4.3.2. Determination of Glycine Betaine Concentration

Glycine betaine was determined as described by Grieve and Grattan [50]. Extracts were diluted 1:1 with 2 N H₂SO₄. Aliquots of 0.5 mL were cooled in ice water for 1 h. A volume of 0.2 mL of cold KI-I₂ was added and the mixture was stirred. Samples were stored for 16 h at 0–4 °C and then centrifuged at 10,000× g for 15 min at 0 °C. After removing the supernatant, the periodide complex was dissolved in 9 mL of 1,2-dichloroethane. Finally, the absorbance was measured at 365 nm after 2 h. Standard solutions of glycine betaine (50–200 µg·mL⁻¹) were prepared in 1 N H₂SO₄.