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The contents of total ASA and total glutathione were determined
by dissolving 0.1 g homogenates in 1ml of 5% mphosphporic
acid solution. The extract was then centrifuged
at 13,000 × g for 10 min at 4 ◦C. The supernatant was used
for total ASA and total glutathione assay. Both total ASA
and the reduced ASA contents were determined spectrophotometrically
at 525 nm using different reacting solutions; the
former was a potassium phosphate buffer (pH 7.4), dithiothreitol
and N-ethlmaleimide, the latter was trichloroacetic
acid, o-phosphoric acid, -dipyridyl in 70% alcohol and
FeCl3 [38]. Total ASA and the reduced ASA contents were
calculated from a standard curve plotted from a known concentration
of ASA. In addition to total ASA and reduced
ASA contents, we also calculated the ratio of oxidized ASA
to reduced ASA as an indication of the redox state of ascorbate
in the roots. The content of oxidized ASA (Vitamin
C) was calculated by subtracting the reduced ASA content
from the total ASA content.
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