3.3.2.9 Determination of Proline
Proline contents were determined spectrophotmetrically adopting the ninhydrin
method of Bates et al. (1973). As much as 300 mg of fresh leaf samples were
homogenized in sulphosalicylic acid. To the extract, 2 mL each of acid ninhydrin and
glacial acetic acid were added. The samples were then heated to 100 ºC. The mixture
was extracted with toluene and the free toluene was quantified spectrophotmetrically
at 528 nm using L-proline as a standard.
3.3.2.10 Determination of Glycine betaine (GB)
Glycine betaine contents were estimated by the method of Grieve and Grattan
(1983). Leaves were weighed and oven dried at 80 ºC and the dried leaves were finely
ground with deionized water at 100 ºC for 60 min. GB concentration was determined
at 365 nm, using aqueous extracts of dry ground leaf material after reaction with
potassium tri-iodide solution.
3.3.2.11 Determination of total soluble sugars (TSS)
Total soluble sugars were extracted following the method adopted by
Homme et al. (1992). Sugar free residues were extracted with 1.5 N H
2
SO
4
following
the method by Naguib (1963). Soluble sugars and those resulting after
polysaccharides hydrolysis were estimated by anthrone reagent (Fairbairn, 1953).
3.3.2.12 Determination of soluble proteins
Soluble proteins were extracted according the method described by Hassanein
(1977). Water insoluble residues remaining after extraction of soluble proteins were
extracted with 1N NaOH. Soluble proteins and those resulting after insoluble residue
hydrolysis were measured by using BIO-RAD protein assay dye reagent according to
the method adopted by Bradford (1976).
3.3.2.13 Assay of antioxidant enzymes
Fully expanded leaves of plant samples were collected for enzymatic analysis
after 30 days of salt stress. The 0.5 g fresh weight of leaves was homogenized in a
pre-chilled mortar under ice-cold conditions in 5.0 ml 50 mM cold sodium phosphate
buffer (pH 7.8). After centrifuging at 13,000×g for 30 min, the supernatants were
stored at 4 ºC and used for measurements of various antioxidant enzymes.
100
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