To begin, grow Nicotiana benthamiana seeds in wet soil within a controlled environment chamber set at 23 degrees Celsius, maintaining 16 hours of light, followed by eight hours of darkness. After transferring the two-weeks-old seedlings to larger pots, grow the plants under the same conditions for four to five weeks to prepare them for agroinfiltration experiments. Streak Agrobacterium to Mephasins EHA105 cells containing desired vectors onto LB agar plates and incubate them at 28 degrees Celsius.
After two days, transfer a single colony from the plate into two milliliters of LB broth and incubate overnight at 28 degrees Celsius with agitation at 250 revolutions per minute. Then, remove one milliliter from the overnight culture and add four milliliters of fresh LB broth to reculture for one hour under the same conditions. Pellet the cells by centrifugation.
After that, determine the cell count of the bacterial suspension at 600 nanometers and adjust the optical density to 0.1 or 0.2 using infiltration buffer. Incubate the bacterial suspension at room temperature for three hours with gentle agitation. Next, infiltrate the abaxial surface of fully expanded leaves from different plants using a one-milliliter needleless disposable syringe.
Mark the infiltrated area, which will appear darker than the surrounding non-infiltrated tissue, with a waterproof pen.
