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Background  Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from  Artemisia annua  is still one of the most effective treatments for malaria. Increasing the artemisinin content of  A. annua  plants by genetic engineering would improve the availability of this much-needed drug.    Methods  In this regard, a high artemisinin-yielding hybrid of  A. annua  produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As  rol  genes are potential candidates of biochemical engineering, genetic transformation of  A. annua  with  Agrobacterium tumefaciens  GV3101 harbouring vectors with  rol B  and  rol C  genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC–MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.    Results  Transgenics of  rol  B  gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4–12-fold increase in artesunate and 1.2–3-fold increase in dihydroartemisinin. Whereas in the case of  rol C  gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the  rol B  gene had higher expression of these genes than  rol C  transformants. TAFR1 was also found to be more expressed in  rol  gene transgenics than wild type  A. annua , which was also in accordance with the trichome density of the respective plant.    Conclusion  Thus it was proved that  rol B  and  rol C  genes are effective in the enhancement of artemisinin content of  A. annua ,  rol B  gene being more active to play part in this enhancement than  rol C  gene.

Enhanced artemisinin yield by expression of rol genes in Artemisia annua