This study outlines a method for the sterile cultivation of plant seedlings and their inoculation with Agrobacterium tumefaciens. The process includes monitoring for bacterial contamination and the subsequent integration of T-DNA into plant cells.
Take a tank containing a sterile growth medium with an acidic pH.
Transfer a mesh screen with plant seedlings into the tank.
Seal with a lid and porous tape to maintain sterility while allowing gaseous exchange.
Incubate with shaking to aerate the medium.
Inspect the medium to check for the absence of cloudiness, indicating no bacterial contamination.
Spot a sample onto a culture plate and incubate it to observe the lack of bacterial growth, confirming the absence of contaminating bacteria.
Inoculate with Agrobacterium, a plant pathogen, and incubate.
Plant root-derived signaling molecules and the acidic pH activate a bacterial membrane-bound sensor protein.
The activation induces gene expression, producing proteins for bacterial attachment to root surfaces.
Following attachment, Agrobacterium delivers a DNA fragment, known as transfer DNA (T-DNA), into the plant cells via a secretion system.
Upon integration into the plant genome, the T-DNA synthesizes signaling molecules that enhance bacterial colonization.
In a sterilized flow hood, pour 18 milliliters of autoclaved liquid MS medium into sterile cylindrical glass tanks with sterile lids.
Next, use sterile forceps to gently transfer each mesh square with 10 to 14 day old seedlings from the Petri dish of semi solid medium to a hydroponical cylindrical tank. The seedlings shown here are alfalfa. Seal the lids onto the tanks using porous surgical tape.
Then, cultivate the seedlings at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Prior to the inoculation, examine the tank with seedlings carefully for any potential contamination. The medium in the uncontaminated tanks should be clear and transparent.
If tanks are cloudy with contamination, discard them and number the rest of the tanks. Sample 20 microliters of hydroponic medium from each numbered tank and spot it on a Petri dish with Luria broth or LB agar. Incubate the dish at 28 degrees Celsius for 28 hours.
Immediately inoculate each numbered hydroponic tank with 50 microliters of the Agrobacterium tumefacians suspension into each numbered hydroponic tank. Co-cultivate the seedlings and A. tumefacians bacterium at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Examine the spotted LB plate after 12 and 28 hours of incubation to verify the sterility of the hydroponic growth medium.
If there is any contamination, do not use the corresponding numbered tank inoculated with bacteria for further downstream assays.
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