Dr1900 spectrophotometer

The total phenolic compounds (TPC) content (mg of gallic acid equivalents (GAE)·100 g⁻¹ FW) was determined in accordance to the Folin-Ciocalteu method proposed by Jałoszyński et al. [87 ] with slight alterations [14 (link),15 (link)]. The fresh and fragmented (using Thermomix) shoots and roots (~2 g) were placed in 50 mL falcon tubes and the aqueous methanol (20 mL, 80%) was added. The test tubes were sonicated (Bandelin Sonorex RK 100 H, Berlin, Germany) for 15 min and centrifuged (10 min, 4500 rpm) (Heraeus Megafuge 40, rotor TX-750, Thermo Scientific). To the supernatants (0.1 mL), the Folin-Ciocalteu’s phenol reagent (0.2 mL) and distilled water (2 mL) were added and left to stand at room temperature in the dark for 3 min. Afterwards, sodium carbonate (1 mL, 20%) was added, and the reaction mixtures were kept in the dark for 1 h. The absorbance at 765 nm was measured (HACH DR1900 spectrophotometer, four replicates).

Total nitrogen content was determined according to the persulfate digestion method⁴³ . In brief, a HI839800 reactor (HANNA instruments) and 10 mL of sulfuric acid were used to digest aerial and root tissues from tomato seedlings at 105 °C for 1–2 h. Persulfate powder pillows were added to p hydroxide digestion reagent vials with 0.5 mL of the digested samples (0.5 mL of deionized water for blank) were added. Then, vials were vigorously shaken for 30 s and placed in a heating reactor for 30 min at 105 °C and let to cool to room temperature. TN Reagent A was added, and vials were shaken for 30 s, while a 3-min reaction happened. After, TN Reagent B was added to the vials which were shaken for 15 s, while a 2-min reaction happened. Then, 2 mL were extracted and added to TN Reagent C vials. Program “394 N, Total HR TNT” was started in a DR 1900 spectrophotometer (Hach company). Finally, blank vial is inserted into the 16-mm cell holder and set as “zero”, then each sample nitrogen content is read in mg/L. The total nitrogen content estimation is converted to grams $(\frac{mg/L}{gtissue/L})$ and multiplied by 1000 to express in grams per kilogram of fresh weight.