Resistance of Microbial Community of Artemisia annua L.to Pathogenic Fungi

[Objectives]This paper was to figure out whether the dominant bacterial community has the role and effect of bacterial community and its defense mechanism against potential pathogenic fungi in Artemisia annua,and thus establish a systematic model of bacteria-fungus-plant.[Methods]Fifty-eight strains of bacteria and one strain of pathogenic fungi,Globisporangium ultimatum,were used for the experiments.These 58 bacterial strains were assembled into a bacterial community,and the bacteria with abundance in the top 1%were reassembled into a dominant bacterial community as measured by 16S rDNA.[Results]The growth of A.annua seedlings inoculated with bacterial communities and pathogenic fungi or dominant bacterial communities and pathogenic fungi was significantly better than that of A.annua seedlings inoculated with pathogenic fungi during in vitro confrontation,which was evident in both enzymatic and non-enzymatic antioxidant assays.[Conclusions]The results suggest that the dominant bacterial community has a crucial role as a representative core microbial community of synthetic bacterial community,which can protect plants by interfering with the growth of phytopathogenic fungi mediated by chemical signals,and can be used as the main synthetic community of biocides to achieve the effect of biocontrol.

Effect of Artemisia annua (Asteraceae) Extracts on Hemolysis in Individuals with G6PD-Deficiency

Individuals with Glucose-6-phosphate dehydrogenase (G6PD) deficiency are susceptible to hemolytic anemia when exposed to pro-oxidant substances. This study investigates the hemolytic impact of Artemisia annua (A. annua) extracts in G6PD-deficient subjects through a mixed experimental approach. In the in vitro phase, red blood cells from G6PD-deficient individuals and rats induced with Dehydroepiandrosterone (DHEA) were exposed to various concentrations of A. annua infusion, with distilled water and physiological saline as positive and negative controls respectively. The in vivo study involved G6PD-deficient Wistar rats divided into three groups receiving A. annua infusion, quinine (positive control), and distilled water (negative control) via gavage. Blood samples were collected for biochemical and hematological analyses. Notably, at a 40% concentration of A. annua infusion, there was a significant increase in the hemolysis rate of G6PD-deficient red blood cells compared to controls (p A. annua exhibited elevated aspartate aminotransferase (129.25 ± 4.55 U/L vs. 80.09 ± 4.03 U/L;p A. annua infusion tested positive for saponins. These findings underscore the risk of hemolysis in G6PD-deficient individuals upon ingesting A. annua.

Effect of Organic Amendment on the Growth of Artemisia annua in the North of Côte d’Ivoire

Malaria causes many deaths around the world, particularly in Africa, which ultimately affects the socio-economic development of African countries. The resistance of Plasmodium falciparum to quinine-based drugs led to new studies showing the efficiency of new artemisin-based drugs. The molecule artemisin is extracted from Artemisia annua a plant from China that has been used for decades in traditional Chinese medicine. The purpose of this study is to improve the production of sweet wormwood (Artemisia annua) using organic fertilizers in the north of Cote d’Ivoire. To do so, a morpho-pedological characterization of the study site was firstly performed to determine the soil type and their fertility level. Then, a randomized complete block system including two factors (the quantity of compost and the plant density) was implemented to test the effect of organic amendment and plant arrangement on the growth of Artemisia annua. Six treatments were set up: a control plot (no compost) where the plants are arranged in square (T0D1) and the plants are arranged in staggered (T0D2). Then, a treatment with compost addition of 25 t/ha where the plants are arranged in square (T1D1) and in staggered (T1D2). A treatment with compost addition of 50 t/ha where plants are arranged in square (T2D1) and in staggered (T2D2). Our results showed that the soils hosting our experimentation are Arenithic Plinthic Ferrasols with a very low level of fertility, prone to leaching and erosion. T1D2 and T2D2 treatments obtained the highest yields of 2.82 t/ha and 3.91 t/ha, respectively. Our findings indicate that a high dose of organic amendment combined with a staggered plant arrangement strongly improves the biomass production of sweet wormwood. This is in agreement with previous studies showing that the addition of organic matter can restore the level of soil fertility by increasing soil porosity and the activity of micro and macroorganisms.

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr.) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae , the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.

