Artemisinin Yield Research Articles

Genetic engineering of artemisinin biosynthesis: prospects to improve its production

Approaches involved in production of Artemisinin in different hosts. Artemisinin, a potent antimalarial natural compound, is obtained from aerial parts of Artemisia annua L. plants. The demand (101–119 MT) for artemisinin is exponentially increasing every year because of increased incidence of drug-resistant malaria throughout the world, especially in Africa and Asia. However, the presence of low concentrations (0.01–1.1 %) of the compound in A. annua L. plants poses a major constraint in the commercialization of artemisinin-based combination therapies (ACTs) recommended by WHO for the treatment of multidrug-resistant and cerebral malaria. Further, the improvement in the yield of artemisinin through conventional breeding, in vitro culture, cell suspension culture and total organic synthesis still poses a challenge. However, possibilities are there to enhance the artemisinin biosynthesis either by overexpression of the genes encoding enzymes associated with the rate-limiting steps of the mevalonate and artemisinin biosynthetic pathways or by the suppression of genes encoding enzymes of other competing pathways. Based on the current understanding of the pathway and cloning of the related genes, efforts have been made for the past few years to increase the artemisinin content in A. annua L., Cichorium intybus L. and microbes through metabolic engineering. In the present review, we have discussed the metabolic engineering strategies in both plant and microbial systems for artemisinin accumulation in bioengineered hosts.

The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.

The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

Artemisia annua as a possible contraceptive agent: a clue from mammalian rat model

In a previous study, we evaluated the maternal and fetal safety of antimalarial herb Artemisia annua with artemisinin yield of 1.09%. Here, we attempted to ascertain the contraceptive claim of A. annua. Sexually matured female Wistar rats (180–220 g) were allotted into four study groups of six rats each. The control group received normal saline, while the A. annua-treated groups received 100, 200 and 300 mg/kg of A. annua for 2 weeks, followed by mating with proven fertile males (1:1). The rats were allowed to carry the pregnancy to term. At birth and weaning periods, selected reproductive outcome and fertility indices were determined. The results showed that A. annua significantly reduced litter size, reproductive outcome and fertility indices compared with the control (p < 0.05). These results imply that A. annua could serve as a prospective contraceptive agent in addition to its antimalarial activity.

Overexpression of artemisinic aldehyde Δ11 (13) reductase gene-enhanced artemisinin and its relative metabolite biosynthesis in transgenic Artemisia annua L.

Artemisinic aldehyde Δ11 (13) reductase (DBR2) is the checkpoint enzyme catalyzing artemisinic aldehyde to form dihydroartemisinic aldehyde directly involved in artemisinin biosynthetic pathway. In the present study, DBR2 was employed to engineer the biosynthetic pathway of artemisinin in transgenic plants of Artemisia annua L. Seven independent transgenic plants of A. annua with DBR2 overexpression driven by the cauliflower mosaic virus 35S promoter were obtained by Agrobacterium-mediated genetic transformation and confirmed by genomic PCR. The results of real-time qPCR analysis showed that the expression levels of DBR2 gene in all the seven transgenic lines were significantly higher than in nontransgenic control. The high-performance liquid chromatography analysis of artemisinin and its relative metabolites demonstrated that the contents of artemisinin and its direct precursor dihydroartemisinic acid were remarkably increased in the transgenic plants of A. annua with DBR2 overexpression. Interestingly, it was also found that the contents of arteannuin B and its direct precursor artemisinic acid in the branch pathway competing against artemisinin biosynthesis were also improved in DBR2-overexpressed A. annua plants. The transgenic results in the present study indicated that DBR2 is a useful structural gene in engineering the artemisinin biosynthetic pathway to develop genetically modified A. annua with the higher yield of artemisinin.

Mutagenic effects of heavy-ion beam irradiation on in vitro nodal segments of Artemisia annua L.

Artemisia annua L. is a commercial source of artemisinin. Nevertheless, artemisinin content within the plant is relatively low and varies depending on genotype and environment. To broaden the genetic variability, the mutation effect of 12C-ion beam irradiation on A. annua was examined. Irradiation at 2.5 Gy had a slight lethal effect to nodal segments while a noticeable lethal effect was observed at 5 and 10 Gy. Furthermore, at higher doses (20 and 50 Gy), a severe lethal effect was observed. Mutations at the DNA level of axillary bud-derived shoots were performed by RAPD. The mutation frequency at 10 Gy was about 1.7 and 2.1 times higher than that at 2.5 and 5 Gy, respectively. After growth and artemisinin production observation of 72 irradiated mutants, around 14 and 7 % of them showed higher artemisinin content and artemisinin yield compared to the controls, respectively. The highest artemisinin content in a mutant was 1.43 % DW, which was 3.2-fold higher than the original wild type. Additionally, the highest artemisinin yield in mutants was 3.68 mg/plant, which was around 1.4-fold higher than in the wild type. Moreover, irradiated mutants exhibited antibacterial activity against S. aureus, but the wild types did not. This study presents an effective application of heavy ion beam irradiation to create variations and improve artemisinin production in A. annua.

