Following passage, the chondrocyte cell suspension (1 × 10 cells/ml) was transferred to six-well tissue culture plates and incubated for 24 48 h in the above medium to allow cells to reach ∼80% confluency.
For the acidic minimal media 2 (AMM-2) culture, cells were first grown in LB medium to stationary phase as described above, rinsed twice with magnesium minimal medium (MgM) pH 5.0, resuspended in an equal volume of MgM pH 5.0, and then incubated at 37°C with shaking (200 rpm) for 4 h.
A total of 5 × 10 cells per well were cultured in duplicate in six-well plates in 2 ml normal growth medium to near confluency as mentioned above.
The TSQ were generated from TSP at 6 days growth by switching the medium to serum-free as described above and all subsequent culturing of TSQ was performed in this medium.
First, single salts were omitted from the medium described above to evaluate their influence on enzyme production.
Regardless of the wheel degradation modes outlined above, medium-to-high-carbon pearlitic steels are still the most popular materials for heavy haul wheels.
When regenerated plants reached 3 to 5 cm in length, they were excised and transferred to secondary regeneration medium (as above, but without Haloxyfop) and incubated at 25° under 16-hour white fluorescent light conditions (approximately 50 μEm−2 s−1) to allow for further growth and development of the shoot and roots.
On day 1, the medium was removed and cells were carefully washed with PBS before being overlaid with 1 mL serum-free, BSA containing medium as above and left to incubate for three more days.
Hairy roots of 3-4 mm in length that developed after 25-30 days were aseptically excised from the infected stems, and transferred to a liquid medium as above but without agar, containing the antibiotic cefotaxime (100 mg/L), as well as 1% of the antioxidant polyvinylpyrrolidone (PVP) for several subcultures.
The cells were plated at a density of 5 × 103 cells per well for 24 h in 96-well plates in a standard growth medium prior to exposure to the above materials.
