Anthocyanin Extraction and HPLC Analysis

The anthocyanins in petals of three chrysanthemum cultivars (‘Arctic Queen’, ‘Nannong Lvdong’, and ‘Ibis Sunny’) and transgenic plants (tobacco and ‘Jinba’) were extracted by using a solution of methanol/distilled water/formic acid/trifluoroacetic acid (70/27/2/1, v/v/v/v) at 4 °C in the dark for 24 h, following a previously described method [25 (link)]. All extracts were filtered through 0.22 μm and then used for HPLC analysis, which was performed using a Shimadzu HPLC system (Kyoto, Japan) equipped with an LC-20AD pump, an SPD-M20A DAD detector, a CTO-20A column oven, a SIL-20A auto-injector, and a C18 column of Inert Sustain (4.6 × 250 mm, 3 μm, Shimadzu GL, Shanghai, China). Anthocyanin contents were measured according to the method described in our previous study [27 (link)]. Each sample was performed with three biological replicates.