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.