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Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

Background The blue pigmentation of Japanese gentian flowers is due to a polyacylated anthocyanin, gentiodelphin, and all associated biosynthesis genes and several regulatory genes have been cloned and characterized. However, the final step involving the accumulation of anthocyanins in petal vacuoles remains unclear. We cloned and analyzed the glutathione S-transferases (GSTs) in Japanese gentian that are known to be involved in anthocyanin transport in other plant species. Results We cloned GST1, which is expressed in gentian flower petals. Additionally, this gene belongs to the Phi-type GST clade related to anthocyanin biosynthesis. We used the CRISPR/Cas9-mediated genome editing system to generate loss-of-function GST1 alleles. The edited alleles were confirmed by Sanger and next-generation sequencing analyses. The GST1 genome-edited lines exhibited two types of mutant flower phenotypes, severe (almost white) and mild (pale blue). The phenotypes were associated with decreased anthocyanin accumulation in flower petals. In the GST1 genome-edited lines, sugar-induced stress conditions inhibited the accumulation of anthocyanins in stems and leaves, suggestvhing that GST1 is necessary for stress-related anthocyanin accumulation in organs other than flowers. These observations clearly demonstrate that GST1 is the gene responsible for anthocyanin transport in Japanese gentian, and is necessary for the accumulation of gentiodelphin in flowers. Conclusions In this study, an anthocyanin-related GST gene in Japanese gentian was functionally characterized. Unlike other biosynthesis genes, the functions of GST genes are difficult to examine in in vitro studies. Thus, the genome-editing strategy described herein may be useful for in vivo investigations of the roles of transport-related genes in gentian plants.

Virus-induced gene silencing (VIGS) systems are effective for rapid analysis of gene functions in plants that require a long period of growth such as Lilium. We successfully developed a VIGS system using the cucumber mosaic virus (HL strain, CMV-HL) vector to induce RNA silencing of the L. leichtlinii phytoene desaturase gene (LlPDS), where at 30 days postinoculation (dpi), photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, white regions appeared on flower tepals that accumulate orange carotenoids. This vector spreads in bulbs, and it could induce silencing on emerged shoots in the following year. The CMV-HL vector can be easily constructed by insertion of a 30-60 nt fragment into the cloning site of the RNA3 genome. In this chapter, we describe how to use the CMV-HL vector system in the context of Lilium plants.

Virus-induced gene silencing (VIGS) systems are effective for rapid analysis of gene functions in plants that require a long period of growth such as Lilium. We successfully developed a VIGS system using the cucumber mosaic virus (HL strain, CMV-HL) vector to induce RNA silencing of the L. leichtlinii phytoene desaturase gene (LlPDS), where at 30 days postinoculation (dpi), photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, white regions appeared on flower tepals that accumulate orange carotenoids. This vector spreads in bulbs, and it could induce silencing on emerged shoots in the following year. The CMV-HL vector can be easily constructed by insertion of a 30-60 nt fragment into the cloning site of the RNA3 genome. In this chapter, we describe how to use the CMV-HL vector system in the context of Lilium plants.

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2′-glucoside (Ch2′G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2′G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2′G by chalcone 2′-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2′G simultaneously to give orange color.

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes.

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.

The spider-type azalea cultivars, Rhododendron kaempferi ‘Kin-kujyaku’ and Rhododendron macrosepalum ‘Seigaiha’, are characterized by extremely narrow leaves and choripetalous flowers with narrow petals. Previous morphological analysis of these cultivars revealed that narrowed organs would be a common mutation in the growth of lateral organs in the transverse plane. In the present study, we focused on the analysis of WUSCHEL-related homeobox (WOX) genes, WOX1 and WOX3, which regulate the laminar width and development of lateral organs. Sequence analysis of the two genes confirmed a fatal mutation in Rk/RmWOX3/SPIDER of ‘Kin-kujyaku’ and ‘Seigaiha’. To demonstrate the role of SPIDER in the spider-type phenotype in azalea, we performed genotyping using DNA markers developed for multiplex PCR that can distinguish between the wild-type and the spider-type allele based on the partial mutated region of RkWOX3/SPIDER in ‘Kin-kujyaku’. The investigation of BC1 population derived from ‘Kin-kujyaku’ confirmed complete co-segregation between spider-type mutation and Rkwox3/spider band pattern of DNA markers. Therefore, we concluded that SPIDER is responsible for the spider-type mutation in azalea.