Elicitation on Artemisinin Biosynthesis in Artemisia annua Hairy Roots by the Oligosaccharide Extract from the Endophytic Colletotrichum sp. B501

The oligosaccharide elicitor from the mycelial wall of an endophytic Colletotrichum sp. B501 promoted the production of artemisinin in Artemisia annua L. hairy root culture. When hairy roots of 22-day-old cultures (later growth phase) were exposed to the elicitor (20 mg/L) for 4 d, the maximum content of artemisinin reached 1.15 mg/g, a 64.29% increment over the control. The electron X-ray microanalysis disclosed the rapid accumulation of Ca 2+ in the elicited cortical cells of hairy root. The electronic microscope observation revealed the high electron density area in vacuole of elicited cells. During the first day of elicitation the peroxidase activity of hairy roots was improved sharply. Some cellular morphological changes including cell shrinkage, condensation of cytoplasm and nuclear fragmentation, coincident with the appearance of DNA ladders, were observed after the third day of elicitation. It was suggested that the oligosaccharide elicitor triggered the programmed cell death, which may provide the substance or chemical signal for artemisinin biosynthesis.

Molecular Cloning, Escherichia coli Expression and Genomic Organization of Squalene Synthase Gene from Artemisia annua

A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.

Cloning, E. coli Expression and Molecular Analysis of a Novel Sesquiterpene Synthase Gene from Artemisia annua

A 1 886 bp full-length sesquiterpene synthase (AaSES) cDNA was cloned front a high-yield Artemisia annua L. strain 001 by a rapid amplification of cDNA end (RACE) strategy. AaSES is 59% identical to Artemisia cyclase cDNA clone cASC125, 50% identical to epi-cedrol synthase from A. annua , 48% identical to amorpha-4, 11-diene synthase from A. annua, 39% identical to the 5-epi-aristolechene synthase from tabacco, 38 % identical to vetispiradiene synthase front H. muticus, 41 % identical to the, delta-cadinene synthase from cotton. The coding region of the cDNA was inserted into a procaryotic expression vector pET-30a and overexpressed in E. coli BL21 ( DE3). The cyclase proteins extracted front bacterial culture were found largely in an insoluble protein fraction. AaSES expresses in leaves, stems a-rid flowers, not in roots as indicated by Northern blotting analysis.

Effects of Ag-carrying Zirconium Phosphate on the Kinetics of Growth of the Roots of Culture Artemisia annua

在植物组织和细胞培养过程中 ,尤其是在生物反应器培养中的染菌问题 ,一直是制约植物细胞培养工业化的难题。通过比较各种防腐剂的抑菌效果 ,确定银型磷酸锆盐抗菌粉为青蒿根培养的最佳防腐剂。银型磷酸锆盐抗菌粉在浓度为 30mg/L时 ,既能降低培养液的染菌几率 ,又不明显抑制青蒿根的生长及青蒿素的生物合成。在添加 30mg/L抗菌粉的培养液中进行的青蒿根生长、pH值变化以及残糖、铵离子和硝酸根离子消耗的动力学研究表明 ,在 4 0d内青蒿根在培养液中生长良好 ,营养成分的消耗和对照呈相似的趋势。

黄花蒿(Artemisia annua L.)提取物对两种病原真菌的生物活性

以石油醚Ⅰ(30℃～60℃)、石油醚Ⅱ(60℃～90℃)、乙醇和丙酮等4种溶剂对黄花蒿的根、茎、叶进行初步提取,采用生长速率法测定不同提取物对玉米小斑病菌、棉花枯萎病菌的抑菌活性。结果表明,黄花蒿提取物对玉米小斑病菌的生物活性好于棉花枯萎病菌;黄花蒿叶的提取物抑菌效果最好,根的抑菌效果最差;叶的石油醚(60℃～90℃)提取物对玉米小斑病菌EC50为156.32mg/L;而叶的丙酮提取物对玉米小斑病菌的EC50为82.37mg/L。

Studies on Acaricidal Bioactivities of Artemisia annua L.Extracts Against Tetranychus cinnabarinus Bois.(Acari:Tetranychidae)

