Environmental Engineering Reference
In-Depth Information
2.4. Proline Content Determination
Proline content was measured according to Bates
et al
. (Bates, Waldren, and Teare, 1973)
with a little modification. Samples were grinded and homogenized in 3ml 5% (w/v)
sulphosalicylic acid extracting for 10 min at 100
℃
. The homogenate was centrifuged at
13000g for 10min, and then the supernatant was collected as extracting solution. 2ml glacial
acetic acid and 3ml ninhydrin reagent were added to 2ml of the supernatant and incubated at
100
℃
for 40min.
After cooling to room temperature, 5ml toluene was added to the reaction liquid and the
mixed liquor would layer, the red material was extracted into the toluene phase. The
absorbance of the toluene phase was measured at 520nm at last. The standard curve was
plotted according to the proline solution of known concentration.
2.5. Measurement of Glycine Betaine Content
The measurement of the glycing bataine was assayed according to Gorham
et al.
(Gorham, McDonnell, and Wyn Jones, 1982). Briefly, the plant tissues were homogenized in
3ml methanol-chloroform-KHCO
3
solution containing methanol: chloroform: 0.2mM
KHCO
3
= 12:5:1. The homogenate was incubated and shaken at 60
℃
for 20min and
centrifuged at 10000g for 10min after cooling to 4
℃
. The supernatant liquid was collected to
a new centrifuge tube and the precipitation was washed again and again. It was washed with
the same extract solution at first, and then was washed with methanol-H
2
O (1:1) solution. All
the supernatant was transferred to the above tube and 2ml chloroform and 3ml distilled water
were added. The mixed liquor was vortexed and centrifuged at 10000g for 10min. Then the
supernatant was the crude extract of glycing bataine. The crude extract was purified further
by passing through ion exchange column and distillation subsequently.
In addition, the residue was dissolved by methyl alcohol and filtered through Millipore
filter. The content of the glycine betaine was measured by HPLC (high performance liquid
chromatograph) at 195nm. The value was obtained according to the standard curve prepared
with pure glycine betaine (Sigma, USA) solutions.
2.6. Measurement of Na
+
, K
+
and Cl
-
Content
The extraction and measurement of Na
+
, K
+
, Cl
-
were performed according to Yu et al.
(Yu, 2001). The samples were killed out at 105
℃
for 30 min and baked at 85
℃
to standing
weight. The dried material was smashed and sifted into powder. 100mg powder was added to
15ml of deionized H
2
O and incubated in boiling water for 2h. The extracting solution was
centrifuged at 10000g for 20min, and then the supernatant was used for the measurement of
the ion content.
The contents of Na
+
and K
+
were detected by atomic absorption spectrophotometer (PAS-
990, PERSEE, China), while the Cl
-
was analyzed through a Cl
-
electrode (Leici, China). The
amounts ions were calculated according to the standard curve prepared with pure NaCl (for
Na
+
and Cl
-
) and KCl (for K
+
) solutions.