Japanese gentian is one of the most important ornamental flowers in Japan. Its most attractive trait is the vivid blue flower, whose color results from polyacylated anthocyanins. Other important traits in gentian breeding include flower shape, flowering time, and overwintering characteristics. To show the molecular mechanism by which these traits are regulated, transgenic gentian plants are often generated by Agrobacterium-mediated transformation. However, this method is not suitable for all gentian cultivars because of the difficulties in regeneration and/or transformation. Also, it takes a long time to obtain transgenic plants and analyze their phenotypes. Therefore, it is important to develop a more efficient method to analyze gene function in gentian. In the present study, we developed a novel viral expression vector for efficient gene functional analysis in gentian using Broad bean wilt virus 2 (BBWV-2) infectious cDNA. First, we expressed two reporter genes, GFP and GUS, in gentian using the BBWV-2 vector. Fluorescence from GFP was observed in various organs including the leaf, flower, root, and overwintering bud. β-Glucuronidase (GUS) activity was detected in the inoculated leaf, but not in upper uninoculated leaves because the GUS insert was unstable in the BBWV-2 vector. Next, we inoculated the BBWV-2 vector carrying GtMYB3, a transcription factor regulating anthocyanin biosynthesis, fused with SRDX, which encodes the EAR transcriptional repression domain. Expression of GtMYB3-SRDX via the BBWV-2 vector partly changed the gentian petal color from blue to white. The expression levels of genes encoding anthocyanin biosynthetic enzymes (flavanone 3-hydroxylase, dihydroflavonol-4-reductase, anthocyanidin synthase, flavonoid 3′,5′-hydroxylase, and anthocyanin 5,3′-aromatic acyltransferase) were markedly lower in the white parts of petals than in the blue parts. These results clearly indicated that GtMYB3-SRDX expressed via the BBWV-2 vector was functional in gentian flowers. This BBWV-2-based gene expression system enabled us to analyze a phenotypic change in gentian flower color within a few months.

The purpose of this study is to address requests for higher quality and productivity improvement through wire electrode coating technology. A prime example of high quality is the improved life of tungsten carbide. When a tungsten carbide material is cut in water, the cobalt binder starts to dissolve. As a result, the material becomes weak. In this study, we experimentally examine the effect of coating the cutting surface with wire brass. We found that the life of molds could be thereby extended. As an example of productivity improvement, we show a method for core processing automation. When the operator completes a rough cut for all shapes leaving uncut parts, the operator must move the axis manually for each shape in order to finish the uncut part and remove the core. We studied the effect of coating the cutting surface with brass wire in this context, as well. Accordingly, we found that brass could be coated on a part 1 mm from its upper face and could hold the cores. The operator can then cut away the coated part by simply tapping on the cores.

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 time-consuming, 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, photo-bleaching 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.

This article has been published with an erroneous version of Table 2. Please find the correct Table 2 in this document.

Floral shape in higher plants typically requires genetic regulation through MADS transcription factors. In Japan, hundreds of azalea cultivars including flower shape mutations have been selected from the diversity of endogenous species and natural hybrids since the early 17th century, the Edo era (1603–1867). The long-lasting trait, known as “Misome-shō” in Japanese, has been identified in several species and cultivar groups of evergreen azaleas (Rhododendron L.) from three hundred years ago in Japan. However, the natural mutation conferring the long-lasting trait in azalea remains unknown. Here, we showed MADS-box gene mutations in long-lasting flowers, R. kaempferi ‘Nikkō-misome,’ R. macrosepalum ‘Kochō-zoroi,’ R. indicum ‘Chōjyu-hō,’ and R. × hannoense ‘Amagi-beni-chōjyu.’ All of the long-lasting flowers exhibited small-sized corollas with stomata during long blooming. In the long-lasting flowers, transcript of the APETALA3 (AP3)/DEFICIENS (DEF) homolog was reduced, and an LTR-retrotransposon was independently inserted into exons 1, 2, and 7 or an unknown sequence in exon 1 in gDNA of each cultivar. This insertion apparently abolished the normal mRNA sequence of the AP3/DEF homolog in long-lasting flowers. Also, long-lasting flowers were shown from F2 hybrids that had homozygous ap3/def alleles. Therefore, we concluded that the loss of function of the AP3/DEF homolog through a transposable element insertion may confer a stable long-lasting mutation in evergreen azaleas.

Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS, from the gentian plant, Gentiana triflora. Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants.

Morphological mutations have been admired and preserved in the classical cultivars of the Japanese evergreen azalea since the Edo era (1603–1867). One such mutation is the presence of narrow leaves and petals. In this study, we investigated the characteristics and inheritance patterns of the narrow leaf-type mutational form of Rhododendron macrosepalum ‘Seigaiha’, R. kaempferi ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant. The leaves of all cultivars showed weak development in the transverse plane and thickened margins. Subsequently, based on this qualitative character, the inheritance of the narrow leaf mutation was investigated. F1 seedlings of cross combinations between normal and narrow leaf-type plants had normal leaf-type. All F1 progenies of cross combinations between narrow leaf-type plants had narrow leaves like their parents. In addition, seedlings obtained from backcross and sib-cross tests using narrow leaf mutants segregated into normal and narrow leaf-types at a 1:1 and 3:1 ratio, respectively. These results indicate that the narrow leaf trait of three different azalea cultivars, ‘Seigaiha’, ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant, is probably a recessive mutation caused by a single, common gene.

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.

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F2 populations were produced by selfing F1 plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F2 populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future.

Summary Many 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 F1 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.

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 time-consuming, 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, photo-bleaching 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.

The morphological traits of the floral mutation cultivars ‘Kagaribi’, ‘Kinshibe’ and ‘Kin-kujyaku’ of Rhododendron kaempferi that characteristically exhibit extremely narrow and/or staminoid petals were investigated. In addition, to confirm the causes of the mutations in these cultivars, the MADS-box C class AG gene homologues in R. kaempferi were isolated, and the MADS-box gene expression patterns in the floral organs of the wild-type plants and cultivars were analyzed. A morphological observation of the epidermis showed that the ‘Kagaribi’ and ‘Kinshibe’ cultivars formed staminoid flag petals and staminoid organs in whorl 2, respectively. The epidermal cells of the staminoid organs of whorl 2 in ‘Kagaribi’ and ‘Kinshibe’ resembled those of the stamens in whorl 3. In ‘Kin-kujyaku,’ the petals in whorl 2 and leaves were extremely narrow. The sepals in whorl 1 were absent, and the style in whorl 4 was split into 5 parts. The epidermal cells of the petals in whorl 2 in ‘Kin-kujyaku’ were dissimilar to the epidermal cells of the stamens in whorl 3. The deduced amino acid sequence of the isolated AG homologue from R. kaempferi was well-conserved among the AG orthologues. The expression levels of RkAG in whorl 2 to whorl 3 of ‘Kagaribi’ and ‘Kinshibe’ was approximately 27% and the same level, respectively. In ‘Kin-kujyaku,’ the expression pattern was similar to that of the wild type. These results suggest that the ‘Kagaribi’ and ‘Kinshibe’ floral mutations might be caused by the expression of RkAG in whorl 2.

To investigate seasonal changes in transcript levels of flowering-related genes corresponding to shoot condition, the endogenous expression patterns of FLOWERING LOCUS T ( FT ), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC 1), LEAFY ( LFY ), TERMINAL FLOWER 1 ( TFL 1), and CENTRORADIALIS ( CEN ) homologs were investigated in evergreen azalea ‘Oomurasaki’. In this plant, in which the floral transition is triggered by the about 25 °C and shortening days of summer under natural conditions, the mRNA levels of the FT , SOC 1, and LFY homologs ( RpFT , RpSOC 1, and RpLFY ) increased in August corresponding to the period of flower bud formation, and the mRNA levels of TFL 1 and CEN homologs ( RpTFL 1 and RpCEN ) increased during July before flower bud formation. RpTFL 1 showed low transcript levels during the period of flower bud formation and development, but RpCEN showed a different expression pattern from RpTFL 1. The expression of RpCEN , RpSOC 1, and RpLFY peaked again during January corresponding to the period of flower bud dormancy. During blooming in May, the expression levels of all the investigated genes were low. In the period of flower bud formation, RpFT expression started to increase, and the expression of RpSOC 1 peaked in mid-July. These results suggest that the shoot apical meristem (SAM) starts to transition from vegetative to reproductive growth in mid-July. Thus, changes in the transcription of RpFT , RpSOC 1, and RpLFY were found to correlate positively with seasonal flowering in the shoot apex of ‘Oomurasaki’, whereas changes in RpTFL 1 and RpCEN transcription correlated negatively with seasonal flowering.

