In vitro Regeneration and Genetically Transformed Culture of Artemisia diffusa

The present study has introduced a simple and rapid tissue culture system aimed at in vitro regeneration of Artemisia diffusa and in vitro artemisinin production in its genetically transformed culture. An in vitro regeneration of A. diffusa was developed using different combinations of plant growth regulators including Naphthalene Acetic Acid (NAA), Indole-3-Acetic Acid (IAA), Thidiazuron (TDZ) and Benzyl Adenine (BA). Also, an efficient genetically transformed root induction system for A. diffusa was developed through Agrobacterium rhizogenes- mediated transformation using four bacterial strains, A4, ATCC15834, MSU440, and MAFF-02-10266. The stem and leaf of one month old sterile plants of A. diffusa were used as explants. Molecular analysis of transformed root lines was confirmed by PCR using primers specific for the rolB gene. The highest regeneration occurrence was obtained using MS medium containing 0.5 mg/L TDZ and 0.1 mg/L IAA (75%). Root induction was obtained on MS medium supplemented with 0.5 mg/L IBA. The results showed a significant increase in transformation frequency when the strain MSU440 was used (80.7%). Approximately 0.05 % artemisinin was detected by High-performance liquid chromatography (HPLC) analysis in root cultures. To the best of our knowledge, this is the first report of A. diffusa in vitro organogenesis and transformation. This study describes an efficient protocol for hairy roots culture of A. diffusa which can be used for scaling up the plant active phytochemicals or for genetic manipulations of the plant.