Hygromycin

Arabidopsis plants were transformed with the floral dip method (Clough and Bent, 1998 (link)) using A. tumefaciens GV3101 carrying the 35S::ZmNBS25 construct or the control vector. Transformed Arabidopsis seeds were sown onto Murashige and Skoog (MS; Sigma-Aldrich) agar plates containing 20 mg mL⁻¹ hygromycin (Roche Molecular Diagnostics) to screen for positive transformants, which were then transplanted for further growth. Genomic DNA was extracted from the leaves of transgenic Arabidopsis lines using the CTAB (cetyl trimethyl ammonium bromide) method (Clarke, 2009 (link)). The DNA extracted from each transgenic Arabidopsis plant was used as template to determine positive transgene integration by PCR. The 2× Taq Master Mix (Dye Plus; Vazyme Biotech Co., Ltd., Nanjing City, PRC) was used for PCR amplification under the following profile: 95°C for 10 s, 32 cycles of 55°C for 30 s, and 72°C for 30 s. Seeds from positive transformants were harvested, subjected to hygromycin screening, and the process was repeated until only seeds capable of growing in hygromycin medium remained. The homozygous T3 generation was selected for subsequent experiments.