ORIGINAL ARTICLE Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L. Tazyeen Nafis • Mohd. Akmal • Mauji Ram • Pravej Alam • Seema Ahlawat • Anis Mohd • Malik Zainul Abdin Received: 15 May 2010 / Accepted: 3 November 2010 / Published online: 27 November 2010 Ó Korean Society for Plant Biotechnology and Springer 2010 Abstract Artemisinin is effective against both chloro- quine-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 tran- scriptional 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 non- transgenic lines. Keywords Artemisia annua L. Á Genetic transformation Á RT-PCR Á HPLC Á Artemisinin Abbreviations 6-BA Benzylaminopurine HMGR 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase MVA Mevalonate NAA Naphthaleneacetic acid NOS Nopaline opine synthase NPT Neomycin phosphotransferase SISM Shoot-induction selection medium SQS Squalene synthase Introduction Artemisinin, a sesquiterpene-lactone isolated from the aer- ial parts of Artemisia annua L. plants, is currently the best therapeutic agent against both drug-resistant and cerebral malaria-causing strains of Plasmodium sp. (Newton and White 1999). It is also effective against other infectious diseases, such as schistosomiasis, human immunodefi- ciency virus (HIV), hepatitis B, and leishmaniasis (Jung and Schinazi 1994; Borrmann et al. 2001; Utzinger et al. 2001; Romero et al. 2005; Sen et al. 2007), and has recently been shown to be effective against a variety of cancer cell lines, including breast cancer, human leukemia, colon cancer, and small cell-lung carcinomas (Efferth et al. 2001; Singh and Lai 2001). Due to its current use in artemisinin based- combination therapy (ACT), the global demand of arte- misinin is continuously increasing, but the relatively low yield of artemisinin from the A. annua L. plant (0.01–1.1%) is a serious limitation to its commercialization (Laughlin 1994; Van Agtmael et al. 1999). This has led to worldwide efforts to enhance its biosynthesis through biochemical and T. Nafis Á Mohd. Akmal Á M. Ram Á P. Alam Á S. Ahlawat Á A. Mohd Á M. Z. Abdin (&) Centre for Transgenic Plant Development, Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi 110062, India e-mail: mzabdin@rediffmail.com 123 Plant Biotechnol Rep (2011) 5:53–60 DOI 10.1007/s11816-010-0156-x