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Chrysanthemums (Chrysanthemum morifolium Ramat.) are ornamental flowers, which are famous worldwide. The mode of inheritance has great implications for the genetic analysis of polyploid species. However, genetic analysis of chrysanthemum has been hampered because of its controversial inheritance mode (disomic or hexasomic). To classify the inheritance mode of chrysanthemums, an analysis of three approaches was carried out in an F1 progeny of 192 offspring using 223 expressed sequence tag-simple sequence repeat (EST-SSR) markers. The analysis included segregation analysis, the ratio of simplex marker alleles linked in coupling to repulsion, as well as the transmission and segregation patterns of EST-SSR marker alleles. After segregation analysis, 204 marker alleles fit hexasomic inheritance and 150 marker alleles fit disomic inheritance, showing that marker alleles were inherited predominantly in a hexasomic manner. Furthermore, the results of the analysis of allele configuration and segregation behavior of five EST-SSR markers also suggested random pairing of chromosomes. Additionally, the ratio of simplex marker alleles linked in coupling to repulsion was 1:0, further supporting hexasomic inheritance. Therefore, it could be inferred that chrysanthemum is a complete or near-complete hexasome.

Flavonoids confer a wide color range to plants, thus influencing the flower quality and commercial value of various ornamental plants. Flavones and flavonols are colorless pigments that are distinct from the colored anthocyanins. Flavones and flavonols are transformed from flavanones and dihydrokaempferol, which are catalyzed by flavone synthase (FNS) and flavonol synthase (FLS), respectively, and play important roles in regulating plant growth and development, and resistance to various stresses, in addition to coloration. However, few studies have been conducted on CmFNS and CmFLS genes in chrysanthemums. In this study, we isolated and identified CmFNS and CmFLS from Chrysanthemum morifolium. CmFNS and CmFLS were constitutively expressed at different levels in various C. morifolium organs, and in vitro catalytic activity of CmFNS and CmFLS was verified. CmFNS- and CmFLS-overexpressing tobacco plants exhibited phenotypes that accumulated more flavones and flavonols, respectively, but less anthocyanins. Moreover, the transcripts of CmFNS were negatively correlated with flower color, whereas CmFLS presented an opposite trend compared to CmFNS in five flower color cultivars with different anthocyanin levels. These findings suggest that CmFNS and CmFLS act as important regulators of flavone and flavonol biosynthesis, respectively, and dicate flower coloration in chrysanthemums.

Indirect defense is an important strategy employed by sessile plants to defend against herbivorous insects by recruiting the natural enemies of herbivores mediated by herbivore-induced plant volatiles (HIPVs). We aimed to determine whether indirect defense occurs in Compositae with Chrysanthemum morifolium as the model and elucidate the mechanisms underlying the biosynthesis of HIPVs. Using two-choice olfactometer bioassays, we showed that C. morifolium plants following infestation by larvae of the tobacco cutworm (Spodoptera litura, TCW) were significantly more attractive to two natural enemies of TCW larvae than control plants, indicating that indirect defense is an active defense strategy of C. morifolium. The chemical cue responsible for indirect defense in C. morifolium was identified as a complex blend of volatiles predominated by sesquiterpenes and monoterpenes. A total of 11 candidate terpene synthase (TPS) genes were identified by comparing the transcriptomes of healthy and TCW-infested plants. Gene expression analysis confirmed that up-regulated CmjTPS genes are consistent with the elevated emission of terpenes after herbivory treatment. Our study showed that the herbivore-induced emission of JA-dependent volatile terpenes attracted both predatory and parasitic enemies of herbivores. Generally, our findings indicate that indirect defense might be an important defense mechanism against insects in C. morifolium.

Characterization of genetic diversity in germplasm collections requires an efficient set of molecular markers. We assessed the efficiency of 36 new SCoT markers, 10 new ISSR markers, and 5 microsatellites for the characterization of genetic diversity in chrysanthemum core collection of 95 accessions (Russian and foreign cultivars). Seven new SCoT (SCoT12, 20, 21, 23, 29, 31, 34) and six new ISSR markers ((GA)8T, (CT)8G, (CTTCA)3, (GGAGA)3, (TC)8C, (CT)8TG) were efficient for the genetic diversity analysis in Chrysanthemum × morifolium collection. After STRUCTURE analysis, most Russian cultivars showed 20–50% of genetic admixtures of the foreign cultivars. Neighbor joining analysis based on the combination of SSR, ISSR, and SCoT data showed the best accordance with phenotype and origin compared to the separate analysis by each marker type. The position of the accessions within the phylogenetic tree corresponded with the origin and with some important traits, namely, plant height, stem and peduncle thickness, inflorescence type, composite flower and floret types, flower color, and disc color. In addition, several SCoT markers were suitable to separate the groups distinctly by the phenotypical traits such as plant height (SCoT29, SCoT34), thickness of the stem and peduncle (SCoT31, SCoT34), and leaf size and the floret type (SCoT31). These results provide new findings for the selection of markers associated with important traits in Chrysanthemum for trait-oriented breeding and germplasm characterization.

