4.4.4. Determination of Proline Content

Proline was determined using Bates et al. [98] method as follows; 0.5 g of fresh plant shoot were homogenized in 4 mL of 3.0% Sulphosalicylic acid. Then the homogenate was centrifuged for 10 min at 1000 rpm. To 1 mL of the supernatant 2 mL of acid Ninhydrin reagent and 2.0 mL of glacial acetic acid were added in a test tube, Then the mixture was incubated in a water bath at 100 °C for 60 min. then the mixture was cooled suddenly in an ice bath. After cooling, 4 mL of toluene were added to the solution mixture and vortex. The chromophore containing toluene (upper layer) was transferred to a new test tube. Finally, the absorbance was read at 520 nm using a spectrophotometer and Toluene as a blank. The concentration of proline was determined using the standard curve and expressed as mg g⁻¹.