Inoculation of ''Arabidopsis thaliana'' with Microbes

True leaves of young Arabidopsis thaliana can be inoculated via either a foliar spray [1] or pipetting [2] of the microbial suspension. A foliar spray would likely coat the leaves more evenly and effectively, but it would also likely inoculate the agar as well, potentially leading to adverse bacterial growths on the agar surface. Pipetting would be more labor intensive and possibly less effective at leaf inoculation, but it would ensure that only the plant received the microbial suspension.

Foliar pipetting

Note: these steps are to be observed under sterile conditions.

1. Obtain a suspension of phyllosphere microbiota - this can be achieved by following steps one and two in Removal and DNA Extraction of Phyllosphere Microbes and then removing the leaves from the mixture.
2. Dilute cell suspension so that "OD600 = 0.02" [3]
3. Pipet "5 to 10 uL" of cell suspension onto the leaves "when plants [are] 2 weeks old."
4. For control plants that will receive no bacteria, pipet "sterile 10 mM MgCl2 solution to plant ... leaves."

Sources:

Foliar spray

Follow the same steps as for Foliar pipetting, but instead of using a pipet, apply 5 to 10 uL of cell suspension using a small 30 mL spray bottle.

Sterile Cotton Q-Tip Application Technique

Note to future researchers: Although we were not able to test this method during the initial phase of the experiment, we hypothesize that applying a bacterial solution to the plant leaves using a sterile cotton swab will be the easiest method for inoculating Arabidopsis with microbes. Experimentation is needed to determine if this is an effective method of transferring microbes, and how many leaves the cotton swab can inoculate before another is necessary.