L cysteine

The reference strains of P. gingivalis W50 (ATCC 53978) and F. nucleatum subsp. polymorphum (ATCC 10953) were incubated in BHI broth (Oxoid, Basingstoke, UK) supplemented with L-cysteine (0.4 g/L) (Duchefa Biochemie, Harleem, The Netherlands), hemin (Calbiochem, Merck, Darmstadt, Germany) (5 mg/mL), and menadione (Sigma-Aldrich, Burlington, MA, USA) (5 mg/mL). For the cultures in the solid medium, blood agar plates were prepared using 1.5% agar and 5% defibrillated sheep blood (Winkler Ltda., Santiago, Chile), which were supplemented with 1% hemin and 1% menadione. Bacteria were incubated in an anaerobic atmosphere of 90% N₂, 5% H₂, and 5% CO₂ at 37 °C for 3–4 days, which was achieved using both anaerobiosis jars and anaerobiosis generators (AnaeroGen).
To grow P. gingivalis and F. nucleatum in the liquid medium (broth culture), isolated colonies (inoculum) were selected with a platinum loop from the blood agar plates supplemented as mentioned above. The tubes were incubated for 20–24 h anaerobically at 37 °C until reaching the required optical density, depending on the experiment to be performed.

The breeder’s seed of desi chickpea variety, DCP92-3 was used for genetic transformation experiments. The protocol utilized modified MS medium consisted of MS salts (Murashige and Skoog, 1962 (link)), B₅ vitamins (Gamborg et al., 1968 (link)), 3% sucrose and 0.8% Agar (pH 5.8). The growth regulators, cytokinins [6-benzyl amino purine (BAP), 6-furfurylaminopurine (kinetin), thidiazuron (TDZ) and 2-isopentenyl adenine (2iP)], gibberellic acid (GA₃), three organic additives [L-cysteine, L-glutamine, and L-arginine (Duchefa Biochemie, Haarlem, the Netherlands)] and antibiotics kanamycin monosulfate, cefotaxim and rifampicin and acetosyringone (Sigma Aldrich, United States) were added to different medium after filter sterilization or autoclaving. The cultures were incubated at 23 ± 2°C under a light regime of 16 h light (100 μmol/m²/s) provided by cool white fluorescent lamps and 8 h dark.