Progress of safflower (Carthamus tinctorius L.) regeneration through tissue culture

Background: Safflower regeneration through tissue culture has long been limited to low frequency and lack of an efficient protocol that suitable for most safflower cultivars. In past decades, researches had been carried out to investigate safflower regeneration through tissue culture and great progress had been made. Objective: To investigate factors that affect safflower regeneration through tissue culture principally. Methods: This article summarized available literatures about advancements in safflower regeneration, especially discussed factors affecting safflower tissue culture in detail. Results: Safflower regeneration was fairly hard than other congeneric plants, such as chrysanthemum. The genotype, seedling age, type of explants, medium components, plant growth regulators and other additives all had specific influences on safflower tissue culture. More deepgoing researches need to be undertaken to establish an effective safflower regeneration system.

Color spaces of safflower (Carthamus tinctorius L.) for quality assessment

Objective:In this study,safflower(Carthamus tinctorius L.)was taken as a representative example to examine the application of color characteristics to evaluate quality.Methods:A computer vision system was established for the objective and nondestructive assessment of color using image processing algorithms.Color parameters were investigated based on the RGB,L*a*b and HSV color spaces.The content of hydroxysafflor yellow A(HSYA),a major bioactive constituent of safflower,was determined by high-performance liquid chromatography.The relationship between HSYA content and color values was investigated by Pearson correlation analysis.A multiple linear regression model was established to predict the HSYA content from color values.Results:The red color and lightness of safflower were found to be significantly related to HSYA content.The prediction equation obtained by multiple regression was reliable with an R2 value of 0.805(P<.01).Conclusion:The results suggest that the computer vision technique could be used as a promising and non-destructive technology for color measurement and quality evaluation of CHM.

Safflower (Carthamus Tinctorius L.) a Potential Source of Drugs against Cryptococcal Infections, Malaria and Leishmaniasis

In this research we present that Carthamus Tinctorius L.(gen.Asteraceae,otherwise known as Safflower)(Fig.1)may contain agents active in Cryptococcal infections,malaria and Leishmaniasis,as treatment options are becoming scarce due to drug resistance development.Phytochemistry and pharmacological activities(antimicrobial,antimalarial,antileishmanial)of C.tinctorius L.were analyzed.The composition of volatile oil of safflower dried flowers was analyzed by gas chromatography-mass spectrophotometry with flame ionization detector(GC-FID)and in vitro sensitivity assays were performed to assess biological activity.8 known and 3 unknown compounds were detected in the extract(Fig.1).Then the Safflower ointment was manufactured and its acute toxicity study on rats was tested.The volatile oil of C.tinctorius L exhibited activity against Cryptococcus neoformans,Plasmodium falciparum and Leishmania donovani.Safflower volatile oil has anticryptococcal,antimalarial and antileishmanial effects.The prepared ointment had an excellent acute toxicity safety profile.

Assay for the developmental toxicity of safflower (Carthamus tinctorius L.) to zebrafish embryos/larvae

Objective:To explore the developmental toxicity and the potential toxicological mechanism of safflower (Carthamus tinctorius L.) on zebrafish embryos/larvae.Methods:Mortality,malformations and increased apoptosis induced by safflower were assessed in zebraflsh embryos from 6 to 96 hours post-fertilization.Enzymes and genes in the anti-oxidative and apoptotic pathways were also assayed.Results:The lethal concentration 50 of safflower to zebrafish embryos was 345.6 mg/L.Hatching inhibition,abnormal spontaneous movement,depressed heart rate,pericardial edema,yolk sac edema,abnormal head-trunk angle,inhibition of melanin release,enlarged yolk,and short body length were observed in safflower-treated zebrafish.Additional apoptotic cells mainly appeared around the heart.Safflower exposure changed the activities of defense enzymes (SOD T,CAT↑,MDA↑,GPX without trend),increased MDA content,decreased caspase-3 activity,and altered mRNA levels of related genes (ogg1 ↓,p53↓,Cu/Zn-sod↑,Mn-sod↓,cat↓,gpx↑).Conclusion:Safflower exhibits developmental toxicity for zebrafish embryos/larvae.The developing heart was speculated as the target organ of toxicity.Oxidative stress and increased apoptosis have roles in the developmental toxicity of safflower.This article provides a novel method to research the teratogenicity and possible mechanisms of toxicity of traditional Chinese medicines that are prohibited or contraindicated in pregnant women.