The variation of flower color of chrysanthemum (Chrysanthemum×morifolium) is extremely rich, and carotenoids, which are mainly stored in the plastid, are important pigments that determine the color of chrysanthemum. However, the genetic regulation of the carotenoid metabolism pathway in this species still remains unclear. In this study, a pink chrysanthemum cultivar, 'Jianliuxiang Pink', and its three bud sport mutants (including white, yellow and red color mutants, 'Jianliuxiang White', 'Jianliuxiang Yellow' and 'Jianliuxiang Red', respectively) were used as experimental materials to analyze the dynamic changes of carotenoid components and plastid ultrastructure at different developmental stages of ray florets. We found that the carotenoid components and plastid ultrastructure of the four color cultivars in the early developmental stage of the chrysanthemum capitulum (S1) were almost identical, and the carotenoids mainly included violaxanthin, lutein and β-carotene, which exist in proplastids and immature chloroplasts. With the development of capitulum, the chloroplasts in 'Jianliuxiang White' and 'Jianliuxiang Pink' were degraded, and the protoplasts did not transform but rather formed vesicles that accumulated trace amounts of carotenoids. The proplastids and chloroplasts in 'Jianliuxiang Yellow' and 'Jianliuxiang Red' were all transformed into chromoplasts and consist of lutein as well as lutein's isomer and derivatives. Using comparative transcriptomics combined with gene expression analysis, we found that CmPg-1, CmPAP10, and CmPAP13, which were involved in chromoplast transformation, CmLCYE, which was involved in carotenoid biosynthesis, and CmCCD4a-2, which was involved in carotenoid degradation, were differentially expressed between four cultivars, and these key genes therefore should affect the accumulation of carotenoids in chrysanthemum. In addition, six transcription factors, CmMYB305, CmMYB29, CmRAD3, CmbZIP61, CmAGL24, CmNAC1, were screened using weighted gene co-expression correlation network analysis (WGCNA) combined with correlative analysis to determine whether they play an important role in carotenoid accumulation by regulating structural genes related to the carotenoid metabolism pathway and plastid development. This study analyzed dynamic changes of carotenoid components and plastid ultrastructure of the four bud mutation cultivars of chrysanthemum and identified structural genes and transcription factors that may be involved in carotenoid accumulation. The above results laid a solid foundation for further analysis of the regulatory mechanism of the carotenoid biosynthesis pathway in chrysanthemum.

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.

Simple sequence repeat (SSR) markers are valuable for genetic and breeding applications, but SSR resources for the ornamental genus chrysanthemum (Chrysanthemum ×morifolium Ramat.) are still limited. Expressed sequence tags (ESTs) are sources of SSRs that represent an opportunity to develop SSRs to accelerate molecular breeding in chrysanthemum. In total, 4661 SSR loci were identified from 3823 SSR-containing unigenes in the chrysanthemum transcriptome with an average of one SSR per 6.98 kb. Of these SSR sequences, trinucleotide repeats (30.0%) predominated, followed by dinucleotide repeats (17.9%). In total, 1584 primer pairs were subsequently synthesized. By screening the parents and six individuals of the F1 progeny, 831 (52.5%) valid EST-SSR markers were identified, of which 361 (43.4%) were polymorphic. The annotation of unigenes containing polymorphic SSRs indicated that 330 (93.5%) demonstrated significant homology to other plant protein sequences. Twenty-five polymorphic EST-SSR markers were further selected for transferability analysis and exhibited 93% amplification in six Ajania species and six other Chrysanthemum species. Based on genotyping of the 59 samples, neighbor-joining analysis revealed six phylogenetic groupings, which was confirmed by population structure analysis and principal component analysis (PCA). Phylogenetic relationships among the 59 samples revealed by SSRs were highly consistent with the traditional taxonomic classification of Chrysanthemum and Ajania. The polymorphism information content (PIC) values ranged from 0.29 to 0.86, with a mean of 0.67, indicating high levels of informativeness. This research reveals the SSR distribution characteristics of chrysanthemum and provides a large number of new EST-SSR markers for further genetic diversity studies, genetic mapping, and molecular marker-assisted selection breeding for chrysanthemum.

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 structure and phylogeny of MADS-box genes HAM91 of sunflower (Helianthus annuus) and CDM115 of chrysanthemum (Chrysanthemum morifolium) were characterized. It is shown that these genes encode MADS-domain transcription factors, which are orthologs of TM6 (Solanum lycopersicum) and APETALA3 (Arabidopsis thaliana), respectively. We obtained two types of transgenic tobacco plants (Nicotiana tabacum) with constitutive expression of HAM91 and CDM115 genes. Both types of plants flowered later than the control plants and formed more flowers and seed pods. The weight of seeds of 35S::CDM115 plants was significantly lower than in the control and 35S::HAM91 plants, which may indicate to a change in the identity of ovules in 35S::CDM115.

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 Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils. Results We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding. Conclusions The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.

