3.5. Chromosome count analysis
The number and morphological appearance of chromosomes for particular species is almost fixed. Basic chromosome structure consists of two arms and a primary constriction known as centromere which attaches the two arms of a chromosome. The location of centromere may vary from median or centre known to be Metacentric compared with other having the centromere at very end of chromosome termed as Telocentric while those which have their centromere slightly shifted away from centre are known as Submetacentric chromosomes.
The prepared slide is viewed under a microscope and scanned for a chromosome spread. The chromosomes in the spread are examined and manually counted in order to obtain the ploidy of the sample. Place the slide under microscope and scan it properly so as to visualise separated, non overlapping chromosomes. Count and record the number of chromosomes and draw the structure of the chromosome as under microscope on a piece of paper.
On the basis of these countings and structure, normal chromosome appearance and deviations from it, if any, which have been occurred due to overlapping of chromosome, deletion or translocation can be easily studied. C-banding Giemsa staining method is followed to study regenerated callus, protoplast and suspension studying the size position and no of bands on each chromosome. In in-vitro regenerated plants mostly from callus and protoplast, the chromosome number and the karyotype is most of the times stabilized. In such cases any variation, if observed, can be due to mechanical procedure of squash prepration.
3. Flow cytometry
Flow cytometry analysis involves the estimation of total nuclear DNA content and not microscopic evaluation of chromosome number. Thus, the terms Ploidy and DNA ploidy should be used to distinguish between karyotype and DNA content analysis, respectively. The details of the flowcytometry is explained in Chapter 19.
Advantages of flow cytometric assay are:
- • Rapidity, precision and convenience (several hundred samples per working day)
• No need for pressing cells
• Non-destructive (requires small amount of tissue)
• Analysis of large populations of cells (detection of subpopulations - mixoploidy)