HPLC-DAD Analysis of Abietane Diterpenes

Lyophilized and powdered plant tissues from control, CCC-treated or transformed HR lines were extracted with acetone for 72 h at room temperature, as previously described (Vaccaro et al., 2014). Briefly, the extracts were evaporated under reduced pressure, the residues dissolved in methanol and loaded (50 μl) on a C8 column (Agilent, ZORBAX Eclipse C8 250 × 4.6 mm) for targeted high-performance liquid chromatography-diode-array detector (HPLC-DAD) analysis (Agilent 1200 Series, G1312A binary pump, G1329A automatic sample injector, G1315D diode array detector). The targeted ADs were detected at 280 nm and their concentration calculated by interpolation of the peak areas on calibration curves obtained using standard purified compounds over the range of 10–200 μg ml^-1. Content of ADs in roots was expressed as mg g^-1 of HR dry weight (DW) in small scale cultures (100 ml) or as mg L^-1 in 1 L of HR culture. Chemical structures of the most abundant S. sclarea ADs and typical chromatographic patterns for control and representative transformed HR lines are reported in Figure S5.