Flower Color Modification Research Articles

The Genome-Wide Identification of the Dihydroflavonol 4-Reductase (DFR) Gene Family and Its Expression Analysis in Different Fruit Coloring Stages of Strawberry.

Dihydroflavonol 4-reductase (DFR) significantly influences the modification of flower color. To explore the role of DFR in the synthesis of strawberry anthocyanins, in this study, we downloaded the CDS sequences of the DFR gene family from the Arabidopsis genome database TAIR; the DFR family of forest strawberry was compared; then, a functional domain screen was performed using NCBI; the selected strawberry DFR genes were analyzed; and the expression characteristics of the family members were studied by qRT-PCR. The results showed that there are 57 members of the DFR gene family in strawberry, which are mainly expressed in the cytoplasm and chloroplast; most of them are hydrophilic proteins; and the secondary structure of the protein is mainly composed of α-helices and random coils. The analysis revealed that FvDFR genes mostly contain light, hormone, abiotic stress, and meristem response elements. From the results of the qRT-PCR analysis, the relative expression of each member of the FvDFR gene was significantly different, which was expressed throughout the process of fruit coloring. Most genes had the highest expression levels in the full coloring stage (S4). The expression of FvDFR30, FvDFR54, and FvDFR56 during the S4 period was 8, 2.4, and 2.4 times higher than during the S1 period, indicating that the DFR gene plays a key role in regulating the fruit coloration of strawberry. In the strawberry genome, 57 members of the strawberry DFR gene family were identified. The higher the DFR gene expression, the higher the anthocyanin content, and the DFR gene may be the key gene in anthocyanin synthesis. Collectively, the DFR gene is closely related to fruit coloring, which lays a foundation for further exploring the function of the DFR gene family.

Integrated Analyses of Metabolome and RNA-seq Data Revealing Flower Color Variation in Ornamental Rhododendron simsii Planchon.

Rhododendron simsii Planchon is an important ornamental species in the northern hemisphere. Flower color is an important objective of Rhododendron breeding programs. However, information on anthocyanin synthesis in R. simsii is limited. In this research, the regulatory mechanism of anthocyanin biosynthesis in R. simsii was performed through the integrated analysis of metabolome and RNA-seq. A total of 805 and 513 metabolites were screened by positive and negative ionization modes, respectively, In total, 79 flavonoids contained seven anthocyanidins, 42 flavanones, 10 flavans, 13 flavones, and seven flavonols. Methylated and glycosylated derivatives took up the most. Differentially accumulated metabolites were mainly involved in "flavone and flavonol biosynthesis", "cyanoamino acid metabolism", "pyrimidine metabolism", and "phenylalanine metabolism" pathways. For flavonoid biosynthesis, different expression of shikimate O-hydroxycinnamoyltransferase, caffeoyl-CoA O-methyltransferase, flavonoid 3'-monooxygenase, flavonol synthase, dihydroflavonol 4-reductase/flavanone 4-reductase, F3'5'H, chalcone synthase, leucoanthocyanidin reductase, and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase genes ultimately led to different accumulations of quercetin, myricetin, cyanidin, and eriodictyol. In flavone and flavonol biosynthesis pathway, differential expression of F3'5'H, flavonoid 3'-monooxygenase and flavonol-3-O-glucoside/galactoside glucosyltransferase genes led to the differential accumulation of quercetin, isovitexin, and laricitrin. This research will provide a biochemical basis for further modification of flower color and genetic breeding in R. simsii and related Rhododendron species.

Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments

BackgroundBetalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri—or wishbone flower—a popular ornamental plant, has not been attempted.ResultsWe report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar “Crown Violet.” Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins—mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected.ConclusionsThis study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Flower color modification through co-overexpression of the VtF3′5′H and RhNHX genes in Rosa hybrida

Roses (Rosa hybrida) are a highly merchandised flower but lack blue varieties. Overexpression of the flavonoid 3′,5′-hydroxylase (F3′5′H) gene can increase the accumulation of blue pigment (delphinidin anthocyanin). However, sometimes the effect of F3′5′H gene alone is inadequate for producing blue flowers. Furthermore, the internal environment of the cell, such as an increase in pH, can also help the conversion of anthocyanins to blue pigments. Nonetheless, genetic engineering methods can simultaneously introduce multiple genes at the same time to regulate the development of blue pigments to achieve the ultimate breeding goal of producing blue color in roses. In the present study, to simultaneously adjust the accumulation of delphinidin and vacuolar pH, we introduced the Viola tricolor flavonoid 3′,5′-hydroxylase (VtF3′5′H) and Rosa hybrida Na+/H+ exchanger (RhNHX) genes into the white rose line “KR056002” using Agrobacterium-mediated transformation. The quantitative real time polymerase chain reaction (qRT-PCR) results showed that the heterologous genes in the transgenic lines were highly expressed in petals and leaves, and simultaneously promoted the expression of related anthocyanin synthesis structural genes. Obvious color changes were observed in both petals and young leaves, especially when petals changed from white to red-purple. The formation of delphinidin was not detected in the petals of control plants, whereas the petals of transgenic lines had higher delphinidin content (135–214 μg/l) and increased pH value (0.45–0.53) compared with those of control plants.

Comparative Transcriptome Analysis of Anthocyanin Biosynthesis in Pansy (Viola × wittrockiana Gams.)

Pansy (Viola × wittrockiana Gams.) is an important and attractive ornamental plant with a wide variety of flower colors. To date, the molecular genetic understanding of its colorful petal pigment patterns remains largely unknown. In this study, we analyzed the bicolor petals of “Mengdie” in cytological, physiological, and transcriptomic aspects. Results showed that the difference of flower colors was mainly determined by the pigment distribution in the epidermal cells. Pigment analysis indicated that anthocyanins had strong correlations with color parameters, which acted as the main factor in flower coloration. Comparative transcriptome analysis found a total of 43,908 unigenes with the mean length of 682 bp. There were 24,323, 16,668, 8507, and 7680 unigenes annotated in the Nr, Swiss-Prot, KOG, and KEGG databases, respectively. Differential expression genes (DEGs) showed that the expression of anthocyanin late biosynthesis genes (LBGs), VwF3′H, VwF3′5′H, and VwUFGT, would be likely to play a major role in the color formation of pansy. The expression patterns of selected DEGs were verified by qRT-PCR. This study contributes an excellent insight into molecular mechanism of pigment biosynthesis and provides some useful information for flower color modification in pansy.

Overexpression of the Raphanus sativus RsMYB1 using the flower-specific promoter (InMYB1) enhances anthocyanin accumulation in flowers of transgenic Petunia and their hybrids

The generation of transgenic floricultural crops for flower color modification requires a transgene that enhances anthocyanin accumulation and a flower-specific promoter with stable expression throughout flowering stage. Here, the anthocyanin regulatory R2R3-MYB transcription factor (TF) RsMYB1 was placed under the control of the stable flower-specific promoter (InMYB1) to enhance anthocyanin accumulation in Petunia hybrida cv. Mirage Rose. RsMYB1 overexpression with the flower-specific promoter enhanced anthocyanin accumulation only in floral tissues via the upregulation of key anthocyanin biosynthetic genes. The target gene introgression to another Petunia hybrida cv. White Hawaiian lacking anthocyanins also changed the flower color from white to intense pink in the F1 hybrids by upregulating the biosynthetic genes. Target gene inheritance in the F2 hybrids also produced diverse flower colors, redder than those of non-inheritance F2 hybrids. RsMYB1 overexpression using the flower-specific promoter (InMYB1) enhanced anthocyanin levels in the petals of transgenic Petunia and its related hybrids. Thus, this system can be used as a promising tool for intensifying the flower color of other commercially important floricultural crops. In addition, the obtained F2MW11 and F2MW12 lines are potential commercial cultivars for the floriculture and landscape industries due to its attractive flower color pattern.

Genetic engineering as a way to obtain ornamental plants with a changed flower color

