2.5 Flavonoids measurement
Flavonoids contents in tobacco tissues were determined as described in our previous studies (Z. Wang et al., 2018; Z. Wang et al., 2020). In brief, fresh tobacco tissues were frozen in liquid nitrogen, and then freeze-dried till constant weight. The tissues were grinded to powder, and suspended with 1.5 ml 80% ethanol (with 0.012 g/L vitexin as internal standard). The mix was ultrasonicated for 30 min, and then centrifuged (14,000 rpm) for 10 min. A 0.22 μm membrane was adapted to filter the supernatant, which was used for flavonoids content determination by HPLC-UV. The experimental parameters are as follows: chromatographic column-ACQUITY UPLC®BEH Phenyl (1.7 μm 2.1mm×150 mm, Waters); injection volume-1μl; gas temperature-350°C; flow-0.3 mL/min; wave length-230 nm, 260nm, 360 nm, and 570 nm; mobile phase-A 100% water, B 100% acetonitrile; gradient elution-8 min 85% A+15% B, 5 min 58% A+42% B, 0.01 min 100% B, 3 min 95% A+5% B, 18 min stop.