MITOSIS IN THE ROOT MERISTEM OF ONION, Allium cepa

Lagua JS, Pacimio JO, Ringor, LDS, Salum, LJ, Simon MJC, and Tambasen, KAA

Group #5: BioBabes: Cellfie Lords

I. Introduction
From a fertilized egg, vital processes that produce the 200 trillion somatic cells
that now make up your body, and the same processes continue to generate new cells
to replace dead and damaged ones, are mitosis and cytokinesis.
Mitosis, the division of the genetic material in the nucleus, is usually followed
immediately by cytokinesis, the division of the cytoplasm. One cell has become two,
each the genetic equivalent of the parent cell.
Mitosis is conventionally broken down into five stages: prophase,
prometaphase, metaphase, anaphase, and telophase. Overlapping with the latter
stages of mitosis cytokinesis completes the mitotic phase (Reece, et al., 2014).
In the laboratory activity, six inquisitive scholars ventured the capabilities of
onion (Allium cepa) to demonstrate the stages of the cell cycle in a living tissue. They
also prepared the specimens (root meristem) that were used in the activity. The
students observed the specimen by staining the root with Fuchsine and transferred it
to a glass slide in order to observe mitosis using a light microscope.

II. Materials and Methods

 Onion roots  Forceps
 Stains  Pipet
 Test tube  Tissue paper
 Scissors  Microscope
 1M of HCl  Scalpel
 Dissecting probe

A. Growing the Roots

A set-up with container filled with water up to the brim and the onion fixated
at the top of the container was prepared by the scholars. The set-up was set aside
for a few days in order to allow the roots to grow. These are the roots hat have
been previously harvested and treated with a fixative to stabilize the cells.
B. Softening the Roots
The roots were softened by the scholars so that it can be spread on a glass
slide. Using scissors, two (2) toot tips of 1cm were cut and then were transferred
into a test tube. The test tube was then carefully filled with 1M HCl such that it is
2/3 full. The tube was then placed in a water bath and the roots were allowed to
incubate for 10 minutes. After the incubation period, the tube was removed from
the water bath.
C. Rinsing the Roots
The root tips were carefully transferred to a small petri dish using forceps.
Using a pipette, the HCl was removed from the tube and was transferred to the
“discard flask”. The roots tips were then rinsed three (3) times with water.
 D. Staining the Chromosomes
After removing the water from the third rinse, the root was covered with the
Feulgen stain/Toluidine blue/Fuchsine/Acetocarmine. The roots were then
incubated in the stain for 10 minutes. During the 10 minute incubation, the roots
began to turn red as the DNA stained the numerous small cells that were dividing
as the tip. Using a pipette, the Feulgen stain was carefully removed and discarded
in the flask as the excess stain were blotted using tissue paper.
E. Preparing the Root Tip Squash
A root was transferred to the center of the clean glass slide and a drop of
water was added. Using a scalpel, the students cut off most of the unstained parts
of the root, and discarded it. The root tip was covered with a cover slip. The cover
slide was then pushed down with the wooden end of a dissecting probe. It was
made sure that it was not twisted nor push sideways. The root tip was spread out
to a diameter about 0.5-1 cm.
F. Observing Mitosis
The slide was scanned under the low power (10x) objective. The region that
had a large nuclei had the cells displaying mitosis. The microscope was switched
to high power (40x) objective to attain closer and clearer observations.
Observations and data were recorded.
III. Results

Table 1. Mitosis observed in root meristem of Allium cepa (Smith, n.d.).

