Rocket accessions were selected from three European gene banks based upon information provided by Elsoms Seeds Ltd. (Spalding, Lincolnshire, UK). In total 19 were sourced; 2 from the Centre for Genetic Resources in the Netherlands (CGN, Wageningen, The Netherlands), 12 from the Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK, Gatersleben, Germany), and 5 from the University of Warwick Crop Centre Genetic Resources Unit (Wellesbourne, UK; formerly Warwick HRI). A further

16 commercial varieties were collected: 13 were independently sourced from retailers, 1 provided by Elsoms Seeds Ltd., and 2 from Bakkavor Group Ltd. (Bourne, Lincolnshire, UK). Three biological replicates of each accession/variety NVP-BGJ398 were germinated under controlled environmental conditions (in Saxcil growth cabinets) after being sown in a random sequence. Long-day selleck inhibitor lighting was used (16 h light, 8 h dark) at an intensity of 200 μmol m−2 s−1 (equivalent to 10,800 Lux of sunlight). Daytime temperatures were set at 20 °C and nighttime temperatures at 14 °C. Seedlings were grown for ten days in seedling trays and then transplanted to larger trays; four plants of each replicate were grown on. Plants were grown for another twenty days and then leaves from the four plants were harvested together. Sampling for each plant took approximately one minute from the cutting of the leaves

at the petiole to being placed in zip-loc freezer bags on dry ice inside a polystyrene container (with lid). For health and safety reasons it was decided that liquid nitrogen would not be used in this process. Thirty days was chosen as the optimum point of harvest as it reflects the typical number of days commercial growers grow their crop after sowing. Bags were placed in a −80 °C freezer immediately after harvest and transport was completed (<30 min). Samples were freeze-dried

Cepharanthine in batches for three days (in a Vertis Bench-top Series). Leaves from each rep were ground into a fine powder using a combination of pestle and mortar and miniature coffee grinder (De’Longhi KG49, Treviso, Italy). All solvents and chemicals used were of LC–MS grade and obtained from Sigma–Aldrich (Poole, UK) unless otherwise stated. The following method was adapted from Pasini, Verardo, Caboni, and D’Antuono (2012), Jin et al. (2009). Three experimental replicates of each biological rep were prepared as follows: 40 mg of ground rocket powder was heated in a dry-block at 75 °C for 2 min, as suggested by Pasini, Verardo, Caboni, and D’Antuono (2012), as a precautionary measure to inactivate as much myrosinase enzyme as possible before liquid extraction. 1 ml of preheated 70% (v/v) methanol (70 °C) was then added to each sample and placed in a water bath for 20 min at 70 °C. Samples were then centrifuged for 5 min (6000 rpm, 18 °C) to collect loose material into a pellet. The supernatant was then taken and put into fresh Eppendorf tubes.