Effect of irradiated sodium alginate and phosphorus on biomass and artemisinin production in Artemisia annua

It is now being realized that irradiation products of natural bioactive agents can also be beneficially utilized to impart value addition in agriculture by converting these bioactive agents into more useful form. Polysaccharides, such as sodium alginate, have proven to be wonderful growth promoting substances in their depolymerized form for various plants. Artemisinin has been increasingly popular as an effective and safe alternative therapy against malaria; also proved effective against the highly adaptable malaria parasite, which has already become resistant to many other drugs. The drug artemisinin can be extracted from the leafy tissues of Artemisia annua. Therefore, experiments were conducted with an aim to evaluate artemisinin production and overall plant development though depolymerized sodium alginate application and nutrient supply. In the present study, sodium alginate, irradiated by Co-60 gamma rays together with various phosphorus doses, was used to study their effect on growth, physiological and biochemical processes and production of artemisinin in A. annua. Among various applied doses of phosphorus fertilizer, P40 (40kg Pha−1) together with ISA80 (80mgL−1) significantly improved all the parameters studied. Increase in plant height as well as weight was noted at this treatment. Dry leaf yield, artemisinin concentration in leaves and artemisinin yield was also significantly enhanced by the treatment.

Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants

The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis.

Optimization of Ultrasound-Assisted Extraction of Artemisinin from Artemisia annua L. by Response Surface Methodology

Artemisinin is a compound extracted from Artemisia annua L. with a remarkable curative effect against malaria. It can be extracted using ultrasound-assisted extraction (UAE) and then detected via HPLC. In this study, response surface methodology (RSM) was used to optimize UAE conditions for obtaining the maximum yield of artemisinin. Three independent variables (ratio of solvent to material, extraction temperature, and ultrasonic power) were evaluated using the Box-Behnken experimental design, with the yield of artemisinin as a response variable. Experimental data were highly fitted to a mathematical-regression model using multiple linear regression (MLR). Based on response surface plots, the three independent variables exhibited interactive effects on the yield of artemisinin. The optimal extraction conditions were as follows: 42.71 mL/g ratio of solvent to material, 41.86°C extraction temperature and 120 W ultrasonic power. The predicted yield of artemisinin by model was 0.7848%, whereas the actual yield in the extracts was 0.7826% ± 0.0790% in adjusted optimal conditions, with a relative error of 0.28%. The results undoubtedly demonstrated that RSM could be used to explore the optimum conditions of artemisinin extraction.

Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production.

Background:Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose.Aim:To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants.Setting and Designs:The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient β-glucuronidase (GUS).Materials and Methods:Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression) on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency.Results:The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of expression than Silwet L-77, were 2.3- and 1.3-fold, respectively. For GV3101, Silwet L-77 was still higher than Silwet S-408, were 1.5- and 1.4-fold, respectively. However, GV3101 produced higher levels of expression than AGL1. The area of GUS expression spots of AGL1, LBA4404, and GV3101 strains was 53.43%, 41.06%, and 30.51%, respectively.Conclusion:A. tumefaciens AGl1 strain was the most effective to be transformed in to A. annua than GV3101 and LBA4404 strain. Surfactant Silwet S-408 produced the highest efficiency of transformation.

The effect of O-methylated flavonoids and other co-metabolites on the crystallization and purification of artemisinin

Methoxylated flavonoids casticin, artemetin and retusin were identified as putative causative factors for low crystallization yields of artemisinin from extracts. Comparative profiling of biomass grown in different countries found elevated levels (∼60% higher) of artemetin in the East African biomass, which also demonstrates poor crystallization yields. The single compound and the combined doping experiments at 0, 25 and 50μgmL−1 doping levels showed that artemetin (at 50μgmL−1) caused a reduction in the amount of artemisinin crystallized by ca. 60%. A combination of the three flavonoids at 50μgmL−1 almost completely inhibited crystallization, reducing the yield by 98%. Treatment of extracts by adsorbents efficiently resolves the problem of low crystallization yield.

