Squash preparation of onion root tip to identify mitotic stages

Squash preparation of onion root tip to identify mitotic stages

Aim

To prepare a slide of onion root tip by squash method

Principle

Squashing technique is adopted in studying materials which are neither sufficiently soft enough to make a smear nor sufficiently hard to be sectioned without embedding in wax.  In materials like root tip, ovules, petals, etc, the pectin cementing material of the middle lamellae is removed either by acid hydrolysis or by enzyme hydrolysis.  In acid hydrolysis, 10% hydrochloric acid is normally added to a previously fixed material.  The cover glass is placed and gently pressed to spread the material.  Enzyme hydrolysis makes use of an enzyme for digesting plant material.  

The genetic information of plants, animals and other eukaryotic organisms resides in several individual DNA molecules, or chromosomes.  During DNA replication, the two strands of the DNA double helix separate, and for each original strand a new complementary strand is produced, yielding two identical DNA molecules.  DNA replication yields an identical pair of DNA molecules known as sister chromatids attached at a region called the centromere.  New cells are formed by the process of cell division which involves both replication of the cell’s nucleus (karyokinesis) and division of the cytoplasm (cytokinesis) to form two genetically identical daughter cells.  There are two types of nuclear division: mitosis and meiosis. Mitosis typically results in new somatic cells.

The phases of plant mitosis are:

Interphase: The nondividing cell is in a stage called interphase. Here the nucleus has one or more dark-stained nucleoli filled with a fine network of threads, the chromatin. Interphase is essential to cell division because the genetic material (DNA) is duplicated (replicated) during this stage.  

Prophase:  Cell division starts with prophase where thickening of the chromatin threads occurs and continues until it is condensed into chromosomes composed of two chromatids which are joined at the centromere. As prophase continues, the chromatids continue to shorten and thicken. In late prophase the nuclear envelope and nucleoli are not visible, and the chromosomes are free in the cytoplasm. Spindle fibers appear in the cytoplasm, attach to the chromosomes and pull them toward the poles of the cell.

Metaphase: At metaphase, the chromosomes are moved to the center of the spindle. One particular portion of each chromosome, the centromere, attached to the spindle. The centromeres of all the chromosomes lie at about the same level of the spindle forming the metaphase plate.

Anaphase: At the beginning of anaphase, the centromere regions of each pair of chromatids separate and are moved by the spindle fibers toward opposite poles of the spindle, dragging the rest of the chromatid behind them. The daughter chromosomes continue pole ward movement until they form two compact clumps, one at each spindle pole.

Telophase: Telophase is the last stage of division marked by a pronounced condensation of the chromosomes, followed by the formation of a new nuclear envelope around each group of chromosomes. The chromosomes gradually uncoil to form the fine chromatin network of interphase, and the nucleoli and nuclear envelope reappear.

Cytokinesis: The cell develops into two new cells. In plants, a new cell wall is laid down between the daughter cells. This division of the cytoplasm is called cytokinesis.

Materials required

Fresh onion root tip was obtained by keeping onin buds over wet cotton or wet sand kept in a petri dish for 2-3 days.

Acetocarmine stain, 1N HCl, Glass slide, Coverslip, Blotting paper, Needles, Microscope

Procedure

1. 1 or 2 mm of the root tip was cut off from the bottom using a sharp blade and placed in a watch glass containing 1N HCl.  

2. The watch glass was heated over the flame of a spirit lamp and the the root tip was kept in them warm HCl for about 5 minutes.  

3. The root tip was washed with distilled water to remove HCl

4. The root tip removed from the watch glass with forceps and placed on a slide.

5. 1 drop of 1% Acetocarmine was added onto the slide and the slide was gently warmed over flame of a spirit lamp.

6. The root tip was teared using a needle.

7. A coverslip was placed on the root tip and the extra stain was carefully removed using a blotting paper.

8. The slide was placed, coverslip down on a paper towel and using a pencil eraser or thumb, pressure was carefully applied to the coverslip in order to squash and spread the root tip tissue.  

9. The slide was observed under microscope.

10. The low power objective was used to look for thin layers of cells and then 40X power objective was used to observe mitotic stages in individual cells.

Observation

The various stages of mitosis were observed

Results

A slide of onion root tip showing the various mitotic stages were prepared.