T60 uv vis

Samples (2 g) were macerated with 5 mL of extraction solvent (9:1 methanol: formic acid, v/v) and incubated overnight at 3 °C, without stirring. The obtained suspension was centrifuged at 3500× g for 15 min (Rolco, CM4080, Ciudad de Buenos Aires, Argentina) and the supernatant was recovered. Three independent extractions (one from each of three plastic boxes containing the carrot samples) were performed for each treatment, accession, and sampling time. Total phenolic content was determined using the Folin–Ciocalteu reagent, according to Singleton and Rossi [37 ] with minimal modifications. Briefly, 50 µL-aliquots of the extracts were mixed with 250 μL of Folin–Ciocalteu reagent, 1000 μL of Na₂CO₃ (20% w/v), and 3700 μL of distillated water. The absorbance at 765 nm was measured in a spectrophotometer (T60 UV/VIS, PG Instruments Ltd., Wibtoft, Lutterworth, UK) after 60 min of reaction. A commercial standard of chlorogenic acid (CGA) (Cayman Chemical, Buenos Aires, Argentina) was used as a reference to estimate TP levels, and results were expressed as CGA equivalents in mg kg⁻¹ of fresh weight (fw). For each extract, three determinations were performed (i.e., three technical replicates).

H₂O₂ amounts were deciphered as previously explained [147 (link)]. Freeze dried samples were crushed and, then, the powdered sample (0.3 g) was homogenized in ice bath with 5 mL 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 rpm for 20 min. The supernatant (0.5 mL) was combined with 10 mM potassium phosphate buffer (0.5 mL, pH 7.0) and 1 M potassium iodide (1 mL). The mixture was stored at 25 °C for 20 min and its intensity was recorded at 390 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
The thiobarbituric acid (TBA) test was used to estimate lipid peroxidation in leaves [148 (link)]. The reaction mixture comprised 0.5 mL of 0.1% TCA extract that was added to 1 mL of 0.5% TBA (prepared in 20% TCA). The mixture was incubated in boiling water (95 °C, 30 min), followed by cooling in ice bath (10 min). Afterwards, the mixture was centrifuged (12,000 rpm, 5 min) and the supernatant absorbance was read at 532 and 600 nm (T60 UV-Vis, PG Instruments Ltd., Wibtoft, UK).
Plant materials were analyzed for their fatty acids content with reference to a formerly published method [149 (link)]. Gas chromatography–mass spectrometry analysis was performed on an Agilent Model 7890A series (Agilent, Dover, DE, USA).