*5.6. Soluble Sugar, Sucrose and Starch Content*

After 14 d of treatment, the fourth trifoliate leaf of alfalfa seedlings was collected for measurement of soluble sugar, sucrose and starch contents. Dried leaf tissues from all plants were ground to obtain homogenous samples, and subsamples were used to determine content of soluble sugar, sucrose and starch.

Soluble sugar and sucrose were extracted from the powdered sample (50 mg) three times, using 80% (*v*/*v*) ethanol at 80 ◦C. The supernatants were pooled and then diluted with 80% ethanol to 25 mL for the measurement of soluble sugar and sucrose content. Soluble sugar content was determined using the anthrone–sulfuric acid reagent method and calculated based on absorbance at a wavelength of 620 nm using the UV spectrophotometer [63]. Sucrose content was measured using the resorcinol method and estimated on the basis of the absorbance at a wavelength of 480 nm using the UV spectrophotometer [64]. The residue obtained after extraction was analyzed for starch, using the perchloric acid digestion method. Following extraction, starch content was determined photometrically in the presence of anthrone–sulfuric acid reagent and estimated on the basis of absorbance at a wavelength of 620 nm using the UV spectrophotometer [63].