Influence of Meta-Topolin on Efficient Plant Regeneration via Micropropagation and Organogenesis of Safflower (<i>Carthamus tinctorius</i>L.) cv. NARI-H-15

The effect of meta-Topolin (mT) was assessed to develop a reliable protocol for efficient plant regeneration of safflower (Carthamus tinctorius L.) cv. NARI-H-15. For micropropagation, 7 - 9 days old shoot-tip explants cultured on MS basal medium supplemented with 3.0 mg/L meta-Topolin (mT) + 0.5 mg/L CPPU showed 97.7% adventitious shoot formation (42.4 shootlets) than node after 45 days of culture. For organogenesis, the seedling explants of immature leaf cultured on 1.5 mg/L CPPU or 1.5 mg/L NAA fortified medium produced high amount of callus than cotyledon and stem calli after 60 days of culture. However, MS basal medium fortified with 4.0 mg/L mT + 1.5 mg/L CPPU was found beneficial to stimulate 100% organogenic response (74.7 shootlets) from immature leaf calli than cotyledon and stem derived calli after 45 days of culture. The healthy plantlets obtained from micropropagation and organogenesis process cultured on 1/4 MS basal salts, 1.5% sucrose (w/v) and 0.8% agar (w/v) medium supplemented with NAA (1.5 mg/L) and mT (0.1 mg/L) produced maximum of 96% (12.8 rootlets) and 84% (7.3 rootlets) adventitious rooting, respectively than mT and CPPU tested medium. However, maximum of 67% and 42% survival rate was noticed when in vitro raised plants from micropropagation and organogenesis were hardened in pots containing soil mix and maintained under green house condition. This optimized regeneration protocol might be helpful in regeneration of new genotypes and cultivars of safflower to improve agronomic traits through in vitro selection process and Agrobacterium-mediated genetic transformation system.

Phosphorus Use Efficiency of Safflower （Carthamus tinctorius L.） and Sunflower （Helianthus annuus L.） Studied in Nutrient Solution

Safflower represents an important oil crop internationally and may have a production potential under low input conditions, but its putatively high phosphorous use efficiency is not sustained. This study aims to directly compare safflower with sunflower in terms of phosphorus use efficiency in nutrient solution under controlled conditions. Growth of both species responded strongly to increasing P supply. Safflower recovers less proportion of added P than sunflower. External P requirement （（g P supply （100 g dry matter （DM） produced）~） was higher in safflower than sunflower. The efficiency of the crops for DM production based on accumulated P （mg P potl, efficiency ratio）, and P concentration in DM （（mg P （g DM）＇I）, utilization index） were interpreted using Michaelis-Menten kinetics as growth response curves. Accordingly, Km constant was lower in sunflower compared to safflower in terms of utilization index, but both were similar in terms of efficiency ratio. High Km constant in safflower in terms of utilization index indicates the high P concentration in tissues to produce 50% of potential maximum DM, consequently less efficient crop. Utilization efficiency contributed more than uptake efficiency in overall PUE in the efficient cultivar and could be the cause of its superiority in PUE. It can be concluded that safflower has a high requirement for P with respect to growth, sunflower is more efficient in terms of uptake and utilization of P at optimal and sub-optimal P supplies indicating that safflower can not be considered a low nutrient input crop compared to sunflower with respect to phosphorus.

红花(Carthamus tinctorius L.)种子醇溶蛋白遗传多样性分析

利用A—PAGE（Acid—polyacrylamide gel electrophoresis）技术对来源于32个国家的53份红花材料进行了种子醇溶蛋白检测。结果表明，这些红花材料具有丰富的醇溶蛋白等位变异，共分离出15条迁移率不同的谱带。其中，每份材料可电泳出5-12条谱带，平均8．5条，所有材料有1条共有带。红花种子醇溶蛋白可作为评价红花遗传多样性的工具之一。不同材料的遗传相似系数（GS）变异范围为0．375—1．000，平均值为0．752。聚类分析结果表明，在GS值为0．752的水平上供试材料聚为6大类，其亲缘关系远近与地理来源关系不大。其中，来自中国的7份材料分别被聚在了3个大类中，表明中国红花种子醇溶蛋白遗传多样性比较丰富。

SRAP Analysis on Genetic Relationship of Carthamus tinctorius L Varieties in Xinjiang

[Objective] This study aimed to analyze the genetic relationship of the Carthamus tinctorius L（safflower） varieties in Xinjiang, and thus to provide a scientific basis for its protection and development. [Method] The genomic DNA of five safflower varieties from Xinjiang and one from Yunnan were analyzed and compared by SRAP molecular marker. [Result] Twelve pairs of SRAP primers were selected to amplify the genomic DNA of the six materials. Among the 171 clear DNA bands finally obtained, 93 were polymorphic, accounting for 54.4% of total. Genetic similarity coefficient of the six safflower varieties ranged from 0.60 to 0.92. [Conclusion]SRAP molecular marker is suitable for safflower varieties research and to guide the molecular breeding.

