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Study Different Stages of Mitosis in Onion Root Tip

Mitosis was studied in onion root tip cells with and without colchicine treatment. Without treatment, normal mitotic stages were observed including aligned metaphase chromosomes and even chromosome separation in anaphase. With colchicine treatment, abnormalities occurred including uneven chromosome pulling and arrested prometaphase chromosomes in C-metaphase. Colchicine disrupts microtubules and arrests the cell cycle in mitosis.

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520 views

Study Different Stages of Mitosis in Onion Root Tip

Mitosis was studied in onion root tip cells with and without colchicine treatment. Without treatment, normal mitotic stages were observed including aligned metaphase chromosomes and even chromosome separation in anaphase. With colchicine treatment, abnormalities occurred including uneven chromosome pulling and arrested prometaphase chromosomes in C-metaphase. Colchicine disrupts microtubules and arrests the cell cycle in mitosis.

Uploaded by

fatema
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Practical in Cell Biology

and Immunology
Study of different stages of Mitosis
Aim Of The Experiment
To study and demonstrate mitosis by preparing the
mount of an onion root tip cells.
 Mitosis
 In mitosis, the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the
splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief
stages:
• Cytokinesis – Cytoplasm division
• Karyokinesis – Nucleus division
 Stages Of Mitosis
 The various stages of mitosis are:
 1. Prophase
• The process of mitosis is initiated at this stage wherein coiling and thickening of the
chromosomes occurs
• Shrinking and hence the disappearance of the nucleolus and nuclear membrane takes place
• The stage reaches its final state when a cluster of fibres organizes to form the spindle fibres.
 2. Metaphase
• Chromosomes turn thick in this phase. The two chromatids from each of the
chromosomes appear distinct
• Each of the chromosomes is fastened to the spindle fibres located on its controller
• Chromosomes align at the centreline of the cell

3. Anaphase
 Each of the chromatid pair detaches from the centromere and approaches the other
end of the cell through the spindle fibre
 At this stage, compressing of the cell membrane at the centre takes place
4. Telophase
 Chromatids have reached the other end of the cell
 The disappearance of the spindles
 Chromatin fibres are formed as a result of uncoiling of daughter chromosomes
 The appearance of two daughter nuclei at the opposing ends due to the
reformation of the nucleolus and nuclear membrane
 At this phase, splitting of the cell or cytokinesis may also occur.

Post mitosis, the next stage is referred to as interphase which is part of the cell
cycle that is non-dividing and between two consecutive cell divisions. A cell spends
most of its life in the interphase. It comprises the G1, S and G2 stages.
Why is onion root tip used to demonstrate
mitosis in this experiment?
 It is because of the meristematic cells that are situated in the tip of the roots that
render the most desirable and suitable raw material to study the different stages of
mitosis. Onion is a monocot plant. Monocotyledonous plants possess large
chromosomes that are clearly visible. Hence, their root tips are used. The period of
time taken for mitosis varies as it is dependent on the cell type and type of species.
 mitosis, the cell cycle is affected by various factors such as time and
temperature.
 Materials Required
• Compound microscope
• Acetocarmine stain
• Water
• Burner
• N/10 Hydrochloric acid
• Filter paper
• Coverslip
• Aceto alcohol (Glacial acetic acid and Ethanol in the ratio 1:3)
• Glass Slide
• Onion root peel
• Forceps
• Blade
• Watch glass
• Dropper
• Needle
• Vial
 Procedure Of The Experiment
• Place an onion on a tile
• With the help of a sharp blade, carefully snip the dry roots of the onion
• Place the bulbs in a beaker containing water to grow the root tips
• It may take around 4 to 6 days for the new roots to grow and appear
• Trim around 3 cm of the newly grown roots and place them in a watch glass
• With the help of forceps, shift it to a vial holding freshly prepared aceto-alcohol i.e., a mixture
of glacial acetic acid and ethanol in the ratio 1:3.This will fix the DNA of the root cells.
• Allow the root tips to remain the vial for one complete day
• With the help of forceps, pick one root and set in on a new glass slide
• With the help of a dropper, allow one drop of N/10 HCl to come in contact with the tip of the
root.This will soften the cell walls. Additionally, add around 2 to 3 drops of the acetocarmine
stain.
• Heat is lightly on the burner in such a way that the stain does not dry up.
• Excessive stain can be carefully treated using filter paper
• The more stained part of the root tip can be trimmed with the help of a blade.
• Discard the lesser stained part while retaining the more stained section.
• Add a droplet of water to it
• With the help of a needle, a coverslip can be mounted on it
• Gently tap the coverslip with an unsharpened end of a needle in order for the
meristematic tissue of the root tip present under the coverslip to be squashed
properly and to be straightened out as a fine cell layer
• The onion root tip cells’ slide is now prepared and ready to be examined for
different stages of mitosis
• Observe and study mitosis by placing the slide under the compound
microscope. Focus as desired to obtain a distinct and clear image.
Observations and Conclusion
 The slide containing the stained root tip cells is placed on the stage of the
compound microscope, changes taking place are noted and sketched.
 The different phases of mitosis, such as prophase, metaphase, anaphase and
telophase can be observed.

