Icap 7000 duo

The concentrations of metallic elements are determined after degreasing according to the method described by Munter and Grande (1981)¹⁵ . About 500 mg of each sample were weighed into a glass tube and dissolved in 6 mL of 70% concentrated HNO₃. The tubes were then placed in a digestion block (QBlock series, Ontario, Canada) and the whole was heated for 60 min at 90 °C. Then 3 mL of H₂O₂ (30%) were added to each tube and the mixture was heated at 90 °C for 15 min, then 3 mL of HCl (6 M) were added. The mineral solution in each tube was cooled and filtered and the solution was adjusted to volume of 10 mL. The concentrations of calcium, copper, iron, magnesium, manganese, sodium, and zinc were determined by inductivity coupled plasma-optical emission spectrometry (ICP-OES); (ICAP-7000 Duo, Thermo Fisher Scientific, France).

0.4 g of each ground sample in triplicates was placed in individual tubes and digested with 2.5 mL of H₂SO₄ at 100 °C for 2H, followed by adding 2 mL of 30% hydrogen peroxide (H₂O₂) carefully to each tube to complete digestion at 330 °C for 2H. Finally, after the sample solutions were cooled down, the volume was adjusted to 75 mL, and then filtered. The concentrations of NPK were determined by flow ionic analysis, Sakalar scan++ system at the Algal Biotechnology Center, Mascir, Morocco. 0.4 g of dry root and shoot were put it into digestion tube placed on aluminium heating block digester in triplicate. The digestion and mineralization of samples was done according to Gupta^{79 (link)} method. Heavy metals and mineral nutrient concentrations were estimated using inductively coupled plasma-optical emission spectroscopy (ICP-OES); (iCAP-7000 Duo, Thermo Fisher Scientific) at the Cereal and Legume Quality Laboratory, ICARDA, Morocco. Bioconcentration factor (BCF) and translocation factor (TF) were used to evaluate the potential of tomato to accumulate H.M within their root and the translocation to the above part. BCF and TF were determined following equation publishes in^{80 (link)}: $$\text{BCF}=\text{Heavy metal content}\left(\text{mg/kg}\right)\text{in the roots}/\text{Heavy metal content}\left(\text{mg/kg}\right)\text{in the soil}$$ $$\text{TF}=\text{Heavy metal content}\left(\text{mg/kg}\right)\text{in a certain above - ground tissue}/\text{Heavy metal content}\left(\text{mg/kg}\right)\text{in the roots}.$$

Mineral concentration was measured using a previously published method (20 (link)). Five hundred milligrams from each sample were weighed into a glass tube and then 6 mL concentrated (70%) nitric acid (HNO₃) was added. The tubes were then placed into a digestion block (QBlock series, Ontario, Canada) and heated for 60 min at 90°C. Three milliliters of 30% hydrogen peroxide (H₂O₂) were then added, and the samples were heated at 90°C for another 15 min, after which most of the residue was digested. Then, 3 mL of 6 M hydrochloric acid (HCl) was added to each sample. After cooling to ambient temperature, the volume was adjusted to 10 mL and then filtered. Fe and Zn concentrations were measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES); (ICAP-7000 Duo, Thermo Fisher Scientific, France). Calibration curves for Fe and Zn were made using serial dilution from 0.1 to 10 mg L⁻¹. Data validation was done using lab references and the NIST standard references.

Proteins content in ACE was determined in 96 well-cell Plate Assay protocol according to Bradford⁶⁶ method. Soluble sugar was quantified following phenol–sulfuric method^{67 (link)}. The ACE was also characterised for NPK determination using Sakalar scan++ system at the Green Biotechnology Center, Mascir, Morocco. For heavy metals and mineral nutrient concentrations were estimated using inductively coupled plasma-optical emission spectroscopy (ICP-OES); (iCAP-7000 Duo, Thermo Fisher Scientific) at the Cereal and Legume Quality Laboratory, ICARDA, Morocco.

Seeds were ground by a Cyclone mill (Twister, 10 mm–250 um, Retsch). Iron (Fe) and zinc (Zn) concentrations were measured using a modified HNO₃ and H₂O₂ method [105 (link)]. In the digestion block (QBlock series, Horiba), 0.5 g of each ground sample was placed in individual tubes and digested with 6 mL nitric acid (HNO₃), followed by heat treatments at 90 °C for 1 h. To each tube, 3 mL of 30% hydrogen peroxide (H₂O₂) was added, and sample digestion was continued by heating for 15 min at 90 °C, and then 3 mL of 6 M hydrochloric acid (HCL) was added. Once samples were cooled, the solutions were filtered and diluted to 10 mL with distilled water. The mineral content analysis was carried out by inductivity coupled plasma-optical emission spectrometry (ICP-OES); (iCAP-7000 Duo, Thermo Fisher Scientific, Waltham, MA, USA) at the Cereals and Legumes Quality Laboratory, ICARDA, Rabat, Morocco.