The complete plastid genome of Chrysanthemum morifolium ‘Orizaba’, a cultivar from Holland, was determined and analyzed in this work. It is a circular chromosome and has a length of 151,060 bp. The LSC and SSC of 82,858 bp and 18,294 bp were separated by two IRs of 24,954 bp. The chloroplast genome of C. morifolium ‘Orizaba’ contains 125 genes, including 83 protein-coding genes, eight ribosomal RNA genes, and 34 transfer RNA genes. Phylogenetic analysis showed that C. morifolium ‘Orizaba’ clustered together with other Chrysanthemum varieties in the family Asteraceae. The plastome is useful for the elucidation of phylogenetics and evolution in the Asteraceae and Chrysanthemum varieties.

Chrysanthemums (Chrysanthemum morifolium Ramat.) are famous ornamental crops with high medicinal and industrial values. The inflorescence and leaf traits are key factors that affect the yield and quality of chrysanthemum. However, the genetic improvement of those traits is slow within chrysanthemum because of its hexaploidy, high heterozygosity and enormous genome. To study the genetic control of the important traits and facilitate marker-assisted selection (MAS) in chrysanthemum, it is desirable to populate the genetic maps with an abundance of transferrable markers such as microsatellites (SSRs). A genetic map was constructed with expressed sequence tag&ndash, simple sequence repeat (EST-SSR) markers in an F1 progeny of 192 offspring. A total of 1000 alleles were generated from 223 EST-SSR primer pairs. The preliminary maternal and paternal maps consisted of 265 marker alleles arranged into 49 and 53 linkage groups (LGs), respectively. The recombined parental maps covered 906.3 and 970.1 cM of the genome, respectively. Finally, 264 polymorphic loci were allocated to nine LGs. The integrated map spanned 954.5 cM in length with an average genetic distance of 3.6 cM between two neighbouring loci. Quantitative trait loci (QTLs) analysis was performed using the integrated map for inflorescence diameter (ID), central disc flower diameter (CDFD), number of whorls of ray florets (NWRF), number of ray florets (NRF), number of disc florets (NDF), number of florets (NF), ray floret length (RFL), ray floret width (RFW), ray floret length/width (RFL/W), leaf length (LL), leaf width (LW) and leaf length/width (LL/W). Overall, 36 (21 major) QTLs were identified. The successful mapping of inflorescence and leaf traits QTL demonstrated the utility of the new integrated linkage map. This study is the first report of a genetic map based on EST-SSR markers in chrysanthemum. The EST-SSR markers, genetic map and QTLs reported here could be valuable resources in implementing MAS for chrysanthemums in breeding programs.

Roots provide anchorage and enable the absorption of water and micronutrients from the soil for plants. Besides these essential functions, roots are increasingly being recognized as an important organ for the production of diverse secondary metabolites. The goal of this study was to investigate the chemical composition and function of terpenoid secondary metabolites in roots of different cultivars of the popular ornamental plant Chrysanthemum morifolium Ramat. Although C. morifolium is known for rich production of secondary metabolites in its flower heads and leaves, the diversity of secondary metabolites in roots remains poorly characterized. In this study, 12 cultivars of C. morifolium were selected for comparative analysis. From their roots, a total of 20 terpenoids were detected, including four monoterpenes, 15 sesquiterpenes, and one diterpene. The cultivar &lsquo, She Yang Hong Xin Ju&rsquo, exhibited the highest concentration of total terpenoids at approximately 730 &micro, g&middot, g&minus, 1 fresh weight. Most cultivars contained sesquiterpenes as the predominant terpenoids. Of them, (E)-&beta, farnesene was detected in all cultivars. Based on their terpenoid composition, the 12 cultivars were planed into four groups. To gain insights into the function of root secondary metabolites, we performed bioassays to assess their effects on growth of three species of pathogenic fungi: Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. Significant variability in antifungal activity of the root extracts among different cultivars were observed. The cultivar &lsquo, Xiao Huang Ju&rsquo, was the only cultivar that had significant inhibitory effects on all three species of fungi. Our study reveals the diversity of terpenoids in roots of C. morifolium and their function as a chemical defense against fungi.

Chrysanthemum (Chrysanthemum morifolium (Ramat.) Kitamura) plants have great ornamental value, but their flowers can also be a source of pollen contamination. Previously, morphological and cytological studies have shown that anthers of some chrysanthemum cultivars such as &lsquo, Qx-115&prime, fail to dehisce, although the underlying mechanism is largely unknown. In this study, we investigated the molecular basis of anther indehiscence in chrysanthemum via transcriptome analysis of a dehiscent cultivar (&lsquo, Qx-097&prime, ) and an indehiscent cultivar (&lsquo, ). We also measured related physiological indicators during and preceding the period of anther dehiscence. Our results showed a difference in pectinase accumulation and activity between the two cultivars during dehiscence. Detection of de-esterified pectin and highly esterified pectin in anthers during the period preceding anther dehiscence using LM19 and LM20 monoclonal antibodies showed that both forms of pectin were absent in the stomium region of &lsquo, anthers but were abundant in that of &lsquo, anthers. Analysis of transcriptome data revealed a significant difference in the expression levels of two transcription factor-encoding genes, CmLOB27 and CmERF72, between &lsquo, and &lsquo, during anther development. Transient overexpression of CmLOB27 and CmERF72 separately in tobacco leaves promoted pectinase biosynthesis. We conclude that CmLOB27 and CmERF72 are involved in the synthesis of pectinase, which promotes the degradation of pectin. Our results lay a foundation for further investigation of the role of CmLOB27 and CmERF72 transcription factors in the process of anther dehiscence in chrysanthemum.

