Integrated breeding strategies for blue lotus based on petal pH, anthocyanin profiling, and gene expression analysis.

Lotus (Nelumbo Adans.) is an important aquatic ornamental plant, yet it exhibits limited color diversity and lacks blue or purple flowers. This study aims to investigate the mechanisms underlying flower coloration and to develop targeted strategies for breeding blue lotus through integrating analyses of petal anatomy, pigment profiles, vacuolar pH, and anthocyanin biosynthetic gene expression. We found that petal pigments in the palisade parenchyma primarily determine color, with epidermal cell structure playing a negligible role. A freeze-induced color shift from red to blue was attributed to a pH increase following vacuole rupture rather than to changes in anthocyanin composition. Anthocyanin profiling verified the presence of delphinidin-based pigments in red cultivars, indicating an intact biosynthetic pathway for blue pigments. Furthermore, gene expression analysis revealed that the absence of anthocyanins in yellow and white flowers was linked to severely reduced expression of 4-coumarate-CoA ligase (4CL), while downstream genes, including the crucial F3'5'H, DFR and ANS, were highly expressed in yellow petals. Using grey relational analysis, we selected nine elite parental candidates suitable for breeding blue lotus. In conclusion, a promising strategy for breeding blue lotus involves hybridizing Asian lotuses (exhibiting high 4CL expression) with American lotus (showing high F3'5'H, DFR, ANS expression), which is anticipated to promote delphinidin accumulation, combined with future efforts to elevate vacuolar pH to near that of the petal sap through genetic techniques. This study provides a theoretical foundation and practical germplasm resources for the molecular breeding of blue lotus flowers.