2.3 Southern blot analysis of transgenic plants of transgenic plants
The southern blot assay was performed as described previously (An et al., 2017). Briefly, mature leaves of wild-type (WT) and transgenic plant were harvested for genomic DNA isolation. Approximately 20 µg of total DNA was digested with EcoRI overnight, followed by separating on a 0.8% (w/v) agarose gel and subsequently transferred to a positively charged nylon membrane (Roche, Mannheim, Germany). The blot was hybridized with the digoxigenin (DIG)-labeled hygromycin phosphotransferase (HPT) gene probe, which was generated by using the PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). According to the manufacturer’s instructions, hybridization, membrane washing, and detection were performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany).