Abstract
Artemisia annua is the chief source of artemisinin, a potent antimalarial agent, in which other bioactive phytochemicals are also present. Due to low levels of bioactive compounds including artemisinin and flavonoids, it is necessary to increase the level of the secondary metabolites by regulating the expression of rol genes in the plant.
A hybrid variety of A. annua (Hyb1209r, Shennong) developed by the Centre for Novel Agricultural Products, University of York, UK, was selected to produce transgenics of rolB and rolC genes. Genetic transformation was carried out via Agrobacterium tumefaciens GV3101 harboring rolB and rolC genes of Agrobacterium rhizogenes cloned separately. HPLC was used for the qualitative and quantitative analysis of flavonoids and artemisinin. Furthermore, thin layer chromatography (TLC) was also used to analyze artemisinin content.
Comparative analysis via HPLC revealed considerable enhancement in the phytochemical content of transgenic A. annua plants as compared to the wild type plant. Transgenics of rolB gene showed an average increase of 321% in rutin, 97.2% in caffeic acid, and 218.4% in myricetin, respectively. In the case of rolC gene transgenics, an average increase of 197.5% in rutin, 76.3% in caffeic acid, and 209.3% in myricetin was observed. Transgenics of rolB and rolC genes showed a 14.3%–28.6% and 2.8%–12.7% increase in artemisinin content respectively by HPLC analysis. TLC analysis showed that an average 142.2% and 110.2% enhancement in artemisinin for rolB and rolC transgenics respectively, compared with the wild type. An enhanced production of total flavonoids (average 30.2% and 25.5% increase in rolB and rolC transgenics, respectively) and total phenolics (average 34.3% and 25.8% increase in rolB and rolC transgenics, respectively) was observed as a result of transformation. Transformed A. annua plants showed improved free radical scavenging activity (average 46.5% and 29.1% increase in rolB and rolC transgenics, respectively) and total reducing power (average 32.7% and 26.4% increase in rolB and rolC transgenics, respectively) compared with untransformed plant.
rolB and rolC genes were effective for developing A. annua plants with an enhanced level of phytochemicals.