Molecular and morphological discrimination of Chrysanthemum indicum using allele-specific PCR and T-shaped trichome.

Chrysanthemum indicum L. is a traditional oriental medicinal herb prepared as a tea from flowers that have been used in China and South Korea since ancient times. It has a long history in the treatment of hypertension, inflammation, and respiratory diseases. Among Chrysanthemum species, C. indicum has more active chemical components as well as better therapeutic effects, and C. indicum is mostly used for medicinal purposes in South Korea. However, the usage of C. indicum has become problematic over the years due to the abundance of adulterated Chrysanthemum and confusion with morphologically related species such as C. morifolium, C. boreale, and Aster spathulifolius. Thus, here we developed a method for molecular authentication using chloroplast universal region rpoC2 and morphological authentication based on T-shaped trichomes of the adaxial leaf surface. By using a species-specific primer derived from the rpoC2 region, we established a multiplex allele-specific PCR for the discrimination of C. indicum. Amplicons of 675 bp for C. indicum and 1026 bp for other Chrysanthemum species were produced using both rpoC2-specific and common primers. These primers can be used to analyze dried samples of Chrysanthemum. Morphological discrimination was performed using T-shaped trichomes present only on the adaxial leaf surface of C. indicum species, and then molecular markers were utilized to authenticate C. indicum products from adulterant samples available in the market. Our results indicate that these molecular markers in combination with morphological differentiation can serve as an effective tool for identifying C. indicum.

GC-MS Analysis of the Composition of the Essential Oil from Dendranthema indicum Var. Aromaticum Using Three Extraction Methods and Two Columns.

Dendranthema indicum var. aromaticum, which is an aromatic plant with a strong and special fragrance throughout the whole plant, is used for the treatment of colds and headaches, and as a mosquito repellant in Shennongjia, Hubei province, China. To analyze the composition of the essential oil from this medicinal herb, we developed a gas chromatography-mass Spectrometry (GC-MS) method including microwave-assisted extraction, hydrodistillation and direct headspace analysis in two different stationary phase columns. In total, 115 volatile compounds were identified, of which 90 compounds were identified using Rxi-5MS and 78 using HP-INNOWAX. Our results revealed that the oil was mainly composed of five categories of compound: oxygenated monoterpenes (28.76-78.10%), oxygenated sesquiterpenes (4.27-38.06%), sesquiterpenes (3.22-11.57%), fatty hydrocarbons (1.65-9.81%) and monoterpenes (0-3.32%). The major constituents are α-thujone, ß-thujone, cis-sabinol, sabinyl acetate and (-)-neointermedeol.However, the essential oil composition in the published literature differs significantly. Therefore, a cluster analysis was carried out using the top ten compositions in the reported literature as well as this study, using Minitab software. To provide detailed information on plant origin, the ITS1-5.8s-ITS2 region was amplified and sequenced (Accession No. MF668250). Besides, in order to provide a macroscopic view of the chemical composition, the biosynthetic pathway of the main components was summarized according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the published literatures.

The complete chloroplast genome sequence of Chrysanthemum indicum.

Chrysanthemum indicum, an important medicinal plant of Asteraceae, had a long history in use for medicine in China. In this study, the complete chloroplast genome of C. indicum was sequenced by a 454 sequencing platform, and the structure of the obtained chloroplast genome was also analyzed. The complete chloroplast genome of C. indicum was 150 972 bp in length and had a pair of inverted repeats (IR, 24 956 bp) separated by a large (LSC, 82 741 bp) and small single copy (SSC, 18 319 bp) regions. Its total GC content was 37.48%. There were 126 chloroplast genes including 83 protein-coding genes, 35 tRNAs and eight rRNAs were successfully annotated. Sixteen genes contained one or two introns. Phylogenetic analyses declared that the chloroplast genome could distinguish C. indicum from its closely related species and might become a potential super barcode for the identification of these species.

Identification of floral scent in chrysanthemum cultivars and wild relatives by gas chromatography-mass spectrometry.

