Carotenoid extraction

Between 5 and 20 mg of powder, depending on the intensity of CSR color, was transferred to a mortar and hydrated. A scoop of Celite powder was added and the mixture crushed with acetone and filtered through a sintered glass funnel. Following three washes with acetone, a sequential transfer of 1/5 of the acetone extract volume was partitioned to 50 mL petroleum ether in a separatory funnel. Slowly, 300 mL of ddH₂O was added, allowing the two phases to separate.

The lower aqueous-acetone phase was discarded by washing five times with ddH₂O. The solvent phase was collected in a volumetric flask after having passed through anhydrous sodium sulfate. This procedure was employed to generate HPLC-DAD profiles and for quantification of specific carotenoids. An alternative extraction procedure was applied for separate samples used for carotenoid quantification by spectrophotometer. This procedure includes hydration of the sample (100 to 2000 mg powder) with ddH₂O, addition of petroleum ether followed by four to six pulses with a Polytron, centrifugation (4000 rpm, 4 °C, 20 min), sonication to disperse any micelles formed, and collection of the solvent phase. This was then filtered through anhydrous sodium sulfate and its volume adjusted with petroleum ether until λmax and maximum absorbance for each sample at 300 to 600 nm were obtained. A total of 23 landraces were processed for different analytical procedures, including total carotenoid content and HPLC-DAD analyses depend on the question addressed in particular experiments.