PRETREATMENT
The pretreatment of meristematic tissue serves two purposes. By preventing the formation of micotubuli, dividing cells are arrested at mitotic metaphase, thus, the number of cells in metaphase is increased. Second, the chromosomes are more contracted and shorter, making chromosome counts easier. Several pretreatments can be used for plant chromosomes:
Ice water pretreatment
Cut roots when they are approximately 1.5 to –2-cm long. Place in glass vials that contain 2-mL tap water cooled to 1°C in an ice-water bath. Add a small piece of paper with the description of the material, cap tube, and return to the ice-water bath. Fix the material after 24 h. The mitotic index after ice water pretreatment is much higher compared to the other treatments, because the cells are arrested in metaphase during 24 h rather than only 3 h as with other pretreatment procedures.
Colchicine pretreatment
Colchicine pretreatment prevents the formation of micotubuli and, thereby, arresting cells in metaphase. The lack of spindle formation also makes squashing easier. Prepare a 0.5 % stock solution by dissolving 0.25 g colchicine (Sigma C-9754) in 50 mL distilled water. Colchicine is an alkaloid that is isolated from the plant Colchicum autumnale. Store the solution in a dark bottle at room temperature.
Prepare the 0.05 % pretreatment solution by diluting the colchicine 0.5% stock solution 1:9 (v:v) with tap water. Pretreat actively growing roots for 3 h with 0.05 % colchicine at room temperature and then fix. For cereals it is convienient to make the pretreatment in 1.5-mL microcentrifuge tubes. Add approximately 1.2 mL 0.05 % colchicine solution to the tubes and place the roots together with a label in the tubes, close and leave at room temperature for 3 h. Shake the vials periodically to make sure the roots are immersed in the colchicine solution. Drain the solution on paper towels and add a freshly-prepared fixative. Store in the refrigerator until use.
The mitotic index after colchicine treatment is much lower compared to the ice-water pretreatment, but colchicine pretreatment preserves chromosome morphology better and is recommended if the chromsomes are to be analyzed by C-banding. Sister chromatids tend to fall apart after this treatment and are connected only by the centromeres. If the treatment is too long, the centromere also will split. However, no anaphase movemenet takes place and thus the sister chromatids are lie close together (ski pairs).
1-monobromonaphthalene pretreatment
1-monobromonaphthalene (alpha-monobromonapthalene) also prevents the formation of the spindle and arrests cells at metaphase. It is used in a saturated aqueous solution, which is obtained by adding a few milliliters of a monobromonaphthalene to 500 mL tap water, shaking, and letting settle. The undissolved 1-monobromonaphthalene can be reused by addiing tap water according to requirements.
8-hydroyquinoline pretreatment
Treatment with 8-hydroyquinoline does not prevent the formation of the spindle but also arrests chromosomes at metaphase. Prepare a 0.002 M solution of 8-hydroyquinoline (Sigma Q-3126) in tap water, and treat actively growing roots with this solution for 3 h at room temperature. This procedure yields a similar mitotic index compared to pretreatment with colchicine and 1-monobromonaphthalene. However, it is especially useful for identifying small secondary constrictions, because these regions become extended after treatment.
