Onion root tip squash preparation to study the mitotic stages -Practical

Aim:To prepare a temporary squash preparation of onion root tips.

Materials required:

1. Fixed onion root tips.

2. Acetic alcohol.

3. Acetocarmine stan.

4. n/10 HCl or 2N hydrochloric acid.

5. Spirit lamp.

6. Watch glass.

7. Clean glass slides.

8. Coverslips.

9. 70% alcohol.

10. Filter paper.

11. Nail polish and a compound microscope.

12. Conical flask.

13. Beakers etc.

Principle:

Cell division in flowering plants takes place in particular regions of the plant called meristems. Cells in

meristems are not specialized for any particular function and divide repeatedly by mitosis. Some of the

daughter cells remain meristematic others cease dividing and become differentiated into appropriate cell

types depending on their position. The root tip meristem is usually a denser white and more rounded than thecut end. Chromosomes in root tip tissue are made visible with the stain. Dividing cells (if present) will show up clearly with chromosomes in different forms according to the stage of mitosis. Individual chromosomes (as tightly-coiled threads) are visible during anaphase. The links between the cellulose walls of plant cells are broken down by the treatment with hydrochloric acid. This ensures that the stain can penetrate the cells and allows the tissue to be squashed out one cell thick.

Procedure:

1. For the growth of onion root tips place the onion bulbs with their root side down in contact with water in a beaker or conical flask.

2. After 3 to 4 days when the roots grow upto a length of 2 to 3cm remove the onion bulbs and cut the root tips and transfer them to a solution of acetic alcohol (acetic acid 1 Part and ethyl alcohol 3 parts v/v.

3. Acetic alcohol acts as a fixative to fix the roots.

4. Keep root tips in this fixative for 12-24 hrs.

5. After fixation transfer these root tips in 70% alcohol until they are used for squash preparation.

6. Transfer the root tips from fixative or 70% absolute alcohol into a watch glass and wash them with water.

7. Drain off water with the help of a pipette and add a few drops of n/10 HCl. Wait for 10 minutes at room temperature or heat for a minute over a spirit lamp flame.

8. After hydrolysis drain off the HCl and wash the root tips in water.

9. After hydrolysis drain off the HCl and wash the root tips in water.

10. Remove the water and cut the root tips to retain only the meristematic region and remove the debris from the slide/watch glass.

11. Take one onion root tip on the clean slide and add a drop of 1% acetocarmine on the root tip and

carefully place a cover slip over it.

12. Gently heat it on a spirit lamp and then coatl, repeat this process 4-5 times.

13. Place the slide between the filter paper and gently press the cover slipdown. Using the flat bottom of a pencil slowly tap on the filter paper(you can tap with your finger also) to obtain a uniform spread of the material.

14. Seal the edges of the cover slip by applying nail polish.

15. Observe the slide under the compound microscope.

Observations:

Different stages of mitosis are as follows:-

1. Prophase:-

i. Appearance of chromosomes due to dehydration and unwindingof chromatin reticulum.

ii. Chromosomes become shorter and thicker.

iii. Each chromosome. Begins to appear as two chromatids with asingle centromere.

iv. Nuclear membrane and nucleolus slowly disappears.

2. Metaphase:-

i. Chromosomes tend to arrange themselves in the middle of thecell called the equatorial plate.

ii. When observed carefully, the sister chromatids of thechromosomes can be seen.

3. Anaphase:-

i. Centromere splits and a single chromosome with two chromatidsbecomes two independent chromosomes.ii. Daughter chromosomes moves towards the opposite poles.

4. Telophase:-

i. Chromosomes reach the poles and undergo coiling and formchromatin reticulum.

ii. The nuclear membrane and nucleolus reappears.

iii. Two daughter nuclei are formed at the poles.

5. Cytokinesis:-

i. Cell plate formation.