Sucrose

Murashige and Skoog [27 (link)] medium (MS) was fortified with BAP, KIN and TDZ ranging from 0.5 to 5.0 mgL⁻¹, and 3% (w/v) sucrose (Himedia) as carbon source and 0.8% (w/v) agar (Himedia, India) as gelling agent. The media pH was maintained at 5.65 to 5.75 using 0.1 N NaOH or 0.1 N HCl. The medium (approx. 20–25 mL) was poured into 50 mL culture tubes or 100 mL culture flasks, before autoclaving at 121 °C and 15 psi pressure for 15–25 min The cultures were incubated at 25 ± 2 °C and 55 ± 2% relative humidity under 16/8 h (light/ dark) photoperiod in cool white fluorescent light (1800–2000 lux) intensity for shoot induction. After inoculation, these cultures were maintained for 4 weeks in incubation room. Regenerated multiple shoots were then transferred to the fresh medium after 4 weeks interval for further proliferation and elongation of developed shoots. The shoot multiplication was observed by counting of proliferated shoots. Further sub-culturing was made only on medium which showed maximum shoot multiplication.

Tannin (gallotannin) was purchased from LOBA Chemie (Mumbai, India), and methyl gallate was purchased from Sigma (Steinheim, Germany). Ingredients such as peptone, yeast extract, malt extract, and carbon sources such as glucose, galactose, sucrose, maltose, lactose, raffinose, and trehalose were purchased from HiMedia (Nashik, India). The genomic DNA of yeast was extracted using a Wizard^® Genomic DNA purification kit (Promega Corp., Madison, WI, United States). Nucleic acid amplifications were performed using TaKaRa Ex Taq^® (Shiga, Japan). Other reagents and solvents were of analytical grade.

Wild-type A. thaliana seeds (Col-0 background) were used in the experiment. The seeds were surface-sterilized with 70% ethanol, washed with autoclaved double-distilled water and sown on plates containing Murashige and Skoog (MS) basal medium (for media composition, see Table 1), sucrose 1.5% (w/v) and agar 0.6% (w/v) (Hi Media Laboratories Pvt. Ltd. Mumbai, India) [44 (link)], supplemented with appropriate rohitukine concentrations: 0 (control), 0.25 mM, 0.5 mM and 1.0 mM concentrations. For foliar treatment, plants were grown in plastic pots filled with 300 g autoclaved soil mixture (soil rite–sand–soil) at the ratio of (3:1:1). Seeds were kept at 4 °C in the dark for 48 h to ensure homogenous germination. After 48 h, the plates containing seeds were transferred to a growth chamber with the following conditions: photosynthetically active radiation (PAR): 680 μmole/m²/s; temperature: 24 °C; photoperiod light/dark cycles: 16/8 h; and relative humidity: 65%, in the Indian Institute of Integrative Medicine, Jammu and Kashmir, India. The plants grown in soil were irrigated with autoclaved distilled water at intervals of 24 h, while 1 mL of quarter-strength nutrient medium was applied at intervals of 48 h up to five weeks.

The biofilm-forming ability of enterococci was evaluated phenotypically using the Congo red (CR) test, as described previously [39 (link)]. In the CR assay, enterococci strains were cultured on Congo red agar (CRA) plates to determine their biofilm-forming abilities. In order to prepare CRA plates, 0.8 g of CR (HiMedia, Maharashtra, India) and 36 g of sucrose (HiMedia, Maharashtra, India) were mixed with 1000 mL of blood agar (HiMedia, Maharashtra, India). The mixture was then incubated at 37 °C overnight to ensure its sterility. Subsequently, enterococci cultures grown overnight were streaked onto CRA plates and incubated at 37 °C for 24 and 48 h. The observable characteristics of the examined isolates were then analyzed to assess their ability to form biofilms. Isolates displaying dry filamentous crusty black colonies, darkening but lacking the presence of dry crystalline structured colonies, almost black colonies, and red colonies were interpreted as strong, intermediate/moderate, weak, and non-biofilm formers, respectively [40 (link),41 ].