To further test the accuracy and rationality of the Chinese classification system for chrysanthemum (Chrysanthemum morifolium), 10 simple sequence repeat (SSR) markers were used to identify a collection of 88 chrysanthemum and its related genera accessions. In total, 42 effective alleles across 88 accessions were detected; 3 429 bands were obtained by PCR amplification, including 2 630 polymorphic bands. The similarity coefficient ranged from 0.53 to 0.88. Cluster analysis based on UPGMA illustrated that the wild species and large-flower cultivars were first divided into two clusters, then the large-flower cultivars formed five distinct groups according to petal type, indicated that petal type can be a classification criterion. In the wild species cluster, C. vestitum and C. zawadaskii grouped with A. trilobata, suggested that the Ajania genera was closely related to the Chrysanthemum genera. ‘Hangbaiju’, ‘Gongju’ and ‘Chuju’ were grouped together, and ‘Boju’, O. longilobus and C. mongolicum constituted another branch, showed a correlation with geographic region of origin. Population structure analysis was subsequently performed with K values ranging from 2 to 10, and the most likely estimate for the population structure is five subpopulations, which is nearly consistent with the clustering results. Principle component analysis was further performed to verify the classification results. The results showed that these SSR markers are very powerful for studying genetic relationships and will be useful tools in the identification and classification of chrysanthemum. Keywords: chrysanthemum, SSR, polymorphism, genetic relationship, classification

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most important floricultural crops in the world. Although the origin of modern chrysanthemum cultivars is uncertain, several species belonging to the family Asteraceae are considered to have been integrated during the long history of breeding. The flower color of ancestral species is limited to yellow, pink, and white, and is derived from carotenoids, anthocyanins, and the absence of both pigments, respectively. A wide range of flower colors, including purplish-red, orange, red, and dark red, has been developed by increasing the range of pigment content or the combination of both pigments. Recently, green-flowered cultivars containing chlorophylls in their ray petals have been produced, and have gained popularity. In addition, blue/violet flowers have been developed using a transgenic approach. Flower color is an important trait that influences the commercial value of chrysanthemum cultivars. Understanding the molecular mechanisms that regulate flower pigmentation may provide important implications for the rationale manipulation of flower color. This review describes the pigment composition, genetics, and molecular basis of ray petal color formation in chrysanthemum cultivars.

Today’s chrysanthemums are highly evolved flowering plants and they are considered as one of the most important ornamental cut flowers. In this research an isopentenyl transferase gene named CmIPT1 was isolated from Chrysanthemum morifolium cv. ‘Jinba’ using RACE and RT-PCR methods. The full cDNA sequence of CmIPT1 was 873 bp which encoded a deduced protein of 290 amino acids. It contained GxxGxGKS which is a conserved sequence of the typical domain of IPT family. The phylogenetic tree analysis of CmIPT1 in Chrysanthemum morifolium cv. ‘Jinba’ shows that it has the closest relationship with CcIPT1 in Cynara cardunculus var. scolymus. Expression of CmIPT1 was higher in stems and apex, whereas it was lower in leaves and roots. And the overexpression of CmIPT1 obviously increased the number of rosette branches in Arabidopsis. Here, in our study, we showed that CmIPT1 is a positive regulator of branch development in Chrysanthemum and may play a key role in regulating lateral branch formation of Chrysanthemum plants.

The complete plastid genome of Chrysanthemum morifolium ‘Anhuishiliuye’, a Chinese traditional cultivar, was determined and analyzed in this work. It had a circular-mapping molecular with the length of 151,059 bp.The LSC and SSC of 82,857 bp and 18,294 bp were separated by two IRs of 24,954 bp. The chloroplast genome of C. morifolium ‘Anhuishiliuye’ contains 125 genes, including 83 protein-coding genes, 34 ribosomal RNA genes and 8 transfer RNA genes. Phylogenetic analysis showed that C. morifolium ‘Anhuishiliuye’ clustered together with other Chrysanthemum species. The data provided would be useful for elucidation of phylogenetics and evolution in Chrysanthemum cultivars.

Floral scent is an important trait that has a significant influence on the reproduction of many flowering plants and the market value of several ornamental crops. The family of Asteraceae is well known for its unique floral structure (capitulum) that consists of many florets. Although the constituents of either floral essential oils or emitted floral volatiles have been reported in many species of Asteraceae, little information is available on the mechanisms that determine floral volatile emission. In the present study, a total of 44 species/varieties of Chrysanthemum were analyzed to determine the relationship between the internal accumulation of floral terpenoids and their release as volatiles. By performing both headspace collection and organic extraction, it has been found that the emission rates of floral terpenoids are largely correlated to their internal concentrations. Particularly, the flowers of cultivated C. morifolium, when compared to their wild relatives, were found to exhibit lower emission rates that contain lowered concentrations of floral terpenoids. The differences were largely determined by six monoterpenes and five sesquiterpenes that were revealed by principal component analysis. Besides, the relationship between concentrations and emission rates of floral terpenoids as well as the sizes of capitulum was studied in detail. Separated into three different parts, disc florets were found to have a larger contribution to floral volatile emission than ray florets, whereas the phyllaries and receptacles are the main parts of volatiles accumulation. Finally, the potential biosynthetic pathway of the floral terpenoids produced in capitula of Chrysanthemum was proposed. In summary, our findings on the diversity and variations of floral terpenoids in Chrysanthemum reveal correlations between their production and emission. These findings can be useful to develop different plant breeding methods to create novel aromatic cultivars of Chrysanthemum.

