## 2 Materials and Methods  
### Seed Selection, Soil Characteristics and Experimental Design  
For the experiment, seeds of the Middle European ecotype of _Solanum nigrum_ L. ssp. _nigrum_ were collected in the City Botanical Garden in Zabrze (Upper Silesia, Poland) (50\({}^{\circ}\)17\({}^{\prime}\)45.81\({}^{\prime\prime}\) N 18\({}^{\circ}\)45\({}^{\prime}\)52.37\({}^{\prime\prime}\) E). These seeds, originated from plants grown freely in the natural unpolluted soil (C\({}_{\text{soil}}\) = 0.22 mg kg\({}^{-1}\) Cd) as weeds, were used as a primary experimental material (N0). Besides, the seeds from the same plants but adapted to high Cd pollution through growing in the soil treated with 50 mg kg\({}^{-1}\) Cd in the previous year (first-generation adapted variety A50) were selected for a comparative study. The experiment was carried out at the Institute of Environmental Engineering, Polish Academy of Sciences in Zabrze. The topsoil (0-20 cm) was collected from the area of seeds origin and analyzed according to the standard methods of soil analysis [19]. The soil was slightly acidic (pH 6.57), total organic carbon TOC 16.73 g kg\({}^{-1}\), total N 0.73 g kg\({}^{-1}\), available P 15.33 mg kg\({}^{-1}\), available K 189.05 mg kg\({}^{-1}\), Cd 0.22 mg kg\({}^{-1}\), Pb 17.15 mg kg\({}^{-1}\), and Zn 37.88 mg kg\({}^{-1}\). Cadmium (Cd) and other PTEs--Potentially Toxic Elements (Pb, Zn) represented a low natural level. The tested soil was brown soil according to the World Reference Base for Soil Resources WRB (IUSS Working Group WRB 2022) [20] classification, of a loamy texture. After sieving through a 1 mm sieve and homogenizing, the soil naturally dried in the open air was divided into 20 kg DW portions, next spiked with CdCl\({}_{2}\) 2.5 H\({}_{2}\)O (added as a solution of superior pure reagent >99.99%, Sigma-Aldrich, St. Louis, MO, USA) and equilibrated for three months. In total, an untreated control sample (T0, 0.22 mg kg\({}^{-1}\) Cd) and four Cd treatments (with nominal concentrations 10, 20, 30, 50 mg kg\({}^{-1}\) Cd) were applied. The actual measured Cd concentrations in the homogenized and equilibrated soil are given in Table 1. The research was conducted as a pot experiment, in three independent biological replicates. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**T-Treatments mg Cd kg\({}^{-1}\)** & **0** & **10** & **20** & **30** & **50** \\ \hline \(\mathbf{Cd_{soil}}\) & **0.22 \(\pm\) 0.08** & **10.52 \(\pm\) 0.87** & **20.44 \(\pm\) 1.96** & **30.09 \(\pm\) 1.52** & **50.49 \(\pm\) 2.29** \\ \hline \hline \end{tabular}  
* **N0** & 2.06 \(\pm\) 0.41 aA & 2.02 \(\pm\) 0.62 aA & 1.59 \(\pm\) 0.48 aA & 1.34 \(\pm\) 0.85 aA & 1.31 \(\pm\) 0.15 aA \\
**A50** & 2.00 \(\pm\) 0.28 aA & 1.89 \(\pm\) 0.08 aA & 1.43 \(\pm\) 0.37 aA & 1.38 \(\pm\) 0.70 aA & 1.41 \(\pm\) 0.33 aA \\ \multicolumn{6}{c}{**Bms\({}_{\text{S}}\)Biomass of shoot (g pot\({}^{-1}\) DW)**} \\
**N0** & 13.00 \(\pm\) 3.51 aA & 11.90 \(\pm\) 2.11 aA & 11.00 \(\pm\) 1.29 aA & 8.26 \(\pm\) 3.96 aA & 9.65 \(\pm\) 0.68 aA \\
**A50** & 12.01 \(\pm\) 1.80 aA & 10.86 \(\pm\) 3.69 aA & 9.98 \(\pm\) 1.51 aA & 9.08 \(\pm\) 4.40 aA & 11.38 \(\pm\) 1.01 aA \\ \hline \hline \end{tabular}  
* Note: (1) T (0 = 50) mean soil treatments with Cd (0, 10, 20, 30, 40, 50 mg Cd kg\({}^{-1}\), respectively; (2) Data in each line marked with the same capital letters are not significantly different at \(p\) < 0.05; Data in each column/section marked with the same lowercase letters are not significantly different at \(p\) < 0.05.  
\end{table} 
Table 1: Effects of different Cd treatments on the biomass of the Middle-European ecotype of _S. nigrum_ L., non-adapted (N0) and adapted to Cd stress (A50). The homogenized and equilibrated soil of each Cd treatment was put into pots \(\varphi\) = 25 cm and H = 20 cm. (2.5 kg per pot, with 3 g (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) Sigma-Aldrich, St. Lois, MO, USA, added as fertilizer). For the experiment, _S.