The flower’s color is regarded as one of the most outstanding features of the rose. Rosa praelucens Byhouwer, an endemic and critically endangered decaploid wild rose species, is abundant in phenotypic diversity, especially in flower color variation, from white to different degrees of pink. The mechanism underlying this variation, e.g., the level of petal-color-related genes, is worth probing. Seven candidate reference genes for qRT-PCR analysis, including tubulin α chain (TUBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H2B (Histone2A), eukaryotic translation elongation factor 1-α (EEF1A), 60S ribosomal protein (RPL37), eukaryotic translation initiation factor 1-α (EIF1A), and aquaporins (AQP), were detected from the transcriptome datasets of full blooming flowers of white-petaled and pink-petaled individuals, and their expression stabilities were evaluated through qRT-PCR analysis. According to stability rankings analysis, EEF1A showed the highest stability and could be chosen as the most suitable reference gene. Moreover, the reliability of EEF1A was demonstrated via qRT-PCR analysis of six petal-color-related target genes, the expression patterns of which, through EEF1A normalization, were found to be consistent with the findings of transcriptome analysis. The result provides an optimal reference gene for exploring the expression level of petal-color-related genes in R. praelucens, which will accelerate the dissection of petal-color-variation mechanisms in R. praelucens.

Gypsophila paniculata is one of the most popular cut flowers in the world whose major cultivars are blooming white. As is well known, blue flowers could be generated via the overexpression of the gene encoding flavonoid 3′5′-hydroxylase (F3′5′H) in species that naturally lack it. In this study, we established the regeneration and a genetic transformation system for the commercial cultivar ‘YX4’ of G. paniculata and introduced the F3′5′H of Platycodon grandiflorus (PgF3′5′H) successfully into ‘YX4’ using the established protocol. A total of 281 hygromycin (Hyg)-resistant plantlets were obtained, and 38 of them were polymerase chain reaction (PCR) positive, indicating a 13.5% transformation efficiency. Shoot apex without meristem was more suitable for explant due to its high regeneration capacity, and the supplement of thidiazuron (TDZ) provided the most efficient promotion of adventitious bud induction, whereas the supplement of 6-Benzyladenine (6-BA) and 1-naphthaleneacetic acid (NAA) did not affect much. Additionally, the combination of 1 day (d) pre-culture, 5 d co-culture, 10 min infection, 30 mg·L⁻¹ additional acetosyringone (AS) supplement, and 10 mg·L⁻¹ Hyg selection formed the optimized system for ‘YX4’ transformation. This reliable and efficient agrobacterium-mediated transformation of the valuable commercial cultivar ‘YX4’ will contribute not only to the creation and improvement of G. paniculata cultivars, but also to the function research of genes associated with important ornamental traits.

Anemone obtusiloba D. Don is a valuable medicinal herb that also possesses considerable ornamental value for its attractive yellow, blue, and white flowers. Previous studies primarily focused on its pharmaceutical properties and the pigmentation of yellow flowers. However, the formation mechanisms underlying blue and white flowers remain unexplored. In this study, we employed transcriptomic and metabolomic approaches to compare the expression profiles of anthocyanin biosynthesis genes and pigment compositions between the blue and white flowers of A. obtusiloba. Key candidate genes were further validated by quantitative real-time PCR (qRT-PCR). Our results showed that six structural genes, such as flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′5′H), anthocyanin synthases (ANSs), dihydroflavonol 4-reductase (DFR) and anthocyanidin 3-O-glucosyltransferase (3GT), exhibited significantly higher expression levels in blue flowers than in white flowers. Among the differentially accumulated metabolites (DAMs), nine pigments were more abundant in blue flowers. Notably, delphinidin-3-O-sambubioside-5-O-glucoside and cyanidin-3-O-glucoside were detected exclusively in blue flowers. We speculate that blue flower coloration is associated with these up-regulated genes, which may contribute to increased pigment content and diversity. Furthermore, a putative R3MYB-like repressor may be involved in suppressing anthocyanin biosynthesis, as its elevated expression in white flowers was associated with lower transcript levels of structural genes, which may contribute to the white flower phenotype in A. obtusiloba.