Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color.

An important part of the assessment of the potential environmental impact from the introduction of a genetically modified (GM) plant is an evaluation of the potential for gene flow from the GM plant to related wild species. This information is needed as part of the risk-assessment process, in the context of whether gene flow to wild species is possible. One method for evaluating gene flow is to use molecular techniques to identify genes in wild species populations that may have originated from a cultivated species. An advantage of this method is that a phenotypic marker or trait is not required to measure gene flow. In the present study we analyzed the seedlings of seeds from three wild native Rosa species (R. multiflora Thunb., R. luciae Rochebr. et Franch. ex Crép. and R. rugosa Thunb.) selected from several locations across Japan where the wild rose was growing in close proximity to cultivated rose plants (Rosaϫhybrida). To determine whether gene flow from cultivated rose had occurred, young leaves of 1,296 seedlings from the wild Rosa plants were analyzed by PCR for the presence of the KSN locus. This locus originated from a sport of R. chinensis Jacq. var. spontanea (Rehd. et Wils.) Yu et Ku and is involved in the recurrent flowering phenotype observed for cultivated rose hybrids, but is absent in Japanese species roses. The KSN locus was absent in all seedlings sampled, indicating no gene flow to wild Rosa species from the cultivated rose had occurred, and providing evidence that the probability of gene flow from cultivated to wild Rosa species in Japan is low or non-existent.

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