In Vitro Cell.Dev.Biol.—Plant (2010) 46:233–238 DOI 10.1007/s11627-009-9249-z
SECONDARY METABOLLISM
Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of Thevetia peruviana Mario Arias Zabala & Mónica Angarita & Juan M. Restrepo & Luis A. Caicedo & Margarita Perea
Received: 16 September 2008 / Accepted: 25 August 2009 / Published online: 14 October 2009 / Editor: D. T. Tomes # The Society for In Vitro Biology 2009
Abstract Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige– Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17±0.2 g g−1 inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyljasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l−1 of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l−1 medium. The current results are the first report of an in vitro peruvoside production system.
M. A. Zabala (*) : M. Angarita : J. M. Restrepo : L. A. Caicedo : M. Perea Universidad Nacional de Colombia sede Medellin, Medellin, Colombia e-mail:
[email protected]
Keywords Plant cell culture . Peruvoside . Thevetia peruviana . Culture media . Elicitation . Methyl-jasmonate
Introduction Thevetia peruviana is a small tree commonly used as an ornamental plant. It belongs to the Apocynaceae family and it can be found in South and Central America, Asia, and Africa. This plant species produces several compounds with industrial application as pharmaceuticals, such as the cardiac glucosides neriifolin, thevetoxin, peruvoside, and thevetin A and B (Arnold et al. 1935). Among these compounds, peruvoside is particularly attractive because it has been used as a digoxin substitute in allergic patients, and it is commercially distributed in Germany for that purpose (Kumar 1992; Abe et al. 1995). On the other hand, thevetin has not been used as a pharmaceutical compound because the margin between the therapeutic and the toxic dose is too small (Watt and Breyer-Brandwijk 1962). Those compounds are usually found in low concentrations in the plant, therefore making the direct extraction difficult and expensive. Additionally, successful procedures for its chemical synthesis have not been found, probably because of their complex structures. As such, the strategy of plant cell culture is attractive for the production of those metabolites. Plant cell culture has several advantages over the traditional cultivation, such as the control of the production conditions, weather independency, and continuous production. It is still necessary to overcome some difficulties such as low productivity and low specific growth rate (Sajc et al. 2000). In vitro culture technology has been proven to be effective in some cases for the production of secondary metabolites such as taxol (Oksman-Caldentey and Inzé 2004). However,
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little attention so far has been put on the development of in vitro cultures from Thevetia peruviana, and most of the studies have been focused on somatic embryogenesis rather than the production of secondary metabolites (Sen and Datta 1981; Kumar 1992; Sharma and Kumar 1994). The establishment of in vitro cell cultures, the cell growth and the accumulation of secondary metabolites can be affected by the illumination conditions (Hata et al. 2000; Chattopadhyay et al. 2002; Yu et al. 2005), the hormonal composition of the culture medium (Zhong et al. 1996; Pasqua et al. 2005) and the culture medium itself. Although the best culture conditions were determined previously, secondary metabolites are usually produced in low concentrations and in some cases not at all, for which it is necessary to develop strategies such as elicitation to stimulate the production of the desired component (Zhao et al. 2005). An elicitor is a compound (natural or synthetic) that can simulate a stress condition in the culture, triggering the same response in the cells that would be induced in the natural environment, usually resulting in the accumulation of secondary metabolites (Zhao et al. 2005; Vasconsuelo and Boland 2007). When studying the elicitation of in vitro cell cultures for the production of secondary metabolites, it is important to take into account three aspects that can affect it: the elicitor type, its concentration, and the time in which it is administrated to the culture (Vasconsuelo and Boland 2007). MeJ has been proved to be able to elicit the production of several compounds in many species (Ignatov et al. 1996; Rijhwani and Shaks 1998; Dong and Zhong 2002; Wang and Zhong 2002; Vasconsuelo and Boland 2007). Its broad effectiveness can be explained by the fact that this molecule acts as a secondary messenger in a wide spectrum of signaling pathways (Van der Fits and Memelink 2000; Zhao et al. 2005). In the present work, cell suspension cultures of T. peruviana were established in four different culture media to study their effect on cell growth. The cell growth dynamics as well as the eventual spontaneous production of extracellular peruvoside during the culture time were determined in Schenk–Hildebrandt (SH) medium. Finally, the effect of different concentrations of MeJ applied at different culture times was studied in cell suspension cultures of T. peruviana established in SH media.
