This involves various steps like choice of treatment, pretreatment, fixation,
staining and squashing, and material choice. Healthy root tips are taken, which
are excised late in the morning. After thorough washing, these root tips are handled directly, or can be subjected to pretreatment.
Treat the materials for cytological studies with physical and chemical agents
like colchicines, 8-hydroxy quinoline, para dichloro benzene, etc. Pretreatment
can be done before or after fixation of the materials.
Objectives of Pretreatment
- Removal of extra deposits on the cell walls, especially waxy or oily deposits.
Otherwise, these extra deposits get in the way of fixation. Chloroform is
recommended for the dissolution of the waxy substance.
- To clear the cytoplasm and make it transparent. This is done by using 1N
HCl. Certain enzymes, like cellulose and pectinases, are also used for
dissolving certain substances.
- To soften the tissues—it involves the dissolution of the middle lamella that
connects the adjacent cells. In plants, 1N HCl is used.
- To increase the frequency of nuclear division. This is done through
colchicine, which induces nuclear division. It also destroys spindles.
- To bring about the differential condensation of chromosome at metaphase.
This refers to the coiling of the chromosomes.
- Demonstration of the heterochromatin in the chromosome for this special
treatment is needed, i.e., low-temperature treatment.
These are the chemicals that bring about the arrest of the mitotic apparatus in
dividing cells and result in scattering of chromosomes. They do not affect the
cell in any other way. A very large variety of chemicals are used. The most
effective of them are colchicine, gammaxene and their derivatives, 8-hydroxy
quinoline and para dichloro benzene.
It is a poisonous alkaloid that occurs in the liliacae plant, colchicual autumnase
(Autumn lilly). It is a small plant with small corn, native to Europe and the UK.
Alkaloid is positive in underground corn and seed. Seeds are said to be the
chief source of colchicine.
Action of Colchicine
It is believed that the organization of the mitotic apparatus depends upon
balance between the elements of cytoplasm and the mitotic apparatus. Any
chemical that disturbs this delicate balance will prevent the formation of the
mitotic apparatus, and colchicine is said to have this property.
Preparation of Colchicine Solution
In the case of onion root tips the strength of the prepared solution is 0.05% (i.e.,
100 mL of H2O, 0.5 mg of colchicine). This treatment to the root tips at room
temperature varies from 1 to 1½ hours.