Agro-infiltration of the RNAi plasmid in soybean seedlings

A. tumefaciens strain LBA 4404 harbouring pFGC5941::AC2-ihp plasmid (comprising the AC2 gene silencing construct) was cultured in LB broth with Kanamycin selection at 28 °C and 200 rpm for a period of 30 h. Bacterial cells were harvested when the liquid culture reached OD₆₀₀ value of 1.0. Harvested cells were resuspended in agro-infiltration medium to obtain a final OD₆₀₀ of 0.2 (Patil and Fauquet 2010). After incubation at room temperature for 3 h, agro-infiltration was performed in leaves 2 days post-MYMIV inoculation using a syringe without a needle. Soybean seedlings were inoculated with MYMIV infectious clones (NRCS-A2X-7 and NRCS-B2X-1); agro-infiltrated soybeans with A. tumefaciens LBA4404 harbouring empty pFGC5941 served as inoculated control for monitoring MYMIV disease development.