Effects of yeast elicitor on the growth and secondary metabolism of a high-tanshinone-producing line of the Ti transformed Salvia miltiorrhiza cells in suspension culture
Introduction
Higher plants are a rich source of various biologically active compounds named secondary metabolites with a total of 119 chemical substances extracted from plants as drugs in Western medicine [1]. Plant cell culture offers an alternative means for producing these pharmacologically important compounds. However, until recently, this technology was far from fully exploited due to the lack of information about the biosynthesis of secondary metabolites and its regulation. For a long time, secondary metabolites were regarded as waste products of plants. In recent years, the ecological functions of secondary metabolites to serve plants per se, such as protections against UV light and ozone, herbivores, and microbial pathogens have become recognized [1], [2], [3].
Salvia miltiorrhiza Bunge (Labiatae) is a famous traditional Chinese medicinal plant. Its roots, called Dan-shen or Tan-shen, contain two groups of biologically active compounds: caffeic acid-derived phenolic acids (e.g. rosmarinic acid) and various tanshinones (e.g. cryptotanshinone), which are diterpene quinones (Fig. 1) [4]. Ti transformed S. miltiorrhiza cell cultures were obtained after infecting the plant with the wild type Agrobacterium tumefaciens strain C58 [5]. From these cell cultures, two cell lines (A and B) were selected. Cell line A produced only a small amount of tanshinones while cell line B contained a high level of tanshinones, mainly cryptotanshinone [5]. Upon elicitation with yeast extract, the production of tanshinones by cell line A could be enhanced greatly from a trace to ca. 20 mg l−1 [5]. In addition, both cell lines A and B constitutively biosynthesized high concentrations of phenolic acids, mainly rosmarinic acid [6], [7]. It was also proposed that preformed rosmarinic acid and induced cryptotanshinone may represent passive and active defense compounds, respectively, in S. miltiorrhiza [7]. In this paper, we report the effects of yeast elicitor on the growth and secondary metabolism of cell line B of Ti transformed S. miltiorrhiza cells.
Section snippets
Cell culture
Experiments were carried out with a high-tanshinone-producing line of Ti C58 transformed S. miltiorrhiza cells (cell line B) [5]. The cell aggregates (teratoma) in suspension culture were propagated in 250-ml shake flasks containing 50 ml of hormone-free 6.7-V medium supplemented with 20 g l−1 sucrose [8] at 14-day intervals. Cultivation was performed on an orbital shaker at 140 rpm in darkness at 25°C. The pH of the medium was adjusted to 5.7 prior to autoclaving at 121°C for 15 min.
Chemicals and standards
Rosmarinic
Effect of yeast elicitor on cell growth
As shown in Fig. 2, the use of yeast elicitor at all four concentrations resulted in a great decrease in biomass accumulation which indicated that defense responses reduced primary metabolism of cells. For example, at day 16 of cultivation, the dry cell weight concentration in elicited cultures was between 6.8 and 7.8 g l−1 while that in the control was 11.3 g l−1. Furthermore, elicitation also reduced the water contents of the cells as indicated by a decrease in fresh/dry weight (FW/DW) ratio
Discussion
In the past 40 years, numerous strategies have been developed to improve the productivity of plant cell culture such as cell line selection, medium optimization, cell immobilization, using differentiated cells, elicitation and more recently metabolic engineering [10]. However, due to the lack of basic information about the raison d’être of plant secondary substances, these approaches were applied only on a trial and error basis. The understanding of the defensive roles of plant secondary
Acknowledgements
The financial support of CRCG (the University of Hong Kong Committee on Research and Conference Grants) is gratefully acknowledged.
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