Protective effect of Salvia miltiorrhiza and Carthamus tinctorius extract against lipopolysaccharide-induced liver injury

AIM:To investigate the hepatoprotective effects and mechanisms of an extract of Salvia miltiorrhiza and Carthamus tinctorius in vivo.METHODS:C57BL/6J mice were randomly assigned to five groups and intraperitoneally administered 0.9% saline,Salvia miltiorrhiza and Carthamus tinctorius extract [Danhong injection(DHI),0.75 and 3 g/kg mixed extract] or reduced glutathione for injection(RGI,300 mg/kg) for 30 min before exposure to lipopolysaccharide(LPS,16 mg/kg). After intraperitoneal LPS stimulation for 90 min or 6 h,the mice were sacrificed by ether anaesthesia,and serum and liver samples were collected. Histological analysis(H&E) and terminal deoxynucleotidyl transferase-mediated d UTP nick end-labelling(TUNEL) staining were performed. Alanine transferase(ALT),aspartate transaminase(AST),total bilirubin(TBil),glutathione-S-transferase(GST),malondialdehyde(MDA),tumour necrosis factor(TNF)-α,interleukin(IL)-6,and caspase-3 levels were measured. Bax,Bcl-2,P-IκBα,IκBα,P-NF-κB p65,and NF-κB p65 protein levels were determined by Western blot. TNF-α,IL-6,caspase-3,Bax and Bcl-2 m RNA expression was measured by real-time reverse transcription-polymerase chain reaction(RT-PCR).RESULTS:Hematoxylin-eosin staining and TUNEL results suggested that DHI(3 g/kg) treatment alleviated inflammatory and apoptotic(P < 0.01) injury in the liver of mice. DHI treatment dose-dependently blunted the abnormal changes in biochemical parameters such as ALT(72.53 ± 2.83 for 3 g/kg,P < 0.01),AST(76.97 ± 5.00 for 3 g/kg,P < 0.01),TBil(1.17 ± 0.10 for 3 g/kg,P < 0.01),MDA(0.81 ± 0.36 for 3 g/kg,P < 0.01),and GST(358.86 ± 12.09 for 3 g/kg,P < 0.01). Moreover,DHI(3 g/kg) remarkably decreased LPS-induced protein expression of TNF-α(340.55 ± 10.18 for 3 g/kg,P < 0.01),IL-6(261.34 ± 10.18 for 3 g/kg,P < 0.01),and enzyme activity of caspase-3(0.93 ± 0.029 for 3 g/kg,P < 0.01). The LPS-induced m RNA expression of TNF-α,IL-6 and caspase-3 was also decreased by DHI. Western blot analysis revealed that DHI antagonised LPS-stimulated decrease of Bcl-2 and increase of Bax protein expression. Furthermore,DHI inhibited LPS-induced IκBα and NF-κB p65 phosphorylation.CONCLUSION:DHI may be a multi-function protectant against acute hepatic injury in mice through its antiinflammatory,anti-oxidative and anti-apoptotic activities.

Molecular mechanism of Salvia miltiorrhiza Bunge and Carthamus tinctorius L. in the treatment of myocardial ischemia based on network pharmacology: a comparative study

Based on network pharmacology and molecular docking technology,the similarities and differences of anti-myocardial ischemia mechanism between Salvia miltiorrhiza Bunge(SM)and Carthamus tinctorius L.(CT)were studied.Firstly,based on the traditional Chinese medicine systems pharmacology(TCMSP),the related compounds of SM and CT were obtained,and the potential targets of these compounds were collected by the target fishing method.Genecards database was used to obtain targets related to myocardial ischemia.The cross targets of CT,SM,and myocardial ischemia were then selected,and the protein-protein interaction(PPI)network was constructed based on the STRING database.The cross targets were imported into the Metascape database for Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Cytoscape software was used to build the topological network diagram of the drug-compound-target path.Finally,the binding ability of the active ingredient and the key target was verified by molecular docking.65 active ingredients and 38 potential targets were screened from SM,and 22 active ingredients and 58 potential targets were excavated from CT.Important targets common to SM and CT were TNF,IL6,VEGFA,AKT1,etc.The common enrichment pathways involved are fluid shear stress and atherosclerosis,IL-17 signaling pathway,pathways in cancer,and toxoplasmosis.The findings suggested that the two traditional Chinese medicines exerted the effect of myocardial ischemia through the characteristics of multiple targets,multiple pathways,and multiple compounds.

