Proline, as one of the secondary metabolites, was measured according to Bates et al. (1973). Fresh leaf material (0.5 g) was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and was centrifuged at 10,000 × g for 10 min.
Afterward, 1 ml of the filtrated mixture was mixed with 1 ml of acid- Ninhydrin and 1 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1 h at 100° C. The reaction mixture was extracted with 4 ml toluene and the absorbance was determined at 520 nm with a spectrophotometer (Model Shimadzu UV- 1601, Shimadzu Corp., Japan).Proline, as one of the secondary metabolites, was measured according to Bates et al. (1973). Fresh leaf material (0.
5 g) was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and was centrifuged at 10,000 × g for 10 min. Afterward, 1 ml of the filtrated mixture was mixed with 1 ml of acid- Ninhydrin and 1 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1 h at 100° C. The reaction mixture was extracted with 4 ml toluene and the absorbance was determined at 520 nm with a spectrophotometer (Model Shimadzu UV- 1601, Shimadzu Corp., Japan).
