tumefaciens GV3101∷pMP90 to obtain the strain GV3101∷pMP90(pPZP-eGFP). The vector pRK415 Selleck CX 5461 (Keen et al., 1988) or the plasmid pRKLACC (Shah et al., 1998), which is pRK415 containing the acdS gene from Pseudomonas putida UW4 under the control of a lac promoter, was electroporated into A. tumefaciens GV3101∷pMP90(pPZP-eGFP) to obtain strain YH-1, which is GV3101∷pMP90(pPZP-eGFP)(pRK415), and strain YH-2, which is GV3101∷pMP90(pPZP-eGFP)(pRKLACC). Agrobacterium strains were grown in Luria–Bertani (LB) (Miller, 1976) or M9 medium (Atlas, 1993) (for ACC deaminase

activity assay) at 28 °C. When required, antibiotics were added at the following concentrations: rifampicin, 50 μg mL−1; gentamicin, 50 μg mL−1; spectinomycin, 50 μg mL−1; streptomycin, 20 μg mL−1; and tetracycline, 2 μg mL−1. An ACC deaminase activity assay was performed as described by Hao et al. (2007). The infection and regeneration protocols were modified from Cardoza & Stewart (2003). The media used are listed in Table 1. Seeds of B. napus cv. Westar, B. napus cv. Hyola 401 and B. napus cv. 4414 RR were surface sterilized by soaking in 70% ethanol for 1 min, followed by 20% commercial bleach for 20 min, and were then

rinsed four times with sterilized distilled water and planted at a density of 10–12 seeds per Petri dish (100 × 25 mm) (Fisher Scientific, Ottawa, ON) on seed germination medium. Seeds were germinated at 22–25 °C in the dark for about 1 week. The seedling PR-171 purchase hypocotyls were cut into about 1-cm pieces and preconditioned for 3 days on a cocultivation medium. Agrobacterium tumefaciens strains YH-1 and YH-2 were grown in 50 mL LB medium until the culture reached OD600 nm≈1. The cells were pelleted, resuspended in the infection medium and normalized to OD600 nm=1 to obtain a 1 × dilution. Serial dilutions were then performed using the infection medium to obtain 10−1× and 10−2× dilutions. The preconditioned explants were infected by soaking in A. tumefaciens culture suspensions for 30 min at room temperature with gentle shaking. The infected hypocotyls were first

cocultured on a cocultivation medium for 48 h, then transferred to a callus induction medium for 2 weeks, Resminostat then to an organogenesis medium with (OA) or without AgNO3 (OB) for another 2 weeks, and then to a shoot induction medium for 3–6 weeks until shoots appeared. The induced shoots were transferred to a shoot elongation medium for 2 weeks and then to a rooting medium for another 2 weeks, and finally, the transgenic plants were transferred to soil and grown in a greenhouse. Plant tissue cultures were maintained in a growth chamber at 25 °C with 16 h of light and 8 h of dark, with a light intensity of 40 μmol m−2 s−1 from cool-white fluorescent lamps. The stable transformation frequency was calculated using the following formula: transformation frequency=the number of transgenic plants obtained/the number of explants used for transformation. After infection with various dilutions of A.