As a medicinal and edible plant, Chrysanthemum morifolium cv. "Hangju" has economic value and is widely used as a Chinese herbal medicine and tea. However, the metabolomic difference between "Hangju" flowers with white and yellow petals is unclear. In this study, Chrysanthemum variety ZXYJ with white petals and its bud mutation strain JJYH with yellow petals were used as research materials. A preliminary study found that the relative content of total flavonoids in JJYH was 45.4% higher than that in ZXYJ. Combined transcriptomic and metabolomic analysis revealed that the phenylpropanoid biosynthetic pathway and the phenylalanine metabolic pathway had the highest degree of differentially expressed genes and significantly changed metabolites enrichment, respectively. The redirection of metabolic flux may contribute to increased accumulation of total flavonoid levels in JJYH, which may begin as early as phenylalanine metabolism. The results of this research provide a scientific basis for the development of products related to different colored medicinal chrysanthemums and references for the breeding of new varieties and the biosynthesis of phenylpropanoids.

Chrysanthemum is one of the most beautiful and popular flowers in the world, and the flower color is an important ornamental trait of chrysanthemum. Compared with other flower colors, green flowers are relatively rare. The formation of green flower color is attributed to the accumulation of chlorophyll, however, the regulatory mechanism of chlorophyll metabolism in chrysanthemum with green flowers remains largely unknown. In this study, we performed Illumina RNA sequencing on three chrysanthemum materials, Chrysanthemum vestitum and Chrysanthemum morifolium cultivars ‘Chunxiao’ and ‘Green anna,’ which produce white, light green and dark green flowers, respectively. Based on the results of comparative transcriptome analysis, a gene encoding a novel NAC family transcription factor, CmNAC73, was found to be highly correlated to chlorophyll accumulation in the outer whorl of ray florets in chrysanthemum. The results of transient overexpression in chrysanthemum leaves showed that CmNAC73 acts as a positive regulator of chlorophyll biosynthesis. Furthermore, transactivation and yeast one-hybrid assays indicated that CmNAC73 directly binds to the promoters of chlorophyll synthesis-related genes HEMA1 and CRD1. Thus, this study uncovers the transcriptional regulation of chlorophyll synthesis-related genes HEMA1 and CRD1 by CmNAC73 and provides new insights into the development of green flower color in chrysanthemum and chlorophyll metabolism in plants.

Age, as a threshold of floral competence acquisition, prevents precocious flowering when there is insufficient biomass, and ensures flowering independent of environmental conditions; however, the underlying regulatory mechanisms are largely unknown. In this study, silencing the expression of a nuclear factor gene, CmNF-YB8, from the short day plant chrysanthemum (Chrysanthemum morifolium), results in precocious transition from juvenile to adult, as well as early flowering, regardless of day length conditions. The expression of SQUAMOSA PROMOTER BINDING-LIKE (SPL) family members, SPL3, SPL5, and SPL9, is upregulated in CmNF-YB8-RNAi plants, while expression of the microRNA, cmo-MIR156, is downregulated. In addition, CmNF-YB8 is shown to bind to the promoter of the cmo-MIR156 gene. Ectopic expression of cmo-miR156, using a virus-based microRNA expression system, restores the early flowering phenotype caused by CmNF-YB8 silencing. These results show that CmNF-YB8 influences flowering time through directly regulating the expression of cmo-MIR156 in the aging pathway., The mechanisms by which plant age regulates flowering remain incompletely understood. Here the authors show that age dependent regulation of SPL transcription factors by miR156 influence flowering via control of NF-YB8 expression in Chrysanthemum.

The morphological traits, essential oil composition, and anthocyanin content of 17 Iranian C. morifolium cultivars were evaluated. Based on Shannon diversity index ( I ), such morphological characters as flower head diameter, ray floret, shape of apex, and main color of ray floret revealed more variability than the other traits. Significant genotypic variations were also observed in essential oil content (ranging from 0.1 to 0.56% (w/w)) and composition. Gas chromatography-mass spectrometry (GC-MS) analysis of the essential oil was conducted to identify 47 compounds in which camphor (0–44.6%), chrysanthenone (0–48.96%), chrysanthenyl acetate (0–16.22%), verbenol (0–11.78%), (+)-5- epi -neointermedeol (0–25.41%), and aromadendrene (0–14.98%) were the main constituents. Results of cluster analysis of essential oil compositions divided the cultivars into four groups. Hybridization among cultivars of divergent clusters led to heterotic effects for flower quality and phytochemical characters. The relationships established between the components revealed high correlations of anthocyanin with the two major aroma compounds of verbenol and chrysanthenyl acetate while they also provided a clue to the association between aroma compounds and color pigmentation. Moreover, a correlation was established between chrysanthenone and flower head diameter (r = −0.502). Based on the results obtained, it might be suggested that smaller flowers are capable of accumulating higher amounts of chrysanthenone. Clearly, the associations between morphological traits and essential oil components provide new insights for improved breeding programs in chrysanthemum.