The aim of this study was to determine the best extraction technique, the most suitable solvent, the optimal plant parts, and the acaricidal activities of Artemisia annua L. The petroleum ether （30-60℃）, petroleum ether （60-90℃）, ethanol, acetone, and water parallel and sequenced extracts were obtained from the leaves, stems and roots of different period of A. annua L. in April, May, June, July and September respectively. And then the acaricidal bioactivities against Tetranychus cinnabarinus of all extracts were determined by the slide-capillary method in the laboratory. The results indicated that the acaricidal bioactivities elevated as the development of A. annua plant at the concentration of 5 mg mL-L The general tendency exhibited the sequence of July 〉 June 〉 May 〉 April, but September decreased comparing to July. However, the most effective extracts in five months were all acetone parallel extract of A. annua leaf, and the corrected mortalities treated after 48 h ranged from 74 to 100%. The median lethal concentrations （LC50） against T. cinnabarinus of acetone parallel extracts ofA. annua leaves in September, July, June, May and April were 0.5986, 0.4341, 0.8376, 0.9443 and 1.3817 mg mL^-1, respectively, treated after 48 h. The 13 groups were isolated from acetone extracts ofA. annua leaves in July by column chromatography, both the 1 lth and 12th groups exhibited strong bioactivities. The median lethal concentrations of the 1 lth and 12th groups against T. cinnabarinus were 0.3683 and 0.1586 mg mL^-1, respectively. The acetone parallel extract ofA. annua leaf in July was the most toxic to T. cinnabarinus and the corrected mortality was 100% after 48 h. The acetone parallel extract of the 1 lth and 12th groupswere the most active components, acted as the emphases in further study.

Dried-leaf Artemisia annua: A practical malaria therapeutic for developing countries?

Artemisinin from the plant Artemisia annua （A. annua） L., and used as artemisinin combination therapy （ACT）, is the current best therapeutic for treating malaria, a disease that hits children and adults especially in developing countries. Traditionally, A. annua was used by the Chinese as a tea to treat “fever”. More recently, investiga-tors have shown that tea infusions and oral consumption of the dried leaves of the plant have prophylactic and therapeutic effcacy. The presence of a complex matrix of chemicals within the leaves seems to enhance both the bioavailability and effcacy of artemisinin. Although about 1000-fold less potent than artemisinin in their antiplasmodial activity, these plant chemicals are mainly small molecules that include other artemisinic compounds, terpenes （mainly mono and sesqui）, favonoids, and polyphenolic acids. In addition, polysaccharide constituents of A. an-nua may enhance bioavailability of artemisinin. Rodent pharmacokinetics showed longer T？ and Tmax and greater Cmax and AUC in Plasmodium chabaudi -infected mice treated with A. annua dried leaves than in healthy mice. Pharmacokinetics of deoxyartemisinin, a liver metabolite of artemisinin, was more inhibited in infected than in healthy mice. In healthy mice, artemisinin serum levels were 〉 40-fold greater in dried leaf fed mice than those fed with pure artemisinin. Human trial data showed that when delivered as dried leaves, 40-fold less artemisinin was required to obtain a therapeutic response compared to pure artemisinin. ACTs are still unaffordable for many malaria patients, and cost estimates for A. annua dried leaf tablet production are orders of magnitude less than for ACT, despite improvements in the production capacity. Considering that for 〉 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, the evidence argues for inclusion of affordable A. annua dried leaf tablets into the arsenal of drugs to combat malaria and other artemisinin-susceptible diseases.

A new sesquiterpene from hairy root culture of Artemisia annua

A new sesquiterpene（Z）-7-acetoxy-methyl-11-methyl-3-methylenedodeca-1,6,10-triene（AMDT） was isolnted and identified from the methanol extract of the hairy root culture of Artemisia annua.The structure of AMDT was determined based on the analysis of spectroscopic data,notably of the 2D NMR spectra.This new compound showed cytotoxicity against human tumor cell lines 95-D and HeLa with IC50 values of 27.08 and 20.12μmol/L,respectively.

Maintaining the artemisinin content through direct and indirect in vitro regeneration and their assessment of variations with the field grown mother plants of Artemisia annua L.

An efficient protocol for maintaining the artemisinin content in tissue culture and high frequency of in vitro direct and indirect regenerations of multiple shoots of high artemisinin yielding genotypes of Artemisia annua has been developed and their comparison with field grown mother plant has been carried out.The eleven elite genotypes(containing more than 1%artemisinin)were tested on seven different modified media formulations.Modified half MS(Murashige and Skoog’s)media containing 100 mg L^(-1) myo-inositol,0.5 g L^(-1) casine hydrolysate,5 mg L^(-1) biotin,2 mL L^(-1) RT(Revised Tobacco)vitamin stock,0.5 mg L^(-1) BAP and 0.01 mg L^(-1) NAA showed best regeneration while,above modified MS medium containing 0.2 mg L^(-1) BAP and 0.2 mg L^(-1) NAA showed maximum shoot multiplication with maintained artemisinin content.Based on the chemical profiles of both the systems,minor difference was observed in their artemisinin content.A large scale culture of these plants maintained the normal growth index along with the artemisinin content and could be a better alternative to maintain the high artemisinin yielding genotypes with their genetic constraint in specific media combinations and also used as base material for further genetic improvement.

