SummaryMany angiosperm families develop spatially regulated anthocyanin spots on their flowers. The Asiatic hybrid lily (Lilium spp.) cv 'Latvia' develops splatter-type spots on its tepals. The splatters arise simply from the deposition of anthocyanin pigments in the tepal epidermis.To determine how splatter development was regulated, we analysed the transcription of anthocyanin biosynthesis genes, and isolated and characterized an R2R3-MYB gene specific to splatter pigmentation.All anthocyanin biosynthesis genes were expressed in splatter-containing regions of tepals, but not in other regions, indicating that splatter pigmentation is caused by the transcriptional regulation of biosynthesis genes. Previously characterized LhMYB12 regulators were not involved in splatter pigmentation, but, instead, a new allele of the LhMYB12 gene, LhMYB12-Lat, isolated in this study, contributed to splatter development. In 'Latvia' and other lily plants expressing splatters, LhMYB12-Lat was preferentially transcribed in the splatter-containing region of tepals. Progeny segregation analysis showed that LhMYB12-Lat genotype and splatter phenotype were co-segregated among the F 1 population, indicating that LhMYB12-Lat determines the presence or absence of splatters.LhMYB12-Lat contributes to splatter development, but not to full-tepal pigmentation and raised spot pigmentation. As a result of its unique sequences and different transcription profiles, this new allele of LhMYB12 should be a novel R2R3-MYB specifically associating with splatter spot development.

BackgroundCRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.).ResultsWe attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene.ConclusionsThese results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1539-3) contains supplementary material, which is available to authorized users.

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Lilium speciosum is one of the most important wild species used for breeding Oriental hybrid lily cultivars. Typical L. speciosum flowers possess red tepals and dark red anthers because of anthocyanin accumulation. However, white-flowered L. speciosum with two distinct phenotypes, one with white tepals and dark red anthers and the other with white tepals and yellow anthers, also exists. To elucidate the molecular mechanisms underlying white colouration, we isolated two regulatory genes and eight biosynthetic genes responsible for anthocyanin pigmentation in L. speciosum and compared these in red-and white-flowered lines. In the white tepal line with dark red anthers, we detected an amino acid substitution in the R2 repeat of LsMYB12, the R2R3-MYB that regulates tepal-specific anthocyanin biosynthesis, which led to reduced anthocyanin biosynthetic gene transcription and no tepal pigmentation. This LsMYB12 sequence was identical to that of an LhMYB12 in the white-flowered Oriental hybrid lily cultivar 'Rialto', indicating that this LsMYB12 sequence is present in lines that have been used for breeding. In addition, a nonsense mutation was detected in the dihydroflavonol 4-reductase (DFR) sequence of the white tepal line with yellow anthers, which resulted in a lack of anthocyanin accumulation in tepals and anthers. This mutated DFR has not been used for lily breeding because white tepal Oriental hybrid lily cultivars usually have dark red anthers. These results suggest that several mechanisms generate white flowers in lilies, and provide a substantial foundation for the production of Oriental hybrid lily cultivars with novel flower colour combinations.

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), and anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3′GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3′AT resulted in the accumulation of delphinidin 3G-5G-3′G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3′GT and the other involving an acylation by 5/3′AT. The Gt5GT, Gt3′GT, and Gt5/3′AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3′-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

Japanese gentians are the most important ornamental flowers in Iwate Prefecture and their breeding and cultivation have been actively conducted for half a century. With its cool climate and large hilly and mountainous area, more than 60% of gentian production in Japan occurs in Iwate Prefecture. Recent advances in gentian breeding and cultivation have facilitated the efficient breeding of new cultivars; disease control and improved cultivation conditions have led to the stable production of Japanese gentians. Molecular biology techniques have been developed and applied in gentian breeding, including the diagnosis of viral diseases and analysis of physiological disorders to improve gentian production. This review summarizes such recent approaches that will assist in the development of new cultivars and support cultivation. More recently, new plant breeding techniques, including several new biotechnological methods such as genome editing and viral vectors, have also been developed in gentian. We, therefore, present examples of their application to gentians and discuss their advantages in future studies of gentians.

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Lilies (Lilium) are among the most important floriculture crops, and to accelerate research regarding lily genetics, the development of reverse-genetics tools is necessary. However, Agrobacterium-mediated transformation in Lilium is timeconsuming, since the plants require several years to progress from acclimation to flowering. Thus, virus-induced gene silencing (VIGS) is an attractive method for assaying gene function. In the present study, we modified a lily-derived strain of Cucumber mosaic virus (CMV-HL) as a VIGS vector and evaluated its effectiveness for inducing gene silencing in Lilium leichtlinii by introducing L. leichtlinii phytoene desaturase (LlPDS) gene fragments into an intercistronic region between the 3a and 3b genes of the CMV-HL RNA3 genome. At 30 days after inoculation (dpi) with LlPDS-containing CMV-HL, photobleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, we observed that the natural orange color in flower tepals had faded. Reduced LlPDS expression and the detection of small interfering LlPDS RNA indicated that the color changes were the result of LlPDS gene silencing. In addition, the leaves also exhibited a mild photo-bleaching phenotype in the following year. Therefore, our results indicate that CMV-HL spreads systemically in the leaves and flowers of Lilium during the first year of infection, as well as in new shoots during the following year, and that the vector system can be successfully applied to induce short-term endogenous gene silencing in lilies.