4.5. Agrobacterium Growth and vir Gene Expression Assay Using LB Broth

For the Agrobacterium growth and vir gene expression assay, LB broth were prepared with nine different supplements (M1–M9, as listed in Table 1 with antibiotic rifampicin and kanamycin both at 50 mg·L⁻¹ but without MS salts). The overnight culture of Agrobacterium strain ATCC 15834 harboring pBI121 was prepared and equal aliquots were centrifuged at 2600× g for 5 min. Agrobacterium pellets were re-suspended with each 100 mL LB broth and the initial OD₆₀₀ values were adjusted to 0.15. The cultures were then incubated at 28 °C in the dark on a rotary shaker (180 rpm). The change in OD₆₀₀ values were recorded after 7 h of incubation. As PVPP was insoluble in the media, the OD₆₀₀ values of the cultures were measured after pelletizing PVPP by centrifugation at 150× g for 5 min. The corresponding media without Agrobacterium were used as references for measuring the OD₆₀₀ of the inoculated cultures. Agrobacterium cells were then harvested and total RNA was isolated using an RNAprep pure Cell/Bacteria Kit (Tiangen Biotech., Beijing, China) according to the manufacturer’s instructions. The RNA was reverse-transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real time quantitative polymerase chain reaction (qPCR) was performed using the top Green qPCR SuperMix Kit (TransGen, China) with gene specific primers for virA, virB1, virB2, virC1, virD1, virD2, virD4, virF, virG, and virK (Table S1). Atu0972, an Agrobacterium chromosomal gene, was used as a reference gene according to Anand et al. [47]. qPCR reactions were performed on a Bio-Rad CFX96 platform and programmed as follows: 3 min at 95 °C, 40 cycles of 10 s at 95 °C, 40 s at 55 °C; followed by a 5 s melting curve analysis of 65–95 °C, in 0.5 °C increments. Transcript data were analyzed using 2^−∆∆Ct method [48] with the CFX Manager™ software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All measurements were conducted in triplicate.