Whatman no 42 filters

For the extraction of heavy metals (i.e., Cd, Pb, Zn, Ni and Cu), 0.5 ± 0.05 g of soil was processed by means of a digester tube using 3 mL of hydrogen peroxide to remove the organic matter. Soil samples were then mineralized with 9 mL of hydrochloric acid (Baker Instra-Analyzed, Fisher Scientific, Waltham, MA, USA) and 3 mL of nitric acid (68%) for 40 min at 180 °C and then filtered by means of Whatman no. 42 filters (pore size: 2.5 µm). An atomic absorption spectrophotometer (SpectrAA 220 FS, Varian, Santa Clara, CA, USA) was used for the determination of heavy metals. The concentrations of heavy metals in the soil samples were determined by means of calibration curves, and procedural blanks were used to ensure the absence of contamination in the laboratory. Calibration curves were prepared by diluting a multi-Element standard mix of heavy metals in 10% HNO₃ (O2Si Smart Solutions, North Charleston, SC, USA).
The chemical analyses were carried out in triplicate.

The roots, flag leaves, and seeds collected from WT, TF1, and TF2 plants during the three different seed developmental stages were weighed (2g) and digested using a modified protocol of dry-ashing digestion (Jiang et al., 2007 ). The acidic ash solution was filtered through Whatman no. 42 filters, and the final volume was brought up to 25ml. The iron and zinc content of the clear filtrate was analysed using an atomic absorption spectrometer (AAnalyst200, Perkin Elmer, USA) with hollow cathode lamps (HCLs, PerkinElmer) at their respective wavelengths of 248.3nm and 213.9nm. Three plants from each line were selected for micronutrient concentration analysis. The concentration of iron and zinc was also measured three times.