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Mutation induction is a feasible and established breeding method for crop improvement and genetic diversity creation to introduce new plant cultivars. The present study was aimed at mutagenesis of four chrysanthemum cultivars (‘Homa’, ‘Fariba2’, ‘Arina’, and ‘Delkash’) using ethyl methanesulfonate (EMS) (0, 0.125, 0.25, and 0.5%) as mutagen and leaf disks as explants to obtain novel variants. In addition, genetic polymorphism among mutants and their parents was detected using inter simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) molecular markers. A total of 2082 plantlets were produced through EMS induced mutagenesis under in vitro conditions and at the end 58 mutants including 28 leaf and 32 flower mutants were analyzed for phenotypic and molecular variation. The explant survival rate decreased by increasing EMS concentration. A wide range of phenotypic leaf and inflorescence variability was obtained in four studied chrysanthemum cultivars confirming the efficiency of EMS to create genetic variation and desired mutants. All generated variants with different inflorescence and leaf shape and color were maintained through cuttings and they expressed same traits in the next generation. The mutants were different in leaf size and shape, plant height, day to flowering, inflorescence head size, ray floret color and ray floret size. The used ISSR and IRAP primers could classify chrysanthemum mutants based on cultivar and somewhat based on used EMS concentration confirming their effectiveness for the discrimination of real variants that allow their earlier selection and reduction of the mutant population size. The in vitro EMS-induced mutation can be a promising tool to assist breeding programs for the generation of new chrysanthemum cultivars. New chrysanthemum mutants with distinct colors and inflorescence shape were obtained in four chrysanthemum cultivars using ethyl methanesulfonate (EMS). IRAP and ISSR could successfully classify the EMS-induced mutants and their relationship with mother plants.

Flowers have become a part of our daily life, and wind up joining the waste stream, after little use and almost practically no reuse. Disposal of the flowers despite playing an eloquent role in religious faith, rituals, wedding ceremony, etc., finishes up mostly at open dumping or in water bodies. An endeavor has been made to valorize the flower waste generated from the flower market in Puducherry. White Marigold Chrysanthemum morifolium was retrieved in bulk from the market waste stream and was thermally treated into valuable low-cost biochar. The products were synthesized by pyrolysis in slow thermal carbonization conditions at 350 °C and 500 °C. Before treating thermally, the collected waste marigold flowers were analyzed for decomposition behavior via TGA. The biochar products were further characterized, analyzed, and compared, for various physiochemical parameters, namely pH, conductivity, moisture content, ash content, and bulk density. Specific physical and chemical properties and the effect of slow pyrolysis at two different temperatures were further investigated by performing FTIR, SEM, XRD, and BET. Biochar pyrolyzed at 500 °C possessed better characteristics for environmental applications than biochar derived at 350 °C.

The inflorescence of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China due to its antioxidant and anti-inflammatory properties. The biosynthesis and regulation of flavonoids, a group of bioactive components, in C. morifolium are poorly understood. Transcriptome sequencing is an effective method for obtaining transcript information. Therefore, single-molecule real-time (SMRT) sequencing was performed to obtain the full-length genes involved in flavonoid biosynthesis and regulation in C. morifolium. High-quality RNA was extracted from the inflorescence of C. morifolium at different flowering stages and used to construct two libraries (0–5 kb and 4.5–10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with a mean length of 2009 bp were obtained. Of these, 2,083 transcripts were annotated to pathways related to flavonoid biosynthesis, and 56 isoforms were annotated as CHS, CHI, F3H, F3’H, FNS II, FLS, DFR and ANS genes. Based on gene expression levels at different stages, we predicted the major genes involved in flavonoid biosynthesis. By phylogenetic analysis, we found two candidate MYB transcription factors (CmMYBF1 and CmMYBF2) activating flavonol biosynthesis. Based on the full-length transcriptomic data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C. morifolium were predicted in our study. The results provide a valuable theoretical basis for the introduction and cultivation of C. morifolium cv. ‘Hangju’.

Natural fiber reinforced green composites have been attracting high attention nowadays, as the green movement in the world forced companies to use green materials instead of synthetic fibre reinforced composites. In this respect, the aim of the study is to investigate usage possibility of undervalued Chrysanthemum morifolium stem fibers as a new reinforcement of composite materials. Chemical, thermal, crystallographic, density, mechanical and morphological characterizations of the C. morifolium fibers were examined. Crystallinity Index, density and tensile strength values were found as 65.18%, 1.33 g/cm3 and 65.12 MPa, respectively. Chrysanthemum morifolium has a low cellulose content of 32.9% while the thermal resistance temperature was determined as 267.5 °C. Although its cellulose content is low, C. morifolium fiber can be a good alternative for many other reinforcement plant fibers in terms of tensile strength. The high tensile strength of the fiber can be attributed to the high crystallinity index and fiber morphology advantage (low lumen diameter and thick cell wall). Hollow fiber morphology can increase the insulation and absorption properties of the fibers and can also create a usage area in lightweight composites by providing low density. This study suggests a novel sustainable ecological reinforcement fiber for green polymer composites with low density, reasonable tensile strength, high surface hydrophobicity and high surface roughness.

In this study, chemical profiling, phenolic and flavonoid contents, as well as the antibacterial and antioxidant activity of 17 Iranian Chrysanthemum morifolium cultivars were evaluated. The high performance liquid chromatography analysis revealed the presence of eight compounds with the major constituents including chlorogenic acid (0–934.7 mg/100 g), ferulic acid (12.7–171.6 mg/100 g), rutin (0–225.8 mg/100 g) and luteolin (2.83–213.5 mg/100 g). The cultivar “Ashna” with 63.6 mg tannic acid equivalents g−1 DW had the highest amount of total phenol, while the highest flavonoid content (13.52 mg quercetin equivalents g−1DW) was observed in cultivar “Shokoh”. The antioxidant activities of the samples were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the reducing power assay. The results showed that the cultivars “Poya3” (IC50 = 385.7) and “Dorna2” (IC50 = 489.4) possessed a higher antioxidant activity than the others in DPPH model system. Antimicrobial activity was also evaluated based on minimal inhibitory concentration (MIC) and minimal bactericidal concentration values. MIC values were in the range of 5–10 mg ml−1 against Salmonella enterica and Bacillus cereus and 10–20 mg ml−1 against Staphylococcus aureus and Escherichia coli. Finally, Chrysanthemum cultivars with high bioactive compounds were introduced for beneficial usage in food and industrial applications.

Chrysanthemum morifolium cv. Hang-ju (HJ) is considered as a healthy food supplement for a long time in Asia. But until now, there are few reports on the biological activity of HJ leaves, which is the main by-products of HJ. In this study, HJ leaves was extracted by water, ethanol and water: ethanol (30: 70), respectively. The results showed that the total polyphenol content of water extract, hydroalcoholic extract and ethanol extract was 75.1 ± 6.9 mg GAE/g, 162.2 ± 5.6 mg GAE/g and 128.5 ± 1.9 mg GAE/g, respectively. However, the ethanol extract has the highest antibacterial activity against the tested strains with a MIC of 0.25 mg/mL compared to water extract (MIC > 8.0 mg/mL) and ethanol extract (MIC = 0.50 mg/mL). The essential oil was extracted from HJ leaves by water distillation (0.5%), which showed higher antibacterial effect on gram-positive bacteria (MIC = 0.125 mg/mL) than ethanol extract. GC–MS was used for the preliminary identification of essential oil composition. The compound (1S,4aS,7R,8aS)-1,4a-dimethyl-7-(prop-1-en-2-yl)decahydronaphthalen-1-ol was first identified from HJ and found to be the most dominant compound. Furthermore, essential oil can significantly deform cell morphology and damage cell wall structure, which was confirmed by LIVE/DEAD® BacLight Viability Kit and scanning electron microscopy observations. The mentioned findings indicated that HJ leaves could be utilized as a natural additive in food industry with significant antimicrobial potential.

