The growth conditions and preparation of A. tumefaciens were the same as those described in Cheng et al. (1997). The A. tumefaciens cell density was adjusted to an optical density of 0.5 (OD660) for inoculation. About 100 PCIEs were transferred into the A. tumefaciens cell suspension in a 6-well plate with 10 ml agro in
each well (Fig. 1b). The inoculation was carried out at 23–25C for 15–30 min. Following the inoculation period, the remaining A. tumefaciens cells were removed. A piece of sterile Whatman No. 1 filter paper was placed inside each 6015 mm petri dish and 200 mml sterile H2O was placed onto each piece of filter paper.
Usually, about 20 explants were grouped as one stack (about 1 cm in size and approx. 60–80 mg/stack), with four to five stacks in each plate. The plates were covered with Parafilm and then cocultured in the dark at approximately 23–25C for approximately2–3 days