油用型红花(Carthamus tinctoris L.)种子醇溶蛋白遗传多样性分析

利用A-PAGE（Acid-polyacrylamide gel electrophoresis）技术对来源于不同国家的79份油用型红花材料醇溶蛋白位点进行检测。结果表明，油用型红花醇溶蛋白位点存在丰富的变异类型，共分离出15条迁移率不同的谱带，每份材料具有3—12条不等，平均8．5条。材料间平均遗传相似系数（GS）为0．5303，变幅为0．1333—1．000，且各洲间遗传多样性大于洲内遗传多样性。在GS值为0．512的水平上，供试材料聚为六大类，聚类结果表明，红花醇溶蛋白图谱类型与其地理分布有一定的相关，但不显著。

Simultaneous determination of four nucleosides in Carthamus tinctorius L.and Safflower injection using high-performance liquid chromatography

We quantitatively determined four nucleosides, including cytidine, uridine, guanosine, and adenosine, in Carthamus tinctorius L. and Safflower injection. Separation was performed on a Zorbax Eclipse XDB-18 column using a gradient elution with mobile phases of 0.05% trifluoroacetic acid （TFA） aqueous solution （A） and methanol （B）. The assay was carried out at a flow rate of 1 mL/min at 25 ℃ with detection at 260 nm. Cytidine, uridine, adenosine and guanosine showed good linearity in the ranges of4.02-503μg/mL （r2= 0.9998）, 9.38-1407 μg/mL （rz = 0.9999）, 80.6-8060μg/mL （r2 = 0.9999） and 2.10---630μg/mL （r2 = 0.9987） with average recoveries of 97.2%, 94.5%, 98.6% and 108.6%, respectively. The contents of cytidine, uridine, adenosine and guanosine in different Carthamus tinctorius L. and Safflower injection were significantly different. This is the first report on the quantitative determination of nucleosides in Carthamus tinctorius L. and Safflower injection.

Effects of Plant Population on Growth, Development and Oil Yield of Safflower

Two field trials were clone to evaluate the effects of plant density on the growth, development and yield of safflower. The results showed that plant density and season of growth had significant （P ≤ 0.01） effects on growth, development, yield components, yield and oil content of safflower. Increasing safflower plant density from 100,000 to 250,000 plants ha^- significantly reduced plant height （13.2%-21.3%）, branch number plant^-1 （37%-54.7%）, leaf number plant^-1 （39%-39.2%）, leaf area （19.5%-53%）, plant spread （39.6%-54.4%）, root length （28.1%-54.4%）, plant biomass （17%-50%）, capitula size （12%-12.7%）, capitula number plant^-1 （39.5%-50.5%）, seed number capitula~ （39%-45%）, capitula weight （3.3%-3.6%）, seed yield （67.9%-69.8%） and seed oil content （14.7%-20.8%）. The reduction in vegetative growth, yield components, yield and oil content of safflower due to increased plant density was attributed to inter and intra-plant competition for light, nutrients and water necessary for growth and development. The differences between winter and summer grown safflower were attributed to difference in day and night temperature （DIF） and the average daily temperature which were optimum for safflower growth in winter. It was concluded that under Botswana conditions or in semi-arid areas, safflower should be planted at 50 cm × 20 cm or wider in order to maximize yield and oil content and allow the plants to express their maximum genetic potential.