An important trend in the field of floriculture is the creation of new varieties of ornamental plants, among which varieties with unusual color are most in demand. To this end, traditional breeding and selection programs have been successfully applied for many years. However, currently genetic engineering is able to offer an alternative way to obtain new forms and varieties. Anthocyanins belonging to flavonoids, betalains and carotenoids are the main types of pigments that are synthesized in the plant and are responsible for the color of flower petals. The modification of pigment biosynthesis pathways using genetic engineering techniques can produce results that cannot be obtained by traditional breeding. This review presents the main advances in the application of genetic engineering techniques in floriculture using the example of flower color modification. There are several main areas of work with the genes of pigment biosynthesis. Among them, the strategy of suppressing gene expression is used most often. Expression of certain genes is suppressed to prevent pigment synthesis, or vice versa, to eliminate factors that hinder color development. The method of additional heterologous genes insertion to plants lacking them in the pathway of pigment biosynthesis is often used. Genomic editing, in particular by using the CRISPR/Cas system, is also used for color modification, but the application of this method to ornamental plants is a relatively recent innovation. Despite the rapid development of biotechnology, there are obstacles to the distribution of genetically modified plants on the world market. By addressing a number of problems, the production of transgenic ornamental plants may become economically more cost-effective and attractive than the development of new varieties exclusively through traditional breeding methods.

A novel R3 MYB transcriptional repressor, MaMYBx, finely regulates anthocyanin biosynthesis in grape hyacinth

R3-MYBs negatively regulate anthocyanin pigmentation in plants. However, how R3-MYB repressors finely modulate anthocyanin biosynthesis in cooperation with R2R3-MYB activators remains unclear in monocots. We previously identified two anthocyanin-related R2R3-MYB activators (MaMybA and MaAN2) in grape hyacinth (Muscari spp.). Here, we isolated a R3-MYB repressor, MaMYBx, and characterized its role in anthocyanin biosynthesis using genetic and biochemical markers. The temporal expression pattern of MaMYBx was similar to that of MaMybA and MaAN2, and it was correlated with anthocyanin accumulation during flower development. MaMYBx could be activated either by MaMybA alone or by MaMybA/MaAN2 and cofactor MabHLH1, and it suppressed its own activation and that of MaMybA promoters mediated by MaMybA/MaAN2 and MabHLH1. Like MaMybA, MaMYBx interacted with MabHLH1. MaDFR and MaANS transcription and anthocyanin accumulation mediated by MaMybA/MaAN2 and MabHLH1 were inhibited by MaMYBx. Overexpression of MaMYBx in tobacco greatly reduced flower pigmentation and repressed the expression of late structural and regulatory anthocyanin pathway genes. Thus, MaMYBx finely regulates anthocyanin biosynthesis by binding to MabHLH1 and disrupting the R2R3 MYB-bHLH complex in grape hyacinth. The regulatory network of transcriptional activators and repressors modulating anthocyanin biosynthesis is conserved within monocots. MaMYBx seems a potentially valuable target for flower color modification in ornamental plants.

An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants

Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy. Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth.

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri

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.

Flavonoid Metabolic Engineering for Modification of Flower Color in Chrysanthemum

Flavonoid Metabolic Engineering for Modification of Flower Color in Chrysanthemum

Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter.

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6–43.2 fold in the PANSBP_DP plants and 2.0–124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation.

Chrysanthemum: rich diversity of flower color and full possibilities for flower color modification

The diversity of chrysanthemum (Chrysanthemum × morifolium) germplasm resources is unique in global horticultural history for the beauty and rich variations of its flower color. Due to the lack of blue and bright red colors, modification of chrysanthemum color has become a hot topic in the study of flower color. Recent advanced studies on flower color of chrysanthemum germplasm and the worldwide possibilities for improvement of its flower coloration are reviewed. Literature analysis showed that intuitional description of chrysanthemum color in the abundant archaic Chinese literature provided clues about the evolution of chrysanthemum color. Moreover, an integrative research system for chrysanthemum color has been successfully constructed, including phenotypic analysis, phytochemicals, plant physiology and genetics progressing. The modification of chrysanthemum color is also developing rapidly. The present understanding of the mechanism of flower coloration provides significant references for future studies on flower color modification in chrysanthemums.

Transcriptomics and Metabolite Analysis Reveals the Molecular Mechanism of Anthocyanin Biosynthesis Branch Pathway in Different Senecio cruentus Cultivars

