. It should also be noted that the observed results specified for calculation are the average results calculated from the collated class data.

To establish the actual validity of the null hypothesis, it is necessary to compare this X² value to the relevant value contained within a probability table. The probabilities referenced within these tables are tabulated in respect to the relative degrees of freedom; which is always one less than the numerical amount of categories present.

 Degrees of Freedom = Number of Classes - 1

 Degrees of Freedom = 4 - 1

 Degrees of Freedom = 3

This must then be comparatively analysed using a tolerance of P = 0.05; helping account for some variation in results caused by natural chance and probability. This may also be referred to as the 95% confidence level, i.e., 95% of repetitions (assuming a great enough sample is collected) should prove to offer the same trend of results. In this manner, any differences between the expected and observed results would be due to the random nature of the occurring reactions and interactions within the course of the investigation.

If X² > P = 0.05 (7.82), the null hypothesis should be rejected.

If X² < P = 0.05 (7.82), the null hypothesis should be accepted.

Because X² is 7.97, which is greater than P = 0.05 (7.82), the null hypothesis should be rejected.

Further Analysis and Evaluation: The Significance of the Findings of the Chi-Squared Test

Although the value returned by the Chi-Squared test is only a relatively small amount over the 0.05 tolerance value of 7.82, this is still significant in the terms of the investigation, demonstrating a rejection of the null hypothesis, and showing statistically the level of difference present between the results expected (which were calculated from a researched ratio of 100:15:10:60, or 20:3:2:12 when simplified) and the average results that were obtained through the experimental procedure completed. This level of difference has been proved to be large enough to show the lack of relationship between these two forms of results, even when the concepts of degrees of freedom and tolerance for the random variation of results has been fully taken into account.

        To expand on this concept, because X² is 7.97, and hence is greater than P = 0.05 (7.82), this signifies that in addition to the rejection of the null hypothesis, the actual hypothesis (in which the physical relative durations were predicted) must also be disregarded as incorrect. This indicates a variety of points of biological interest. Firstly, one possibility is that this difference expressed between expected and observed results is due to the cytological differences between the cells analysed, which are from not only different species of organism, but different classes of wildlife; the expected results originate from a source concerned with the phase duration of (animal) embryonic grasshopper cells, and the observed results concern (plant) Allium root tip cells. This concept will be further explored below. Another point to reference is the difference in environmental factors between the conditions present during the experimental procedure researched, and that conducted.

Furthermore, from the relatively small difference between the 0.05 tolerance level and the returned Chi-Squared X² value, it can be assumed that the environmental and biological conditions in which mitotic nuclear division occurs in, in both the medium of the expected results (i.e. in the undifferentiated embryonic grasshopper cells at 38°) and in the medium of the observed results (i.e. in the meristematic Allium root tip cells) differs in some form, but is not fundamentally different. This could be due to a number of possible factors, including the temperature in which the investigations were conducted, and the effect of the staining and preparation process on the cells mounted on the slide, in both the investigation conducted to obtain this background research, and in the experimental procedure observed in this investigation.

---

Evaluation: Limitations and Errors (See relevant column for the descending order of significance, i.e. #1 is the most significant in the investigation)

---

Overall Investigative Evaluation

As it has been expressed in the above table, the experimental procedure followed throughout the investigation has a number of flaws that would require improvement to allow the experiment to reach full levels of accuracy, reliability, precision, safety and above all, objectivity. Irrespective of these imperfections in the procedure given, overall it must be concluded that the method followed was sound, offering the means to observe and record a range of results concerning the issue that is under investigation. The procedure concerning the sampling and staining processes generally were highly proficient and capable of giving an accurate and reliable range of results that have been utilised to form a further, advanced range of analyses and investigative conclusions.

The Accuracy and Reliability of Obtained Results

Generally, it must be assumed that the accuracy of the results observed and collected throughout the course of the experimental procedure is to a high enough degree to form a range of analyses and conclusions. Although there are certain issues that must be addressed concerning the level of subjectivity in which the stages of mitosis have been observed, and concerning the repetition in counting of individual cells, these are minor flaws in the analytical process that may or may not have occurred during the process of observing the cells and collecting results. The lack of objectivity present in the procedure is almost unavoidable, and could only truly be solved either by using a range of people to analyse and come to a conclusion on the stage of mitosis the cell is currently undergoing (this concept has also been explored in the above table).

        Similarly, the idea of the level of reliability of obtained results must be evaluated to some degree. This has been explored more fully in the table, but should also be considered in terms of anomalous results. This form of evaluation is presented below.

Evaluation of Results: Identification of Experimental Anomalies

In order to compensate for possible anomalous results, a series of data has been collected; each from a different source concerning this experimental procedure. Using this method has a distinct number of advantages over using a single dataset of results; accuracy, reliability and the ability to produce an informed conclusion with basis on actual biological relationships and observed investigational results. In addition to these advantages, collating a range of data has the added benefit of allowing a more representational and hence accurate average to be calculated, in addition to making it easier to identify and explain the possible reasons behind any anomalous results present in the investigation.

Plotting the data in the form of a number of graphs increases the ease (and accuracy) at which anomalous and incongruous pieces of data can be identified. Please note that on the scatter graph, the lines connecting the results are intended purely to serve as a graphical means of observing anomalies and do not represent any form of biological trend or relationship.

(On the above chart, an anomalous result is indicated by a red circle.)

