| Popis: |
The chromosome number can be established in cells during mitotic or meiotic cell division. Counting of mitotic chromosomes is easier and faster. Root tips are the most convenient source of mitotic cells. When roots are not available, young buds, leaves or callus can be used. Cytological procedures of chromosome preparation and staining are modified depending upon plant species. Nevertheless, basic principles for handling the mitotic chromosomes of all plant species are similar and consist of collection of material, fixation and chromosome staining. Critical for chromosome counting is proper chromosome preparation. It is important to obtain sufficient number of well spread metaphase plates and good physical separation of the chromosomes. There are two types of chromosome preparation: one is staining of intact root meristem or other tissue prior to squashing and other is preparation of chromosome samples before staining. Method of chromosome staining applied for ploidy level analysis depends on plant species and chromosome size. Generally, methods presented here can be used for all species. For example Feulgen staining, which is based on reaction of Schiff s reagent with the aldehyde groups of DNA, previously exposed by acid hydrolysis, is effective in each species but in plants with small genome size (low amount of nuclear DNA) can give weak staining result. For species with small chromosomes such as Arabidopsis, Beta, Brassica, Oryza, Solanum and others, fluorescent staining with DAPI is specially recommended. The method is simple and quick, can be applied for each species but required a fluorescent microscope. Both, Feulgen method and DAPI stain only chromosomes while cytoplasm remains clear. Detail modifications for some plant species are included in protocols for DH. |