The cyanidin (Cy), pelargonidin (Pg), and delphinidin (Dp) pathways are the three major branching anthocyanin biosynthesis pathways that regulate flavonoid metabolic flux and are responsible for red, orange, and blue flower colors, respectively. Different species have evolved to develop multiple regulation mechanisms that form the branched pathways. In the current study, five Senecio cruentus cultivars with different colors were investigated. We found that the white and yellow cultivars do not accumulate anthocyanin and that the blue, pink, and carmine cultivars mainly accumulate Dp, Pg, and Cy in differing densities. Subsequent transcriptome analysis determined that there were 43 unigenes encoding anthocyanin biosynthesis genes in the blue cultivar. We also combined chemical and transcriptomic analyses to investigate the major metabolic pathways that are related to the observed differences in flower pigmentation in the series of S. cruentus. The results showed that mutations of the ScbHLH17 and ScCHI1/2 coding regions abolish anthocyanin formation in the white and the yellow cultivars; the competition of the ScF3′H1, ScF3′5′H, and ScDFR1/2 genes for naringenin determines the differences in branching metabolic flux of the Cy, Dp, and Pg pathways. Our findings provide new insights into the regulation of anthocyanin branching and also supplement gene resources (including ScF3′5 ′H, ScF3′H, and ScDFRs) for flower color modification of ornamentals.

Red Anthocyanins and Yellow Carotenoids Form the Color of Orange-Flower Gentian (Gentiana lutea L. var. aurantiaca).

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.

Transient hairpin RNAi-induced silencing in floral tissues of Dendrobium Sonia ‘Earsakul’ by agroinfiltration for rapid assay of flower colour modification

Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants, and transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for colour modification assay in floral tissues of Dendrobium Sonia ‘Earsakul’, a tropical hybrid orchid with purple and white bi-coloured flowers. Portions of the D. Sonia ‘Earsakul’ anthocyanin-related genes chalcone synthase (DseCHS-B) and dihydroflavonol 4-reductase (DseDFR) which are required for anthocyanin synthesis, were cloned into the hairpin-based RNAi vectors, pSTARGATE and pWATERGATE, under the control of the maize ubiquitin and Arabidopsis Rubisco small subunit (ARbcS) promoters, respectively. Characterisation of DseCHS-B and DseDFR expression profiles revealed that their expression was developmentally regulated in the flower and associated with flower pigmentation. Transient RNA silencing of DseCHS-B and DseDFR in the floral tissues of D. Sonia ‘Earsakul’ was induced by delivering 436-bp DseCHS-B and 470-bp DseDFR hairpin RNAs (hpRNAs) into the sepals and petals of flower buds at early developmental stages using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the sepals and petals. Silencing of the endogenous DseCHS-B and DseDFR mRNAs was highly effective at the early stages of mRNA synthesis and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in D. Sonia Earsakul and other orchids through gene suppression.

Bioinfomatics Analysis of the <i>An</i><i>thocyanidin s</i><i>y</i><i>nthase</i> Gene in Tree Peony

Anthocyanin mainly responsible for flowers color in many plant species, it also accumulated in response to lots of environmental stress to reduce the damage to plant cell. Anthocyanin synthesis (ANS) protein is an important synthetase participated in anthocyanin biosynthetic pathway. In this study, we isolated the PsANS gene from transcriptome database built by our previous study. The PsANS gene contain an 1050bp open reading frame encoding 349 amino acid, phylogenetic analysis revealed that PsANS was segrated into a group with ANS from others plant species. Secondary and thri-dimension structure prediction also revealed that it may have similar function with ANS in others plant species. The identified PsANS gene would be helpful for further research in flower color modification and resistance breeding.

ANALYSIS OF FLAVONOID-BIOSYNTHESIS GENE EXPRESSION IN THE FLOWERS OF MAJOR LILY CULTIVARS

Flavonoids biosynthesis pathway mostly determines the flower colour pheno-types in plants. This study was carried out to determine the presence and expression rates of the flavonoids biosynthesis-related genes in major lily cultivars. Primers of major colour genes were prepared by comparing the homologous genes of other species like sorghum, onion, and rice, and used for RT- PCR of eight lily cultivars. The results of transcriptional rate analyses showed that, in pink-flowering lily cultivars like, 'Pirax', red-flowering cultivar 'Atika', yellow-flowering cultivar 'Gironde', and white cultivar 'Siberia', all the genes tested were expressed. However, F3'H and DFR genes were not expressed in yellow-flowering cultivar 'Yelloween'. In white-flowering cultivars 'Samarinde' 'Richmond' and 'White Elegance', gene expressions varied among cultivars, especially in the genes CHS, F3H and ANS. We assume that the results on the presence and/or expression rates of the major flavonoid-biosynthesis genes shall be useful in the hybridization and transformation breeding in lilies for flower colour modification.