Virus-Induced Gene Silencing Optimization in Petunia

Virus-Induced Gene Silencing (VIGS) elicits RNA interference, which can be used to down regulate genes of interest within plants. The efficiency of VIGS is dependent on the proliferation of viral infection and systemic movement. Although VIGS has been successfully used in petunia (Petunia x hybrida), it has not been thoroughly optimized. Here we report the findings of VIGS optimization using the Tobacco Rattle Virus (TRV) in five areas: 1) inoculating methods, 2) evaluating petunia cultivars, 3) measuring silencing efficiencies among plants grown under three different growing temperatures, 4) developing a control that overcomes the empty vector viral symptoms, and 5) determining an effective developmental stage for inoculating petunias. From our research, we utilized a simple shoot apical meristem inoculation method that was both effective and consistent in silencing. This method displayed silencing 3 days before any other method evaluated. From an evaluation of 11 species, we identified a compact petunia variety, 'Picobella Blue,' that exhibited a 1.8-fold higher efficiency in chalcone synthase silencing than any other variety tested. We also determined that a lower daytime temperature of 20° with a night temperature of 18 °C provided an environment that induced stronger gene silencing than 23 °C and 26 °C daytime temperatures. Severe viral symptoms of the empty vector control confounded experimental results. To overcome this, we created a recombinant TRV vector that contains a fragment of the green fluorescent protein gene which eliminated viral symptoms and served as a viable control. Our research also determined that efficient silencing can occur on petunia seedlings from 3 to 4 weeks old. This research provides essential information on the development of an efficient method of VIGS for petunia that is both simple and consistent, as this does not require the formation of stably-transformed genetic lines. We will use this method for high throughput characterization of genes associated with production timing and postproduction quality.