The flower color has drawn extensive attention in rapeseed breeding for its ornamental value. However, the color formation and precise design are still elusive. Here, we successfully introduced betalain biosynthesis pathway into rapeseed and achieved constitutive betalain production by overexpressing RUBY. The varying expression levels of RUBY and the flower colors of the receptor materials jointly determined the final color presentation. When RUBY was expressed in yellow-flowered rapeseed (cultivar R10), the flower color turned into different shades of orange. In white-flowered background (cultivar R2), RUBY created red flowers. However, RUBY overexpression led to dark-red leaves and decreased photosynthesis. To recover normal photosynthesis, we created orange flowers with green leaves using petal-specific XY355 promoter in yellow-flowered R10. We further verified that white flower is dominant to yellow and created green leaves with shining red flowers by crossing orange flower (XY355:RUBY expressed in yellow background) with white flower (R2). Given that the widespread carotenoid, betalain, and anthocyanin can produce the three major colors of yellow, red, and blue, respectively, we provide a promising approach for creating derivative colors in Brassica napus by employing bioengineering approaches to precisely regulate the pigment biosynthesis.

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