To begin, add 150 milliliters of clean five-millimeter glass beads to the clean jar and close the lid. Cover the closed jar with enough aluminum foil to conceal the lid to jar junction and autoclave at 121 degrees Celsius for 20 minutes. When the seedlings are ready to transfer into the jar on the sterile bench, remove the aluminum foil and lid of the jar.
Add 35 milliliters of half-strength Murashige and Skoog medium of pH 5.7 to 5.9 to the jar, and stir the beads to coat evenly with the medium. Introduce three to five seedlings into the jar, ensuring the root systems are placed between the glass beads. Adjust the beads with sterile forceps or spoons to cover the roots and lift the leaves out of the medium.
Secure two strips of 1.25 centimeter micropore tape across the top of the jar and gently place the lid on top. Seal the gap between the lid and jar with 2.5-centimeter micropore tape to maintain sterility while permitting air exchange, and place the jar into a growth chamber. When the plants reach the desired age, remove the micropore tape and lid from the jar.
Aliquot 20 microliters of growth medium onto LB Agar Plate for sterility testing. Using a 25-milliliter volumetric pipette, remove as much growth medium as possible without damaging the roots. Add 50 milliliters of collection medium along the jar wall to prevent wetting the leaves.
Close the jar with a lid and incubate in sterile conditions for two hours. After incubation, remove the desired amount of collection medium into tubes. Measure the weight of the root and shoot from the plant in one jar to normalize the root exudates by plant weight.
Arabidopsis thaliana plants looked healthy across two different laboratories. The glass jar system proved adaptable for various plant species and developmental stages. Wheat had the highest chute weights, followed by sorghum and tomato.
While sorghum had the highest root weights, followed by wheat and Medicago truncatula. Inoculation of Arabidopsis thaliana with commensal bacteria did not alter the plant phenotype, and inoculated bacteria persisted in the system for at least 12 days with a slight increase in colony-forming units.