The objective of this study was to identify the major volatile compounds and their relative concentrations in flowers of different chrysanthemum cultivars and their wild relatives. The volatile organic components of fresh flowers were analyzed using a headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. In total, 193 volatile organic components were detected; the major scent components were monoterpenoids and oxygenated monoterpenoids, which accounted for 68.59%-99.93% of the total volatiles in all tested materials except for Chrysanthemum indicum collected from Huangshan, in which they accounted for only 37.45% of total volatiles. The major volatile compounds were camphor, α-pinene, chrysanthenone, safranal, myrcene, eucalyptol, 2,4,5,6,7,7ab-hexahydro-1H-indene, verbenone, ß-phellandrene and camphene. In a hierarchical cluster analysis, 39 accessions of Chrysanthemum and its relatives formed six clusters based on their floral volatile compounds. In a principal component analysis, only spider type flowers were located closely on the score plot. The results of this study provide a basis for breeding chrysanthemum cultivars which desirable floral scents.

[The analyses and identification of Flos rhododendri mollis and Flos chrysanthemi indici via infrared spectroscopy].

In this study, major chemical components of Flos rhododendri mollis and Flos chrysanthemi indici were characterized using Fourier transform infrared spectroscopy (FTIR). For Flos rhododendri mollis, the bands at 1,648 and 1,543 cm(-1) were attributed to amide I and amide II , respectively, indicating that it contained proteins probably resulting in immunization. In case of Flos chrysanthemi indici, stretching vibration of C==O function group was responsible for the bands at 1,734 and 1,515 cm(-1), as a result of essential oils, lipids, etc. Since FTIR spectra of Flos rhododendri mollis and Flos chrysanthemi indici are almost identical and it is difficult to discriminate them, two-step identification was investigated via secondary derivative of the FTIR spectra. The bands at 1,656 and 1,515 cm(-1) corresponds to flavonoides in Flos rhododendri mollis and Flos chrysanthemi indici. In the secondary derivative of the FTIR spectrum of Flos chrysanthemi indici, characteristic bands of inulin were present at 1,163, 1,077, 1,026, 986 and 869 cm(-1), and therefore Flos chrysanthemi indici contained inulin as well. Tri-step identification was carried out for Flos rhododendri mollis and Flos chrysanthemi indici by means of comparing their 2D-IR correlation spectra in different wave number range. In the characteristic range of flavonoides (1,700-1,400 cm(-1)), Flos rhododendri mollis exhibited 3 obvious autopeaks, while 10 autopeaks were visualized in the 2D-IR correlation spectrum of Flos chrysanthemi indici Moreover, in the characteristic range of glucoside (1,250-900 cm(-1)), 10 and 9 autopeaks were present in the 2D-IR correlation spectra of Flos rhododendri mollis and Flos chrysanthemi indici, respectively. Therefore, the tri-step identification of FTIR is a time-saving; accurate, cost-saving and convenient method to effectively distinguish traditional Chinese medicines.

[Study on inflorescences microscopic characteristics of five wild medicinal plants from Chrysanthemum genus in Anhui].

OBJECTIVE: To probe into the disparities in the microscopic characteristics of each part of inflorescences of five wild medicinal plants from Chrysanthemum genus in Anhui Province, and explore the correlation between the distribution of plants and ecological environment. METHODS: The transverse sections of peduncles of the five Chrysanthemum species were observed; The surface piles of the involucre, ray flower, and tubular flower were compared; The ultra-morphology of pollen was observed. RESULTS: There were evident differences, which were associated with ecological environment, among the structures of peduncles of the five species. The morphology of non-glandular hairs on the external involucre presented significant differences, but the morphology of non-glandular hairs tended to be similar gradually from the external to the internal layer of the involucre, and the morphology of surface piles of each part of the inflorescences also tended to converge from the external to the internal. The disparities of glandular hairs among different species were their sizes; As for each species, the size of glandular hairs grew bigger from the involucre to ray flower, and to tubular flower, the density of glandular hairs decreased from the external to the internal layer of the involucre, while it increased on ray flower and tubular flower. The ultra-morphology of pollen of C. zawadskii was distinctly different from that of other species, and disparities of pollen morphology also existed between C. nankingense and C. indicum. CONCLUSION: The structure of peduncles and the disparities in morphology of non-glandular hairs on the surface of external involucre can serve as significant basis for differentiating the inflorescences of Chrysanthemum plants. The categorizing of designating C. nankingense as an separate species is supported.

Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization.

The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known.

[Rapid identification system for seedlings of medicinal Chrysanthemum morifolium].

OBJECTIVE: To achieve the rapid identification for seedlings of medicinal Chrysanthemum morifolium, the discriminant equation was established and the software for rapid identification was designed. METHOD: Leaf structure of medicinal Chrysanthemum of 12 cultivars was analyzed to establish the discriminant equation based on variance analysis and discriminant analysis. On this basis, the identification program and software (based on the python language) were designed. RESULT: Through the analysis of variance and multiple comparisons for the 11 leaf parameter index data of 12 different cultivars, it was found that that the leaf parameters were significant different from each other and reached significant levels. The discriminant equation and the rapid identification software were set up based on the analysis of various indicators. CONCLUSION: The rapid identification system of seedlings of medicinal Chrysanthemum could be achieved through the establishment of discriminant equation combined with computer technology.

[Study on hydroxyproline tolerance of different cultivars of Chrysanthemum morifolium in vitro].

OBJECTIVE: To investigate hydroxyproline tolerance of Chrysanthemum morifolium plantlets included " Boju", "Huaiju", "Chuju", "Gongju" and "Hangju",and provide references basis for excellent cultivar and breeding of Ch. morifolium. METHOD: Plantlets in vitro from five kinds of Ch. morifolium were inoculated on medium added with different concentrations of hydroxyproline. Free proline in leaves from plantlets was determined, then the damage index and survival rate were compared. RESULT AND CONCLUSION: The results showed that hydroxyproline tolerance of " Boju" and "Huaiju" were superior, the survival rates and free proline of them were higher, but the damage index was inferior. The hydroxyproline tolerance of "Hangju" was the worst, and the survival rate was minimum. The survival rate of "Chuju" and "Gongju" was between "Boju" and " Hangju", and the hydroxyproline tolerance of them was also medium.

Chemical variations of the essential oils in flower heads of Chrysanthemum indicum L. from China.

The volatile compositions of hydrodistilled essential oils in the flower heads of Chrysanthemum indicum L. from eight populations in China were analyzed by GC/MS. A total of 169 compounds representing 88.79-99.53% of the oils were identified, and some remarkable differences were found in the constituent percentages of the eight populations. The predominant components of the essential oils were 1,8-cineole (0.62-7.34%), (+)-(1R,4R)-camphor (0.17-27.56%), caryophyllene oxide (0.54-5.8%), ß-phellandrene (0.72-1.87%), (-)-(1S,2R,4S)-borneol acetate (0.33-8.46%), 2-methyl-6-(p-tolyl)hept-2-ene (0.3-8.6%), 4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-yl acetate (0.17-26.48%), and hexadecanoic acid (0.72-15.97%). The chemotaxonomic value of the essential-oil compositions was discussed according to the results of cluster analysis (CA) and principal-component analysis (PCA). The eight populations were divided into five groups as different chemotypes (Groups A-E), and the scores together with the loadings revealed clearly different chemical properties of each population. In conclusion, GC/MS in combination with chemometric techniques provided a flexible and reliable method for characterizing the essential oils of different populations of C. indicum L.

[Comparative analysis on the configuration of vegetative organs of medicinal Chrysanthemum from different original locations and species].

OBJECTIVE: Compared the difference of the configuration of vegetative organs of medicinal Chrysanthemum in the same habitats but from various original locations and species, in order to provide scientific basis for the study of introduction, differentiation within the species, breeding and genetic diversity. METHODS: The experimental plot were divided into some groups randomly in design and the configuration of vegetative organs were observed, measureed and analysed. RESULTS: The differences of the type and shape of the leaves were obvious. All the Chrysanthemum morifolium cv. 'Gongju' had deep notch long-leaves; Chrysanthemum morifolium cv. 'Boju', Chrysanthemum morifolium cv. 'Chuju', Chrysanthemum morifolium cv. 'Hang bai ju' and Chrysanthemum morifolium cv. 'Hang huang ju' had deep notch correctitude-leaves; Chrysanthemum nakingense and Chrysanthemum indicum had long-leaves; Chrysanthemum indicum had clump-leaves. All the 12 medicinal Dendranthema material had auricle except Chrysanthemum morifolium cv. 'Boju' and Chrysanthemum morifolium cv. 'Chuju'. The young leaves of Chrysanthemum morifolium cv. 'Gongju', Chrysanthemum morifolium cv. 'Gongju' and Chrysanthemum morifolium cv. 'Hang bai ju' did not split and the rest were all splitting. CONCLUSION: The differences of the configuration of vegetative organs were obvious and the Key of Vegetative organs was built.

