Artemisinin is effective against both chloroquine-resistant and -sensitive strains of Plasmodium species. However, the low yield of artemisinin from cultivated and wild plants is a serious limitation to the commercialization of this drug. Optimization of artemisinin yield either in vivo or in vitro is therefore highly desirable. To this end, we have overexpressed the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthus roseus L. in Artemisia annua L. and analyzed its influence on artemisinin content. PCR and Southern blot analyses revealed that the transgenic plants showed stable integration of the foreign hmgr gene. The reverse transcriptase-PCR results suggested that the hmgr was expressed at the transcriptional level in transgenic lines of Artemisia annua L., while the high-performance liquid chromatography analysis showed that artemisinin content was significantly increased in a number of the transgenic lines. Artemisinin content in one of the A. annua transgenic lines was 38.9% higher than that in non-transgenic plants, and HMGR enzyme activity in transgenic A. annua L. was also higher than that in the nontransgenic lines.

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Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by Artemisia annua (A. annua). The lower in planta content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular. Abbreviations ART Artemisinin A. annua Artemisia annua miRNA MicroRNA TF Transcription factor Artemisia annua (A. annua), an important medicinal herb is the only natural source of artemisinin (ART). ART, a sesquiterpene lactone is the most reliable and widely used drug to quell Malaria, which is one of the most devastating diseases and a leading cause of deaths worldwide. According to WHO, there were a staggering 228 million cases and 405,000 deaths reported in 2018 alone, despite the disease being preventable as well as curable 1. WHO has recommended the use of ART only in combination of other drugs, to slow down the development of resistance in Plasmodium sps. against it and to maintain its efficacy. The combination treatment is widely known as artemisinin-based combination therapies (ACTs). In wild type A. annua, ART is present in the aerial parts, in extremely low quantities (0.02-1.07%) 2. Numerous efforts were made to generate ART synthetically or semi-synthetically, but does not meet industrial practices due to high production cost. The insufficient supply of ART produced by plants or synthetic methods, resulted in the lack of ACTs available to the patients and causes approximately half a million mortalities every year 3. Despite various efforts to produce semi-synthetic ART at low cost, the natural source of ART remains the main provider. Thus, researchers are investigating effective strategies to enhance in planta content of ART. The Ian Grahm lab and Bill and Malinda gates foundation invested huge fund in production of high ART yielding hybrids an...

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Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.