Cytogenetical Techniques

                                                                                                          Professor R. S. Sangar

Chromosomes are gene-strings, bearers of heredity, that control life in its three aspects of reproduction, heredity, and development. Therefore, the ability of gene to exist, function and move or inherit in a normal way, depends heavily upon the well being of the chromosome to which it belongs. Any substantial structural or functional abnormality in a chromosome affects normal existence as well as behaviour of some or all the genes carried by it. Thus, explanation of several genetic phenomena or causes, especially those arising due to alterations at chromosomal level, can be obtained by the correlated study of behaviour of chromosomes and the genes present on them.

This correlated or combined study of various aspects of chromosomes with those of genes has led to development of a science known as cytogenetics. Cytogenetics deals with the study of heredity through the methods of cytology and genetics. It is concerned with the structure, number, function and movement of chromosomes and the numerous variations of these properties as they relate to the transmission, recombination, and expression of genes.

Since the basic theme of plant improvement programmes is to improve the characters through manipulation and modification of genes or gene combinations, a number of such modifications or manipulations can be achieved with the help of changes in structure and number of chromosomes. In support of this statement, we wish to remind the reader about the famous work of Sears (1956) in which he used translocations induced by X-radiation to transfer the leaf rust resistance from agronomically poor Triticumumbellulata (2n = 14) to T. aestivum (2n = 42) through a bridging species, T. dicoccoides. Numerous such examples of transfer of desirable genes through chromosomal manipulation techniques are available in literature. Another important application of chromosomal aberrations and aneuploids is in the area of gene mapping and chromosome map construction. The techniques for chromosomal studies at cytological level also aid in study of various aspects of euploidy, aneuploidy and chromosomal aberrations, especially in identification and characterization of these events. Two types of cytological techniques, namely, the conventional staining methods and banding pattern analysis, which are used frequently in cytogenetical studies are described in Art. 3.1 and 3.2, respectively.

STAINING METHODS

In this Art., various steps in staining methods used for chromosomal studies are discussed. The steps are:

• Pretreatment
• Fixation
• Staining
• Observations
• Pretreatment

Pretreatment is carried out for following objectives: (a) clearing of cytoplasm, (b) separation of middle lamella causing softening of the tissue or (c) bringing about scattering of chromosomes and helping constrictions and other peculiar regions become visible. The pretreatment alters the viscosity of the cytoplasm causing destruction of spindle making chromosomes free. The pressure applied during squash preparation results in the easy scattering of the chromosomes. The various constriction regions of chromosomes appear appe well defined due to differential hydration of different segments. The pretreatment may be done by putting material in specific concentration of pretreatment agents at certain temperature for some period.

Fixation

Fixation is the process by which tissues or their components are fixed selectively to a desired extent. Fixation increases the visibility and stainability of chromosomes. It also clears up the details of chromosome morphology, such as euchromatic and heterochromatic regions and the primary and secondary constrictions. A number of fixatives are available for cytogenetical studies and commonly used fixatives, Carnoy's fluid I and II can be prepared in laboratory.

Carnoy's fluid I. It can be made by mixing glacial acetic acid with absolute ethyl alcohol in 1: 3 ratio. The period of fixation required varies from 15 minutes to 24 hours in cold or at room temperature depending upon the study material. The material should be taken out from fixative at the end of fixation period and washed with 70 or 90 per cent alcohol. It is an effective fixative for all plant and animal materials for squash preparation. However, various modifications are used for specific or difficult materials where glacial acetic acid and absolute ethyl alcohol may be present in 1: 1,1:2 and 1:4 proportions.

Carnoy's fluid II. Glacial acetic acid, chloroform and absolute ethylalcohol are mixed in 1:3:6 proportions to make this fluid. Certain other modifications of the proportions such as 1:3: 4,1:1:3 and 1:1:1 may be used in special cases. The period of treatment needed ranges from 15 minutes to 24 hours. In both these fixatives propionic acid may be effectively substituted for acetic acid. A small amount of iron acetate may be added to the fixative in cases where staining is poor particularly for small chromosomes.

Processing

The processing is done to prepare the plant material suitable for proper staining and observations. Smear and squash techniques are preferred for cytogenetical studies as compared to sectioning methods. The smear or squash technique has a definite advantage over sectioning in that the entire process is rapid and more suitable for critical observations. Cell being released of its components undergoes much enlargement in volume and observations can be carried out on separated single cells. In smear, the cells are directly spread over the slide prior to fixation, while in squash sometimes special treatments are required for the separation of individual cells. For pollen mother cells, anther after fixation can be directly squashed in aceto-carmine solution, by tapping over the coverslip and then applying necessary pressure. For mitotic cells, usually softening technique is first applied. The most useful agent for softening of the tissues is N hydrochloric acid which causes hydrolysis. The hydrolysis of the material understudy is carried out at 60°C for a period of 6 to 20 minutes which varies from material to material. Hydrolysis helps in separation of cells and clearing of cytoplasm.