Materials and Methods Plant material. The initial plant material for the establishment of callus cultures was obtained from the flesh of young fruit of plants grown on the campus of the Universidad Nacional de Colombia sede Medellín. The fruit was disinfected using ethanol (70%) for 3 min and
NaClO (10%) for 5 min. In between and after the disinfectants, the fruit was rinsed three times with distilled water. The fresh explants were extracted aseptically from the fruit in a laminar flow hood and finally sowed in SH medium. The conditions for the establishment of callus cultures are described below. Establishment of callus and cell suspension cultures. The established calluses were maintained in SH medium supplemented with 2 mg l−1 of 2,4-D, 0.5 mg l−1 of kinetin, 7 g l−1 of agar and sucrose at a concentration of 30 g l−1. The pH of the medium was adjusted to 5.8 before autoclaving at 121°C and 20 psi for 15 min. The cultures were maintained in a photoperiod of 12 h light:12 h darkness and 25°C. Subcultures were made every 3 wk. For the establishment of cell suspension cultures, inocula of approximately 10 g of friable callus were transferred to 50 ml of SH medium supplemented with 2 mg l−1 of 2,4-D and 0.5 mg l−1 of kinetin in 250 ml flasks. The pH of the medium was adjusted as described above. The cultures were maintained in an orbital shaker at 110 rpm under a photoperiod of 12 h light:12 h darkness and 25°C. Subcultures were made every 3 wks. Culture media. The effect of the culture media on the growth index (GI) of cell suspensions was studied using four different basal media with macro, micro, and vitamin compositions as described in the following references: MS (Murashige and Skoog 1962), half MS (MS media diluted in water, even the carbon source), B5 (Gamborg et al. 1976) and SH (Schenk and Hildebrandt 1972). In all the cases, sucrose was used as carbon source at a concentration of 30 gl−1. The pH was adjusted to 5.8 before autoclaving. The experiment was developed using an approximate inoculum of 5 g of cells in 20 ml of medium in 100 ml flasks. The rest of the culture conditions were maintained as described above. After 17 d the total weight was determined and the GI was calculated according to Eq. 1. The experiment was conducted in triplicate. Wf Wi GI ¼ ð1Þ Wi Wf is the final weight and Wi is the initial weight. To determine fresh weight, cells were harvested by centrifugation at 4,000 rpm for 5 min, the supernatant was carefully removed and the pellet was weighed. The morphological characteristics of the cells in suspension were observed using an optical microscope. Growth curve. To determine the growth kinetics, a cell growth curve was made. The experiment was developed in 100-ml flasks using an approximate inoculum of 5 g of cells in 20 ml of SH medium supplemented with 2 mg l−1
PERUVOSIDE PRODUCTION IN CELL SUSPENSION CULTURES OF T. PERUVIANA
of 2,4-D and 0.5 mg l−1 of kinetin. The rest of the culture conditions were maintained as described above. The cell growth was determined by measuring dry weight (DW) every 2 d for 22 d. DW was determined by harvesting individual flasks, the content was filtered through a filter paper and rinsed three times with distilled water; finally it was dried to constant weight in an oven at 60°C. The experiment was conducted in triplicate. Elicitation by MeJ. The cell suspensions used for this experiment were prepared using an approximate inoculum size of 2 g of cells in 20 ml of SH medium in 100-ml flasks under the same conditions described above. MeJ, provided by Sigma/Aldrich St. Louis, MO, USA, was prepared in ethanol (99%); concentrations of 0, 1, 10, and 100 mg l−1 of MeJ in the medium were evaluated. To determine the best time of addition of MeJ to the culture, three times were evaluated at 0, 7, and 14 d after the experiment was initiated. These times correspond to lag, exponential, and stationary phases of the culture, respectively. The experiment was concluded on the 21st day, and it was conducted in triplicate. Analysis of peruvoside production. The analysis of the extracellular peruvoside production was made every 2 d during the whole growth kinetics and at the end of the elicitation experiment. The samples were centrifuged at 4,000 rpm for 5 min, and then the supernatants were microfiltered using micro-filters of 0.2 μm. The measurements were made in triplicate. The micro-filtered samples were analyzed by HPLC using a LiChrospher® 100RP-18 (4×250 mm, 5 μm) column and the method described by Kyerematen et al. (1985). The production of peruvoside was determined by comparison with external standard of peruvoside provided by Sigma/Aldrich USA that appeared at a retention time of 8.862 min. Statistical analysis. All the data obtained were analyzed by the statistical program SAS 9.1 using a completely randomized design. When studying the effect of culture media, a one-way ANOVA (Montgomery 1997) was applied. In the case of the elicitation experiment, a factorial arrangement (4×3) was used; the factors evaluated were: MeJ concentration (0, 1, 10, and 100 mg l−1) and time of application (0, 7, 14 d).; The level of significance was 0.05, and differences among treatments were evaluated using the LSD test (Montgomery 1997).
Results and Discussion Selection of the adequate culture medium. After studying the effect of the culture medium composition on cell GI of
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cell suspension cultures of T. peruviana, we obtained the results shown in Table 1. The statistical analysis showed significant differences (p value