This study aimed to compare effects of different plant growth regulators on multiple shoot production of Chrysanthemum morifolium Ramat. Nodal segments and shoots of chrysanthemum were cultured on (a) Murashige and Skoog (MS, 1962), (b) MS supplemented with benzylaminopurine (BAP), (c) MS supplemented with BAP and indole acetic acid (IAA), (d) MS supplemented with IAA and BAP, (e) MS supplemented with kinetin (Kin) and indolebutyric acid (IBA) in different concentrations. The shoot cultured on MS supplemented with 0.5 mg L-1 BAP showed the highest multiple shoots (4.91 shoots segment-1). Micropropagation using shoots is the most desirable and safe technique, as it does not only minimize genetic variation but also helps in the formation of healthy shoots. It also provides high rate of multiplication. The seedlings induced from shoots cultured on modified ½MS supplemented with activated charcoal showed higher growth rate than the shoots cultured on modified ½MS without activated charcoal.

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), causes major losses in agricultural and horticultural crops worldwide. The volatile compounds methyl isonicotinate, p-anisaldehyde, eugenol, and linalool are known as olfactory attractants, and salicylaldehyde is known as a repellent for F. occidentalis under clean-air conditions in laboratory experiments. In the present study we assessed the responses of F. occidentalis to these compounds when presented alone, in combination, and in the presence of background odours emanating from cucumber (Cucumis sativus L., Cucurbitaceae), capsicum (Capiscum anuum L., Solanaceae), chrysanthemum (Chrysanthemum morifolium Ramat., Asteraceae), clove basil (Ocimum gratissimum L.), and lavender (Lavandula angustifolia L., both Lamiaceae) plants in a Y-tube olfactometer. In the presence of any background plant odour, the attractiveness of pure methyl isonicotinate to F. occidentalis proved to be consistently significant. Compared to clean-air conditions, a slightly lower percentage of thrips chose the Y-tube arm loaded with 10% p-anisaldehyde in the presence of cucumber leaf odour. With non-flowering clove basil plants in the background, F. occidentalis responses to 1% eugenol, a constituent of clove basil essential oil, were neutral, and the same applied to responses to pure linalool, a constituent of lavender essential oil, in the presence of flowering lavender plants. Also, thrips responses to pure or diluted salicylaldehyde were clearly influenced by plant background odours. We simulated a push-pull situation and found a trend indicating that the percentage of F. occidentalis choosing the airflow loaded with the attractant methyl isonicotinate was higher when the airflow in the other arm of the Y-tube was loaded with the repellent salicylaldehyde compared to clean air, and vice versa. We showed interactions between attractive or repellent volatile compounds and the environmental odours in the chemical ecology of F. occidentalis and the potential of a combined use of these compounds in thrips pest management.

Plants of Chrysanthemum morifolium (sun species) and Spathiphyllum lanceifolium (shade species) were used to study the effects of chilling stems under high illumination. The stress conditions resulted in a greater accumulation of H2O2 in C. morifolium than in S. lanceifolium, and in the down-regulation of photosynthetic linear electron transport in both species. However, only a slight decrease in the maximal quantum yield of PSII was observed under unfavorable conditions in both species, suggesting that mechanisms exist in the chloroplasts that dissipate excess excitation energy and prevent damage to the photosynthetic apparatus. Additionally, changes were observed in the PGR5 polypeptide involved in cyclic electron flow around PSI and in chlororespiratory enzymes (plastidial NDH complex and PTOX). The level of PGR5 increased significantly only in chilled plants of C. morifolium, whereas the levels of the PTOX and NDH-H polypeptide of the plastidial NDH complex and the NDH activity increased significantly only in chilled plants of S. lanceifolium. These findings suggest that the cyclic electron flow involving PGR5 is more active in C. morifolium, while in S. lanceifolium, other mechanisms involving chlororespiratory enzymes are stimulated in response to chilling and high light, resulting in less H2O2 being accumulated in leaves.

The flowers of Chrysanthemum morifolium Ramat. (family: Asteraceae) are used in Traditional Chinese Medicine to treat fever, cold and swelling. However the aerial part is considered as agriculture waste and the chemical and pharmacological information is scanty. From the stems of this plant, four new compounds named as morineoliganosides A (1), B (2), C (3) and heterophyllol-1-O-β- d -glucopyranoside A (4) together with 27 known isolates (5–31), including flavonoids and caffeoylquinic acids were isolated. Their structures were elucidated by chemical and spectroscopic methods The inhibitory effects on xanthine oxidase of these compounds have been determined. This research provided a scientific development and utilization of C. morifolium stems.

