The techniques and steps to be followed for the chromosome preparation are described in the following:
The specimen should be actively growing so that maximum number of dividing cells are obtained. In most cases roots are used as the experimental material. For this purpose roots may be collected from germinating seeds, young plants seedling and young adventitious roots (e.g. Sugarcane). Young shoots, buds and tendrils may also be used for mitotic studies. In Case of Allium Cepa 1-2 cm long root tips of 2-3 days old should be collected. It should be kept in mind that roots should touch water, otherwise the mitotic index will be very low.
Pre-treatment of roots is an essential step for the study of somatic chromosomes. It performs several purposes: stops the formation of spindles, increases the number of metaphase cells by arresting the chromosomes at the metaphase plate, contracts the chromosome length with distinct constrictions, and increases the viscosity of the cytoplasm. The commonly used chemicals or methods for pretreatment are as follows:-
i. Colchicine (0.05 % – 0.2 %):- It was first isolated from the roots of Colchicum autumnale. It is soluble in water. Colchicine brings about a change in the colloidal state of the cytoplasm, causing spindle disturbance. Colchicine can arrest metaphase of nearly all plant organs. Duration of treatment varies from only 15 minutes to 4 hours. In case of Allium cepa 3.5 to 4 h treatment is needed. Preferably suitable temperature lies between 8-160 C.
ii. α- Bromonapthalene: – Saturated solution of Bromonapthalene is used. Duration and temperature requirements are similar for that of Colchicine.
iii. Ice cold water: Keeping the roots in ice cold water for 24-48 hours helps in contraction of chromosomes. This method is suitable for mostly monocotyledonous plants
i. Should have the property to precipitate the chromatin ii. Should have rapid penetration capability to kill the tissue instantly iii. Able to check autolysis of proteins iv. Able to prevent decomposition by maintaining an aseptic condition in which bacterial decay can’t take place The most used fixatives are: i) Carnoy’s Solution 1
• 1 part glacial acetic acid
• 3 parts ethanol (95 to 100%)
Note: This fixative is prepared fresh each time and is used for the fixation of roots and anthers. For anthers, a trace (1 g/100 mL fixative) of ferric chloride (FeCl2·6H2O) is added to the fixative as a mordant, if acetocarmine is used as a stain. The material should be kept in the fixative at least 24 h.
ii. Carnoy’s Solution 2
• 1 part glacial acetic acid
• 3 parts chloroform
• 6 parts ethanol (95 to 100%)
A modification of 1:3:4 has also been used for wheat chromosomes.
iii. Propionic Acid Alcohol Solution
This fixative is good for plants with small chromosomes. It provides clear cytoplasm and optimal staining for chromosomes.
After 24 h of fixation, roots are transferred to staining solution.
Haematoxylin itself is not a dye, and it has to be oxidised by a mordant to haematein, which is a dye, before it can be used. This process is called ripening.
Haematoxylin works better where tissues are treated with fixative and kept stored for long.