Lyophilization and Metabolite Extraction

The biomass collected from the different treatments was lyophilized using LABCONCO^® Freezone1^® equipment, with a vacuum of 0.017 mBar and a temperature of −35 °C for five days until the desired dryness was achieved. This allowed the obtaining of ~100 mg of total dry biomass per sample. Subsequently, the samples were macerated until a fine powder was obtained. To extract secondary metabolites, the dry biomass previously processed was used, and we analyzing the shoot (rosette) and the root separately. For this, the assisted extraction equipment Dionex ASE 350 (Accelerated Solvent Extractor) was used, using SST (stainless steel)-type cells, at a temperature of 60 °C for 5 min, a static time of 5 min, a washing volume of 30%, and a purge time of 90 s, using only HPLC (high-performance liquid chromatography)-grade methanol as the solvent, and performing two extraction cycles per sample. Once the above was done, the extracts were stored at −20 °C until later use. Subsequently, the previously obtained supernatants were concentrated with a rotary evaporator (Büchi, R100) to complete dryness, and later, by re-suspension, recovered in 300 µL of 50% methanol [20 (link),21 (link)].