Short term UV-B radiation-mediated transcriptional responses and altered secondary metabolism of in vitro propagated plantlets of Artemisia annua L.

In vitro propagated Artemisia annua plantlets were exposed to low dose (2.8 W m−2) of UV-B (280–315 nm) radiation for different short-term (1, 2, 3 and 4 h) durations that resulted in 103 and 100 % enhanced artemisinin and flavonoid yield respectively after 3 h of UV-B irradiation. UV-B irradiation resulted in reduced chlorophyll a, b and carotenoids while the concentrations of UV-B-absorbing anthocyanins and phenolics were induced. Under UV-B radiation, superoxide radical formation increased in a time dependent manner and was maximum at 4 h. This lead to differential responses of enzymatic antioxidants (superoxide dismutase, catalase, peroxidases and glutathione reductase) to withstand the UV-B radiation-induced reactive oxygen species burst. The transcript analysis through RT-PCR revealed the significant up-regulation of HMGR, DXR, IPPi, FPS, ADS, CYP71AV1 and RED1 gene transcripts leading to higher artemisinin accumulation. Differential expression of stress regulated genes (AOX1a, NDB2, UPOX, PAL, LOX) helped the plant towards better adaptability at least up to 3 h UV-B irradiation period. Simultaneous clustering of genes and UV-B treatments using complete linkage and a euclidean distance matrix revealed that 3 h irradiation period is optimum for high artemisinin yield with better tolerance. Histological studies revealed the larger trichome size in UV-B treated plants as compared to untreated plants. The present study conclude that short term UV-B treatment to in vitro raised A. annua may be a safe approach for continuous supply of high artemisinin producing plantlets with simultaneous action of stress- regulated genes to keep the plant healthy.

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.

Arsenic, chromium and NaCl induced artemisinin biosynthesis in Artemisia annua L.: A valuable antimalarial plant

Effect of As(III), Cr(VI) and NaCl on plant growth, antioxidant enzymes, SOD, TBRAS, protein, cDNA amplification of key genes of artemisinin pathway and artemisinin biosynthesis have been investigated to explore the actual changes in total herb and artemisinin yield in a crop cycle of Artemisia annua. Enhanced TBARS and SOD activity (4Umg−1), decreased catalase activity and total cholorophyll content were observed under metal(loid) and NaCl stress. Accumulation of As (III; µgmg−1 DW) was higher in roots (10.75±0.00) than shoot (0.43±0.00) at 10µgml−1. While Cr(VI; µgml−1 DW) accumulated more in shoots (37±9.6, 41.1±7.2 and 52.71±19.6). cDNA template of these treated plants along with control were amplified with HMGR, ADS and CYP71AV1 genes (artemisinin biosynthetic pathway genes); showed very low expression with Cr(VI) while As(III) (5 and 7.5µgml−1) showed higher expression than control. The results obtained from this study suggest that A. annua can grow well with favoring artemisinin biosynthesis with treatment of As(III) 5, 7.5µgml−1 and NaCl, while 10µgml−1 As(III) and all doses of Cr(VI) affect artemisinin synthesis. Finally some evidence also suggests that As(III), Cr(VI) and NaCl induces stress affects on total herb yield of plant.

Cumulative role of irradiated sodium alginate and nitrogen fertilizer on growth, biochemical processes and artemisinin production in Artemisia annua

Being the world's most severe parasitic infection, malaria causes more than a million deaths annually. Artemisinin and its derivatives are effective against malaria without any reputed cases of resistance. The drug artemisinin can be extracted from the leafy tissues of Artemisia annua L. Application of ionizing radiation to degrade natural bioactive agents, such as sodium alginate and then using them as growth promoting substances is a novel emerging technology to exploit full genetic potential of crops in terms of growth, yield, and quality. In the present study, sodium alginate, irradiated by Co-60 gamma rays together with various nitrogen doses, was used to study their effect on growth attributes, physiological and biochemical processes and production of artemisinin in A. annua. Of the various applied doses of nitrogen, N80 (80kgNha−1) together with ISA80 (80mgL−1) proved superior in positively regulating almost all the parameters studied. Most importantly, an increase of 38.1 and 80.5% in artemisinin content and yield respectively, was noticed at this treatment.