Prophase Metaphase Anaphase Telophase

Figure 1. Onion root Figure 2. Onion root Figure 3. Onion root Figure 4. Onion root
cell in prophase cell in metaphase cell in anaphase cell in telophase
stage. stage. stage. stage.
In prophase, the In metaphase, the In anaphase, the In telophase, there is
individual cells' chromosomes aligned a redistribution of the
chromatin begins to attached to chromosomes daughter
organize and kinetochore began to separate chromosomes that
condense into thick microtubules align in into two sister arrived at the spindle
strands of the metaphase plate chromatids as a full- poles into chromatin
chromosomes. at some place in the pledged but in a less
These center of spindle chromosome and condensed form.
chromosomes each poles. Each sister each migrate in Cytokinesis or the
having their chromatids' opposite poles by division of cytoplasm
centromere then kinetochore is the shortening of by cleavage is also
forms specialized attached to kinetochore under way at thus
protein complexes kinetochore microtubules. The phase. Each
called kinetochores. microtubules coming separation is also daughter cell will be
The mitotic spindle from opposite poles. aided by non- covered by a nuclear
begins to form The kinetochore envelope as it starts
 outside the nucleus photomicrograph microtubules which to reform resulting to
at opposite ends of above depicts the elongates the cell as new cell nuclei. The
the cell and aligned it lengthens. At the photomicrograph
eventually extends chromosomes at a end, the two cells above captured an
as spindle plate, ready for have and equal and early telophase
microtubules. These separation. complete set of wherein the
complexes attaches chromosomes. The chromosomes was
to some spindle photomicrograph being redistributed to
microtubules which above illustrates the a chromatin.
then be termed initial stage of
kinetochore anaphase wherein
microtubules. At this chromosomes are
point the nuclear just becoming
envelope also completely
disrupts the separated.
photomicrograph
above shows the
earliest form of
chromosome as it
coils up to a more
condensed
structure.
IV. Discussion

Features of the Mitotic Phases

Mitosis is a type of cell division in which one cell (the mother) divides to produce two
new cells (the daughters) that are genetically identical to itself. In the context of the cell
cycle, mitosis is the part of the division process in which the DNA of the cell's nucleus is
split into two equal sets of chromosomes (Raven, 2014). Mitosis is consisting of four
basic phases: prophase, metaphase, anaphase, and telophase.

Figure 5. The four stages of mitosis (The cell cycle, n.d.)

Mitosis can be easily observed in onion roots because growth there is greatest
therefore the number of duplicating cells is relatively high and it is easy to find various
stages of mitosis. Each stage can be observed and determined because of a visible
feature. The table below shows the distinguishing visible feature of each stage of mitosis.

Table 2. Visible feature of each stage of mitosis

Stage Visible Feature

Prophase A condensed structure of chromosomes and the nuclear envelope is

still visible that will eventually disappear.

Metaphase Aligned chromosomes in one plate - called metaphase plate

Anaphase Chromosomes separated into half

 Telophase Two sets of condense chromosomes being distributed into chromatin
to form a new cell

In onion root cells, it has 16 diploid chromosomes meaning it has eight pairs of
chromosomes (Johnson, n.d.).

Continuity of Mitosis

Mitosis is not a series of separate events but a continuous process because all
cell changes not all at the same time but at different times showing that mitosis is a multi-
step continuous process and are constantly dividing. Also because the process keeps
repeating it is a continuous process that does not end (Friedman, 2013).

Each stages in mitosis are interdependent on each other. A stage depends on a

completion of whatever stage that comes before it showing that each stage needs the
other to occur.

Processes during the Interphase

The cell cycle has two main phases - interphase and mitosis. Many of the cells
are not undergoing mitosis, rather they are in a stage called interphase. Interphase takes
much longer time than mitosis and it is where preparations for mitosis takes place.

In interphase prior to the onset of mitosis, a process of DNA replication occurs. DNA are
the carriers of genetic instructions used in the growth, development, functioning and
reproduction of all living organisms.

Interphase is made-up of three phases - G1 phase, S phase and G2 phase along

with a special phase called G0. G1 phase is where the cell makes proteins necessary for
the DNA replication. S phase or synthesis phase is where the cell starts to replicate its
DNA and G2 phase is where cell organelles are copied in preparation for mitosis.

Figure 6. Cell in early G1 and late G2 phases (The cell cycle, n.d.).

As cell division ends, the cells will exit the cell cycle and enter the G0 phase. G0 is
the phase of cell cycle, often termed as the resting phase and in this phase the cell is
arrested and does not proceed further for cell division (Jain, 2016). G0 phase maintains
the standard number of cells in a specific tissue and decreases the probability of a cell
becoming cancerous.
Processes during the G0 Phase
Some scholars tend to say that once a cell enters the G0 phase, it is already
“resting” but it is incorrect to say that these cells are "resting" because the cells is
functioning normally and actively preparing for next cell division. The cells are still
metabolically active and going through transcription and translation. Transcription is the
process by which the information in a strand of DNA is copied into a new molecule of
messenger RNA (mRNA) while translation is a step in protein biosynthesis wherein the
genetic code carried by mRNA is decoded to produce the specific sequence of amino acids
in a polypeptide chain (Carroll, 2014).

V. Conclusion

In conclusion, mitosis is a type of cell division in which the cell divided in order to
produces two new daughter cells which are genetically identical from the parent cell. The
laboratory activity used the root meristem of an onion (Allium cepa) and observes the
occurrence of mitosis using a light microscope.