The Main Insect Pests of Safflower on Various Plant Parts in Iran

Safflower （Carthamus tinctorius） has its own limited factors, especially insect pests. There are several injurious insect pests reported from various safflower growing areas in Iran, causing severe and economical losses on various parts of safflower plants. They are safflower Budfly and capsulfly （Acanthiophilus helianthi）, aphid （Uroleucon carthami）, green leafhopper （Empoasca decipiens）, ground bug （Oxycarneus pallens）, grampod borer and capsule borer or bordered straw （Heliothis （and or Heliocaverpa） peltigera）, red spider mite and two spotted spider mite （Tetranychus urtica）, beet armyworm （Spodoptera exigua）, scarab beetle （Tropinota （and or Epicometis） hirta）, Egyptian cotton leaf （Spodoptera littoralis）, cotton boll worm （Heliothi absouleta） and so on. The other reported safflower insect pests from other countries are wireworms （Limonius spp.）, lygus bugs （Lygus hesperus）, flower thrips （Eastern or Western） or onion thrips （Thrips tabaci）, and seed corn maggots （Delia platura）, sunflower moths （Helianthus annuus） and Caterpillar （Perigaea capensis） are the serious pests of this crop. The other reports showed that some stored-product insects are capable of establishing infestations in safflower seeds and seed meal under suitable conditions. Only Lasiaderma serricorne, Stegobium penliceum, and the four species of Trogodema were to be as the infectious pests. Safflower is also attractive to several beneficial insects, including lady beetles and lacewings that may provide insect control in surrounding fields.

Reproductive Components of Safflower Genotypes Submitted of Bulk Density Levels in the Brazilian Cerrado

Nutrient absorption in crops can decline and their development can be hindered by increased bulk density. This study aimed at assessing the manner in which bulk density levels affect the reproductive structures of the safflower genotypes in the Brazilian Cerrado. The completely randomized design was adopted with four replications for the experiment, which was conducted in a greenhouse using Oxisol collected from 0.0 to 0.2 m depth from the region supporting Cerrado vegetation. The treatments included ten safflower genotypes (PI 237538, PI 248385, PI 250196, PI 301049, PI 305173, PI 305205, PI 306520, PI 306603, PI 560202 and PI 613366) and five bulk density levels (1.0, 1.2, 1.4, 1.6 and 1.8 Mg·m-3). Evaluations were done at 90 days after emergence, in terms of the number, diameter and dry mass of the heads. The data were submitted to the analysis of variance. The means were grouped using the Scott-Knott test at 5% probability. The diameter and dry mass of the chapters were influenced by the mean bulk density of 1.10 Mg·m-3. A notable interaction was evident between the safflower genotypes and bulk density levels for the diameter and dry mass of the head alone, revealing the high degree of genetic variability that environmental changes induce among the genotypes. The PI 250196, PI 301049, PI 305173 and PI 305205 genotypes exhibited greater stability to the bulk density variations compared with the others. Mean bulk density of 1.2 Mg·m-3 was found to impair the development of the reproductive components of the safflower genotypes.

Phytometric and Productive Characteristics of Safflower Submitted to Phosphate Fertilization in the Oxisol of the Brazilian Cerrado

The safflower (Carthamus tinctorius L.), in particular, has been soaring in significance as an alternative for oil and biodiesel production. In this context, the goal of this study was to assess the influence exerted by phosphate fertilization on the phytometric and productive characteristics of the safflower raised on the Oxisol of the Cerrado. The experiment was performed in a greenhouse, using pots of 3 dm-3?in volume. Adopting the completely randomized design, five doses of phosphorus (P2O5: 0, 90, 180, 360 and 540 mg·dm-3) and six replicates were included. The following variables were estimated, viz., number of leaves, plant height, and stem diameter, besides chlorophyll index (SPAD index), number of chapters and shoot dry matter. Barring the shoot dry matter and number of chapters, all the other variables were evaluated on days 30 and 45 after the plants were transplanted. Data were submitted to analysis of variance at 1%, 5% and 10% probabilities, and regression analysis, at 1% and 5% probabilities, respectively. All the variables revealed significant differences and adjustments to the linear regression model (P < 0.1), excepting the SPAD index on day 30 after the plant emergence which was adjusted to the quadratic regression model. The highest findings of 24.47 for leaf number, 34.97 cm for plant height, 3.76 mm for stem diameter, 64.76 for chlorophyll index, 5 units for the number of chapters and 3.45 g for dry shoot mass were recorded in response to the highest phosphorus dose in the experimental range (P2O5: 540 mg·dm-3). Phosphate fertilization was observed to affect the phytometric characteristics and boost the development of the safflower plants cultivated in the Cerrado Oxisol, substantiating that safflower could be a viable cash crop that can be established in the Brazilian Cerrado.

