Agrobacterium-Mediated Transient Expression in Nicotiana benthamiana Leaves

Refresh bacteria containing the construct of interest from glycerol stock on agar YEP (or any other bacterial media such as LB) medium with an appropriate antibiotic . Incubate at 28°C for two days. If A. tumefaciens GV3101 is used add rifampicin .

Inoculate 5 mL of liquid YEP medium with appropriate antibiotic in a test tube. Grow at 28°C for 24 hours

with shaking at 250-300 rpm. Be sure to grow bacterial strain with p19 9 as a silencing inhibitor as well!

Measure the OD₆₀₀ using 100 µL of bacterial culture.

Calculate the culture volume needed for an OD₆₀₀of 0.2 - 0.5 per construct for a 4 mL total volume of Infiltration medium. 4 mL are used for test spot infiltration. The specific OD₆₀₀ to use depends on how

well expressed your construct is in N. benth h, the general range is 0.1 – 0.5. Use an O₆₀₀ of 0.3 for P19.

(I use 0.3 for all constructs and it works well).

Pipette the appropriate volume of each strain together in 2 mL tubes.

Centrifuge combined bacteria at 8000× g for 2 min at room temperature. Carefully remove all supernatant.

Add 200 mM acetosyringone to the Infiltration medium at a 1:1000 ratio.

Note: acetosyringone is light-sensitive, keep out of light until ready for use.

Add 2 mL of infiltration medium with acetosyringone to each pellet, and resuspend. Leave in the dark at room temperature for 1–2 hrs (or even longer but not overnight).  

Infiltrate 4-week-old N. benthamiana (pre-flowering) leaf using 1 mL syringe.

Grow plants for 2-3 days after infiltration (up to 5 days, depending on expression level of constructs).

Punch 1 cm leaf discs from leaves and observe under the microscope to visualize the fluorescence.