2.2.3. DAPI staining
Regarding the octoploid and diploid plants, the fixed root
tips (2–3 mm) were softened using the same procedure with 1N
HCl and the enzyme cocktail solutions, as described previously.
Then root tips were expelled onto a glass slide by using a
Pasteur pipette, and treated with a drop of 60% acetic acid for
5 min. The root tips were tapped with the tip of a pair of fine
forceps into invisible particles using a fresh drop of 60% acetic
acid. A cover slip was then placed on the invisible particle; it
was warmed using an alcohol lamp for a few seconds. Then the
cover slip was tapped gently with the tips of the fine forceps and
pressed using the thumb. The chromosomes were viewed under a light microscope
(cover-018; Olympus Optical Co. Ltd.). Subsequently, the
prepared slides were frozen at 80 8C for at least 5 min; then
the cover slips were removed using a razor blade. The
specimens were air-dried, soaked for 5 min in a series of
alcohol (75, 80, and 90%) solutions, air-dried, then stained with
2.0 mg ml1 40,6-diamidino-2-phenylindole (DAPI) in the dark
at room temperature.