1. Disruption of Mitosis in Onion Root cells
by Colchicine Treatment
Samirah Mariam Boksmati
California University of Pennsylvania

2. Introduction:
Normal cell division is due to the Cell Cycle. The Cell Cycle’s function is to duplicate
chromosomal DNA, which subsequently segregates into two genetically identical daughter cells.
The four major phases of this process include G1 phase, S-phase, G2 phase, and M phase. The
two most important phases are S-phase, which is where DNA replication occurs, and M-phase,
which is where mitosis occurs. Mitosis can also be broken down into several phases including,
prophase, metaphase, anaphase, and telophase. Regulation of the Cell Cycle is crucial, and
without it, damage to the genes could not be detected or repaired, and cells would divide
uncontrollably, causing cancer. (Wikipedia.org) Certain alkaloid drugs have been studied with
the hopes of inhibiting this uncontrolled division of cells. Colchicine is a drug isolated from a
plant known as Colchicum autumnale, or “meadow saffron”. Colchicine is currently used in the
treatment of gout, pericarditis, familial Mediterranean fever, and Behcet’s disease, and is being
investigated for its use as an anti-cancer drug. It has the ability to inhibit microtubule
polymerization by binding to the protein tubulin, which leads to microtubule degradation. This
disrupts mitosis and leads to cell death because microtubules make up the mitotic spindles,
which allow eukaryotic cells to separate their chromatids during cell division. Without
microtubules, the cell could not divide. (Wikipedia.org) This could be very beneficial in the
treatment of cancer because colchicine treatment could possibly stop cancerous cells from
dividing. The goal of this experiment was to see what effects, if any, colchicine had on the
mitosis of onion root tip cells. I hypothesize that there will be a much higher percentage of cells
undergoing stages of mitosis in the untreated cells than there will be in the colchicine treated
cells. Untreated cells should be able to divide normally; while colchicine treated cells will die of
microtubule degradation. I will test this hypothesis by creating squashes of untreated root tips
and of root tips treated with Colchicine. I will examine the slides to determine the number of
cells in each stage of mitosis. Each of 10 groups will complete the experiment on their own and
all data will be combined at the end. In a previous experiment that tested the effects of colchicine
on onion root mitosis, evidence showed that “The meristemic region of onion roots without
colchicine treatment, used as a control shows a normal mitotic distribution….most of the actively
diving cells were in prophase and a few in metaphase, anaphase and telophase stages of cell
division. The metaphase chromosomes were lined up at the equator and were pulled evenly
towards spindle poles for the cells at anaphase. No abnormal chromosome was observed.

3. Abnormalities in a metaphase stage were observed with increased concentration of
colchicine…many cells had abnormal mitotic organization with chromosomes being pulled
unevenly……shows an inhibition of mitosis in onion root tip comparable with that of standard.”
(Bharathi et al, 2006) I predict that the class data will show similar abnormalities.
Methods:
Pre-treatment:
First, the untreated onion root tips were preserved. They were fixed overnight in Carnoy’s
solution and then transferred to 70% Ethanol Alcohol. For colchicine treated onion root tips, we
soaked them for two hours with 0.05% aqueous colchicine solution, and then immediately
transferred to Carnoy’s solution overnight and then transferred to 70% Ethanol Alcohol.
Preparing root tip squashes:
One watch glass was filled with 2-4 ml of HCL and another with 2-4 ml of Carnoy’s solution.
Forceps were used to transfer 2 root tips to the HCL solution for 4 minutes. (2 root tips were
used in case the first slide was not prepared correctly.) The root tips were then transferred to
Carnoy’s solution for 4 minutes. I then placed 1 root tip on a slide and cut the very tip off with a
razor blade. (The tip is the narrow, pointy end with an opaque white color.) The tip measured
1mm after cutting. The rest of the tip was discarded. The tip was then covered with 1 drop of
toluidine blue solution and left for 2 minutes. Excess toluidine was blotted off and the root tip
was covered with a drop of water. The coverslip was then added. In a folded paper towel, I took
the eraser end of a pencil and pressed it firmly on top of the root tip beneath the cover slip.
The same process was repeated for the colchicine treated cells.
Examination:
The slides were first examined with 10x and then 40x objectives. The slides were then examined
with the 100x objective. The number of total cells in the field of view was counted, and then the
number of mitotic cells visible in the same field was counted. The mitotic index was calculated.
Then I determined how many mitotic cells were in each stage of mitosis, and calculated the
percentage of cells in each mitotic stage.