The results show that: in prophase, the chromatin beings to condense into thick
strands of chromosomes; in metaphase, the chromosomes attached to kinetochore
microtubules align in the metaphase plate at some place in the center of spindle poles; in
anaphase, the aligned chromosomes separate into two sister chromatids and migrate in
opposite poles accompanied by the shortening of kinetochore microtubules; and in
telophase, there is a redistribution of the daughter chromosomes that arrived at the spindle
poles into chromatin, alongside the occurrence of cytokinesis.

It also helped the scholars to further understand the concept of mitosis wherein
each phase poses a different feat that helps the scholars identify the onset of the next
phase. Furthermore, the activity also showed the continuity of mitosis due to the
interdependence of the steps and the constant division as a result of repetition of the cycle.
The processes during the interphase and the G0 phase was also presented to the scholars.

From the laboratory activity, a sources of error could be identified. The error that
the group has identified is the water used while culturing the root meristem of the Allium
cepa. Considering that tap water was used, the water may contain a lot of impurities that
results to mutation of the cell of the root meristem. Similar to past researches, when tap
water was used, the cells mutate and therefore do not perform mitosis properly which
results to improper observations of the behavior of the meristem in the laboratory.

Lastly, the group opted to use pictures of the occurrence of mitosis in the root
meristem of Allium cepa from the internet because the process of mitosis was not clear
and visible during the observation in the laboratory. Nonetheless, the errors were indicated
and were jotted down in order to prevent further occurrences. To sum up, the objectives
of the activity were met and the scholars were able to apply the concepts of mitosis learned
from the classroom discussions.
VI. References

Bailey, R. (2018, October 12). The Stages of Mitosis and Cell Division. Retrieved November
5, 2018, from https://www.thoughtco.com/stages-of-mitosis-373534

Carroll, J. (2014). Transcription/ DNA transcription. Retrieved from

https://www.nature.com/scitable/definition/transcription-dna-translation-87

Davidson, M. (2004). Mitosis in onion root tips. Retrieved from https://sciencing.com/happens-

interphase-cell-cycle-20315.html

Editors (n.d.). Meristem. Retrieved November 5, 2018, from

https://biologydictionary.net/meristem/

Friedman, S. H. (2013). Mitosis. Retrieved from https://quizlet.com/38169751/mitosis-flash-

cards

Jain, G. (2016, April 8). What is the importance of the G 0 phase of the cell cycle?. Retrieved
fromhttps://www.quora.com/What-is-the-importance-of-the-G_0-phase-of-the-cell-
cycle

Johnson, C. ( n.d. ). Onion diploid number of chromosomes. Retrieved from

https://www.chegg.com/homework-help/questions-and-answers/1-onion-diploid-
number-16-many-chromosomes-visible-cell-anaphase-meiosis-asiatic-lily-dipl-
q1589029

OpenStax College, Biology. (2016, May 27). The cell cycle. In OpenStax CNX. Retrieved
November 4, 2018, from http://cnx.org/contents/GFy_h8cu@10.53:1tJ55Ot6@7/The-
Cell-Cycle.

Raven, P. H., Johnson, G. B., Mason, K. A., Losos, J. B., and Singer, S. R. (2014). How cells
divide. In Biology (10th ed., AP ed., pp. 187-206). New York, NY: McGraw-Hill.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B.
(2011). The cell cycle. In Campbell biology (10th ed., pp. 232-250). San Francisco,
CA: Pearson.

Smith, M. (n.d.). Onion Root Mitosis. Retrieved November 4, 2018, from

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-
uk.org.uk/mag/artnov04macro/jronionroot.html

VII. Appendices

Definition of Terms
 Plant Tissue - a cell or a group of cells dividing, to give rise to large number of cell,
which is collectively referred as tissues. They are structurally and functionally similar
to these cells.
 Meristem - is the area of tissue from which new growths are formed. The plant cells
are continually proliferating and are not yet differentiated at it.
 Root Apical Meristem - found below ground, is responsible for the growth and
development of a plant’s roots. It produces cells in a bilateral direction, meaning that it
yields two types of tissues at the same time.
  Cell Cycle - an ordered series of events involving cell growth and cell division that
produces two new daughter cells. Cells on the path to cell division proceed through a
series of precisely timed and carefully regulated stages of growth, DNA replication,
and division that produces two identical (clone) cells.
Phases of Cell Cycle
 Interphase – the phase where cell undergoes normal growth processes while also
preparing for cell division. In order for a cell to move from interphase into the mitotic
phase, many internal and external conditions must be met.
 Mitotic Phase - is a multistep process during which the duplicated chromosomes are
aligned, separated, and move into two new, identical daughter cells. The first portion
of the mitotic phase is called karyokinesis, or nuclear division. The second portion of
the mitotic phase, called cytokinesis, is the physical separation of the cytoplasmic
components into the two daughter cells.

