The day before cocultivation, liquid cultures of A. tumefaciens were inoculated from colonies on agar plates or frozen glycerol stock. After growth at 28°C in 2 mL LB medium with appropriate antibiotics (25 μg/mL streptomycin plus 50 μg/mL kanamycin or 100 μg/mL spectinomycin, or both) for 18–24 hr, 1.6 mL saturated culture was diluted the next day into 10 mL fresh YEB medium (5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/L MgCl2) to OD600 = 0.3 and was grown until the OD600 reached more than 1.5. Bacteria cells were harvested by centrifugation at 6,000 g for 5 min and washed once with 10 mL washing solution containing 10 mM MgCl2 and 100 μM acetosyringone. After centrifugation at 6,000 g for another 5 min, the pellet of bacteria cells was resuspended in 1 mL washing solution. We also tested a simplified protocol where A. tumefaciens cells were directly scraped from agar plates and resuspended into wash solution at a final OD600 = 0.3. This simplified procedure resulted in similar transformation efficiency as the protocol described above.
In a clean Petri dish (100 × 20 mm), 30–40 4-day-old Arabidopsis (or tobacco) seedlings, 10–15 4-day-old tomato seedlings or 5-day-old rice (or swichgrass) seedlings were soaked with 20 mL cocultivation medium containing 0.25 × MS (pH 6.0, Caisson Laboratories), 1% sucrose, 100 μM acetosyringone, 0.005% (v/v; i.e. 50 μL/L) Silwet L-77 and A. tumefaciens cells at final density of OD600 = 0.5 (6 × 108 cfu/mL). Cocultivation was carried out in darkness at the same temperature as seedling growth for 36–40 hr for Arabidopsis seedlings, 36–60 hr for tobacco (or tomato) seedlings or 6 days for rice (or switchgrass) seedlings before microscopic observation or other analysis was performed. For cocultivation assay in 96-well plate, 2–3 4-day-old Arabidopsis seedlings geminated directly on 30 μL MS-agar (0.8%) plus 1% sucrose in each well were cocultivated with A. tumefaciens cells in 100 μL cocultivation medium.
In a clean Petri dish (100 × 20 mm), 30–40 4-day-old Arabidopsis (or tobacco) seedlings, 10–15 4-day-old tomato seedlings or 5-day-old rice (or swichgrass) seedlings were soaked with 20 mL cocultivation medium containing 0.25 × MS (pH 6.0, Caisson Laboratories), 1% sucrose, 100 μM acetosyringone, 0.005% (v/v; i.e. 50 μL/L) Silwet L-77 and A. tumefaciens cells at final density of OD600 = 0.5 (6 × 108 cfu/mL). Cocultivation was carried out in darkness at the same temperature as seedling growth for 36–40 hr for Arabidopsis seedlings, 36–60 hr for tobacco (or tomato) seedlings or 6 days for rice (or switchgrass) seedlings before microscopic observation or other analysis was performed. For cocultivation assay in 96-well plate, 2–3 4-day-old Arabidopsis seedlings geminated directly on 30 μL MS-agar (0.8%) plus 1% sucrose in each well were cocultivated with A. tumefaciens cells in 100 μL cocultivation medium.
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