The complex capitulum of Chrysanthemum morifolium is often comprised of bilaterally symmetrical ray florets and radially symmetrical disc florets. The TCP transcription factor clade CYCLOIDEA2 (CYC2) appears to play a vital role in determining floral symmetry and in regulating floral organ development in Asteraceae. Our previous study identified six CmCYC2 genes from chrysanthemum and showed that CmCYC2c participated in the regulation of ray floret identity. However, the functions of other CmCYC2 genes and the underlying molecular mechanism of CmCYC2-mediated floral development regulation in chrysanthemums have not been elucidated. In this study, we analysed the function of CmCYC2 genes by ectopic expression of CmCYC2 in Arabidopsis. Then, we examined the protein-protein interaction using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Finally, we analysed the protein-DNA interaction using yeast one-hybrid (Y1H) and dual-luciferase reporter assays. We found that ectopic expression of CmCYC2 genes in the Arabidopsis tcp1 mutant changed its floral symmetry and flowering time. Y2H and BiFC assays confirmed three pairs of interactions between CmCYC2 proteins, that is, CmCYC2b-CmCYC2d, CmCYC2b-CmCYC2e and CmCYC2c-CmCYC2d, suggesting that heterodimeric complexes may form between CmCYC2 proteins to increase their functional specificity. The results of Y1H and dual-luciferase reporter assays indicate that CmCYC2c can bind to the promoter of ClCYC2f. Our findings provided clues that CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in C. morifolium. KEY MESSAGE: CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in Chrysanthemum morifolium.

Plasmid pCAMBIA2201-pta, containing the selectable marker neomycin phosphotransferase gene (npt II), Pinellia ternata agglutinin (PTA) gene and β-glucuronidase gene, were transformed into leaf discs of Chrysanthemum (Chrysanthemum morifolium Ramat.) via Agrobacterium tumefaciens-mediated transformation. It was confirmed by GUS (β-glucuronidase) and PCR analysis that the PTA gene had been successfully inserted into the plant genome. Hemagglutination assay was carried out and the expression of PTA gene in transgenic plants in vitro and the aphid-resistance assay result showed that the inhibition rates of the transformed plants were improved than the control. T1 progenies generated through the cuttage breeding from T0 generation had preserved the aphid-resistance and the inhibition rates of T1 generation had no significant difference with T0 generation (P > 0.05). The results suggest that PTA gene can improve aphid resistance of chrysanthemum and its aphid resistance can be generated to T1 generation stably.

Knowledge of genetic diversity and relatedness of cultivars; is essential for a successful breeding scheme. The present study was conducted to assess the genetic diversity of 30 Iranian Chrysanthemum morifolium cultivars using sequence-related amplified polymorphism (SRAP) molecular markers and morphological traits. For this purpose, 30 cultivars were planted in a completely randomized block design with three replications at the Research Farm of Isfahan University of Technology in 2015. The evaluation of morphological traits during the two consecutive years showed a wide range of variations among different cultivars. The results obtained by the analysis of variance also revealed the significant effects of cultivar, environment and cultivar × environment interaction on all studied traits. Correlation analysis of variables further showed that flowering durability was affected by plant morphology. Results of cluster analysis based on the phenotypic data divided the cultivars into six groups. Molecular analysis using SRAP markers produced a total of 213 identifiable fragments with an average of 21.3 polymorphic bands per combination. The polymorphic information content (PIC) value was 0.46. Along with the Bayesian structure analysis which showed that the highest value of the maximum delta log likelihood (ΔK) was K = 3, cluster analysis also grouped the cultivars into three main clusters. The high value of admixture revealed ancestral relationships between the studied cultivars. Overall, our findings revealed the potential of SRAP markers and morphological traits for the analysis of genetic diversity in Chrysanthemum. This can be, in turn, used in selecting the desired parents in the hybrid breeding programs as well as developing superior cultivars for ornamental applications.

Carotenoids are the most important pigments determining the color of C. × morifolium; however, it is still unknown whether the changes of plastid ultrastructure affect carotenoids accumulation. In this study, we compared the change of carotenoid composition, content, and the plastid ultrastructures in the different developmental stages of capitulum among fourteen C. × morifolium cultivars from seven color groups. We found that the carotenoids and plastids detected at the early stage of capitulum development were similar in all cultivars, including violaxanthin, lutein, and β-carotene, which were present in proplastids and immature chloroplasts. Immature chloroplasts were degraded completely, forming loosely broken plastids during the development of the capitulum in white and pink cultivars. Meanwhile, a number of lipid vesicles appeared at proplastids, which resulted in only trace amounts of carotenoid accumulation in these cultivars. For yellow, orange, red, and brown cultivars, a great number of chromoplasts were found, which contained diverse ultrastructures, such as plastoglobules, tubules, and lipid droplets; these chromoplasts were derived from proplastids or chloroplasts. Compared with the early stage of capitulum development, these cultivars accumulated large amounts of carotenoids, primarily including lutein, lutein derivatives, and their isomers. In green cultivars, proplastids and immature chloroplasts were completely transformed into mature chloroplasts. These chloroplasts mainly contained violaxanthin, lutein, β-carotene, and two new components, (9Z)-lutein and (9'Z)-lutein, compared with carotenoid components presented in proplastids and immature chloroplasts. This research will be helpful for understanding the mechanisms of carotenoid metabolism of C. × morifolium. Furthermore, we found that two different chromoplast transformation patterns could be present in the same tissue cell, which contributed to the research on plastid differentiation and development in higher plants.

A new neolignan glucoside has been isolated from Chrysanthemum morifolium ‘Baekma’ cultivar through repeated silica gel and octadecyl silica gel (ODS) column chromatographies. On the basis of spectroscopic data including NMR, MS, and IR, the chemical structure of the new neolignan glucoside was determined to be (7S,8S)-7-O-[1′-(4′,5′-dihydroxy-3′-methoxyphenyl)propanol]guaiacylglycerol 4-O-β-D-glucopyranoside (1), named baekmaoside A.

