Method: The proline content in conifer needles was determined using the ninhydrin colorimetric method. Plant samples were collected and weighed to 500 mg for reaction. Five milliliters of 3% sulfosalicylic acid (5 μL/mg fresh weight) were added, and the samples were ground on ice. The mixture was then boiled in a water bath for 10 minutes. After filtration, 2 mL of the supernatant was collected, and a reaction mixture was prepared with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin. The centrifuge tubes were well mixed, and the reaction mixture was incubated in a 96°C water bath for 1 hour before being terminated by placing it on ice. Toluene was used to extract the samples; 4 mL of toluene were added to the reaction mixture, and the sample was vortexed for 30 seconds and left to stand for 5 minutes to allow separation of the organic and aqueous phases. The toluene-containing chromophore was transferred to a new tube, and the absorbance at 520 nm was measured using toluene as the reference. The concentration was expressed in mg/g FW or micromoles/g FW.

Standard Curve: Successful Toluene Extraction

Needle Extraction Failure