Staining

In order to make the chromosomes visible under the microscope, staining is necessary because other advanced methods like phase contrast and interference microscopy are neither easily usable nor available in laboratory. For squash preparation of mitotic and meiotic chromosomes, the procedures of preparation of commonly used stains are explained below:

(a) Fuchsin.The dye, basic fuchsin is used to prepare the Feulgen or Schiff's reagent which is considered as the most effective stain. The steps for preparation of Feulgen solution as leuco-basic fuchsin are outlined below:

1. Dissolve 1 g basic fuchsin in 200 ml of boiling distilled water.
2. Shake well and cool the solution to 50°C.
3. Filter the solution and then add 30 ml N HCI to filtrate.
4. Further add 3 g K2S2O5 or Na2S2O5 in filtrate solution.
5. Allow the solution to bleach for 24 hours in a tight stoppered bottle in dark.
6. Add 0.5 g decolourising carbon. Some more carbon may be added if solution is not colourless.
7. Shake well for a minute and filter rapidly through coarse filter paper.
8. Store in tightly stoppered bottle in dark and cool place.

(b) Carmine.This is one of the most widely used dyes for cytogenetical studies and is applied as aceto-carmine or propiono-carmine. Carmine is prepared from the bodies of colchineal insects and its staining property is due to carminic acid.

1. Mix 45 ml glacial acetic acid or pure propionic acid with 55 ml of distilled water.
2. Heat to boiling and add 0.5 to 1 g of carmine.
3. Shake well and boil further for 2-3 minutes for appearance of red colour.
4. Filter the solution when cool and store it in tightly stoppered brown bottle in dark and cool place.

(c) Orcein.This deep purple coloured dye is used for chromosomal study in form of aceto-orcein. Orcein is soluble in water as well as in alcohol and is obtained from orcinol by action of hydrogen peroxide and ammonia.

1. Take 45 ml glacial acetic acid and add 55 ml distilled water.
2. Add 1 gorcein to the solution and shake well.

However, due to deterioration in dilute acid, the stain is best kept as 2.2 per cent stock solution in glacial acetic acid which can be diluted to 45 per cent when required.

Observations

Once the plant material is stained, it becomes ready for taking observations. Observations are taken under microscope by inspecting the slide developed from stained material. The method of preparation of slide varies with materials and techniques used.

Study of Mitotic Cell Division

For study of mitotic cell division the root tips are preferably used, though meristematic tips or buds may also be used. The fresh root tips may be collected from the seedlings grown in pots or field. Alternatively, tips may be obtained by germinating seeds on moist blotting paper placed in petridishes which are kept in incubators for a few days maintaining suitable temperature and moisture for germination. The range of temperature for proper germination of most of the crops is between 20-30°C. When the roots develop about 20 mm in size, a 4-6 min long portion of root tip is cut for study. Several alternative ways for further processing and study of material may be followed depending upon the nature of root material and the techniques to be used. Three commonly used techniques are outlined below for this purpose.

(a) Feulgen technique. The various steps are:

1. Pretreatment.Take 4-6 mm long fresh root tips and treat them with saturated solution of p-dichlorobenzene at 10-12°C for 3-4 hours.
2. Fixation. Transfer the root tips to 1: 3 mixture of glacial acetic acid and absolute ethyl alcohol (Carnoy's fluid I) and leave them in solution for 24 hours at room temperature.

If the root tips are to be preserved for conducting study in future, take out the root tips from fixative and then transfer further to 80% alcohol for preservation and long time storage.

1. Hydrolysis. Take out the material from fixative or preservative and wash it in distilled water. Then put the root tips in N HCI at 60°C for 8 minutes for hydrolysis.
2. Staining. Transfer root tips to leuco-basic fuchsin solution (Feulgen solution) for 15 minutes to one hour.
3. Squashing. Transfer a root tip to a drop of 45% acetic acid or aceto-carmine, cut out the tip region only and discard the rest. Place a coverslip carefully over the material with help of needle so that air bubbles do not develop inside. The slide is gently heated once or twice because heating causes dehydration which deepens the colouring of chromosomes and helps in squeezing out of the air bubbles. Tap and squash applying uniform pressure with thumb on a piece of blotting paper placed on over the coverslip. The coverslip margins may be sealed using warm wax to check the entry of air bubbles later.
4. Observation. Study the slide with help of microscope.

(b) Aceto-orcein technique

1. Pretreatment. As outlined earlier in Feulgen technique.
2. Fixation. As outlined in Feulgen technique.
3. Staining and hydrolysis. Staining and hydrolysis are done simultaneously in this method. Take out the root tips from fixative or preservative and place them in a glass vial containing 2% aceto-orcein solution and N HCI mixture in the proportion of 9: 1 and heat gently over flame for 5-10 seconds without boiling the liquid.
4. Squashing. Squash the root tips in a drop of 1% aceto-orcein solution following the method outlined in Feulgen technique.
5. Observation. Study the slide in microscope.

(c) Aceto-carmine technique

1. Pretreatment.Take 4-6 mm long fresh root tips and place in 5% solution of colchicine for 2 hours.
2. Fixation. Remove the tips from colchicine solution and wash them in distilled water. Follow the procedure of fixation as explained in Feulgen technique.
3. Hydrolysis. Put the root tips in N HCl solution for 35 minutes for hydrolysis.
4. Staining. After hydrolysis root tips are placed in 2 per cent aceto-carmine solution for 4 hours.

1. The hydrolysis and staining may also be combined in this method as given above in aceto-orcein technique. Tips should be placed in 9: 1 mixture of 1 per cent aceto-carmine and N HCl solution for 5-10 seconds followed by gentle heating.

1. Squashing. Squash the root tips in a drop of 45% acetic acid or 1% aceto-carmine solution following the method outlined in Feulgen technique.
2. Observation. Study the slide in microscope.

Writer: Professor R. S. Sengar, Dean College of Biotechnology and Director Training and Placement, Sardar Vallabhbhai Patel University of Agriculture and Technology, Modipuram, Meerut.