Although some species that accumulate only cyanidin (Cy) in nature can produce blue flowers through iron ions, there has been no evidence of blue chrysanthemums being generated in this manner. This study revealed that flavonoid extracts from the ray florets of the chrysanthemum cultivar ‘Wandai Fengguang’ turned blue when exposed to Fe³⁺. Samples that could turn blue were labeled as CB (Cy-determined blue flowers), while samples that did not turn blue were labeled as CN (Cy-determined non-blue flowers). After a series of experiments, a stable screening system was established using flavonoid extracts containing NaAc buffer at pH 5.5 and a total anthocyanin concentration (TAC) of 30 μmol·L⁻¹, and the addition of Fe³⁺ from 0 to 0.25 μmol·L⁻¹ allowed for the selection of five CB samples from 39 chrysanthemum cultivars. All five CB samples exhibited flower color phenotypes that belonged to Cluster-I with redness (a*) values ranging from 29.03 to 45.99, yellowness (b*) values from −11.31 to 3.77, and brightness (L*) values from 29.07 to 45.99. Additionally, the ratio of TAC to total luteolin concentration (TLC) was found to be a critical factor for distinguishing between CB and CN samples. To realize the desired blue hue in the flavonoid extracts with the participation of Fe³⁺, a TAC to TLC ratio of 2.25 and above is required. Moreover, the protoplasts and ray florets of CB samples that turned blue with the involvement of Fe²⁺ showed great potential for cultivating blue chrysanthemums through ferric–anthocyanin chelate. Overall, this study reveals that blue flowers can be cultivated through the increase in the iron ion concentration, combined with the accumulation of Cy.

Virus-induced gene silencing (VIGS) is a genetic tool used to assess gene function. Tobacco rattle virus (TRV) is a VIGS vector commonly used to induce endogenous gene silencing in plants. However, there is no VIGS system established for Centaurea spp. We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase (PDS) as a reporter gene in Centaurea cyanus. Three methods including pressure-, vacuum- and apical meristem-infiltration were tested to infect C. cyanus seedlings. Photobleached leaves were only obtained using apical meristem-infiltration after a 14 d treatment. The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV. Four C. cyanus cultivars were tested to detect their VIGS responses, and ‘Dwarf Tom Pouce Blue’ was the most sensitive. The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing. Seedlings with four true leaves and infiltration with an Agrobacterium density of OD₆₀₀ 0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype. The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C. cyanus.