Seedlings of Chrysanthemum, cultivar ‘Puma Sunny’, were grown under a range of shading regimes (natural full sunlight, 55, 25, and 15% of full sunlight) for 18 days. Here, we characterized effects of varying light regimes on plant morphology, photosynthesis, chlorophyll fluorescence, anatomical traits, and chloroplast ultrastructure. We showed that leaf color was yellowish-green under full sunlight. Leaf area, internode length, and petiole length of plants were the largest under 15% irradiance. Net photosynthetic rate, water-use efficiency, PSII quantum efficiency, and starch grain were reduced with decreasing irradiance from 100 to 15%. Heavy shading resulted in the partial closure of PSII reaction centers and the CO2 assimilation was restricted. The results showed the leaves of plants were thinner under 25 and 15% irradiance with loose palisade tissue and irregularly arranged spongy mesophyll cells, while the plants grown under full sunlight showed the most compact leaf palisade parenchyma. Irradiance lesser than 25% of full sunlight reduced carbon assimilation and led to limited plant growth. Approximately 55% irradiance was suggested to be the optimal for Chrysanthemum morifolium.

The flowers of Chrysanthemum morifolium Ramat. have been used as an herbal tea and in traditional medicine, and the plant has been developed to produce horticultural cultivars of various colors and shapes. In this study, a new chrysanthemum cultivar with dark purple petals (C. morifolium cv. ARTI-Dark Chocolate; ADC) was developed by radiation-induced mutation breeding of its original cultivar with purple striped white petals (C. morifolium cv. Noble Wine, NW). The phenolic profile and antioxidant property of ADC were investigated and compared with NW and the commercially available medicinal herb, C. morifolium with yellow petals (CM), in order to find a scientific support to produce a new source of natural antioxidant. Flavonoid and phenolic acid profiles of the ethanol extracts of the three flowers were analyzed by high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS), while antioxidant properties were evaluated using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay. Among the tested flowers, ADC possessed the strongest antioxidant capacity and the highest phenolic contents. Flavonoids (acacetin, apigenin, luteolin, acacetin-7-O-β-glucoside, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, and linarin) and phenolic acids (chlorogenic acid and mixture of 1,4-, 1,5-, and 3,5-dicaffeoylquinic acids) were identified and quantified.

The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.

Several species of Alternaria are identified as the main cause of ?blight? in the chrysanthemum crops, in the Province of Corrientes, Argentina. To clarify this situation, samples taken from the production area have been tested in Concepción and nearby areas. After that, pathogenic tests have been implemented and have been identified through positive pathogenic analysis. Making a cross-checking of the ITS rDNA sequence of monosporic strain with molecular databases, it was observed a percentage of 100% similarity with sequences of Alternaria alternata, confirming the results of the morphological studies. In addition, a distance tree genetic analysis confirmed the taxonomic identity of the Alternaria strain from C. morifolium samples as A. alternata. Fil: Gauna, Mónica Patricia. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Departamento de Produccion Vegetal.; Argentina Fil: Gutiérrez, Susana Alejandra. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Departamento de Produccion Vegetal.; Argentina Fil: Bich, Gustavo Angel. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Castrillo, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina Fil: Zapata, Pedro Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina Fil: Cabrera, María Graciela. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Departamento de Protección Vegetal; Argentina

BackgroundAphid (Macrosiphoniella sanbourni) stress drastically influences the yield and quality of chrysanthemum, and grafting has been widely used to improve tolerance to biotic and abiotic stresses. However, the effect of grafting on the resistance of chrysanthemum to aphids remains unclear. Therefore, we used the RNA-Seq platform to perform a de novo transcriptome assembly to analyze the self-rooted grafted chrysanthemum (Chrysanthemum morifoliumT. ‘Hangbaiju’) and the grafted Artermisia-chrysanthemum (grafted ontoArtemisia scopariaW.) transcription response to aphid stress.ResultsThe results showed that there were 1337 differentially expressed genes (DEGs), among which 680 were upregulated and 667 were downregulated, in the grafted Artemisia-chrysanthemum compared to the self-rooted grafted chrysanthemum. These genes were mainly involved in sucrose metabolism, the biosynthesis of secondary metabolites, the plant hormone signaling pathway and the plant-to-pathogen pathway. KEGG and GO enrichment analyses revealed the coordinated upregulation of these genes from numerous functional categories related to aphid stress responses. In addition, we determined the physiological indicators of chrysanthemum under aphid stress, and the results were consistent with the molecular sequencing results. All evidence indicated that grafting chrysanthemum ontoA. scopariaW. upregulated aphid stress responses in chrysanthemum.ConclusionIn summary, our study presents a genome-wide transcript profile of the self-rooted grafted chrysanthemum and the grafted Artemisia-chrysanthemum and provides insights into the molecular mechanisms ofC. morifoliumT. in response to aphid infestation. These data will contribute to further studies of aphid tolerance and the exploration of new candidate genes for chrysanthemum molecular breeding.

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.

