Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study

In vivo genetic methods are required to study the biological function of genes in tropical perennial species and horticultural tree crops. However, many of these species are recalcitrant to transformation every generation. The scope of this research is to develop a simple and efficient genetic transformation method applicable to large-sized plants.

Our illustrative case mediate the genetic transformation of plants with difficult to infiltrate tissues has been made possible using a vacuum pump. However, the protocol currently used are limited to a small such plants due to the physical and economic constraints of submitting large-sized plants to vacuum training. The size of the vacuum chambers, which are usually laboratory desiccators, prevents the baking infiltration of plants that are too large to fit into the desiccators as is the case of tropical wooded plants.

We have developed a protocol for efficient localized baking infiltration of tropical wooded plants, making In Vivo genetic transformation studies of large plant species possible. This protocol allow us to overcome plant size limitations for agrobacterium mediated baking infiltration in horticultural tree crops such as cacao and avocado. Digi-culture, free agro bacteria mediated transformation of attached leaves by localized vacuum infiltration can be performed in a rapid, straightforward, and gas effective manner by laboratories that already use vacuum infiltration.

Since the same pump and vacuum vessel can be used with no need to add a costly equipment. To begin, streak transformed 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, LBA 4404 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. Re-suspend 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 vacuum 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 betalene accumulation on the leaves, which can be easily observed as bright red pigmentation with the naked eye. Betalene accumulation was observed to vary with the tissue viability.