1. Introduction

Cytology is a scientific discipline that deals with the study of cells, their physiological and structural properties. Knowing the components of cells and how cells undergo divisions is the main aim of cytology. It helps to understand various chromosomal mutations and genetic rearrangements. The gross structural changes in chromosomes can be evaluated by cytological procedures, which involve staining the chromosomes and evaluating the morphology at metaphase stages of mitosis and meiosis. Cytological staining is particularly used to predict the total content of DNA or ploidy of an individual cell within the mixed population of cells. The determination of ploidy level is an essential technique in plant breeding, genetics and plant tissue culture. The ploidy analysis can be performed either by chromosome counting or by flow cytometry.

 

2. Chromosome counting

Counting of the chromosomes is the most definitive way to differentiate ploidy of an organism. In this technique, smears and squashes of the tissues are prepared which are then observed under the microscope for studies on chromosome counting, morphological differences and karyotyping. Although this technique retains the morphological information, it is tedious and requires longer time for analyzing. The sample taken should be of small quantity for better analysis. The meiotic chromosome smears can be prepared using pollen mother cells isolated from immature anthers dissected from flower buds. Alternately, mitotic chromosomes can be counted from root-tip or axillary-bud or shoot-tip squash preparations. A schematic representation of squash preparation is shown in Scheme 16.1 . Following stains are used to enhance the contrast of the microscopic images:

 i. Propidium iodide is a fluorescent nucleic acid dye which binds only to double stranded nucleic acids. As a counter stain, propidium iodide stains the nucleus light to dark red in colour.

 ii. Hoechst stains are a family of blue fluorescent dyes used to stain DNA. These dyes are excited by ultraviolet light at around 350nm, and emit blue / cyan fluorescent light around an emission maximum at 461nm.

 iii. Aceto-carmine is a saturated solution of carmine (1-2%) prepared in 45% glacial acetic acid by boiling gently. It is used especially for the rapid staining of fresh unfixed chromosomes. Stained chromosomes are distinguished from other organelles, as it gives the chromosome a red colour.

 iv. Aceto-orcein is extracted from two species of lichens, Rocella tinctoria and Lecanora parella. Orcein is also available in synthetic form, but the natural form is preferred for chromosome analysis because it gives better contrast. Orcein is used in the form of 1% solution prepared in 45% glacial acetic acid by boiling gently.

 v. DAPI (4',6-diamidino-2-phenylindole) is a cationic fluorescent dye, which specifically binds to adenine – thymine rich DNA. It is used for cytofluorometric determination of the DNA base content in chromosomes.

 vi. Acridine orange is a nucleic acid and also a fluorescent cationic dye. When bound to DNA it shows an excitation maximum at 502 nm and an emission maximum at 525 nm (green).