Growth Response of Artemisia annua by Effect of Types and Composition of Organic Fertilizer in Lowland

Artemisia annua is a plant used to cure malaria diseases. Artemisia plant contains artemisinin as secondary metabolite that used to eliminate parasite that caused malaria, such as Plasmodium falciparum. Artemisia growth affects production of artemisinin content in plant. Therefore, necessary environment conditions and appropriate organic manure application are needed to support the growth of Artemisia. This research aimed to determine the effect of fertilizer type and proportion in the medium on the Artemisia growth. This research was conducted at greenhouse of Faculty of Agriculture, Universitas Sebelas Maret, Surakarta, in October 2015 to January 2016. This research used a completely randomized design （CRD）, consisting of two factors of treatment with three replications. The first factor was type of fertilizer that consists of three types： horse manure fertilizer, compost filter press mud and cow manure fertilizer. The second factor was proportion of fertilizer with media consisted of five levels： fertilizer as media, proportion of fertilizer with media 4：1, 3：2, 2：3 and 1：4. Data were analyzed using analysis of variance and Duncan＇s multiple range test with level of 5%. It can be concluded that treatment with compost filter press mud provided the highest of plant height, root length, days to flowering, root volume, fresh weight and dry weight of crop.

Effect of Artemisia annua L. as Substitute to Sulfonamides (Sodium Sulfadimerzine) on Coccidiosis and Growth Performance in Rabbits

Coccidiosis is a disease caused by protozoa of the genus Eimeria which seriously affects young rabbits. Treatment based on the use of anticoccidial drugs is increasingly ineffective due to the rapid emergence of resistant strains of coccidia and the high cost of drugs. Consumer demand for rabbit products without chemical residues led to a growing interest in the use of medicinal plants as an alternative treatment for coccidiosis. The present study was carried out during the period of August to December 2020 to assess the anticoccidial effect of hydro-ethanolic extract of leaves of Artemisia annua L., in young rabbits. The antiparasitic efficacy of Artemisia extract was tested on 15 young rabbits (whose age varied between 7 and 9 weeks) divided into 5 lots of 3 animals. The average weight of these animals was 790 g. The results of this study show that the feces samples and the weight of young rabbits before administration of the treatment and the coprological examination (every 7 days for 4 weeks) show a fecal excretion reduction rate (FECRT) of 55.13% in the lot treated by sulfonamide. On the other hand, in animals received treatments extract of the leaves of Artemisia annua L., the average FECRT is evaluated at 69.64%, 79.22%, and 96.36% for respective doses of 400, 800 and 1200 mg/kg bodyweight and proves their anticoccidial effect. Furthermore, the variation in mean Eggs Per Gram (EPG) of coccidia and the average weekly weight gain (AWWG) of each lot were significant in the lots treated with hydro-ethanolic extract (P 0.05). The greatest reductions in oocystal excretion and weight gain obtained were those of lot 5, treated at 1200 mg/kg of hydro-ethanolic leaves extract of Artemisia annua L.

Artemisia annua: A New Version of a Traditional Tea under Randomized, Controlled Clinical Trial for the Treatment of Malaria