Expansin (EXP) plays an important role in plant root formation. The EXP genes associated with chrysanthemum roots have not yet been reported. Here we isolated a root-specific EXP gene in chrysanthemum (Chrysanthemum morifolium), namely CmEXPA4. Bioinformatics analysis showed that CmEXPA4-encoded protein has a conserved DPPB (Double-Psi Beta-Barrel) domain in the N-terminal with a series of Cys residues, an HFD (His-Phe-Asp) motif in the central region, and a pollen allergen domain in the C-terminal. The protein also has a specific α-insertion of WCNP (Trp-Cys-Asn-Pro), which suggests that it belongs to the A-subgroup of the EXP family. In the present study, we cloned the 1,129 bp promoter region upstream of CmEXPA4, and the analysis revealed an abundance of cis-acting elements associated with hormones, light and stress-related responses, and some root-specific regulatory elements in particular. Subcellular localization results indicated that CmEXPA4 locates in the cell wall. Exogenous indole butyric acid induced the up-regulation of CmEXPA4 expression, whereas exogenous abscisic acid inhibited its expression. Tissue expression analysis showed that CmEXPA4 was preferentially expressed in the roots and was synchronized with the rapid emergence of the root. These results suggested that CmEXPA4 may act on the growth and development of chrysanthemum roots. The function of CmEXPA4 was further tested by virus-induced gene silencing, and the results showed that CmEXPA4 silencing inhibited the normal development of the chrysanthemum root system. The roots appeared thinner and shorter, and several important root parameters, including total length, average diameter, surface area, total volume, and root tip number, decreased significantly. The cortical cells of the transgenic plant roots were significantly smaller and shorter than those of the control. Collectively, our results demonstrated that CmEXPA4 gene plays a key role in the growth and development of chrysanthemum roots and affects the root system by acting on the individual cells.

The CmTFL1c gene of Chrysanthemum morifolium inhibits flowering, regulates inflorescence architecture and floral development. The timing of flowering is an important ornamental trait of chrysanthemum. The gene TERMINAL FLOWER1 (TFL1) has been shown to be involved in the regulation of meristem fate and flowering time regulation. Here, a TFL1 gene named as CmTFL1c, was cloned from Chrysanthemum morifolium and further characterized. The open reading frame of CmTFL1c comprises 522 bp, which encodes a polypeptide of 173 amino acids. Phylogenetic analysis revealed that CmTFL1c belongs to the CEN/TFL1 clade. CmTFL1c protein localizes to the nucleus as well as to plasma membrane, which suggests that CmTFL1c may be a transcription factor. The CmTFL1c gene was most highly expressed in vegetative stems, and weakly expressed in leaves and flower buds; both shoot apices and stems had sensitivity to photoperiod. Overexpression of CmTFL1c in wild Arabidopsis and tfl1-13 mutant led to late flowering and altered architecture, including increased secondary branching, and abnormal inflorescences and flowers. The CmTFL1c gene negatively regulated flowering by inhibiting the up-regulation of the AtFT, AtLFY and AtAP1. The biological function of CmTFL1c was further characterized in C. morifolium via Agrobacterium-mediated transformation, which showed that CmTFL1c not only delayed flowering and promoted axillary bud formation, but also played an important role in inflorescence formation of chrysanthemum. These results showed that the CmTFL1c affects flowering time and regulates inflorescence architecture.

The authors investigated whether biogas digestate could be used as a nutrient solution for rockwool culture. To do this, the authors examined growth performances of chrysanthemum cultured in rockwool and fed the filtrate of nitrified biogas digestate (f-NBD) derived from organic fraction of municipal solid waste and investigated whether application of the digestate caused nutrients to leach from the rockwool substrate. Plants that had been fed the f-NBD showed 33% lower fresh shoot weights and produced 35% fewer flowers than those that had been fed a commercial liquid fertilizer, whereas there were no significant differences in lengths of the shoots, chlorophyll contents of the leaves, numbers of days to first flowering, and maximum diameters of the flowers. Also, there were no significant differences in any of the growth parameters between plants that were cultured with the f-NBD supplemented with Mg and the commercial liquid fertilizer. These results indicate that the f-NBD could be used as a nutrient solution for chrysanthemum in rockwool culture if supplemented with Mg. In addition, a considerable amount of Fe was leached from the rockwool substrates following the application of the f-NBD, which could mitigate the expected Fe-deficiency symptoms in chrysanthemum.

AP2/ERF transcription factors (TFs) represent valuable targets for the genetic manipulation of crop plants, as they participate in the control of metabolism, growth and development, as well as in the plants' response to environmental stimuli. Here, an ERF TF encoded by the chrysanthemum (Chrysanthemum morifolium) genome, designated CmERF110, was cloned and functionally characterized. The predicted CmERF110 polypeptide included a conserved DNA-binding AP2/ERF domain. A transient expression experiment revealed that the protein was deposited in the nucleus, and a transactivation experiment in yeast suggested that it had no transcriptional activity. The gene was transcribed in the chrysanthemum root, stem and leaf, with its transcript level following a circadian rhythm under both long and short days. The effect of constitutively expressing the gene in Arabidopsis thaliana was to accelerate flowering. Transcriptional profiling implied that its effect on floral initiation operated through the photoperiod pathway.

Functional suppression of two types of class-C genes caused transformation of pistils and stamens into petaloid organs that exhibit novel phenotypes, which gives a distinct gorgeous impression in the florets of chrysanthemum. The multiple-petal trait is a breeding objective for many horticultural plants. The loss of function of class-C genes causes the multiple-petal trait in several plant species. However, mechanisms involved in the generation of the multiple-petal trait are unknown in Chrysanthemum morifolium (chrysanthemum). Here, we isolated 14 class-C AGAMOUS (AG) genes, which were classified into two types of class-C genes, in chrysanthemum. Seven of these were categorized into CAG type 1 genes (CAG1s) and seven into CAG type 2 genes (CAG2s). Functions of class-C genes were co-suppressed by chimeric repressors and simultaneously knocked-down by RNAi to produce the multiple-petal phenotype in chrysanthemum. The expression of chimeric repressors of CAG1s and CAG2s caused morphological alteration of the pistils and stamens into petaloid organs in the ray and disk florets. Interestingly, the reproductive organs of the disk florets were transformed into petaloid organs similar to the petals of the disk florets, and those of the ray florets were transformed into petaloid organs such as the petals of the ray florets. Simultaneous knockdown of CAG1s and CAG2s expression by RNAi also exhibited a petaloid phenotype as observed in transgenic plants obtained by chimeric repressors. These results showed that CAG1s and CAG2s play important roles in the development of pistils and stamens, and the simultaneous repression of CAG1s and CAG2s resulted in a multiple-petal phenotype in chrysanthemum.