Function of acetocarmine dye.


They bind permanently to the nucleoprotein component of chromatin. That is the
reason why chromatin can be visualized by treating it with the dyes. The dyes are
very active and small quantities of them can be very effective to observe chromatin
material.
observations
Effect of colchicine on mitosis
Introduction.
Colchicine is isolated from Colchicum autumnale with anti-gout and anti-
inflammatory activities.. Colchicine binds to tubulin, thereby interfering with the
polymerization of tubulin, interrupting microtubule dynamics, and disrupting
mitosis.
The effect of colchicine on the course of mitosis is entirely specific. The prophase stages
take place normally: the chromosomes divide, condense, and assume metaphase
appearance. They are, however, not arranged into an.equatoria1 plate. Instead they are all
the time scattered over the cell in a diakinesis-like manner. This condition lasts for a long
time after- the disappearance of the nuclear membrane. The halves of each chromosome
are seen to be coiled around each other in a relational spiral (Fig. 1 a-h). This spiral is then
slowly uncoiled, and during this process the chromosomes assume a whole series of shapes
and never occurring normal.
 The minimum concentration required for a mitotic block of animal
and plant cells differs by several orders of magnitude. Thus, animal
cells are about 100,000 times more sensitive to colchicine than plant
cells (Deysson, 1975). Colchicine is usually lethal to dividing animal
cells, even at low concentrations necessary to block mitosis, yet plant
cells can survive in a state of colchicine induced mitotic arrest (C-
mitosis) for several days and return to division after colchicine is
removed (Rieder and Palazzo, 1992). Plant microtubules are more
resistant to colchicine treatment than vertebrates microtubules, due
to the low affinity of plant tubulin dimers for the drug and they can
be completely disrupted by extremely high concentrations of
colchicine (Morejohn and Fosket, 1991).
MATERIALS AND METHODS
Onion root tip cells preparation –
 Meristematic root tips of length 1 to 2 mm were harvested with a sharp
razor blade and placed immediately in fixative ethanol: chloroform: Acetic
acid (1:2:1) for 5 min and then washed with distilled water. The tips were
transferred to another fixative, Hydrochloric acid: Ethanol (1:1) for 2 min
and washed with distilled water and finally fixed in 70% ethanol. The root
tips were treated Colchicine at a concentration of 10 µg/ml for 1 hour before
observing under microscope.
 A control experiment is performed without treating the cells with colchicine.
 Sample preparation for microscope: Meristematic root tips of 3 mm long
were cut and placed on microscopic slides and gently tapped to make squash.
Sufficient acetocarmine stain was added and kept for 8 to 10 min. Coverslips
were placed over the squash and flattened the cells by rolling a glass rod
over it. Slides were observed under light microscope to observe the different
stages of mitosis.
RESULTS
 The meristematic region of onion roots with out cochicine treatment, used as
control shows a normal mitotic distribution. Most of the actively dividing cells
were in prophase and 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.
 Abnormalities in a metaphase stage were observed with the root cells treated
with colchicine. Many cells had abnormal mitotic organization with chromosomes
being pulled unevenly . Some cells exhibited C-metaphase with condensed
chromosomes arrested in their prometaphase stage for the same treatment.

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