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Study of Polyploidy in Onion Root Tip Cells by Colchicine Treatment

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1K views3 pages

Study of Polyploidy in Onion Root Tip Cells by Colchicine Treatment

Uploaded by

bah292827
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Study of Polyploidy in Onion Root Tip Cells by Colchicine Treatment

Objective:

The objective of this experiment is to induce polyploidy in onion (Allium cepa) root tip cells using
colchicine treatment, to observe the changes in chromosome number, and to study the effects of polyploidy
using Canoy’s solution for fixation.

Introduction:

Polyploidy is the condition in which an organism has more than two complete sets of chromosomes. While
most organisms are diploid (2n), with two sets of chromosomes, polyploidy occurs when there is an increase
in the number of chromosome sets, leading to conditions such as tetraploidy (4n), hexaploidy (6n), and so
on. Polyploidy plays a crucial role in plant evolution and breeding, contributing to increased size, vigor, and
sometimes enhanced resistance to disease and environmental stress. It can occur naturally, as seen in many
plant species, or can be induced artificially in the laboratory.

One of the most common methods for inducing polyploidy is through the use of colchicine. Colchicine is a
widely used agent for inducing polyploidy, derived from the plant Colchicum autumnale. Currently, it is
employed in the treatment of various conditions, including gout and Behçet's disease, and is being explored
for its potential as an anti-cancer therapy. The drug functions by binding to the protein tubulin, which
inhibits microtubule polymerization and leads to their degradation. This disruption of microtubules
interferes with mitosis, ultimately resulting in cell death, as mitotic spindles are essential for cell division.
This mechanism may offer significant advantages in cancer treatment.

In this experiment, onion (Allium cepa) root tips are used as a model system to study polyploidy. Onion
root tips are ideal for such studies because their cells undergo rapid mitosis, and their large chromosomes are
easily visible under a microscope. The use of colchicine in onion root tips will allow us to observe the
induction of tetraploidy (4n) in comparison to the normal diploid condition (2n).

Fixation of the root tip cells is a critical step in preserving the cell structure at the moment of mitosis.
Canoy’s solution, a fixative composed of acetic acid and ethanol, is used to stop further cell division and
preserve the integrity of the chromosomes for accurate analysis. By staining the chromosomes with aceto-
orcein, they can be made visible under a microscope, allowing us to observe the chromosome count in
treated and untreated cells.

This experiment provides an opportunity to explore the process of polyploidy induction, understand the role
of colchicine in chromosome doubling, and observe how polyploidy influences cell division and
chromosome number in plant cells. The findings from this experiment can have important implications in
plant breeding, where polyploidy is often used to create plants with superior traits, such as larger fruit sizes,
improved resilience, or better yields.

Materials Required:

1. Onion bulbs (Allium cepa) 8. Petri dishes


2. Colchicine (1% solution) 9. Scalpel
3. Distilled water 10. Forceps
4. 70% ethanol (for fixation) 11. Glass slides and cover slips
5. 1N Hydrochloric acid (HCl) (for 12. Dissecting microscope
hydrolysis) 13. Light microscope (with high magnification
6. Canoy’s solution (Fixative: 1 part acetic for observing chromosomes)
acid + 3 parts ethanol) 14. Water bath (set at 60°C)
7. Aceto-orcein stain (for chromosome 15. Laboratory gloves and goggles (for safety)
staining)
Procedure:

Part 1: Preparation of Onion Bulbs and Colchicine Treatment

1. Soak the Onion Bulbs:


o Soak the onion bulbs in distilled water for 24 hours to stimulate root growth. After soaking,
place the bulbs in a dark, warm environment (around 20-25°C) to promote further growth of
roots.
2. Prepare Colchicine Solution:
o Prepare a 1% colchicine solution by dissolving 1g of colchicine in 100mL of distilled
water. Stir the solution well until colchicine is fully dissolved.
3. Colchicine Treatment:
o After soaking, remove the onion bulbs from the water and place them in a container with the
1% colchicine solution for 4-6 hours at room temperature. Ensure that the roots are
completely submerged in the solution.
4. Post-treatment Care:
o After the colchicine treatment, rinse the roots thoroughly with distilled water to remove any
residual colchicine.
o Return the bulbs to a dark, moist environment for 2-3 days to allow further root growth.