[Characterization of chemical components of essential oil from cupitulum of Chrysanthemum morifolium cultivated in Tongxiang city].

OBJECTIVE: To analyze the chemical components of the essential oil extracted from five cultivars' Hangjuhua (Zaoxiaoyangju, Chixiaoyangju, Dayangju, Yizhongdabaiju and Xiaotangju), which were breed came from the Chrysanthemum morifolium Ramat, and provide scientific basis for quality control. METHOD: The essential oil of five cultivars' Hangjuhua were extracted by water-steam distillation and separated by GC capillary column chromatography. The components were quantitatively determined with normalization method, and identified by GC-MS. RESULT: From the Zaoxiaoyangju, Chixiaoyangju, Dayangju, Yizhongdabaiju and Xiaotangju, 179, 167, 209, 211 and 270 chromatographic peaks were detected, among them 54, 56, 60, 55 and 65 components were identified, which were composed of 73.40%, 64.14%, 65.19%, 53.55% and 62.89% of the total essential oil, respectively. CONCLUSION: The common chemical constituents were juniper camphor, 1, 3, 3-trimethylcyclohex-1-ene-4-carboxaldehyde, borneol, alpha-curcumene, alpha-bisabolol, cis-caryophyllene, benzyl benzoate, methyl beta, beta-dimethylbenzenepropanoic acid ester, 2, 4-decadienal and heneicosane in the essential oil of the five cultivars' Hangjuhua. Among them, juniper camphor is most which were composed of 11.96%, 10.51%, 10.95%, 13.28% and 10.77% of the total essential oil, and methyl beta, beta-dimethylbenzenepropanoic acid ester is second, which were composed of 1.51%, 1.86%, 2.52%, 4.89% and 2.51% of the total essential oil, respectively. There are marked differences in the compounds contained in the five samples. The juniper camphor is a characteristic constituent in the essential oil of Hangjuhua. The resultant data provided the evidences to establish Hangjuhua' cultivars and scientific basis for quality control of Hangjuhua.

[Study on quality of flavone in various cultivars of Chrysanthemum morifolium for medicine].

OBJECTIVE: To study on dynamic accumulation of various flavone in Chrysanthemum morifolium, in order to appraise the quality of flavone. METHOD: The extract was hydrolyzed and the flavonoid components were determined by RP-HPLC. RESULT AND CONCLUSION: In the samples from Tongxiang, Tezhongboju showed the highest flavonoids content with the total flavone 9.81%, quercetin 4.17 per thousand and apigenin 17.02 per thousand. In the samples from Sheyang, Hongxinju showed the highest content of luteolin with 6.84 per thousand. In the samples from Jiangning, Tezhongboju showed the highest content of total flavone with 7.59%, Yizhongdabaiju showed the highest content of quercetin with 2.78 per thousand, Sheyangxiaobaiju showed the highest content of luteolin with 4.27 per thousand, Sheyanghongxinju showed the highest content of apigenin with 11.20 per thousand. There were differences in the content of flavonoids among the different cultivars, and the same cultivars showed also different when planted in different areas.

[Comparative study on botanical morphologic character of different cultivated types medicinal Chrysanthemum morifolium].

OBJECTIVE: Botanical character of different cultivated types medicinal Chrysanthemum morifolium were observed and compared, which can offer reference for its genetic diversity research and breeding. METHOD: Based on the random blocks field experiments design, the main character of leaf and capitulum of medicinal C. morifolium were observed, it was studied on that the botanical character of different cultivated types medicinal C. mortfolium in China were comparatively study with statistical means. RESULT AND CONCLUSION: There are some different of botanical character among different cultivated types medicinal C. morifolium in China, which can be divided into 7 colonies according as its diversity of the botanical and morphological character. The sawtooth number of lamina, leafstalk length and leaf division number are the important laminae morphologic character of different types medicinal C. morifolium, which are relative independent characters.

