To begin, streak transform Agrobacterium tumefaciens cell culture onto selective antibiotic supplemented Yeast Mannitol Agar plates. Incubate the plates overnight at 28 degrees Celsius in a standing incubator. The next day, inoculate colonies from the culture on 12.5 milliliters of Yeast Mannitol Luria-Bertani broth mix, and incubate on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM.
Inoculate 12.5 milliliters of the Agrobacterium strain LBA4404 in 112.5 milliliters of Yeast Mannitol broth with antibiotics. Incubate the culture on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM. Adjust the overnight culture to an optical density of 0.4 with the culture broth.
Then add 20 micromolar of acetosyringone before incubation. Once the incubated culture reaches an optical density of one, centrifuge to pellet the cells. Resuspend the pellet in suspension solution to an optical density of 0.6, and add 200 micromolar acetosyringone before incubation.
For agroinfiltration, pick woody cacao plants that have branches with leaves. Add 250 micromolar jasmonic acid to the Agrobacterium suspension. Next, set up a desiccator with a vacuum gauge as a vacuum chamber to measure the pressure inside.
Place a jump ring to allow the plant branch to pass through. Place the beaker with Agrobacterium culture inside the desiccator, and place the branch between the desiccator and its lid, with the desired leaves submerged in the Agrobacterium culture. Next, use silicone impression material to seal the gaps between the jump ring, the plant branch, and the desiccator.
Once the silicone material has polymerized, close the desiccator, leaving no gaps, then connect it to the vacuum pump. Start the vacuum pump until it reaches minus 0.07 megapascals. When the desired pressure is reached, close the pressure valve, and turn off the pump to maintain the pressure for five minutes.
Open the pressure valve gradually and steadily to restore the chamber pressure. After repeating infiltration two more times, take the branch off the cell suspension. Clean the infiltrated leaves with distilled water, then place the plants at 25 degrees Celsius in the dark for 48 hours.
After incubation, expose the infiltrated tissue to a 16 hour light and eight hour dark photo period. Infiltrated leaves of the plants appear darker in certain parts, indicative of Agrobacterium penetration. The reporter system used produced betalain accumulation on the leaves, which can be easily observed as bright red pigmentation with the naked eye.
Betalain accumulation was observed to vary with the tissue viability.