Background Chrysanthemum morifolium is one of the most popular ornamental crops. The capitulum, which is the main ornamental part of chrysanthemum plants, consists of ligulate marginal ray florets, an attractive corolla (petals), and radially hermaphroditic disc florets, but no stamens. In Asteraceae species, the zygomorphic ray florets evolved from the actinomorphic disc florets. During this process, the zygomorphic ligulate corolla arose and the stamens were aborted. Although molecular genetic research has clarified ray floret development to some extent, the precise molecular mechanism underlying ray floret development in chrysanthemum remained unclear. Results A CYC2-like gene, Cyc2CL, was cloned from C. morifolium ‘Fenditan’. Subsequent analyses revealed that the alternative splicing of Cyc2CL, which occurred in the flower differentiation stage, resulted in the production of Cyc2CL-1 and Cyc2CL-2 in the apical buds. Prior to this stage, only Cyc2CL-1 was produced in the apical buds. A fluorescence in situ hybridization analysis of labeled Cyc2CL-1 and Cyc2CL-2 RNA indicated that Cyc2CL-2 was first expressed in the involucre tissue during the final involucre differentiation stage, but was subsequently expressed in the receptacle and floret primordia as the floral bud differentiation stage progressed. Moreover, Cyc2CL-2 was highly expressed in the inflorescence tissue during the corolla formation stage, and the expression remained high until the end of the floral bud differentiation stage. Furthermore, the overexpression of Cyc2CL-1 and Cyc2CL-2 in transgenic Arabidopsis inhibited stamen and petal development. Therefore, both Cyc2CL-1 and Cyc2CL-2 encode candidate regulators of petal development and stamen abortion and are important for the ray floret development in chrysanthemum. Conclusion In this study, we characterized the alternatively spliced transcripts of the CYC2-like gene that differ subtly regarding expression and function. The data presented herein will be useful for clarifying the regulatory mechanisms associated with the CYC2-like gene and may also be important for identifying the key genes and molecular mechanisms controlling the development of ray florets in chrysanthemum.

Background MADS-box transcription factors (TFs) are the key regulators of multiple developmental processes in plants; among them, a chrysanthemum MADS-box TF CmANR1 has been isolated and described as functioning in root development in response to high nitrate concentration signals. However, how CmANR1 affects root and shoot development remains unclear. Results We report that CmANR1 plays a positive role in root system development in chrysanthemum throughout the developmental stages of in vitro tissue cultures. Metabolomics combined with transcriptomics assays show that CmANR1 promotes robust root system development by facilitating nitrate assimilation, and influencing the metabolic pathways of amino acid, glycolysis, and the tricarboxylic acid cycle (TCA) cycle. Also, we found that the expression levels of TFs associated with the nitrate signaling pathways, such as AGL8, AGL21, and LBD29, are significantly up-regulated in CmANR1-transgenic plants relative to the wild-type (WT) control; by contrast, the expression levels of RHD3-LIKE, LBD37, and GATA23 were significantly down-regulated. These results suggest that these nitrate signaling associated TFs are involved in CmANR1-modulated control of root development. In addition, CmANR1 also acts as a positive regulator to control shoot growth and development. Conclusions These findings provide potential mechanisms of MADS-box TF CmANR1 modulation of root and shoot development, which occurs by regulating a series of nitrate signaling associated TFs, and influencing the metabolic pathways of amino acid and glycolysis, as well as TCA cycle and nitrate assimilation.

Following publication of the original article [1], the author reported an error in the reference citation. In Results and Discussion section, Zhang et al. [2] should be cited after the first and third sentences of the first paragraph under the sub-section “Determination of main components in different processed products of Boju”. The reference has been included in the list.

Severe water shortage limits horticultural crop growth and development, thereby compromising plant quality. Novel tools to enhance stress tolerance in medicinal horticultural crops are crucial to cope with growing environmental challenges to world crop performance. In this study, water solutions of robinin (25, 100, and 200 ppm) and/or foliar sprays of chitosan (0, 50, and 200 ppm) were applied to Chrysanthemum morifolium Ramat subjected to a 2 (2DWI) or 6 day (6DWI) irrigation intervals for 6 weeks. Morphological, physiological, and genetic markers associated with plant-response mechanisms to water stress were explored. Robinin + chitosan-treated plants showed increased morphological performance associated with enhanced chlorophyll, carbohydrates, proline, K+, Ca+2, phenols, leaf water potential, antioxidants, and leaf water content. Superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) enzymes were more active in robinin + chitosan-treated plants, while H2O2 accumulation was diminished. Higher expression levels of the Chrysanthemum antioxidant gene of zinc-finger transcription factor gene (Cm-BBX24), Chrysanthemum roots fu (DREB1A-1), Chrysanthemum heat shock protein CgHSP70, pyrroline-5-carboxylate synthetases (P5CS), pyrroline-5-carboxylate reductase (P5CR), and proline dehydrogenase (ProDH) were found in robinin- and chitosan-treated plants. Robinin + chitosan treatment stimulated the accumulation of carbohydrates, K+, Ca+2, proline, and chlorophylls to achieve osmotic adjustment and maintain turgor pressure. Accumulation of reactive oxygen species was controlled by enzymatic and non-enzymatic means, as well as the overexpression of stress-related genes (Cm-BBX24, DREB1A-1, CgHSP70, P5CS, P5CR, and ProDH) in robinin + chitosan-treated plants. Plant-response mechanisms for enhanced drought resistance interacted under robinin + chitosan treatment to improve plant performance under stress conditions.

Chrysanthemum is ranked second among the most economically important cut flowers after rose. This flower is considered very significant from different aspects of commercial herbs and traditional medicine. In this study, 30 simple sequence repeat (SSR) and 8 start codon targeted polymorphism (SCoT) markers were used to assess the genetic diversity of 32 cultivated varieties of C. morifolium. In total, 127 alleles were detected. The average number of alleles per marker locus was 2.13 for SSR (ranging from 1 to 4) and 7.78 for SCoT markers (ranging from 1 to 18). A positive significant correlation between similarity matrices was obtained by the two markers. Average PIC values for SSR and SCoT markers were 0.37 and 0.34, respectively. The results of clustering for both marker systems divided the genotypes into four clusters. These findings revealed that the efficiency of SSR and SCoT markers was relatively the same in fingerprinting of chrysanthemum genotypes. The authors performed combination of SCoT and SSR markers as a novel method to assess genetic diversity in chrysanthemum. Further implications of the results could be used in germplasm conservation and enhancing the genetic base of the breeding programs of C. morifolium.

Volatile oil in Chrysanthemum morifolium Ramat (C. morifolium) was extracted by the method of water vapor distillation and its chemical components was identified by gas-chromatography coupled with mass spectrometry (GC–MS). The volatile oil are evaluated for antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella enteritids, Pseudomonas aeruginosa and Bacillus subtilis. Effects of surfactant, temperature, pH and ultraviolet light on antibacterial activity stability of volatile oil were analyzed too. Total 56 compounds were identified in C. morifolium volatile oil. The main constituents in C. morifolium volatile oil were monoterpenes and sesquiterpenes compounds, including hydrocarbons, esters, aldehydes, ketones, phenols and organic acids. α-curcumene was the most abundant volatile component (12.55%). The volatile oil showed promising antibacterial activity against 5 selected strains. The inhibitory effect on P. aeruginosa exhibited maximum inhibition zone diameter 20.43 mm, and E. coli showed 12.29 mm. The volatile oil treated with surfactant Tween 20 showed the strongest antibacterial activity, followed by Tween 80 and the SDS lowest, which showed the lowest. pH also had different effect on antibacterial activity stability of the C. morifolium volatile oil. No significant difference effect on antibacterial activity stability of volatile oil was observed with temperature and UV treatment.

