Overexpression of VtF3′5′H and RhNHX Genes Alters Flower Color and Plant Morphology in Transgenic Rose ‘Red Farm’

Roses (<i>Rosa hybrida</i>) are among the most highly valued ornamental plants worldwide, with flower color serving as a major determinant of consumer preference and commercial success. However, the absence of the flavonoid 3',5'-hydroxylase (<i>F3'5'H</i>) gene limits delphinidin biosynthesis, making it difficult to achieve blue or purple pigmentation. Vacuolar sodium/proton antiporters (<i>NHX</i>) regulate vacuolar pH and are also implicated in color stability. In this study, we introduced <i>Viola tricolor F3'5'H</i> (<i>VtF3'5'H</i>) and <i>Rosa hybrida NHX</i> (<i>RhNHX</i>) into the rose cultivar 'Red Farm' using Agrobacterium-mediated transformation. The non-native <i>VtF3'5'H</i> gene was detected in transgenic plants but not in the wild type, while <i>RhNHX</i> expression was relatively higher in transgenic plants. Petal anthocyanin content was significantly increased in T1-T4 compared to the wild type, and petal pH was also higher than that of the wild type. Growth and floral traits were also altered. Transgenic plants exhibited shorter stems, reduced stem diameter, more lateral branches, fewer prickles, and more than threefold higher petal numbers. Expression analysis showed reduced GA20-oxidase (<i>GA20ox1</i>) and GA3-oxidase (<i>GA3ox</i>) levels and increased GA2-oxidase (<i>GA2ox</i>) and GA2-oxidase6 (<i>GA2ox6</i>), particularly in stems, suggesting enhanced gibberellin (GA) inactivation. Overexpression of <i>VtF3'5'H</i> and <i>RhNHX</i> led to simultaneous changes in floral pigmentation and plant morphology. These findings indicate that both genes play functional roles in color development and growth regulation in roses.