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.
Key words
activation tagging -
Agrobacterium tumefaciens
- gene trapping - green fluorescent protein -
Salvia miltiorrhiza
- Lamiaceae - tanshinones
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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
Telefon: +886-4-2330-4921
eMail: hstsay@cyut.edu.tw