4. Results:
Figure 1: The Number of treated and untreated Cells in Varying Stages of
Mitosis
Figure 1: The number of Onion Root Cells in each of the stages of mitosis. According to the
data, there were more Colchicine-treated cells in Prophase, but less in every other stage of
mitosis. However, most treated and untreated cells alike were not dividing.
0
50
100
150
200
250
300
350
400
450
500
550
Prophase Metaphase Anaphase Telophase # of Mitotic
Cells
Total Cell
Count
NumberofCells
Stages of Mitosis
Untreated Onion Root Cells Vs. Colchicine-treated
Cells
Untreated Cells
Colchicine-treated Cells

5. Figure 2: The Percentage oftreatedand untreated cells in eachstage of
Mitosis
Figure 2: The percentage of Untreated and Colchicine cells in each stage of mitosis and the
percentage of cells not dividing. There are more mitotic cells that have been treated with
Colchicine.
Table 1: The Percentage of Treated and Untreated cells in Varying Stages of
Mitosis
Percentage of Cells in Each Stage Untreated Cells Colchicine-Treated Cells
% Prophase 10.1593 15.8582
% Metaphase 1.7928 1.3059
% Anaphase 1.7928 0.7462
% Telophase 1.3944 0.3731
% Cells not Dividing 84.8605 81.7164
Table 1: The percentage of Untreated and Colchicine-treated cells in each stage mitosis, plus the
percentage of cells not dividing.
0
10
20
30
40
50
60
70
80
Prophase Metaphase Anaphase Telophase Cells not Dividing
%ofCells
Stages of Mitosis
Untreated Onion Root Cells Vs. Colchicine-treated
Cells
Untreated
Treated

6. Table 2: The Number of Treatedand Untreated Cells in eachstage of Mitosis
Type of Cell Prophase Metaphase Anaphase Telophase
# of
Mitotic
Cells
Total
Cell
Count
Untreated Cells 51 9 9 7 76 502
Colchicine-treated
Cells 85 7 4 2 98 536
Table 2: The number of Untreated and Colchicine-treated cells in each stage of mitosis in
relationship to the total number of Mitotic cells and the total number of all cells.
Mitotic Index of Untreated Cells: 76/502=0.1513
Mitotic Index of Colchicine-Treated Cells: 0.1828
According to Figure 1, most treated and untreated cells were not dividing. However, most cells
that were undergoing mitotic activity were in prophase. In Figure 2, a greater percentage of
colchicine-treated cells were dividing compared to untreated cells. From the data In Table 1, it is
clear that there has been an effect due to the Colchicine. The treated cells are able to enter
prophase, but are less successful at moving on to later stages of mitosis. There is a steady
decrease with the amount of cells in each later stage of mitosis. In Table 2, we see that 98 out of
536 total treated cells were experiencing mitotic activity, or 18.3%, while only 76 out of 502
total untreated cells were undergoing mitosis, or about 15.1%.
Discussion:
The data shows that Colchicine had an effect on mitosis. Colchicine did not inhibit mitosis
overall, or cause any obvious significant damage to the cells, however the percentage of cells
going through different phases of mitosis has changed. You can compare the percentage of
untreated cells in Anaphase to be 1.7928% compared to only .7462% for the treated cells. Also,
1.3944% of untreated cells are going through telophase, while only a mere 0.3731% of the
treated cells are in telophase. It looks as if Colchicine-treated cells are able to enter into
prophase, but since their microtubules have become weak, they are unable to advance to later
stages of mitosis. I will have to reject my original hypothesis. If Colchicine would have
inhibited mitotic activity, then the mitotic index for the Colchicine-treated cells would have been
much less than the mitotic index for the untreated cells, but instead we have a greater mitotic
index of the colchicine-treated cells, of 0.1828, compared to only 0.1513 for the untreated cells.
There should have been less treated cells undergoing mitosis than untreated cells. Although, it
should be noted that there is a significant difference in the stages of mitosis the treated cells are
in. In future experiments, more than one concentration of colchicine should be used, and it might

7. be useful to compare data from more than one type of root. Also, having the data collected by 10
different groups could pose an issue because researchers may have varying methods of counting
cells/determining what cells are in each stage of mitosis, and this could lead to incorrect
readings. More tests will certainly need to be conducted in order to have the most reliable data.
Overall, if it is proven that colchicine does in fact inhibit mitosis, this could have drastic benefits
for cancer patients because treatment may be able to slow or inhibit the growth of cancerous
cells.

8. Bibliography:
Wikipediacontributors."Colchicine." Wikipedia,TheFree Encyclopedia.Wikipedia,The Free
Encyclopedia,21Feb.2013. Web.10 Mar. 2013.
Wikipedia contributors. "Cell cycle." Wikipedia, The Free Encyclopedia. Wikipedia, The Free
Encyclopedia, 1 Mar. 2013. Web. 10 Mar. 2013
Bharathi, P.,Phiilomina, D.,Chakkaravarthi, S., 2006. Antimitotic Effect of Colchicine from Six
Different Species of Gloriosa in Onion Roots (Allium cepa).