In vitro regeneration and transient expression of recombinant sesquiterpene cyclase (SQC) in Artemisia annua L.

Abstract

In the present study, we developed an efficient and reliable protocol for in vitro plant regeneration and transient gene expression in medicinal plant Artemisia annua L. For in vitro regeneration, leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.5mg/l NAA plus 2mg/l BA (86.6%) and MS medium supplemented with 1mg/l IAA plus 2mg/l BA (80%). Root induction was obtained on MS medium supplemented with 0.1mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. annua L. in short period via adventitious shoot induction approach. Based on the sequence of a putative sesquiterpene cyclase from A. annua, 1.7kb fragment was amplified by PCR from Iranian variety of this plant after cDNA synthesis. Sesquiterpene cyclase gene was used for sub-cloning into pBI121 between XbaI and SacI sites to allow the expression of the gene driven by CaMV35S promoter. The recombinant plasmids were transferred to competent cells of Agrobacterium tumefaciens strain GV 3850 for vacuum agro-infiltration. HPLC results showed 30.09% increase in atremisinin content (0.15%) in the agroinfiltrated samples (0.67%) in comparison to the control (0.51%).