We find that the DREB subfamily transcription factor, CmERF053, has a novel function to regulate the development of shoot branching and lateral root in addition to affecting abiotic stress. Dehydration-responsive element binding proteins (DREBs) are important plant transcription factors that regulate various abiotic stresses. Here, we isolated an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor from chrysanthemum (Chrysanthemum morifolium 'Jinba'), CmERF053, the expression of which was rapidly up-regulated by main stem decapitation. Phylogenetic analysis indicated that it belongs to the A-6 group of the DREB subfamily, and the subcellular localization assay confirmed that CmERF053 was a nuclear protein. Overexpression of CmERF053 in Arabidopsis exhibited positive effects of plant lateral organs, which had more shoot branching and lateral roots than did the wild type. We also found that the expression of CmERF053 in axillary buds was induced by exogenous cytokinins. These results suggested that CmERF053 may be involved in cytokinins-related shoot branching pathway. In this study, an altered auxin distribution was observed during root elongation in the seedlings of the overexpression plants. Furthermore, overexpress CmERF053 gene could enhance drought tolerance. Together, these findings indicated that CmERF053 plays crucial roles in regulating shoot branching, lateral root, and drought stress in plant. Moreover, our study provides potential application value for improving plant productivity, ornamental traits, and drought tolerance.

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.

The development, reproduction, and population growth of Thrips nigropilosus Uzel (Thysanoptera: Thripidae), reared on leaves of Chrysanthemum morifolium Ramat (Asteraceae), were studied at seven different constant temperatures. Temperature and development rate of T. nigropilosus from egg to adult were linearly related between 15 and 25 °C, allowing the calculation of a lower threshold of 9.26 °C and a thermal constant of 238.1 degree-days. At 35 °C, eggs failed to hatch and larvae did not develop into adults. Adult longevity decreased with increasing temperature between 15 and 35 °C and females lived longer than males. Mean fecundity was highest at 30 °C, with 42.4 eggs per female. The intrinsic rate of natural increase was highest at 30 °C 0.173. During the late summer to spring, which is the main cultivation season for chrysanthemum in Okinawa, a maximum of 11 generations of T. nigropilosus could occur according to our calculations based on meteorological data from 2015 to 2016.

Characterizing the genetic diversity present in a working set of plant germplasm can contribute to its effective management and genetic improvement. The cut flower chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important ornamental species. With the repeated germplasm exchange and intensive breeding activities, it remains a major task in genetic research. The purpose of the present study was to characterize the genetic diversity and the population structure of a worldwide collection of 159 varieties, and to apply an association mapping approach to identify DNA-based markers linked to five plant architecture traits and six inflorescence traits. The genotyping demonstrated that there was no lack of genetic diversity in the collection and that pair-wise kinship values were relatively low. The clustering based on a Bayesian model of population structure did not reflect known variation in either provenance or inflorescence type. A principal coordinate analysis was, however, able to discriminate most of the varieties according to both of these criteria. About 1 in 100 marker pairs exhibited a degree of linkage disequilibrium. The association analysis identified a number of markers putatively linked to one or more of the traits. Some of these associations were robust over two seasons. The findings provide an in-depth understanding of genetic diversity and population structure present in cut flower chrysanthemum varieties, and an insight into the genetic control of plant architecture and inflorescence-related traits.

Background Chrysanthemum is among the top ten traditional flowers in China, and one of the four major cut flowers in the world, but the growth of chrysanthemum is severely restricted by high temperatures which retard growth and cause defects in flowers. DREB (dehydration-responsive element-binding) transcription factors play important roles in the response to abiotic and biotic stresses. However, whether the DREB A-6 subgroup is involved in heat tolerance has not been reported conclusively. Result In the present study, CmDREB6 was cloned from chrysanthemum (Chrysanthemum morifolium) ‘Jinba’. CmDREB6, containing a typical AP2/ERF domain, was classed into the DREB A-6 subgroup and shared highest homology with Cichorium intybus L. CiDREB6 (73%). CmDREB6 was expressed at its highest levels in the leaf. The CmDREB6 protein localized to the nucleus. Based on the yeast one hybrid assay, CmDREB6 showed transcription activation activity in yeast, and the transcriptional activation domain was located in the 3 ‘end ranging from 230 to 289 amino acids residues. CmDREB6 overexpression enhanced the tolerance of chrysanthemum to heat. The survival rate of two transgenic lines was as high as 85%, 50%, respectively, in contrast to 3.8% of wild-type (WT). Over-expression of CmDREB6 promoted the expression of CmHsfA4, CmHSP90, and the active oxygen scavenging genes CmSOD and CmCAT. Conclusion In this study, DREB A-6 subgroup gene CmDREB6 was cloned from chrysanthemum ‘Jinba’. Overexpression of CmDREB6 enhanced heat tolerance of chrysanthemum by regulating genes involved in the heat shock response and ROS homeogenesis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1400-8) contains supplementary material, which is available to authorized users.

Understanding the genetic architecture is a prerequisite for crop improvement. The current research aimed to characterize the extent of genetic variation of drought tolerance harbored in a global collection of 159 chrysanthemum cultivars over 2 years. An average subordinate function value (ASFV), integrating the wilting index, the fresh weight retention rate, and the survival rate after re-watering under two drought-stressed trials, was used to quantify the level of drought tolerance. The performance of ASFV was generally correlated between the 2 years; and a high magnitude (0.95) of broad-sense heritability, coupled with the moderate genetic advance, was estimated for the ASFV. By applying MLM model with both population structure and kinship matrix as covariates association mapping identified 16 markers linked to drought tolerance, with the proportion of the phenotypic variation explained by an individual marker ranging from 4.4 to 7.6%. Of the eight markers predictive across the 2 years, four (E11M24-9, E3M2-8, E1M5-5, and EST-SSR34-3) were identified as favorable alleles for drought tolerance. Several cultivars that carry at least three of the four favorable alleles were identified as potential donor parents for future improvement of the drought tolerance. The findings provide an insight into the genetic basis of the drought tolerance in chrysanthemum and will, therefore, aid in developing new cultivars with enhanced tolerance against drought stress.

Phytoplasma suspected symptoms of phyllody, witches’ broom, leaf yellowing, stunting and little leaf were observed in Chrysanthemum morifolium, Bougainvillea glabra, Jasminum sambac and Callistephus chinensis during survey of flower nurseries and experimental ornamental fields at Delhi, Maharashtra, Tamil Nadu and Karnataka from 2014 to 2016. Pleomorphic bodies typical to phytoplasma structures were observed in the phloem sieve elements of ultrathin sections of all the four symptomatic ornamental plants (stem tissue) in transmission electron microscope. Amplification of 1.8 and 1.2 kb phytoplasma DNA products was observed in all the four test plants in PCR assays using universal primer pairs P1/P7 followed by nested primer pair R16F2n/R16R2, respectively. Pairwise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the association of two phytoplasma subgroups (16SrI-B and 16SrII-D) in four ornamental plant species. ‘Ca. P. aurantifolia’ subgroup D (16SrII-D) was found associated with chrysanthemum phyllody and leaf yellowing at Delhi and Tamil Nadu, bougainvillea little leaf and yellowing at Delhi and Chinese aster phyllody at Bengaluru, Karnataka. However, jasmine little leaf and yellowing at Bengaluru, Karnataka and chrysanthemum stunting at Pune were found to be associated with ‘Ca. P. asteris’ subgroup B-related strains (16SrI-B). The identification of 16SrII-D subgroup phytoplasma infecting bougainvillea and 16SrI-B subgroup infecting jasmine are the new reports to the world. Besides weed species, Cannabis sativa showing witches’ broom in jasmine fields at Bengaluru and Parthenium hysterophorus showing witches’ broom symptoms in chrysanthemum fields at Delhi were identified to be caused by phytoplasma strains classified under subgroups 16SrI-B and 16SrII-D, respectively, by PCR assays and 16Sr DNA sequence comparison analysis. Among the three major leafhopper species identified, only Hishimonas phycitis was identified positive for 16SrI-B and 16SrII-D subgroups of phytoplasmas from chrysanthemum fields at Delhi and jasmine fields at Bengaluru, respectively. The identity of similar phytoplasma strains infecting ornamental species in leafhopper and the weed species in the present study suggested that H. phycitis and weeds may act as potential natural sources for secondary spread of the identified phytoplasma strains.

