Southern Blot Hybridization

Genomic DNA was extracted from the leaves of transgenic plants using Plant DNAzol reagent (Invitrogen^TM, Lot No. 10978021). Southern hybridization was performed according to the standard method (Southern, 2006). In brief, about 4 μg of total DNA was digested overnight with EcoR I and then separated by electrophoresis in a 0.8% agarose gel using 1× TAE buffer. After electrophoresis, the gel was denatured and transferred onto a Hybond^TM-N⁺ membrane (GE Healthcare^TM, Lot No. RPN1576). The blot was hybridized with the DIG-labeled Hygromycin phosphotransferase (Hpt) gene probes (for the XL22 lines) and the phosphinothricin acetyl transferase (Bar) gene probes (for the CA1-OE and SAPK6-OE lines) and exposed to X-ray film for signal detection. DNA probe preparation, hybridization, membrane washing, and signal detection were performed according to the protocols within the PCR DIG Probe Synthesis Kit (Roche^TM, Lot No. 11636090910). The primers used for probe amplification are listed in Supplementary Table S1.