Introduction: The traditional antimalarial tea Artemisia annua, indicated for centuries in China to treat fevers, is again arousing interest for the treatment of malaria due to improvements attained in the plant composition by a few Institutions throughout the world, including the State University of Campinas (UNICAMP), Brazil, increasing its principal component by more than 100 times as from standard varieties, giving 1% in artemisinin and an expressive biomass yield such as 2 tons of dried leaves/hectare. Clinical trials carried out with this material in African countries have proven its therapeutic potential for a new generation of Artemisia tea in the treatment of falciparum malaria. In addition to artemisinin, recent studies have identified and quantified other compounds present in the crude extract and characterized their contributions to the anti-malarial efficacy, including their action against chloroquine-resistant strains. The majority of the clinical trials carried out with Artemisia tea in African countries have shown that the control of the parasitaemia is efficient in the initial treatment period, but few trials have followed the patients up to the 28th day. This first clinical trial carried out in Brazil with the A. annua infusion, after toxicological trials that defined the safety of this form of medication. Methods: The therapeutic efficacy of the tea was measured in patients with falciparum malaria over 28 days, comparing it with the current first-line treatment namely artemether-lumefantrine (Coartem?). The trial was carried out in controlled groups according to official protocol approved by the National Ethics in Research Committee (CONEP: 77/ 2011) and a rigorous control of the 17 patients with non-serious cases of falciparum malaria, recruited in the following three municipalities of the State of Pará, Brazil: Tucuruí, Goianésia do Pará and Anajás. The tea group received the infusion prepared in the proportion of 1.25 g of dry leaves of the variety CPQBA with 1% artemisinin for 250 mL of just boiled water, taken every 6 hours for 7 days, giving a total of approximately 175 mg of artemisinin, whilst the artemether-lumefantrine (Coartem?) group received a total of 525 mg of artemisinin equivalent to artemether. Results: The parasitaemia by the tea treatment became negative in the first days, even though it was administered with a dose that was one third of the recommended dose of artemisinin. However, as in the case artemisinin or artesunate monotherapies, 57.1% of the patients treated with the A. annua tea presented type I resistance, with a return of the parasitaemia around the 14th or 21st day. The other patients in the tea group showed type II/III resistance without manifestation of any serious signs or symptoms. In these cases, according to the protocol, the patients were redirected for treatment with artemether-lumefantrin (Coartem?) with subsequent negativity of the parisitaemia. Discussion: The fact that the efficacy of the tea with 1/3 of the dose of artemisinin was similar to that of the full dose of this medication infers that other compounds present in the crude extract, probably flavonoids, had contributed to the negativity of the parasitaemia at the start of the treatment. Considering that the positive control group, where the compound derived from artemisinin (artemether) was associated with another antimalarial agent (lumefantrine), presented excellent efficacy throughout the entire control of the cure, future trials with the A. annua tea should use the same strategy of association with another antimalarial agent, preferably from A. annua itself, in order to extend its therapeutic action during the whole control period. The Artemisia annua tea in the form standardized and used in this research, should not substitute the most efficient treatment, but could be considered as an emergency therapeutic resource in the first hours of symptoms as a function of its availability, anti-inflammatory action and lack of side effects. Other regimes and standardizations deserve investigation, mainly those with a high content of arteannuin B, as occurs in the initial cultivation phase.

盐池湾自然保护区黄蒿(Artemisia annua)挥发油成分研究

采用水蒸气蒸馏法从盐池湾黄蒿(Artemisia annua)中提取挥发油,分析盐池湾地区不同生长阶段6月(A样品)和7月(B样品)黄蒿挥发油的主要化学成分,利用气相色谱-质谱联用法对其化学成分进行测定,采用归一法计算各组分的含量.从两个不同生长期黄蒿的挥发油中分别鉴定出了21和23种成分,其中相同成分8种.分子式为C15H24的1H-Cycloprop[e]azulene,1a,2,3,4,4a,5,6,7b-octahydro-1,1,4,7-tetramethyl-,1aR-(1a.alpha.,4.alpha.,4a.beta.,7b.alpha.)为样品A化学成分中含量最高的单一成分,匹配度达到98%,含量达到11.502%.桉油精(C10H18O)为样品B化学成分中含量最高的单一成分,匹配度达到96%,含量达到7.913%.两个生长阶段的黄蒿虽然其挥发油主要组成部分的种类及含量很相近,但化学成分种类及数量方面存在明显差异.盐池湾地区的黄蒿所检测出的化合物中只有3种与其他产地黄蒿相同,其他化合物均属首次检测到.

Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in <i>Artemisia annua</i>L., as Reported by a Promoter-GUS Fusion

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination <i>Artemisia annua</i>Progeny and Artemisinin Formation in Regenerated Plants

To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.

Comparative Study on Population Ecological Distribution and Extracellular Enzyme Activities of Endophytic Fungi in <i>Artemisia annua</i>

The endophytic fungi in different tissues of Artemisia annua was isolated and purified to explore their ecological distribution and tissue preference, and the extracellular enzyme activities of dominant endophytic fungi were determined to characterize the metabolic function of endophytic fungi. The results showed that a total of 67 endophytic fungi were obtained from Artemisia annua tissues. The number and species of endophytic fungi in different tissues were significantly different. The number, colonization rate (CR) and isolation rate (IR) of endophytic fungi in root were significantly higher than those of stem and leaf. The dominant endophytic fungi, diversity and similarity coefficient of endophytic fungi also showed significant difference among tissues. The extracellular enzyme activities of endophytic fungi in different tissues are significantly different. The enzyme activities of endophytic fungi isolated from root are significantly higher than those isolated from stem and leaf. The research results showed that the endophytic fungi in Artemisia annua had significant tissue preference, and the metabolic function of endophytic fungi showed significant difference among tissues. This will lay a foundation for further research, development and utilization of endophytic fungi, and also provide a theoretical basis for screening functional endophytic fungi in Artemisia annua.