Aphid, Macrosiphoniella sanbourni, is a major insect pest that adversely affects ornamental quality and production of chrysanthemum, thus it is critical to develop new cultivars resistant to aphid. However, the genetic mechanism governing aphid resistance is thus far not thoroughly investigated in chrysanthemum. This study aimed to characterize the genetic variation of the aphid resistance in a global collection of 80 chrysanthemum entries, during summer and autumn under greenhouse condition, and to identify the molecular markers for aphid resistance by association mapping. The performances of aphid resistance, quantified by the average damage index of aphid, was significantly correlated (r = 0.93, P

Background Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. Results To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3′-UTR, and 5′-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. Conclusion We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4061-3) contains supplementary material, which is available to authorized users.

Replication and cellular movement of viroids are dependent on host plant components. Low temperatures reduce the physiological activity of host plants and thus suppress the levels of viroids. Chrysanthemum stunt viroid (CSVd) concentration was reduced in infected chrysanthemum (Chrysanthemum morifolium) plants subjected to low-temperature treatment compared with untreated plants. In this study, we investigated the distribution patterns of CSVd in the developing shoot tips of CSVd-infected chrysanthemum plants subjected to one-month, three-month, and six-month low-temperature treatment using in situ hybridization. After one month of low-temperature treatment, strong CSVd signals were detected in leaf primordia (LP) and the apical dome (AD); after three months, weak CSVd signals were detected in LP and the AD; while after six months, CSVd signals were not detected in LP or the AD, but only in tissues within the vascular bundle directly beneath viroid-free LP and AD areas. These changes in the distribution of CSVd in shoot tips subjected to low-temperature treatment might result from a reduction in viroid replication and/or the inhibition of viroid intra- and intercellular movement.

Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in 'Borami' collected from Chilgok and Gyeongsan and in 'Secret Pink' from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the 'Yellow Cap' collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

CmWRKY17 was induced by salinity in chrysanthemum, and it might negatively regulate salt stress in transgenic plants as a transcriptional repressor. WRKY transcription factors play roles as positive or negative regulators in response to various stresses in plants. In this study, CmWRKY17 was isolated from chrysanthemum (Chrysanthemum morifolium). The gene encodes a 227-amino acid protein and belongs to the group II WRKY family, but has an atypical WRKY domain with the sequence WKKYGEK. Our data indicated that CmWRKY17 was localized to the nucleus in onion epidermal cells. CmWRKY17 showed no transcriptional activation in yeast; furthermore, luminescence assay clearly suggested that CmWRKY17 functions as a transcriptional repressor. DNA-binding assay showed that CmWRKY17 can bind to W-box. The expression of CmWRKY17 was induced by salinity in chrysanthemum, and a higher expression level was observed in the stem and leaf compared with that in the root, disk florets, and ray florets. Overexpression of CmWRKY17 in chrysanthemum and Arabidopsis increased the sensitivity to salinity stress. The activities of superoxide dismutase and peroxidase and proline content in the leaf were significantly lower in transgenic chrysanthemum than those in the wild type under salinity stress, whereas electrical conductivity was increased in transgenic plants. Expression of the stress-related genes AtRD29, AtDREB2B, AtSOS1, AtSOS2, AtSOS3, and AtNHX1 was reduced in the CmWRKY17 transgenic Arabidopsis compared with that in the wild-type Col-0. Collectively, these data suggest that CmWRKY17 may increase the salinity sensitivity in plants as a transcriptional repressor.

Factors affecting shoot regeneration were optimized in order to develop an efficient protocol for shoot regeneration in leaf explants of the chrysanthemum (Chrysanthemum morifolium Ramat) cultivar Shinma. The maximum number of shoots per explant (9.7) was regenerated in leaf explants of 6-week-old donor plants cultured on Murashige–Skoog medium containing a combination of 0.5 mg L−1 6-benzyladenine and 0.5 mg L−1 α-naphthaleneacetic acid with initial dark treatment for 7 days. Among the different auxins tested, indolebutyric acid was the most effective for root induction and development, whereas the presence of ethylene inhibitors strongly suppressed shoot regeneration. Ploidy levels were analyzed by flow cytometry: there was no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The new protocol will facilitate genetic transformation and micropropagation of chrysanthemum cv. Shinma.

Two high-resolution strain discrimination methods based on variable number tandem repeat (VNTR) analysis were devised for the plant soft-rotting pathogens Dickeya dianthicola and Dickeya solani and used to investigate their diversity and epidemiology. Analysis of D. dianthicola identified 19 VNTR profiles (P1-P19). Diversity was observed in the earliest strains analysed (from 1957), which included four profiles from European strains isolated from Dianthus caryophyllus (P4, P7, P8 and P11) and one from Chrysanthemum morifolium (also P7) originating from the USA. Eleven D. dianthicola VNTR profiles were identified from potato dating from 1984. The time period between which specific D. dianthicola VNTR profiles were first isolated from ornamental hosts and then from potato, was between 8 and 16 years respectively. Identification of two profiles (P2 and P8) that occurred in both ornamental hosts and potato supported cross-infection between these hosts. Limited variation in VNTR profile was identified from strains of D. solani, from which only three VNTR profiles were discriminated by variation at only one of five loci, which may reflect the short time period and/or limited number of D. solani introductions since the pathogen’s first emergence in Europe. Shared profiles between D. solani isolates from hyacinth and potato strains, is consistent with cross infection between hosts grown for horticultural production and potato.

We investigated the effect of tissue source selection in combination with ion beam irradiation on generating flower-color mutants in Chrysanthemum morifolium. Flower color mutation in the chrysanthemum cultivar YoMystery (purple flowers) was used as an index to estimate mutation frequency and the spectrum of mutations. Somaclones regenerated from petal and leaf tissues and ion-beam irradiated clones derived from the two tissues were analyzed for stem length and flower color mutation. Three Gy of argon ion beams was regarded as an appropriate dose for irradiation because the resulting color mutants maintained an adequate stem length suitable for commercial use even with high mutation frequencies. Statistical analysis of 7,258 individuals from three separate experiments revealed that (1) somaclones from petal tissue had a higher mutation frequency (2.91 %, σ = 0.55) than leaf somaclones (2.01 %, σ = 0.26); (2) the effect of tissue source on the frequency of flower color mutations was significantly enhanced when combined with ion-beam irradiation in that the mutation frequency of ion-beam irradiated petal clones was 6.47 % (σ = 0.77), whereas that of ion-beam irradiated leaf clones was 3.89 % (σ = 0.52); and (3) the spectrum of carotenoid color mutations was remarkably increased by ion-beam irradiation in that both leaf and petal ion-beam irradiated clones had brown, red and yellow flowers in contrast to the somaclones that only produced yellow flowers. These results suggest that intentional or directed induction of flower color mutants with practical value is possible by combining tissue source selection with ion beam irradiation.