Part 2: Collection of Root Tips

1. Harvesting Root Tips:


o After the post-treatment period, select onion roots that are approximately 2-3 cm long, as
they are ideal for observing mitosis.
o Using a sterile scalpel, carefully cut the root tips and place them in a Petri dish containing
distilled water.
2. Control Group: lab 2 result
o In addition to the colchicine-treated roots, collect root tips from untreated onion bulbs
(soaked in water only, without colchicine). These untreated roots will serve as the control
group.

Part 3: Fixation Using Canoy’s Solution

1. Fixation with Canoy’s Solution:


o To preserve the cells and halt further cell division, transfer the root tips to Canoy’s solution
(1 part acetic acid + 3 parts ethanol) for 12-24 hours. This solution effectively fixes the cells
at the metaphase stage of mitosis.
o The fixation step stops further chromosome movement and preserves the cell structure for
accurate analysis.
2. Hydrolysis:
o After fixation, transfer the root tips to 1N hydrochloric acid (HCl) for 5-10 minutes at room
temperature. This step softens the tissues and makes the chromosomes more visible.
3. Heat Treatment:
o After hydrolysis, place the root tips in a 60°C water bath for 5-10 minutes to further soften
the tissues and prepare them for staining.

Part 4: Staining and Slide Preparation

1. Staining:
o After heat treatment, transfer the root tips to aceto-orcein stain for 15-20 minutes. This stain
will bind to the chromosomes, making them visible under the microscope.
o After staining, rinse the root tips with distilled water to remove excess stain.
2. Slide Preparation:
o Place a single stained root tip on a clean glass slide.
o Using forceps, gently squash the root tip under a cover slip. Apply gentle pressure to spread
the cells without damaging the chromosomes.
3. Microscopic Observation:
o Observe the prepared slide under a light microscope. Start with a low magnification (10x or
40x) to locate the cells.
o Switch to high magnification (100x) to observe the chromosomes in metaphase, the stage
where chromosomes are most clearly visible and aligned in the center of the cell.

Expected Observations:

 Control Group (Untreated): In untreated onion root tip cells, you should observe 2n = 16
chromosomes in metaphase.
 Experimental Group (Colchicine-treated): In colchicine-treated root tip cells, you should observe
4n = 32 chromosomes, indicating the induction of polyploidy.

Activity Questions:

1. What is polyploidy, and how does it differ from diploidy?


2. Explain the mechanism of action of colchicine during mitosis. How does it prevent chromosome
separation and result in chromosome doubling?
3. Why is Canoy’s solution used for fixation in this experiment?
o What are the benefits of using Canoy’s solution to fix the onion root tip cells? How does it
preserve the chromosomes for observation?
4. What are the expected chromosome numbers in the control (lab number 2) and experimental
groups?
5. Why is the onion root tip an ideal model for studying polyploidy?
6. How can you differentiate between diploid and polyploid onion cells under the microscope?
o What specific characteristics will you look for under the microscope to differentiate between
diploid and polyploid cells?
7. What are the potential applications of polyploidy in plant breeding and agriculture?
8. What precautions should be taken when handling toxic chemicals like colchicine and fixatives
in the lab?
o List the safety precautions necessary when handling colchicine, ethanol, and hydrochloric
acid in this experiment.
9. What would happen if the colchicine treatment was extended too long?
o Predict the possible effects of overexposure to colchicine. Could this lead to problems such as
cell death or incomplete chromosome doubling?
10. What other methods can be used to induce polyploidy in plants besides colchicine?

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