The techniques and steps to be followed for the chromosome preparation are described in the following:

1. Growing and Collection of the Specimen

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.

2. Pretreatment or Prefixation

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

• Fixation and Storage
  Fixation may be defined as the process by which tissue or their components are fixed selectively at a particular stage to desired extent. The science of chromosome study depends upon good fixative. The function of a fixative is to fix, or stop the cells at a desired stage of cell division without causing distortion, swelling, or shrinkage of the chromosomes.

3.Prerequisite of a fixative:

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.

1. Hydrolysis
   Hydrolysis is done for softening the tissue by dissolving the middle lamellae, clearing the cytoplasm and bringing about the scattering of the chromosomes in the cells. The different methods of hydrolysis are as follows:
   i. IN HCl and heating for 2-3 minutes over the flame:
   HCl hydrolysis greatly softens the cell wall, allowing a better spread of cells and chromosomes; as a result the cytoplasm becomes extremely transparent. On the other hand, it often hinders observation of the nucleolus. When the nucleolus must be observed, the hydrolysis step should be tentatively suppressed or reduced to as short a time as possible. After hydrolysis, roots should be washed carefully that chromatin staining with haematoxylin can be greatly harmed in the presence of HCl vestiges.
   ii. Enzyme treatment (e.g. Pectinase) iii. Alkali treatment (e.g. NaOH)
2. Mordanting
   Mordant is a substance which have polyvalent metal ion and forms coordination complexes with certain dyes, which then attaches to the tissue or chromosomes. It may be used for intensifying stains in cell or chromosome preparations.
   The commonest metals used in cytology are aluminium and ferric iron, both with valencies of three. Their attachment to dyes is by a covalent and a coordinate bond. This is otherwise known as chelation, and is a relatively common phenomenon. The word chelation is apparently derived from the name of the large claw, or chela, of a lobster. Gripping a metal atom by two different bonds has a fanciful similarity to gripping food with the two parts of this claw. Among the mordant, iron-alum [Ammonium iron (III) sulfate- FeNH4(SO4)2 ] solution is most used where haematoxylin is used a dye.
   The process of using mordant is called mordanting. The adherence of a dye to the tissue may be accelerated through the process of mordanting. In case where mordants are used prior to staining, they evidently modify the chromosome surface in such a manner that the dye may adheres strongly to the chromosomes. Certain metallic hydroxides form compounds with the dye which attach the dye to the tissue and are called the ‘lake’ for the particular dye.
3. Staining
   The structure and behavior of chromosomes can be made visible by staining. Staining can be done by various methods such as –
   i. Haematoxyline method:
   Haematoxylin is a natural dye which is extracted from the heartwood of the tree Haematoxylum campechianum.The genus names Haematoxylum is derived from two Greek words: haimatos which means blood, and xylon which means wood. The two words together mean “wood of blood” or “bloodwood”, a reference to the colour of the tree’s heartwood from which haematoxylin is
   extracted. Chemical formula of haematoxyline is C16H14O6.

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.

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