2.2.2. Lacto-propionic orcein staining
In diploid plants, the root tips were softened by hydrolyzation
in 1N HCl solution at room temperature for 2 h and at
60 8C for 10 min, then short-rinsed in distilled water, as
described by Iwatsubo and Naruhashi (1991). The fixed root
tips (2–3 mm) of the octoploid plants were hydrolyzed in the
1N HCl solution at room temperature for 2 h, and then digested
using the enzyme mixture of 4% cellulose Onozuka RS (Yakult Co. Ltd., Tokyo), 0.3% pectolyase Y-23 (Seishin Pharmaceutical
Co. Ltd., Tokyo), 2.1% macerozyme R10, and 1 mM
ethylene diamine tetra-acetic acid (EDTA) pH 4.2 at 42 8C for
25 min. After that, they were short-rinsed in distilled water.
Root tips of octoploid and diploid plants were stained with
1.5% lacto-propionic orcein for a few minutes. Then root tips
were selected using a Pasteur pipette with a minimal amount of
staining; the root tips were expelled onto a glass slide. The root
tip was tapped with the tip of a pair of fine forceps into invisible
particles using a fresh drop of stain. 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.
2.2.2. Lacto-propionic orcein staining
In diploid plants, the root tips were softened by hydrolyzation
in 1N HCl solution at room temperature for 2 h and at
60 8C for 10 min, then short-rinsed in distilled water, as
described by Iwatsubo and Naruhashi (1991). The fixed root
tips (2–3 mm) of the octoploid plants were hydrolyzed in the
1N HCl solution at room temperature for 2 h, and then digested
using the enzyme mixture of 4% cellulose Onozuka RS (Yakult Co. Ltd., Tokyo), 0.3% pectolyase Y-23 (Seishin Pharmaceutical
Co. Ltd., Tokyo), 2.1% macerozyme R10, and 1 mM
ethylene diamine tetra-acetic acid (EDTA) pH 4.2 at 42 8C for
25 min. After that, they were short-rinsed in distilled water.
Root tips of octoploid and diploid plants were stained with
1.5% lacto-propionic orcein for a few minutes. Then root tips
were selected using a Pasteur pipette with a minimal amount of
staining; the root tips were expelled onto a glass slide. The root
tip was tapped with the tip of a pair of fine forceps into invisible
particles using a fresh drop of stain. 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.
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