Planta Med 2008; 74(7): 780-786
DOI: 10.1055/s-2008-1074527
Biochemistry and Molecular Biology
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

T-DNA Activation Tagging as a Tool to Isolate Salvia miltiorrhiza Transgenic Lines for Higher Yields of Tanshinones

Chen-Yu Lee1 , Dinesh C. Agrawal2 , Chang-Sheng Wang1 , Su-May Yu4 , Jeremy J. W. Chen3 [*] , Hsin-Sheng Tsay2 [*]
  • 1Department of Agronomy, National Chung-Hsing University Taichung, Taiwan
  • 2Institute of Biotechnology, Chaoyang University of Technology, Taichung, Taiwan
  • 3Institute of Biomedical Sciences, and Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan
  • 4Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
Further Information

Publication History

Received: April 4, 2007 Revised: June 3, 2007

Accepted: March 18, 2008

Publication Date:
26 May 2008 (online)

Abstract

The study of functional genomics has paved the way for directed approaches to the generation of genetically modified plants that produce novel and/or improved yields of pharmaceuticals. In the present study, an activation tagging mutagenesis (ATM) population of Salvia miltiorrhiza Bunge, a medicinal plant, was established by Agrobacterium-mediated transformation. The optimum conditions for Agrobacterium transformation were determined by the expression of green fluorescent protein. Under these optimized conditions, we isolated 1435 ATM cell lines with our initial antibiotic selection. Of these 1435 ATM cell lines, six lines (T1 - T6) showed a red color on a selective medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), which is used as a phenotypic model system to identify the accumulation of tanshinones. 700 out of 1435 ATM cell lines were tested with a β-glucuronidase (GUS) assay, 35 showed GUS activity. Southern blotting analysis revealed that the T1 - T7 ATM cell lines have a single copy of the T-DNA insertion. Comparative analysis by high-performance liquid chromatography of the tanshinones expressed by non-transformed and ATM-transformed calli revealed varying quantities of tanshinones. There were negligible tanshinones in non-transformed white calli induced with 2,4-D. ATM lines T1 - T6 showed significant increases in the yields of tanshinone-I (up to 43-fold), tanshinone-IIA (up to 26-fold) and cryptotanshinone (up to 104-fold) compared with those of the non-transgenic lines on 2,4-D medium. Interestingly, the yield of cryptotanshinone from line T4 on 2,4-D medium was two times higher than that of the non-transgenic lines on trans-zeatin riboside medium. To the best of our knowledge, this is the first report of a quantitative and qualitative improvement in quinoid diterpene production achieved in a medicinally important plant species by activation tagging.

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1 J. J. W. C. and H.-S.T. contributed equally to this work and are joint corresponding authors.

Prof. Hsin-Sheng Tsay

Institute of Biotechnology

Chaoyang University of Technology

Taichung 41349

Taiwan

Republic of China

Phone: +886-4-2330-4921

Email: hstsay@cyut.edu.tw