[Identification of sulphur residue in chrysanthemum by Fourier transform infrared spectroscopy].

Qi and Huai chrysanthemum samples processed in different ways were discriminated by means of Fourier transform infrared (FTIR) spectroscopy. It was shown that different processing methods may result in the variation of relative content of effective components in chrysanthemum. The variation of chemical structure may also occur. And the variations can be reflected differently on the transform infrared spectra. The dry chrysanthemum smoked with sulphur had different characters in the vibration frequency and shape of IR from other samples at 1 600 and 1 060 cm(-1), and showed static characteristic absorptions at 922, 818 and 777 cm(-1), which were stronger than others. The chrysanthemum processed in different ways showed differences in the Fourier transform infrared spectra. The sulphur residue in chrysanthemum can be identified by FTIR without separation and abstraction. This method is not only quick and precise but also convenient and direct. Different sorts of chrysanthemum can be identified in this way.

[Comparative study on inner quality of various Chrysanthemum morifolium cultivated in Tongxiang city].

OBJECTIVE: To study the quality and the chemical components of Chrysanthemum morifolium from Tongxiang city. METHOD: Chemical constituents of nine cultivars were compared in three types of index: chlorogenic acid, flavonoid and volatile oil. RESULT AND CONCLUSION: The content varied significantly. The content of chlorogenic acid in Jinjuerhao was 6.66%, the highest among the samples. Yizhongdabaiju showed the highest flavonoid and volatile oil with 9.49% and 3.30 mL x kg(-1) respectively.

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line.

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.

[The application of infrared spectra and clustering analysis in the discrimination of geographical origin of Flos Chrysanthemi].

OBJECTIVE: To identify the origin of Flos Chrysanthemi. METHODS: FTIR (Fourier transform infrared spectroscopy) and SIMCA (sofe independent modeling of class analogy). RESULTS: The correctness of every classification was higher than 60%. CONCLUSION: This approach was proved to be a reliable and practicable method for the discrimination of geographical origin of Chrysanthemum.

[RAPD analysis for genetic diversity of Chrysanthemum morifolium].

OBJECTIVE: To investigate the genetic diversity of Chrysanthemum morifolium at the level of molecular biology. METHOD: The total genomic DNA was extracted from medicinal chrysanthemums by 2% CTAB method. And the genetic diversity of 22 C. morifolium accessions was tested by RAPD marks. The NTSYS software was used to analyze the marks. RESULT: 26 10-mer arbitrary primers were found to acquire polymophic results. A total of 233 bands were amplified, of which 89.7% bands were found to be polymophic. 8.04 polymophic bands were amplified by each primer on the average. The results of cluster analysis by using UPGMA method showed that all the tested accessions could be differentiated by RAPD marks. CONCLUSION: There actually existed much genetic diversity at the molecular level among the germplasm resources of C. morifolium. RAPD marks could be effective tools to construct DNA fingerprintings of C. morifolium. The differences between the tested chrysanthemums are related to the environments. However, it was affected by genetic facters more significantly.

[Analysis of volatile components from the flowers of Chrysanthemum morifolium by GC-MS with solid-phase microextraction].

OBJECTIVE: To study a method for extraction and analysis of volatile components from Chrysanthemum morifolium 'gonghuangjv' cv. nov (CM GHJ) and C. morifolium 'gongbaijv' cv. nov (CM GBJ) by solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). METHOD: The volatile components were extracted in different temperature, different balance period and different extraction fiber using head space solid-phase microextraction (HS-SPME), and were identified by CGC-MS. The variety in integral area of each component was observed in different conditions and its relative content was determined by normalization of area. RESULT: The better condition of SPME for C. morifolium was that the sample was extracted using 100 microm polydimethylsiloxane (PDMS) extraction fiber after it had been balanced for 6 hours at 75 degrees C. 53 components from CM GHJ and CM GBJ were identified, and there were 35 same components in CM GHJ and CM GBJ. CONCLUSION: HS-SPME-GC-MS is convenient, rapid and reliable for analysis of volatile components in C. morifolium.