In evergreen azalea, unique old mutational cultivars, whose flower have split figure, have been collected and is called “sai-zaki” in Japanese. The morphology of leaves and flowers of the wild-type and sai-zaki cultivars ‘Hanaguruma’, ‘Gin-no-zai’, and ‘Seigaiha’ of R. macrosepalum was investigated. In contrast to the gamopetalous corolla of the wild type, all sai-zaki cultivars formed the choripetalous corolla type, which consisted of 5 independent petals. The width of the leaf, sepal, and petal of all sai-zaki cultivars, except for the sepal of ‘Gin-no-zai’, all tended to be narrower than the wild type; in particular, all lateral organs of ‘Seigaiha’ were extremely narrow compared with the wild type. In contrast to the normal pistil of the wild type, ‘Hanaguruma’ and ‘Gin-no-zai’, normal and malformed pistils we’re observed in ‘Seigaiha’. Epidermal cell size was not related to the narrowness of lateral organs in the wild-type and sai-zaki cultivars. These results indicate that the sai-zaki trait of materials, at least ‘Seigaiha’, would be a common mutation in the growth of lateral organs in the transverse plane. All sai-zaki cultivars showed fertility. The sai-zaki trait of old Japanese cultivars from the Edo era is expected to provide new breeding material that offers the opportunity to generate additional morphological novelties.

The trait of long-lasting flower corolla with temporal change of colour exists in Japanese evergreen azalea. This trait called "misome-sho" has been found in several species and cultivar groups from Edo era. For the purpose of application of this trait for breeding, we investigated several characteristics of these cultivars. Wild species of Rhododendron kaempferi, R. indicum and R. macrosepalum and long-lasting flower cultivars were used in this study. All long-lasting flower cultivars have smaller corolla compared to wild types and have stomata in the corolla surface, while wild types do not have stomata in their surface. Pot planted long-lasting flower cultivars kept their corolla more than 100 days and corolla colour was changed from red or purple-red in anthesis to green later. In contrast, wild type plants in pot finished flowering in about 20 days. Microscopic observation of the longitudinal section of flowers revealed that the abscission layer in the basal portion from wild type corollas is not observed in long-lasting flower cultivars. These cultivars have high pollen fertility and stabilized seed set ability. Inheritance of this trait is investigated using cross progenies. The long-lasting trait of corolla would be derived from the sepaloid nature of petals caused by homeotic gene mutation.

Transcription of anthocyanin biosynthetic genes is usually controlled by transcription factors such as basic-helix-loop-helix (bHLH) and R2R3-MYB. To know the regulatory mechanisms of anthocyanin accumulation in Asiatic hybrid lily (Lilium spp.), cDNA clones encoding bHLH transcription factors were isolated from tepals and their expression was examined in this study. This is the first report about cDNAs for bHLH genes among the species in monocots other than Gramineae. Two cDNAs corresponding to LhbHLH1 and LhbHLH2 genes were isolated; the former encoded the protein having the similarity to AmDELILA and PhJAF13 and the latter one did to PhAN1. LhbHLH1 and LhbHLH2 were expressed in tepals, stems and leaves. In filaments and pistils, only LhbHLH2 was transcribed. During tepal development, transcriptions of LhbHLH genes were always detected, and the peak of LhbHLH2 expression preceded the peak of LhDFR expression. When lily buds were exposed to light, anthocyanin accumulation was induced in leaves and peaked at 4 days after light exposure. The transcription of LhbHLH2 peaked at 2 days after light exposure and decreased after that. LhbHLH1 transcription was affected by light to a lesser extent than LhbHLH2 at 2 days. These expressional changes preceded the change of anthocyanin amount. Similarly in tepals, both anthocyanin amount and the transcription of LhDFR and LhbHLH2 decreased in the dark after 6 days, but LhbHLH1 transcription was not affected. These results suggest that LhbHLH1 and LhbHLH2, especially LhbHLH2, are involved in anthocyanin biosynthesis, and that response to light exposure was different between LhbHLH1 and LhbHLH2.

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.