Stages of Interphase
1) G1 Phase (First Gap) – it
is the first stage of interphase
because, from a microscopic
aspect, little change is visible.
However, during the G1 stage, the
cell is quite active at the
biochemical level. The cell is
accumulating the building blocks of
chromosomal DNA and the
associated proteins as well as
accumulating sufficient energy
reserves to complete the task of
Figure 7. The cell cycle consists of interphase and
replicating each chromosome in
the mitotic phase (The cell cycle, n.d.).
the nucleus.
2) S Phase (Synthesis of DNA) - DNA replication can proceed through the mechanisms
that result in the formation of identical pairs of DNA molecules -sister chromatids - that
are firmly attached to the centromeric region. The centrosome is duplicated during the
S phase. The two centrosomes will give rise to the mitotic spindle, the apparatus that
orchestrates the movement of chromosomes during mitosis. Centrioles help organize
cell division. Centrioles are not present in the centrosomes of other eukaryotic species,
such as plants and most fungi.
3) G2 Phase (Second Gap) - the cell replenishes its energy stores and synthesizes
proteins necessary for chromosome manipulation. Some cell organelles are
duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic
phase. There may be additional cell growth during G2. The final preparations for the
mitotic phase must be completed before the cell is able to enter the first stage of
mitosis.
 Figure 8. The Different Phases of Cell Cycle (Bailey, 2018).

Stages of Mitotic Phase

1) Mitosis or Karyokinesis - the phase of the cell cycle where chromosomes in the nucleus
are evenly divided between two cells. When the cell division process is complete, two
daughter cells with identical genetic material are produced. It is divided into a series of
phases (prophase, prometaphase, metaphase, anaphase, and telophase).

i. Prophase - During prophase, the chromosomes supercoil and the fibers of the
spindle apparatus begin to form between centrosomes located at the pole of
the cells. The nuclear membrane also disintegrates at this time, freeing the
chromosomes into the surrounding cytoplasm.

ii. Prometaphase - During prometaphase, some of the fibers attach to the

centromere of each pair of sister chromatids and they begin to move toward
the center of the cell.

iii. Metaphase - At metaphase the chromosomes have come to rest along the
center plane of the cell.

iv. Anaphase - During anaphase, the centromeres split and the sister chromatids
begin to migrate toward the opposite poles of the cell.

v. Telophase - During telophase, the chromosomes at either end of the cell

cluster begin to cluster together, which facilitates the formation of a new nuclear
membrane. This also is when cytokinesis occurs, leading to two separate cells.
One way to identify that telophase has begun is by looking for the formation of
the cell plate, the new cell wall forming between the two cells.

2) Cytokinesis - or “cell motion,” is the second main stage of the mitotic phase during which
cell division is completed via the physical separation of the cytoplasmic components into
two daughter cells. Division is not complete until the cell components have been
apportioned and completely separated into the two daughter cells. Although the stages of
 mitosis are similar for most eukaryotes, the process of cytokinesis is quite different for
eukaryotes that have cell walls, such as plant cells.

G0 Phase
Not all cells adhere to the classic cell cycle pattern in which a newly formed daughter
cell immediately enters the preparatory phases of interphase, closely followed by the
mitotic phase. Cells in G0 phase are not actively preparing to divide. The cell is in
a quiescent(inactive) stage that occurs when cells exit the cell cycle. Some cells enter
G0 temporarily until an external signal triggers the onset of G 1. Other cells that never
or rarely divide, such as mature cardiac muscle and nerve cells, remain in
G0 permanently.

DOCUMENTATION:

Figure 9 | The materials needed for the Figure 10 | The Fuchsine was being poured to
experiment were being arranged. the specimen (root tip) using a laboratory
dropper.

Figure 11 | The test tube containing the root Figure 12 | The HCl was being poured to the
tips were being heated or incubated. test tube containing the root tips about 2/3 full.
Figure 13 | The root tips on the glass slide Figure 14 | An image of the root tip/s
was being observed through the microscope. observed through the microscope.