We investigated the effect of radiation damage on in vitro mutation induction in chrysanthemum. White petals of chrysanthemum (Chrysanthemum morifolium Ramat cv. Youka) were selected to induce mutation by gamma radiation. Calli produced were irradiated with gamma rays at 0, 10, 15 and 20 Gy. We found that the plants from the irradiated calli were different from control plants in number of leaves, leaf length & width, number of flowers, flower diameter, petiole diameter and petiole length after transplanting into the greenhouse for almost 70 days. Three mutants in flower color and shape were found in 15 Gy-treated plants. First type of mutant (M.1) has tubular petals. The second (M.2) and third (M.3) ones both have yellow flowers, while one of them has spooned shaped ray florets similar to the original cultivar and the other one has flat shaped florets. Semi-quantitative RT-PCR showed that most of carotenoid-biosynthesis related genes, except for violaxanthin deepoxidase (VDE) and lycopene e-cyclase (LCYE), showed similar expression patterns in petals of the original ‘Youka’ and its mutants (M2 & M3). VDE and LCYE results showed high expression levels in M3 and M2 & M3 respectively, comparing with the control. On the other hand, expression patterns for VDE were similar in control and M2. These yellow mutants were maintained vegetatively and proved to be true-to-type in one successive generation. It can be concluded that gamma radiation with 15 Gy dose can be used for in vitro induction of flower color and shape mutations of chrysanthemum cv. Youka.

Intergeneric hybridization is an effective way of improving crop resistance against biotic and tolerance to abiotic stresses. Here, we describe an intergeneric hybrid between Chrysanthemummorifolium ‘Nannongxiaoli’ and Artemisia vulgaris ‘Variegata’ using ovule rescue. Chromosome counting confirmed that out of the 124 regenerated plantlets, 19 were genuine hybrids, carrying the expected hybrid number of somatic chromosomes (45, representing 27 from ‘Nannongxiaoli’ and 18 from ‘Variegata’). The 19 genuine hybrids were vigorous, flowered normally and possessed the cut flower characteristics of ‘Nannongxiaoli’. The hybrids were more resistant against both aphid infestation and Alternaria leaf spot inoculation than the ‘Nannongxiaoli’ parent, but less than the ‘Variegata’ parent.

Chrysanthemum morifolium ‘Hangju’ is a valuable medicinal plant. We previously reported cryopreservation of shoot tips of C. morifolium ‘Hangju’. The present study further evaluated greenhouse performance, assessed genetic stability and analyzed biochemical compounds in the greenhouse-grown plants regenerated from cryopreservation. The results showed that although some minor alternations were detected in early vegetative growth, there were no differences in major parameters of vegetative growth and flower production between the plants regenerated from cryopreservation and in vitro shoots (the control). Morphologies of leaves and flowers were identical between the two types of the plants. No polymorphic bands revealed by ISSR and RAPD, and no alternations at ploidy levels analyzed by FCM were found in the cryo-derived plants. The types and number of biochemical compounds analyzed by UPLC-MS/MS were identical between the two types of the plants. Quantitative analyses by HPLC showed no differences in the contents of the five selected biochemical compounds produced between the plants regenerated from cryopreservation and in vitro shoots. Therefore, cryopreservation would provide a technical platform for establishment of cryo-banking of Chrysanthemum germplasm with medicinal values.

The interaction of stem cell factor (SCF) with its cognate receptor c-Kit is closely associated with the survival and maturation of melanocytes. To investigate novel depigmentation agents, we screened 2,000 plant extracts for c-Kit inhibitors to identify active small molecules by using time-resolved fluorescence enzyme assays. For the active extracts identified as inhibitors of c-Kit enzyme, we evaluated the effects of the active extracts and isolated flavonoids on c-Kit phosphorylation in MO7e/melanocytes. Anti-melanogenic activity was also examined in melanocytes and melanoderm model. The flavonoids such as diosmetin, apigenin, acacetin and luteolin isolated from Chrysanthemum morifolium were found to be active in inhibiting c-Kit both at enzyme and cellular levels. In addition, these flavonoids attenuated SCF-induced proliferation of human primary melanocytes without toxicity and suppressed ultraviolet (UV) B irradiation-mediated melanin synthesis significantly. Among the active flavonoids, diosmetin was found to inhibit SCF-induced melanogenesis in a human melanoderm model. These results strongly suggest that C. morifolium extract and diosmetin have potential to suppress SCF-/UVB-induced melanogenesis, and could be developed as anti-pigmentation agents.

A novel approach for fingerprint analysis of Chrysanthemum morifolium Ramat was developed by combining chemometrics methods such as similarity analysis, hierarchical cluster analysis, and principal component analysis with ultra-performance liquid chromatography. The chromatographic separation was accomplished by gradient elution with mobile phase consisting of acetonitrile and phosphate buffer in 10 min prior to conventional liquid chromatography which usually takes over 1 h. Good reproducibility, precision, and accuracy were obtained with relative standard deviations below 2.10 % of six typical components. Consistent results were obtained in accordance with their cultivated sources. The proposed method above was confirmed to be more rapid and suitable, thus providing important criteria for further quality control of C. morifolium Ramat.

Cutting rot in Chrysanthemum is caused by the fungus Plectosporium tabacinum and was found on cuttings of ‘Jimba-No.2’ in 2008. The results of inoculation tests indicated that P. tabacinum infects major Chrysanthemum cultivars in Japan and that cuttings can serve as carriers and propagules of the pathogen already in the soil can serve as inoculum. Other P. tabacinum isolates may also infect Chrysanthemum, and P. tabacinum can be present as an endosymbiont in the plant. In tests of various fungicides, captan, 2,4,5,6-tetrachloroisophthalonitrile, and thiophanate-methyl effectively controlled cutting rot in Chrysanthemum.

Using embryo rescue, we generated an intergeneric hybrid between Chrysanthemum × morifolium ‘Maoyan’ and Artemisia japonica Thunb. Cytological tests confirmed that regenerated plantlets were all genuine hybrids possessing 45 chromosomes, with 27 chromosomes inherited from C. × morifolium (2n = 6x = 54) and the other 18 derived from A. japonica (2n = 4x = 36). Hybrid plant flowered normally. The shape and color of the hybrid flowers and leaves resembled those of chrysanthemum, while leaf width, leaf length, plant height, and inflorescence diameter were intermediate between those of the parents. Hybrid plant had higher levels of chlorophyll and free proline, and lower concentrations of malondialdehyde and Na+, than the maternal parent (C. × morifolium), and these levels were correlated with the hybrid’s enhanced salt tolerance. These results clearly demonstrate that intergeneric hybridization is an effective method of cultivar improvement in chrysanthemum.

