Construction and optimization of TRV-mediated VIGS silencing system of Helianthus annuus

To analyze gene function in plants lacking a stable genetic transformation system, virus-induced gene silencing (VIGS) is essential. Sunflower (Helianthus annuus), the world’s fourth most important oil crop and a significant cash crop in China, was selected as the experimental material. Three specific fragments of the H. annuus phytoene desaturase gene (HaPDS) were amplified and cloned into the pTRV vector to construct the recombinant vector pTRV-HaPDS for VIGS. Confectionery sunflower cultivars Huiyuan119, LD5009, and 3936, as well as oilseed sunflower cultivars R5 and GK2002, were used to optimize silencing conditions. Agrobacterium tumefaciens-mediated infiltration was performed, and HaPDS gene silencing was evaluated by considering factors such as the position of the silencing fragment within the gene, vacuum treatment, A. tumefaciens co-cultivation time, A. tumefaciens concentration (OD₆₀₀), and plant cultivation temperature. Silencing efficiency was statistically analyzed, and HaPDS expression levels were quantified by qPCR. After various trials, it was determined that seed vacuum infiltration followed by 6 h of co-cultivation produced the most effective VIGS results. The optimal conditions involved silencing the HaPDS3 fragment near the 3′ end, an OD₆₀₀ value of 1.0, and a plant ambient temperature of 22°C, resulting in relatively high silencing efficiency in Huiyuan119 and GK 2002. This study aims to develop an efficient and stable gene silencing system for sunflower, establishing a foundation for the subsequent validation of gene functions in this species.