Plasmid construction and transformation
ThePtrWRKY75cDNA was cloned into the pSUPER1300(+) vector (containing the enhanced green fluorescent protein (eGFP) gene) under the control of the Super Promoter (Zhao et al ., 2011). The hygromycin phosphotransferase of pSUPER1300(+) was used to determine the validity of the positive selection system in the transformation of poplar. The construct was inserted by heat shock into Agrobacterium tumefaciens strain GV3101 and then transformed into wild-type triploid white poplar using the leaf disc method (Li et al., 2012; Wang et al. , 2016). The leaves of triploid white poplar, which were cut into discs, were pre-cultured on MS medium supplemented with 0.02 mg/L TDZ, 0.1 mg/L NAA, and 0.6% (w/v) agar for 3 days, dipped in the diluted Agrobacterium culture for about 15 min and cultured on pre-cultivation medium for an additional 3 days in the dark. Then, the leaf discs were transferred to MS medium (pH 5.8) supplemented with 0.02 mg/L TDZ, 0.1 mg/L NAA, 200 mg/L Timentin, 3 g/L hygromycin phosphotransferase, and 0.6% (w/v) agar for shoot induction and selection. Next, the leaf discs were transferred onto MS medium supplemented with 0.5 mg/L 6-BA, 200 mg/L Timentin, 3 mg/L hygromycin phosphotransferase, and 0.6% (w/v) agar for elongation and selection of adventitious buds. The regenerated shoots were individually separated from the callus and inserted into a selective rooting medium [1/2 MS medium supplemented with 0.05 mg/L NAA, 300 mg/L carbenicillin, 1 g/L hygromycin phosphotransferase, and 0.6% (w/v) agar]. The rooted plantlets were acclimatized in pots and then transferred to a greenhouse at 22 °C under a 16 h/8 h (light/dark) photoperiod and 40%–45% relative humidity.