As the above graphs appear to demonstrate, there are a number of anomalies present throughout the investigation that merit further identification and explanation. Although additional slight anomalies are present, those identified on the above graph are those that depart from the concepts that are described and explained throughout the hypothesis, analysis and therefore background information concerning the reasons for the relative lengths of the stages of mitosis. Students 2 and 4 appear to have obtained results that contradict the trend expressed by the other students; the data provided for the stages telophase and metaphase are essentially reversed. This could be due to a number of factors, but a likely explanation is the student’s perception of the phase of division the cell is in; they have made a subjective decision that may be inaccurate or misrepresentational of the rest of the cell population. The results obtained concerning the prophase stage of the investigation by students 9 and 11 also seem to be erroneous in nature; the phase length does not agree with numerous other sources, with the analysis or with the expected ratio of cells in each stage of mitotic nuclear division. This again could be caused by a number of independent factors, but a likely cause of this anomaly is either some form of repetition in the observed cell counts of the other stages, or again, the misperception of the phases by the investigators. To repeat a previous point, this subjectivity in perceiving accurately the stage of mitosis that is occurring in the meristematic may be resolved by obtaining multiple opinions on the stage that is occurring, analysing each and coming to an informed conclusion on the stage of the questionable cell.

Appendix: Background Knowledge/Research

The Cell Cycle & Mitotic Nuclear Division: Abstract

Mitosis is the process of cell division in which eukaryotic cells undergo nuclear division, producing two genetically identical daughter cells. Biologically, mitosis has great significance as a means for the process of nuclear division to occur during cellular growth, repair and asexual reproduction. The nuclei of the two daughter cells formed contain the same number of chromosomes as the parent nucleus and are hence genetically identical; an ideal growth mechanism. This process of growth may occur through many tissues present throughout the organism, or may (as in the root tip area of the Allium Sativum plant) only be confined to certain meristematic regions of the organism.

Another major physical application of mitosis is the replacement of cellular structures and hence, the repair of tissues. Many organisms comprise of cells that require constant renewal, whereas others possess the capability ‘regenerate’ whole organs, body parts or even limbs. Mitosis is also the active biological process involved in reproduction by asexual means; the production of genetically identical new individuals of a species from the genetic information provided by one parent organism. In unicellular organisms, mitosis effectively forms the majority of this reproductive process; the two resultant cells are considered the new organisms, whereas in more complex, multicellular organisms, individuals may be produced through one of the numerous forms of ‘budding’; the body of which is created through mitotic nuclear division, and cytokinesis.

The cell cycle consists of four distinct phases: G1 phase, S phase and G2 phase (which are collectively known as Interphase) and M phase. M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm physically divides. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase, while cells that have permanently stopped dividing due to age or accumulated DNA damage are said to be ‘senescent’. The component of the cell cycle that is of greatest interest to the investigation is the process of nuclear division through mitosis, which is stimulated in the Interphase section of the cycle by messages received from extracellular bodies. The process of chromosomal replication upon which mitosis occurs happens during the S phase.  

---

The Relative Length of Each Stage of Mitotic Nuclear Division

“Although the stages of mitosis are necessarily shown as static events, it must be emphasized that the process is a continuous one and the names “anaphase”, “metaphase”, etc., do not imply that the process of mitosis comes to a halt at this juncture. Moreover, the stages shown are not selected at regular intervals of time, e.g. in the embryonic cells of a particular grasshopper the timing at 38°C is as follows: prophase 100 min, metaphase 15 min, anaphase 10 min, telophase 60 min.”  

These specified times essentially form the ratio that will be used throughout the investigation, and allow a range of hypotheses to be formed.

The Process of Mitosis in Allium Root Tip Cells

                

           Interphase                                    Prophase                                    Metaphase

                

           Anaphase                                   Telophase                        Cytokinesis and Daughter Cells

The above light micrographs have been included to offer some insight into the actual, physical appearance of the varying stages of mitotic nuclear division. The images above originated from a high-magnification, high resolution optical, light microscope however, and are larger and offer a greater clarity (ability to resolve two points), than should be expected from a conventional, medium power light microscope. From the slides, it is clear what stage of mitosis the cell is currently undergoing (with the possible exception of Interphase and prophase - this will be discussed later in the evaluation section of the investigation). In reality, the image should be expected to be somewhat like the micrographs provided below.

        

---

Allium Sativum & Related Histology

The region of root tissue that is of importance during this investigation is the meristematic region (“zone of multiplication”), found in the area directly between the root cap and the zone of elongation. It is in this area that there is a range of cells undergoing mitotic cellular division, of which it should be possible to identify a number of cells in each mitotic phase. A meristem is a tissue in plants consisting of undifferentiated cells (meristematic cells) and found in zones of the plant where growth can take place - the roots and shoots. Differentiated plant cells generally cannot divide or produce cells of a different type. Therefore, cell division in the meristem is required to provide new cells for expansion and differentiation of tissues and initiation of new organs, providing the basic structure of the plant body. This area of meristematic growth is protected by the root cap region, (an area of differentiated cells), and hence is not directly exposed. Unlike most multicellular animals, in which growth occurs in multiple (and often many) tissues and organs throughout the organisms, it is common in plant-like organisms for there only to be two meristematic regions of cellular growth, often these are present in the ‘root and shoot’ of the organism.

Please Note: In place of a definitive bibliography at the end of the document, full accreditation has been offered for the use of sources of information, diagrammatic representations and micrographs as a footnote at the bottom of each corresponding page.

---