OF PLANT METAPHASE CHROMOSOMES
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):
water for 24 h
for 3-6 h at room temperature or 10-24 h at 4°C
for 1-2 h at room temperature, then 1-2 h at 4°C
-bromonaphthalene saturated water for 2-6 h at room temperature.
blot material and transfer to fixative (Note 5).
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.
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.
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
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.
should not be contaminated with water, so careful blotting or an extra
rinse in fixative is advised.