Reagents The following steps are carried out in small glass or plastic containers (5-10 ml) or 1.5 ml microcentrifuge tubes. Use generous amounts of solutions: typically 1 ml per specimen. Material is transferred carefully by clean forceps or a pipette (Note 2). To accumulate metaphases, treat excised root tips (5-20mm long) or other material with one of the metaphase arresting agents as follows (Note 3):
ice water for 24 h
colchicine for 3-6 h at room temperature or 10-24 h at 4°C
8-hydroxyquinoline for 1-2 h at room temperature, then 1-2 h at 4°C
a -bromonaphthalene saturated water for 2-6 h at room temperature.
- Quickly blot material and transfer to fixative (Note 5).
- Fix for at least 16 h at room temperature. If fixed material is to be kept (up to several months), leave for 2 h at room temperature and then transfer to new fixative (or 70% or 96% ethanol) and store at -20°C.
The response to the metaphase accumulation reagents is different from species to species and has to be established by trial and error. Some guidelines for choosing are given; Dolezel et al . (1996) discusses alternative reagents, including spindle poisons used a herbicides.
- It is very important not to expose seedlings, roots and plants during germination and metaphase-arrest to chemicals and fumes, particularly fixatives (e.g. in a cold room also used for chemical storage) and to use clean labware with tight lids (disposable plastic is ideal), clean forceps, and distilled water.
- Root tips from germinating seeds, and plants grown in controlled conditions, often show waves of cell division that may follow internal or environmental rhythms (e.g. light) or correlate with root length. At certain times, there may be no divisions at all, so it may be helpful to make several fixations.
- Representative times are given. For best results fix material after different times of treatment, experiment with different reagents and check the mitotic index by making chromosome preparations. Treating material for too long in arresting agents, particularly colchicine, results in over-condensation of the metaphase chromosomes which might be desirable for counting chromosomes, but not for in situ hybridization where spatial resolution along chromosomes is wanted.
- Fixative should not be contaminated with water, so careful blotting or an extra rinse in fixative is advised.