| Summary: | <p>Abstract</p> <p>Background</p> <p>Tropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine are a group of important anticholinergic drugs with rapidly increasing market demand, so it is significant to improve TA production by biotechnological approaches. Putrescine <it>N</it>-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme involved in TA biosynthesis. However, there is no report on simultaneous introduction of <it>PMT </it>and <it>TRI </it>genes into any TA-producing plant including <it>Anisodus acutangulus </it>(<it>A. acutangulus</it>), which is a Solanaceous perennial plant that is endemic to China and is an attractive resource plant for production of TA.</p> <p>Results</p> <p>In this study, 21 <it>AaPMT </it>and <it>AaTRI </it>double gene transformed lines (PT lines), 9 <it>AaPMT </it>single gene transformed lines (P lines) and 5 <it>AaTRI </it>single gene transformed lines (T lines) were generated. RT-PCR and real-time fluorescence quantitative analysis results revealed that total <it>AaPMT </it>(<it>AaPMT T</it>) and total <it>AaTRI </it>(<it>AaTRI T</it>) gene transcripts in transgenic PT, P and T lines showed higher expression levels than native <it>AaPMT </it>(<it>AaPMT E</it>) and <it>AaTRI </it>(<it>AaTRI E</it>) gene transcripts. As compared to the control and single gene transformed lines (P or T lines), PT transgenic hairy root lines produced significantly higher levels of TA. The highest yield of TA was detected as 8.104 mg/g dw in line PT18, which was 8.66, 4.04, and 3.11-times higher than those of the control (0.935 mg/g dw), P3 (highest in P lines, 2.004 mg/g dw) and T12 (highest in T lines, 2.604 mg/g dw), respectively. All the tested samples were found to possess strong radical scavenging capacity, which were similar to control.</p> <p>Conclusion</p> <p>In the present study, the co-expression of <it>AaPMT </it>and <it>AaTRI </it>genes in <it>A. acutangulus </it>hairy roots significantly improved the yields of TA and showed higher antioxidant activity than control because of higher total TA content, which is the first report on simultaneous introduction of <it>PMT </it>and <it>TRI </it>genes into TA-producing plant by biotechnological approaches.</p>
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