Smartspec plus spectrophotometer

The MDA concentration was estimated according to the method of Health and Packer [60 (link)]. Fresh tubers (0.2 g) were homogenized with 10% trichloroacetic acid (1 mL) and centrifuged at 10,000 rpm for 10 min. Thereafter, 1 mL of the supernatant was mixed with 20% trichloroacetic acid containing 0.25% thiobarbituric acid and was heated at 95 °C for 30 min., quickly cooled in an ice bath, and then centrifuged again at 10,000 rpm for 10 min. The absorbance of the supernatant was read at 532 and 600 nm (SmartSpecTM Plus spectrophotometer, Bio-Rad, Hercules, CA, USA). The MDA concentration was calculated using an extinction coefficient of 155 mM⁻¹cm⁻¹ and expressed as nmol g⁻¹ FW.
Pro was determined according to Bates et al. [61 (link)]. Fresh tissue of stored tubers (0.5 g) was extracted with 2.5 mL of boiled water. Subsequently, 2.0 mL extract was mixed with an equal volume of ninhydrin solution (1.25 g ninhydrin dissolved in 30 mL glacial acetic acid, and 20 mL of 6 M phosphoric acid) and glacial acetic acid. The samples were then incubated at 100 °C for 1 h and the reaction was terminated by cooling the tubes in an ice bath. After cooling, Pro was determined at 522 nm (SmartSpecTM Plus spectrophotometer, Bio-Rad, Hercules, CA, USA).

The methodology developed by Velikova et al. [40 (link)] was used to determine hydrogen peroxide (H₂O₂). Fresh leaf material (200 mg) was crushed under liquid nitrogen followed by homogenizing in 1.5 mL 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000× g for 15 min at 4 °C. A 0.5 mL aliquot of supernatant was supplemented with 0.5 mL of 10 mM potassium phosphate (pH 7) and 1 mL of 1 M KI, followed by vortex mixing. The H₂O₂ concentration was determined by reading the absorbance at 390 nm (SmartSpecTM Plus spectrophotometer, BioRad) and using a standard curve.

The herbicide tolerance of bacterial strains was assessed in triplicate by observing their growth on liquid LB medium supplemented with different Sekator^® Turbo herbicide concentrations (0.25 mL L⁻¹ (Hb50), 0.375 mL L⁻¹ (Hb75), and 0.5 mL L⁻¹ (Hb100)). The bacterial growth in liquid LB media was monitored by measuring the optical density at 600 nm using SmartSpecTM Plus spectrophotometer (Bio-Rad, Hercules, CA, USA). The significant growth of bacterial strains in the presence of herbicide during 24 h at 37 °C (180 rpm) was considered as herbicide tolerant.