Conceptual Process Synthesis for Recovery of Natural Products from Plants: A Case Study of Artemisinin from Artemisia annua

A systematic method of conceptual process synthesis for recovery of natural products from their biological sources is presented. This methodology divides the task into two major subtasks namely, isolation of target compound from a chemically complex solid matrix of biological source (crude extract) and purification of target compound(s) from the crude extract. Process analytical technology (PAT) is used in each step to understand the nature of material systems and separation characteristics of each separation method. In the present work, this methodology is applied to generate process flow sheet for recovery of artemisinin from the plant Artemisia annua (A. annua). The process flow sheet is evaluated on the basis of yield and purity of artemisinin obtained in bench scale experiments. Yields of artemisinin obtained in individual unit operations of maceration, flash column chromatography, and crystallization are 90.0%, 87.1% and 47.6%, respectively. Results showed that the crystallization step is dominant to the overall yield of the process which was 37.3%.

Purification of artemisinin from quercetin by anti-solvent crystallization

In the present work, anti-solvent crystallization of artemisinin from four different organic solvents (methanol, ethanol, acetonitrile, and acetone) was studied. Water was used as anti-solvent. The effect of an impurity (quercetin) on the performance of anti-solvent crystallization of artemisinin was investigated. The fundamental process data such as solubility of artemisinin in pure organic solvents and their binary mixtures with varying composition water were measured at room temperature. The solubility of quercetin was measured only in pure organic solvents at room temperature. Anti-solvent crystallization experiments were designed based on the fundamental process data determined. Firstly, the anti-solvent crystallization of artemisinin without impurity was performed from all four organic solvents and then the experiments were repeated with addition of an impurity (quercetin) while keeping all other process parameters constant. Two different concentrations of impurity, i.e., 10% and 50% of its solubility, in the respective organic solvents at room temperature were used. The effect of impurity on performance of anti-solvent crystallization was evaluated by comparing the yield and purity of the artemisinin obtained with those in the absence of impurity. Results of the present work demonstrated that the presence of quercetin in the solution does not affect the final yield of artemisinin from the solution of each of four organic solvents used. However, the purity of artemisinin crystals were reduced when quercetin concentration was 50% of its solubility in all solvents studied.

Study of Various Factors for Enhancement of Artemisinin in Artemisia Annua Hairy Roots

Artemisinin is an alternate anti-malarial drug which is widely used in the cure of multi-drug resistant Plasmodium falciparum malaria. In this study, enhancement of artemisinin content by using several elicitors and precursors was attempted initially by using one at a time (OVAT) approach. The most potent elicitor(s) & precursor were thereafter identified by the detailed analysis of the responses with respect to biomass and bioactive compound with respect to the concentrations of different effectors. Central Composite Design (CCD) was thereafter used to identify the antagonistic or synergistic effects of high and low concentrations of different precursors and elicitors. The maximum artemisinin content obtained was 3.45 mg/g on 15d by using the elicitor methyl jasmonate (40 µg/l) and the precursors casein acid hydrolysate (50 µg/l), sodium acetate (500 µg/l). Effect of addition time of elicitors and precursors on overall optimum biomass growth and artemisinin yield and productivity was also established.

Effect of Soil Type and Seasonal Variation on Growth, Yield, Essential Oil and Artemisinin Content of Artemisia Annua L

Aims: This experiment was carried out to study the effects of soil type and seasonal variation on growth, yield, essential oil and artemisinin content of Artemisia annua plant. Methods: Two field experiments were carried out during two successive seasons in two different types of soil, loamy clay soil and sandy loam soil. Artemisia seeds were cultivated in nursery plots at 15th December then transplanted at age of 90 days in a distance of 60cm in apart and 50cm in between plants under organic system then plant samples were collected every 30 days through growth season. Results: Significant increases in vegetative growth characters were observed for plants cultivated in clay loamy soil comparing to those cultivated in sand loamy soil. The plant essential oil percentage of those cultivated in sand loamy soil were increase more than those cultivated in clay loamy soil while essential oil yield of plants grown in clay loamy soil was more than those grown in sand loamy soil. Essential oil % of A. annua aerial parts in the two soils increased with increasing plant age to reach its maximum values at full flowering stage then this tended to decrease towards the end of plant life. The major compound in the essential oil was found to be artemisia ketone followed by 1,8 cineole in both soils. A change in the order of the followed compounds according to the soil type was observed. Artemisinin % and artemisinin yield (kg/fed) of plants cultivated in sand loamy soil was increased in comparing to plants cultivated in clay soil and showed the maximum values of artemisinin % at the flowering stage in August in both types of soil. Conclusion: Cultivation of A. annua in sandy loam soil resulted in increasing essential oil percentage whereas clay loam soil increased oil yield as it increased all vegetative parameters. Artemisia ketone and 1,8 cineole are major compounds in essential oil and their percentage in essential oil for sandy loam soil plants were more than of clay loam plants.

The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua.