*2.2. In Vitro Culture and 2D Root Morphology Analysis*

The in vitro culture was conducted in a growth chamber at a constant temperature of 21 ◦C and a photoperiod of 16 h light (40 μmol photons m−<sup>2</sup> s−1)/8 h darkness. Seeds of 55 cultivars were sterilized in 2 mL microtubes after sequentially adding 70% (*v*:*v*) ethanol for 10 min, 20% (*v*:*v*) hypochlorite solution for 5 min and rinsing twice with sterile water. Then, seeds were plated on square (12 cm × 12 cm) dishes filled with a modified Murashige-Skoog (MS) medium containing 0.010 mM KNO3 + 9.090 mM KCl (hereby referred as low nitrate treatment, N−) or 10 mM KNO3 (high nitrate, N+) and 2% (*w*:*v*) plant agar (Duschefa, Haarlem, the Netherlands) [40,41]. Seeds were stratified during two days at 4 ◦C. After three days, seedlings were transferred to a fresh growth medium of identical composition. The top portion (~3 cm) of the agar medium was cut and four

seedlings were inserted between the gel matrix and the bottom of the plate (Figure 1a). Three plates containing four seedlings per cultivar and per N condition were prepared. Seedlings grew during four days prior to harvest, corresponding to seven days after germination. Root and shoot organs were separated and dried at 60 ◦C. The pooled dry weights of four organs from one plate were measured. For root morphology observation, the dishes were scanned at a resolution of 400 dpi. The scans were annotated using the RootNav image analysis software [42].

**Figure 1.** Culture systems used to grow oilseed rape cultivars. (**a**) Vertically placed agar plates (size: 12 cm × 12 cm); (**b**) Hydroponic containers (capacity: 8 L); (**c**) Column filled with soil (d: 5 cm, h: 30 cm).
