cell & molecular biology

Experiment 7

Introduction

• • A process by which a parent cell divides into two or more daughter cells is called cell division. Cell division is a small part of the cell cycle. In normal eukaryotic cells, the type of cell division is known as mitosis.

  • Another type of cell division is present in reproductive cells of eukaryotes and is known as meiosis. Cell cycle is mainly classified into two segments: M-phase and Interphase. Interphase is the longer period of cell division. During this phase the cell prepares for its next stage.

  • This is a period of diverse activities and these activities are a prerequisite for the next mitotic phase. Interphase is mainly divided into three phases: G1 phase, S phase and G2 phase. S phase is the period of replication. G1 and G2 are the two gap phases during which the cell grows, producing proteins and preparing the cells. These phases also have certain check points and the whole cell cycle is strictly regulated.

  • M phase of the cell cycle stands for Mitosis or nuclear division. In eukaryotes, DNA replication is followed by a process called mitosis which separates the chromosomes in its cell nucleus into two identical sets, in two individual nuclei.. Mitosis is followed by cytokinesis. The process of Mitosis is divided into four stages: Prophase, Metaphase, Anaphase and Telophase.

  • Prophase: During this stage, the chromosomes super coil, condense and become visible for first time during the cell cycle. The spindle fibers start forming. The nuclear membrane starts disintegrating.

  • Metaphase: During this stage, the spindle fibers reach and attach to centromere of each sister chromatids. The chromosomes align along the center plane of the cell. The nuclear membrane disintegrates completely.

  • Anaphase: During this stage, the centromeres start splitting and the sister chromatids begin to migrating towards the opposite poles of the cell.

  • Telophase: During this stage, the chromosomes are clustered on the either end of the cell. The nuclear membrane starts reforming. The cell plate (new cell wall) starts to form between the two daughter nuclei. This will be followed by cytokinesis.

Mitosis in Onion Root Tip

• • The genetic information of all organisms resides in the individual DNA molecules or chromosomes. An onion cell possesses eight chromosomes lwhereas human cells possess forty six chromosomes.

  • In 1842, C. Nägeli first saw chromosomes and in 1888 W. Waldeyer named them. Walther Flemming studied and named the process of cell division as mitosis. Cell division occurs rapidly in growing root tips of sprouting seeds or bulbs.

  • The most commonly used root tips in labs to study mitosis are onion, wheat, lentil, barley and alfalfa. An onion root tip is a rapidly growing part of the onion and thus many cells will be in different stages of mitosis. The onion root tips can be prepared and squashed in a way that allows them to be flattened on a microscopic slide, so that the chromosomes of individual cells can be observed easily. The super coiled chromosomes during different stages of mitosis present in the onion root tip cells can be visualized by treating with DNA specific stains, like Feulgen stain and Acetocarmine stain.

Mitotic Index

• • The percentage of cells undergoing mitosis or it is defined as the ratio of no. of cells in the dividing phase to the total number of cells observed. This will help to identify the region of most mitotic activities. Mitotic index helps us to quantify the cell division. Mitotic index decreases with increasing distance from root tip. That means gradual decrease in cell division as it moves from the zone of cell division to the zone of cell elongation. The meristematic region in the root tip is the actively growing region and thus the mitotic index is high.

Mitotic index = n/N × 100

• • Mitotic index is used to quantify the differences in cell division when environmental parameters are changed. Studies have already proved that, the plants grown in space in microgravity have a greater mitotic index than plants grown on the ground. The gravity sensing signals in the root cap are unable to send proper orientation signals which inhibit growth in the cells that are distant from the root tip and root cap junction when kept in zero gravity. This leads to mitosis in greater number of cells and plants also produce secondary roots at a high rate.

Materials required

1. Onion plant with root

2. Feulgen stain

3. 1 N HCl

4. Scissors

5. Forceps

6. Razor blade

7. Pasture pipette

8. 1.5 ml microfuge tubes

9. Dissection probe with wooden back

10. Microscopic slides and cover slips

11. Water bath

12. Light Microscope

Procedure:

1. Take the onion plant with newly sprouted roots and cut two root tips using scissors and transfer them into a plastic microfuge tube.

2. Fill 2/3 of the tube with 1N HCl using a dropper.

3. Place the tube in a 60°C water bath and incubate the tube for 12- 15 minutes.

4. Remove the tube from the water bath after the incubation.

5. Discard the HCl from the tube using a Pasture pipette to the running tap water.

6. Add some drops of distilled water into the tube and rinse the root. Then remove the water from the microfuge tube using the Pasture pipette. (Rinse the roots at least three times).

7. After the washing step add 2-3 drops of Feulgen stain into the tube with root tips and incubate the roots for 12-15 minutes. (During the incubation, the very tip of the root will begin to turn red as the DNA stains the numerous small actively dividing cells at the time).

8. After the incubation remove the stain using a Pasture pipette.

9. Again rinse the root tips with distilled water. (Rinse the roots at least three times).

10. Transfer a root from the tube to the centre of the microscopic slide and add a drop of water over it.

11. Take a razor blade and cut most of the unstained part of the root.

12. Cover the root tip with a cover slip and then carefully push down on the cover slide with the wooden end of a dissecting probe. (Push hard, but do not twist or push the cover slide sideways).

13. Observe it under a compound microscope in 10x objective. Scan and narrow down to a region containing dividing cells and switch to 40x for a better view.

Result

Reference Material

Mitosis in onion root tip:https://vlab.amrita.edu/?sub=3&brch=188&sim=1102&cnt=1

Questions

1. How many chromosomes are there in onion cells?

2. What is the use of 1 N HCl during staining procedure?

3. The actively growing part of plant is ?

4. The term mitosis was first introduced by ?