3. Procedure for sample preparation for chromosome counting
3.1. Materials and equipments
Meristematic region of root-tips is the most widely used plant material but when the root material is fine and shows only primary root region in such cases other regions such as shoot-tip and young flower buds can be used.
Phase contrast microscope or high resolution microscope, waterbath, vials, staining dishes, rotar shaker, slide holder, glacial acetic acid, ethanol, colchicines, hydrochloric acid, activated charcoal, sorbitol, NaCl, basic fuchsin, Giemsa, orcein, carmine.
3.2. Fixative preparation
Carnoy's fixative is used for fixation and the reagent consist of 60% ethanol, 30% chloroform and 10% glacial acetic acid . The pre-treated material is transferred to the freshly prepared cool, carnoy's fixative for about 2-4 hrs and finally stored in deep freezers until used. For longer storage, fixative can be replaced with 70% ethanol and stored at 4°C.
3.3. Preparation of various stains
Staining of plant chromosomes prior to squash preparation is usually done by using Feulgen stain, aceto-carmine or aceto-orecin.
3.3.1. Feulgen stain preparation
1 gm of basic Fuchsin is dissolved in 200ml of boiling distilled water followed by cooling at 50ºC and filtering. Add 20 ml of 1N HCL and 1 gm of potassium metabisulphate to the filtered fuchsin solution. Leave the solution in dark for about 12 hrs. Finally add 0.5 gm of activated charcoal, shake well and store in a dark bottle at 4°C.
Feulgen staining protocol
- 1. The plant material pre-fixed in fixative is rinsed properly with distilled water
2. Hydrolyze the material in 5N HCl at 20ºC and leave for 40 mins.
3. Wash the material again with distilled water and transfer it to Feulgen stain for a duration of 1-2 hrs.
4. Wash with distilled water
5. Place the material under a phase contrast microscope, add a drop of 45% acetic acid, cover with cover slip and squash.
Root tips, Callus, Protoplast and suspension culture material can be very accurately visualised by using this staining technique, besides it is also suitable for study of somatic cell preparations of cereals, Brassica and Medicago sativa .