AMITY INSTITUTE OF BIOTECHNOLOGY

AMITY UNIVERSITY MUMBAI

CELL BIOLOGY and GENETICS LAB

B. Tech Biotechnology, Semester 1
Experiment Number: 3

Title: Mitosis in Allium cepa. (Onion Root Tip)

Aim: Squash preparations for studying mitotic cell division in Allium cepa

Principle:
Eukaryotic cells pass through a series of stages known as cell cycle: Gl, G2, S
phase (synthesis) in which genetic material is duplicated, and M phase in which
mitosis partitions the genetic material and the cell divides.

✓ Gl phase - Metabolic changes prepare the cell for division at a certain point
(restriction point). The cell is committed to divide and move to S phase.
✓ S phase_ - DNA synthesis replicates the genetic material. Each chromosome now
consists of two sister chromatids.
✓ G2 phase - Metabolic changes assemble the cytoplasmic material necessary for
mitosis and cytokinesis.
✓ M phase - A nuclear division (mitosis) followed by a cell division (cytokinesis).
The period between mitosis division i.e. G1, G2 and S is known as interphase.

THE STAGES OF MITOSIS:

1) 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 in the surrounding cytoplasm. During prometaphase, some of
the fibers attach to the centromere of each pair of sister chromatids and they
begin to move towards the cell.

2) METAPHASE
The chromosomes have come to rest along the centre plane of
the cell.

3) ANAPHASE
During anaphase the centromere splits and the sister chromatids
begin to migrate towards the opposite pole of the cells.

4) TELOPHASE
 Chromosomes at either end of the cell begin to cluster together
which facilitates the formation of new nuclear membrane. This is also when
cytokinesis occurs leading to two separate cells.

REGIONS OF ONION ROOT TIP

There are 3 cellular regions near the tip of an onion root:-

1. The root cap contains all cells that cover and protect the underlying growth
region as the root pushes through the soil.
2. The region of cell division or Meristem - The cells are actively dividing but
not increasing significantly in size.
3. Region of cell elongation - Cells are increasing in size but not dividing.

Requirements: Onion root tip

Acetocarmine stain
Watch Glass
Pipette
1 normal HCI
Coverslip
Slides
Petri dish
Distilled water
Matchbox
Forceps
Burner
Tissue paper
Microscope

Procedure: 1. To soften the roots so that they can be easily spread on the slide, use scissors to
cut root tips about 1 cm long and tr fer it to watch glass.
2. Fill the watch glass with 1 normal HCI.
3. Place the watch class on a burner and allow the roots to incubate for 2 to 3
minutes.
4. After 3 minutes of incubation remove the watch glass from burner.

5. Using forceps carefully transfer the root tip to a small watch glass Petri dish.
6. Using plastic squeezed pipette carefully remove the HCI.

7. Rinse the root tip 2 or 3 times with distilled water.

8. After removing water, cover the root tip with Acetocarmine stain.

9. Incubate the roots in the stain for 2 to 3 minutes. During this time the root tip
begins to turn red as the DNA stains the numerous small actively dividing cells at
the tip.

10. Cover the root tip with cover slip and tap the cover slip with the wooden tip of
 dissecting needle. The root tip should spread out to a diameter of 0.5 to 1 cm.

Observations: PHASES TALLY CLASS TOTAL % OF

TOTAL CLASS
PROPHASE 52 660 7.88
METAPHASE 3 32 9.37
ANAPHASE 2 34 5.88
TELOPHASE 2 18 11.12
TOTAL 59 744 7.93

Result : The percentage of total class of various stages of mitosis was observed to be 7.93

Conclusion : All stages of mitosis were clearly observed in onion root tip under the microscope.