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).