Soil Water Availability on Growth and Development of Safflower Plants

Safflower (Carthamus tinctorius L.) is a promising culture to be widespread in Brazil. However, the lack of basic knowledge about cultivation techniques, such as water demand by the culture, is still obstacle to the expansion of safflower in that country. The objective was, then, to evaluate the effect of the soil water availability on growth and development of safflower in the Cerrado soil of Mato Grosso, Brazil. The experiment was conducted in a greenhouse, in a completely randomized design with five water availabilities (25%, 50%, 75%, 100% and 125% of the maximum water holding capacity in the soil) and four replications. Maintenance soil moisture was performed by gravimetric method with daily weighing of experimental units. The variables analyzed were: plant height, stem diameter, number of leaves, number of heads, heads diameter, dry mass of shoots, heads, and roots. The results were submitted to analysis of variance and regression test at 5% probability by SISVAR program. All variables set to the quadratic regression model, showing the best results in the water availability between 64% and 76%. Safflower is shown to be more sensitive to water stress with increased tolerance to water deficit in the soil than to flooding.

Soil Water Availabilities in the Content and Accumulation of Nitrogen and Chlorophyll Index in the Safflower

The safflower (Carthamus tinctorius L.) is adapted to grow in adverse conditions, is tolerant to salinity and water deficit, however, its productivity increases when grown in areas with greater availability of water and in fertilized conditions, showing that the knowledge of crop management can bring income gains for producers. It is known that extreme conditions of soil moisture damage the crop development. Given this context, the objective was to evaluate the influence of water availability on the nutritional characteristics of the safflower. The experiment was conducted in a greenhouse in a completely randomized design with five water availabilities (25%, 50%, 75%, 100% and 125% of the maximum water holding capacity of the soil) in four replications. Maintaining soil moisture was performed by gravimetric method with daily weighing of experimental units. The variables analyzed were SPAD reading (chlorophyll content), concentration and accumulation of nitrogen in the shoot and in the sections. The results were submitted to analysis of variance and regression test at 5% probability by Sisvar program. There was adjusting of variables to linear and quadratic regression models. Water availabilities between 65% and 75% have greater potential to promote better nutrition for safflower plants. Both deficit and excess of water in the soil are detrimental to the absorption of nitrogen and reduce the chlorophyll content. Safflower is less tolerant to excess than to deficit of water.

Potential for Safflower Cultivation under Temperate Conditions

Safflower is a Mediterranean-subtropical oil crop, with temperate climate. To assess the suitability of safflower may have certain production potential in organic farming in regions cultivation under temperate climate in low input organic farming, ten cultivars were grown in two years experiment in Lindhoft-Kiel in Northern Germany in clay/sand soil. Pearson correlations reveal that oil yield exhibited tight positive correlations with plant height, plant dry matter, capitula plant-1, achenes capitulum-1 and thousand achene mass （TAM）, but was not correlated with days to full flowering or oil concentration. Yield component analysis reveals that achenes capitulum1 had the major influence on the variation of oil yield of all studied cultivars, followed by capitula plant1 and TAM, while the oil concentration had slightly negative influence, and the plant density was negatively associated with oil yield. Although two years field experiment is not enough to analyse the performance of safflower adaptability to certain region, it can be concluded that the most adaptive cultivars are PI-209286 originated from Romania, and cultivar CART-19/89 originated from Poland, while the less adaptive cultivars were DO-13/03 and DO-15/03 with German origin in addition to the Canadian cultivar PI-572475/Saffire. The most striking yield component that contributes to the high yield is achene capitulumt, which can be used as a base for selecting cultivars adaptive to humid cold conditions. The growth period between full flowering and harvest influencing this yield component must be given attention to avoid low temperature and rainfall by tuning sowing date to improve yield at these conditions.

Cloning,expression and activity analysises of chalcone synthase genes in Carthamus tinctorius

Objective:Flavonoids are the bioactive compounds in safflower(Carthamus tinctorius),in which chalcone synthase(CHS)is the first limiting enzyme.However,it is unclear that which chalcone synthase genes(CHSs)are participated in flavonoids biosynthesis in C.tinctorius.In this study,the CHSs in the molecular characterization and enzyme activities were investigated.Methods:Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C.tinctorius.Chalcone biosynthase genes in C.tinctorius(CtCHSs)were cloned from cDNA of flowers of C.tinctorius.The cloned gene sequences were analyzed by bioinformatics,and their expression patterns were analyzed by real-time PCR(RT-PCR).The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot.A recombinant vector of CtCHS was constructed.The CtCHS recombinant protein was induced and purified to detect the enzyme reaction(catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone).The reaction product was detected by HPLC and LC-MS.Results:Two full-length CtCHS genes were successfully cloned from the flowers of safflower(CtCHS1 and CtCHS3),with gene lengths of 1525 bp and 1358 bp,respectively.RT-PCR analysis showed that both genes were highly expressed in the flowers,but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers.WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers.HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.Conclusion:CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.