Proline determination
Proline was determined according to the method described by Bates et al. (1973). Approximately 0.5 g of fresh plant material was homogenized in 10 mL of 3% aqueous sulfosalicylic acid and filtered through Whatman's no. 2 filter paper. Two milliliters of filtrate was mixed with 2 mL acid-ninhydrin and 2 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 chromophore containing toluene was aspirated, cooled to room temperature, and the absorbance was measured at 520 nm with a Shimadzu UV 1601 spectrometer. Appropriate proline standards were included for calculation of proline in the sample.