Plant mitotic chromosome pressing experiment

Summary

Conventional pressing techniques for plant chromosomes can be used to observe plant chromosomes commonly used.

Operation method

Plant mitotic chromosome pressing experiment

Principle

Mitosis is a continuous and dynamic process of cellular change, but it can be artificially divided into phases and studied comparatively by its morphological changes, especially the behavior of chromosomes in the nucleus. In the natural state, a large group of cells in all phases of division are mixed together. The concept of the cell cycle must be established by careful observation to find cells with typical morphological features in all phases of the mitotic process. Cells in the meristematic tissues of plants (e.g., root tip meristematic zone, stem tip growth point, etc.) are able to increase their number through mitosis. Determine the period of time that a plant cell is in based on the changes in the form, number, and location of chromatin or chromosomes in the cell at each period of the cell division cycle. To see the chromosomes or chromatin, stain them with a basic dye.

Materials and Instruments

Rye (Secale cereale), barley (Hordeum vulgare) seeds Onion (A llium cepa) bulb
P-dichlorobenzene saturated solution Methanol Glacial acetic acid 70 % alcohol 1mol L hydrochloric acid Carbonitrile Magenta dye solution
Constant temperature incubator Constant temperature water bath Microscope Slides and coverslips

Move

1. Taking materials: first soak the seeds for a number of hours, and then transferred to a petri dish padded with moist filter paper, placed in a constant temperature incubator at 25 ℃ germination, when the young roots grow to 1 ~ 2cm to take materials; or bulbs placed in a petri dish of water, placed in a constant temperature incubator at 25 ℃, when the root tip grows to 2cm or so to take the tip of the root (apical 0.5 ~ 1cm)

2. Pre-treatment: put the root tip into a penicillin bottle containing p-dichlorobenzene saturated aqueous solution, and soak it for 3~4h.

3. Fixation: After pretreatment, the root tips were washed with water and fixed with methanol-glacial acetic acid (3:1) fixative for 6-24 h. The fixative material could be transferred to 70% alcohol and stored in a refrigerator at 4℃.

4. Dissociation: Pour off the fixative, rinse with distilled water for 2~3 times, then put into preheated 1 mol/L hydrochloric acid, dissociate in 60℃ water bath for 8~12min, then rinse with distilled water for 2~3 times.

5. Staining: put the root tip on the slide, cut off the tip 1～2mm, add drops of magenta carbolic acid staining solution for staining, 5min or so.

6. Pressing: cover the coverslip, gently tap the coverslip with the handle of forceps, and the cells of the meristematic tissue will be spread into a thin layer; then use the filter paper to absorb the excess staining solution, and use the eraser tip of the pencil to tap, so that the cells and chromosomes will be dispersed.

7. Microscopic observation: observe carefully under the microscope, look for chromosomes with clear outlines, moderate staining, dispersed and not overlapped in the middle phase of division.

8. Sealing: put the pressed chromosome specimens in the freezer, then use a blade to lift the coverslip quickly, coverslip and slide at the same time in the oven at about 37 ℃ drying. The slides and coverslips were transparent with xylene and then sealed with a drop of neutral gum to make a permanent film. The slides and coverslips were sealed with new coverslips and slides.

9. The chromosome number of rye, barley and onion is 2n=14. The better split phase was selected and photographed with a digital camera equipped on the microscope.

Caveat

Most of the cells seen in one field of view of the microscope are in the interphase of cell mitosis, because interphase occupies 90% to 95% of the entire cell cycle, and the time is proportional to the number of cells. In addition, it is often not easy to find all the cells at all stages of mitosis in a single field of view, and it is possible to move the mount slowly and look for cells from neighboring meristematic zones.

Common Problems

Significance of mitosis:

i. Maintain normal growth and development of the individual (consistency of genetic composition between tissues and cells);

ii. to ensure continuity and stability of the species (consistency of genetic composition between individuals and generations of unicellular and asexually reproducing organisms).

Source: Laboratory Course in Genetics

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