Daminozide is a well-known chemical inhibitor of the gibberellic acid biosynthesis pathway regulating the vegetative growth of potted chrysanthemums (Chrysanthemum morifolium Ramat.). However, the precise mechanism underlying daminozide-related floral color loss is unknown. To investigate the latter, in two separate greenhouse experiments, bronze flowering chrysanthemum cultivars ‘Baton Rouge’ and ‘Pelee’ were treated weekly with consecutive (0 or 5,000 mg l−1) foliar daminozide spray applications at early, intermediate, and late stages during the short-day photoperiod. The ray florets of both cultivars were sampled, and the effect of daminozide application on anthocyanins and their biosynthetic precursors were determined by HPLC. Daminozide applied to ‘Baton Rouge’ plants at early developmental stages was correlated with partial loss of red color, and HPLC analysis determined that this was associated with a 75 % reduction in ray floret anthocyanins. Conversely, a near complete loss of red coloration in daminozide-sprayed ‘Pelee’ relative to control plants was associated with as much as a 98 % decline in anthocyanins, irrespective of the time of application. HPLC analysis determined that daminozide application was associated with a 22–50 % increase in the flavones apigenin 7-O-rutinoside, acacetin 7-O-rutinoside, diosmetin 7-O-rutinoside, and eupatorin, and a 68 % increase in the flavonol quercetin 3-O-glucoside, in ray florets of ‘Pelee’ relative to control plants. There was no relative change in ‘Baton Rouge’ flavone and flavonol levels. The accumulation of bronze C. morifolium flavones and flavonols following foliar daminozide application suggests that red color loss is associated with inhibition of anthocyanidin synthase of ‘Pelee’ ray florets.

The photosynthetic response was investigated on Chrysanthemummorifolium under dynamic light conditions in the 20–35 °C temperature range to evaluate the effect of climatic variables on photosynthetic induction. The plant material was grown under uniform, controlled conditions and its gas exchange was analyzed. The gas exchange measurements were used to investigate the rate of induction, momentary induction state, and the opening of stomata. At the varying temperature ranges and under dynamic light conditions, C. morifolium reached a quasi-steady-state induction equilibrium (ISeq(PAR,T)) within 14–45 min. For the same level of photosynthetically active radiation (PAR), the equilibrated level of steady-state induction increased as the temperature increased. It was highest approximately at 30 °C. The induction state was equilibrated at a lower level as the temperature increased to 35 °C. The interaction effect of PAR and temperature on induction state was not significant. The rate of photosynthetic induction and the time required at which the induction reached its 90 % value (t90) was influenced by PAR significantly. The light history of a leaf had a significant effect on t90, indicating that the time to reach a steady-state induction is different depending on the light environment and the period at which the leaf was exposed to light. The velocity of the photosynthetic induction was not affected by the temperature. It was associated with stomatal conductance of the leaf prior to the onset of light (gSini).

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.

Background Chrysanthemum morifolium is one of the most important global cut flower and pot plants, and has been cultivated worldwide. However, limited genomic resources are available and the molecular mechanisms involved in the two morphologically distinct floret developmental cycles in chrysanthemum remain unclear. Results The transcriptomes of chrysanthemum ray florets, disc florets and leaves were sequenced using Illumina paired-end sequencing technology. In total, 16.9 G reads were assembled into 93,138 unigenes with an average length of 738 bp, of which 44,364 unigenes showed similarity to known proteins in the Swissprot or NCBI non-redundant protein databases. Additionally, 26,320, 22,304 and 13,949 unigenes were assigned to 54 gene ontology (GO) categories, 25 EuKaryotic Orthologous Groups (KOG) categories, and 280 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. A total of 1863 differentially expressed genes (DEGs) (1210 up-regulated and 653 down-regulated) were identified between ray florets and disc florets, including genes encoding transcription factors and protein kinases. GO and KEGG pathway enrichment analyses were performed on the DEGs to identify differences in the biological processes and pathways between ray florets and disc florets. The important regulatory genes controlling flower development and flower organ determination, as well as important functional genes in the anthocyanin biosynthetic pathway, were identified, of which two leucoanthocyanidin dioxygenase-encoding genes showed specific expression in ray florets. Lastly, reverse transcription quantitative PCR was conducted to validate the DEGs identified in our study. Conclusions Comparative transcriptome analysis revealed significant differences in patterns of gene expression and signaling pathways between ray florets and disc florets in Chrysanthemum morifolium. This study provided the first step to understanding the molecular mechanism of the differential development of ray florets and disc florets in chrysanthemum, and also provided valuable genomic resources for candidate genes applicable for the breeding of novel varieties in chrysanthemum. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2733-z) contains supplementary material, which is available to authorized users.

Ethylene has an inhibitory effect on flowering in a short-day (SD) plant chrysanthemum (Chrysanthemum morifolium Ramat.). In this study, we used a hexaploid chrysanthemum ‘Sei-Marine’ and found that its transgenic lines transformed with a mutated ethylene receptor gene mDG-ERS1(etr1–4), which conferred reduced ethylene sensitivity (J. Plant Biol. 51: 424–427, 2008), opened flowers earlier than the non-transformed control. We examined whether the accelerated flower induction in the transformant occurred through the enhanced expression of chrysanthemum genes homologous to FLOWERING LOCUS T (FT), a floral inducer gene in Arabidopsis. We cloned three cDNAs for FT homologs (CmFTL1, CmFTL2, and CmFTL3) from ‘Sei-Marine’. CmFTL2 putatively encodes a non-functional gene product due to a frame shift caused by a 2 bp-deletion in the coding region. RT-PCR analysis revealed differential expression patterns of CmFTL genes in the transgenic and control lines, suggesting that these genes might be under the control of ethylene. CmFTL1/2 mRNA level was lower in a SD condition than a long-day (LD) condition. CmFTL3 mRNA accumulated abundantly under SD condition as compared with LD condition in the transgenic line. These results suggest the association of increased expression of CmFTL3 gene with the accelerated flowering in the transgenic line with reduced ethylene sensitivity.

It has long been proposed that white-flowered chrysanthemums (Chrysanthemum morifolium Ramat.) have a single dominant gene that inhibits carotenoid formation or accumulation in ray petals. However, the precise function of the proposed gene was unknown. We previously isolated a gene encoding carotenoid cleavage dioxygenase 4, designated CmCCD4a, which is specifically expressed in the ray petals of white-flowered chrysanthemums. Because CmCCD4a was a strong candidate for the single dominant gene, we analyzed the relationship between CmCCD4a expression and carotenoid content in two sets of petal color mutants. Here, we show that CmCCD4a represents a small gene family containing at least four members. Two of them, CmCCD4a-1 and CmCCD4a-2, were highly expressed in ray petals of two taxa with low carotenoid levels. In petal color mutants derived from these taxa, increases in carotenoid levels accompanied decreases in CmCCD4a expression levels in ray petals. Two different circumstances reduced the levels of CmCCD4a expression in the mutants: either a CmCCD4a gene was lost from the genome or the expression of a CmCCD4a gene was suppressed. In the latter case, suppression may be caused by the loss of a function that normally enhances CmCCD4a transcription. A stepwise decrease in the amount of CmCCD4a expression in either L1 or L2 resulted in a corresponding stepwise increase in the carotenoid content in ray petals. From these results, we propose that CmCCD4a expression is the key factor that controls the carotenoid content in ray petals of chrysanthemum.