The hardy garden mum Chrysanthemum, or “mum” (Chrysanthemum × morifolium Ram.), is a popular flowering herbaceous perennial that is commonly grown for fall sales. In October 2011, suspected wilt disease was observed in potted hardy garden mums (cv. Guiin) grown in greenhouses in Jinju, South Korea. Symptoms included unilateral chlorosis of leaves at the stem apex. Wilted leaves occurred initially on the most severely affected side of the plant, but as the disease progressed, the entire plant wilted and died. Black necrosis and vascular discoloration at the base of stems always developed. Five fungal isolates, successfully isolated from 10 infected stems on potato dextrose agar (PDA), yielded rapidly growing floccose to felt-like colonies, initially white, but turning peach colored. The microconidia were ellipsoid, ovoid, and cylindrical, and measured 3 to 12 × 1 to 3 μm. The macroconidia were falcate, lunate, and measured 8 to 30 × 2 to 4 μm, and had 1 to 5 septa. Pathogenicity was studied in inoculated, potted plants in a greenhouse. A representative isolate of the fungus was grown on PDA at 20°C for about 10 days before inoculation. To obtain conidial suspensions, 10 ml of sterile distilled water (SDW) was added to the culture plates and scraped with a paintbrush to dislodge conidia. The suspension from the culture plates was filtered through cheesecloth and diluted to 2 × 104 micro- and macroconidia/ml with SDW. Nine 3-month-old hardy garden mums were planted in 20-cm-diameter plastic pots containing fine sand. After 10 days, the roots were cut to a depth of 5 cm on two sides of each plant at a distance of 2 cm from the stems. Then, 10 ml of conidial suspension were poured into each pot above the cuts roots, followed by 20 ml 12 days later. Three mums treated with SDW served as controls. Plants were fertilized twice weekly with 100 ml/pot of a nutrient solution (1) that lowered the soil pH and enhanced wilt development. Thirty days after inoculation, all of the artificially inoculated plants had wilted. The control mums remained healthy. The fungus was successfully reisolated to complete Koch's postulates. On the basis of the morphological characters, the fungus was identified as Fusarium oxysporum (3). To identify the isolated fungus, the complete internal transcribe spacer (ITS) rDNA and translation elongation factor 1-alpha (EF1-α) sequences were amplified using the primers ITS1/ITS4 and EF1/EF2, respectively, and sequenced. The resulting sequences were deposited in GenBank (Accession Nos. KC491873 and KC491875). A BLAST search of ITS rDNA (544 bp) and EF1-α (712 bp) sequences against a database of fungal isolates found 100% and 99% similarity to those of F. oxysporum, respectively. Fusarium wilt caused by F. oxysporim on C. morifolium has been previously recorded in North America and India but, to our knowledge, this is the first report of F. oxysporum causing wilt in hardy garden mum in Korea (2). F. oxysporum isolates causing wilts are specific to certain hosts and even to host varieties or cultivars. Further work is required to determine to which forma specialis and race the pathogen belongs. References: (1) A. W. Engelhard and S. S. Woltz. Proc. Fla. State Hort. Soc. 84:351, 1971. (2) H. C. Huang et al. Plant Pathol. Bull. 1:57, 1992. (3) C. V. Subramanian. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 217, 1970.

The genus Aphelenchoides (Fischer, 1894) includes a diverse group of species, some of which are of economic importance. A. bicaudatus (Imamura, 1931) Filipjev and Schuurmans Stekhoven, 1941 is reported for the first time from South Korea, with a detailed redescription of the species. Specimens were collected from chrysanthemum (Chrysanthemum morifolium) leaves and shoot tips in South Korea. The species was identified by morphological traits and molecular sequencing. A bifurcated tail distinguishes A. bicaudatus from its congeneric species. To confirm species identification, we determined the partial 18S ribosomal DNA sequence of the specimens and compared with those obtained from other Aphelenchoides species available on GenBank.

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.

Nano Silver has been proven to be effectively applied in the field of biotechnology. In the area of ​​plant biotechnology, nanoparticles have positive and negative effect on the growth of plants. The impact of nanoparticles on plant depends on the composition, content, size, chemical and physical propreties of nanoparticles as well as the plant species. The impact of silver nanoparticles on the problem of infection during culturing, the growth of plants in microponic systems and acclimatization of the plant in greenhouse were studied. In this work, the microponic medium supplemented with of 7.5 ppm silver nanoparticles showed the highest growth rate of Chrysanthemum after 2 weeks in culture. Results of qualitative and quantitative microbial content in microponic culture medium by 4 testing methods including Bergey's Manual of Determinative Bacteriology, ISO 16266, ISO 21527-1 for bacteria and NHS-F15 for fulgi also showed that concentration of 7.5 ppm nanoparticles reduces the microbial content of the 8 species of bacteria (Corynebacterium sp., Enterobacter sp., Arthrobacter sp., Agrobacterium sp., Xanthomonas sp., Pseudomonas sp., Bacillus sp. and Micrococcus sp.) and three species of fungi (Aspergillus sp., Fusarium sp. and Alterneria sp.). The growth of Chrysanthemum plant in a concentration of nanoparticles is better than the other levels after 4 weeks in the greenhouse such as high survival rate (100%), plant height (13.3 cm), number of leaves per plant (14.67 leaves), number of roots per plant (26.67 roots), leaf length (4.03 cm), leaf width (3.77 cm), fresh weight (3816 mg) and dry weight (216 mg).