Genomic DNA Extraction and Southern Blot Analysis

Total genomic DNA was extracted from the leaves of 5-week-old wild-type and transgenic rice plants by using the CTAB (2% CTAB, 20 mM EDTA, 1.4 M NaCl, 2% PVP-40, and 100 mM Tris-HCl, pH 8.0) method. Total genomic DNA was digested with HindIII restriction enzyme (Thermo Scientific, United States) and separated by electrophoresis on a 0.7% agarose gel. Then, the DNA on the gel was transferred to a Hybond-N nylon membrane (GE Healthcare, United Kingdom) by the capillary transfer method, and the blot was hybridized to a ³²P-labeled hygromycin B phosphotransferase (Hph) probe as described by Byun et al. (2018) (link). The autoradiography signals were visualized using a BAS2500 Bio-Imaging Analyzer (Fuji Film, Japan).