Phalaenopsis is one of the most popular flowers worldwide. It has special agronomic traits for abundant flower colors including white, yellow and red-to-purple. However, after many years of breeding, Phalaenopsis orchids still lack ‘true-blue’ color flowers. It has become an important target for orchid breeders to breed ‘true-blue’ flowers. Our previous results showed that the cyanidin-based F3’H gene was highly accumulated, but delphinidin-based F3’5’H gene was not expressed in the violet-to-purple Phalaenopsis flowers. Transient overexpression of PeMYB2 and DgF3’5’H showed 53.6% of delphinidin and 46.4% cyanidin, and resulted novel blue-hue color formation in white-color Phalaenopsis flowers. The aim of my research was to enhance the ‘true-blue’ flowers in Phalaenopsis orchids combining the virus-induced gene silencing (VIGS) to reduce ‘red-to-purple’ color by silencing F3’H, and then overexpressing F3’5’H in the F3’H-silenced plant. To select an effective F3’5’H gene, blue-color Vanda Pachara Delight was used, since delphinidin was accumulated to a higher level than the cyanidin in Vanda Pachara Delight than in Vanda coerulea flower as determined by HPLC analysis. Transient overexpression of PeMYB2 and VpF3’5’H showed 4.5% of delphinidin and 95% cyanidin and resulted pink-purplish color formation in white-color Phalaenopsis flowers. These results suggest that the VpF3’5’H was weaker than DgF3’5’H for producing the ‘true-blue’ color. For VIGS, the endogenous cyanidin F3’H was silenced with a Cymbidium virus-based vector in white-color P. Sogo Yukidian ‘V3’ plant (V3). The F3’H-silencing vector was injected to leaf and floral stalk during flower bud stage. Ectopic overexpression of DgF3’5’H with PeMYB2 was performed by injection of the constructs into F3’H-silenced plants on the 5th, the 6th and the 7th weeks post silencing in various flower bud number 7, 8 and 9 of V3 plants, consecutively. After the flowers bloomed, the fully opened flower petals displayed bluish color formation. Significantly reduced F3’H expression was detected by using qRT-PCR analysis in the F3’H-silenced plants for the 5th, the 6th and the 7th week post silencing as compared to that of the mock-treated flowers with empty Cymbidium virus-based vector. This led to reduction of cyanidin to 23.4% and increase of delphinidin up to 76.5%, and displayed bluish flower color phenotype. In summary, the approach of overexpressing a selected effective DgF3’5’H accompanied with silencing the F3’H accumulation allowed the blue color formation in white-color Phalaenopsis flowers.

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