Plant Cell, Tissue and Organ Culture 62: 11–19, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens B. Massot, S. Milesi, E. Gontier, F. Bourgaud∗ & A. Guckert Laboratoire Agronomie Environnement ENSAIA-INRA, 2 Avenue de la Forêt de Haye, 54500 Vandoeuvre les Nancy, France (∗ requests for offprints; E-mail:
[email protected]) Received 12 October 1999; accepted in revised form 20 June 2000
Key words: psoralen, Ruta graveolens, Rutaceae, secondary metabolism, shoot culture
Abstract Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l−1 of NaH2 PO4 ), nitrate (2527 mg l−1 of KNO3 ), carbon source (10 g l−1 of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 µM and benzylaminopurine (BAP) 50 µM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (tD equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems.
Introduction Psoralen and its derivatives: 5-methoxypsoralen (bergapten), 8-methoxypsoralen (xanthotoxin), 5,8dimethoxypsoralen (isopimpinellin) belong to the furanocoumarins family. They are currently used for the treatment of skin deseases (i.e. vitiligo and psoriasis) and against several cancers (Edelson, 1988; TiolyBensoussan et al., 1988). They are also studied with neurological affections like multiple sclerosis (Koppenhöfer, 1995). Nowadays, pharmaceutical industry uses bergapten as a side product from Citrus bergamia (bergamot tree) essential oil. However this culture is declining and other sources need to be found. Plants belonging to the Rutaceae family are possible candidates for the production of such metabolites (Mc Cloud et al., 1992). In Rutaceae, these compounds are involved as phytoalexins (Aliotta et al., 1994). Investigators report the amount and the localization of these molecules in entire plants. The level of 720 µg g−1
FW of furanocoumarins was quantified by Zobel and Brown (1991) in entire plants of Ruta graveolens L. They also noted that furanocoumarins were localized on the leaf surface and in mature fruits. This large interest for furanocoumarins led us to investigate the possibility of a production by in vitro cultures of Ruta graveolens. Several types of plant material can be used to produce furanocoumarins with Ruta graveolens. The first objective of this work was to determine the most interesting plant material for furanocoumarin production. In a second step, different factors were studied with the objective to optimize both biomass production and furanocoumarin concentration in plant tissues. Various nitrate (NO3 − ), phosphate (PO4 3− ) and carbon sources (sugars) concentrations as well as different phytohormone balances were tested. The effect of light was also investigated by comparison with cultures under dark conditions. Experiments using autoclaved Verticillium dahliae were conducted with the objective to elicitate furanocoumarin synthesis.
12 A concluding experiment compared furanocoumarin production with shoots cultivated in optimized medium (result of the present studies) and standard B5 medium.
Materials and methods Plant material cultures Entire plant Leaves were harvested from Ruta graveolens fieldgrown plants (seeds obtained from Etablissement Bertrand, Orléans, France) for furanocoumarin assessment in entire plants. Standard conditions for micropropagated shoot culture Micropropagated shoots were obtained from sterile seeds of Ruta graveolens. They were maintained in standard Gamborg’s B5 (Gamborg et al., 1968) medium supplemented with 3% sucrose (w/v), kinetin (9 µM) and 2,4-D (9 µM). The 250 ml flasks were placed on an orbital shaker at 100 rpm. The culture took place in phytotron at 24 ◦ C. The light intensity was 145 µmol m−2 s−1 . For experiments, each flask contained 100 ml of culture medium. Callus establishment Calli of Ruta graveolens were obtained from leaves of entire plants cultivated in greenhouses. They were cultivated on semi solid Gamborg’s medium supplemented with 3% sucrose (w/v) and 2,4-D (9 µM). Semi solid medium contained 0.3% (w/v) Phytagel (Sigma – St Louis, USA). Other culture conditions were the same as applied for shoot cultures in B5 standard medium.
flask. The same culture medium was asepticly refilled in the flask after the measurement. Dry weight was measured after freeze-drying for 48 hours. Extraction and high performance liquid chromatography (HPLC) analysis of furanocoumarin compounds Micropropagated shoots were removed from flasks, frozen at −30 ◦ C for 24 h, freeze-dried and ground. For each sample, 10 to 20 mg of powder was extracted using one ml of solvent (50/50 EtOH/H2 O). After 30 minutes, the extract was centrifuged 5 min at 1200 rpm. This extraction method is derived from studies led on Psoralea cinerea (Bourgaud et al., 1994). The supernatant was filtered (0.45 µM) and analyzed by HPLC. The column used was a Nucleosil 100 C18 reversed phase – 125∗4 mm (Merck, Darmstadt, Germany) and the mobile phase was composed of acetonitrile, methanol and water. The gradient used for a run was: acetonitrile/methanol/water 30/30/40 (v/v/v) to 50/50/0 (v/v/v) in 30 min with a concave curve. Furanocoumarins were determined by EI-MS and MS/MS methods (Bourgaud et al., 1994) by comparison with authentic spectra published by Drewes (1974). Routine identification was made with a diode array detector working between 190 and 400 nm by comparison with authentic samples. Psoralen, bergapten and isopimpinellin were purchased from Extrasynthese (Lyon, France). Xanthotoxin was purchased from Sigma (St Louis, USA). Carbon sources analysis The three sugars present in culture medium were analyzed using an enzymatic method from Boehringer Mannheim (Ref. 716260). Experiments
Analytical methods
Phosphate and nitrate experiments Four concentrations of KNO3 (N1=632, N2=1264, N3=2527 and N4=5055 mg l−1 ) and four concentrations of NaH2 PO4 (P1=37.5, P2=75, P3=150 and P4=300 mg l−1 ) were used with Gamborg’s medium. N3 and P3 are the concentrations of standard B5 culture medium. N1, N2, N4 and P1, P2, P4 are multiple of N3 and P3 respectively.
Biomass analysis Fresh weight for each treatment was measured after aseptically draining the culture medium out of the
Phytohormone experiments Eight culture media were prepared with Gamborg’s medium supplemented with sucrose 3% (w/v) and
Cell suspension culture Cell suspensions were obtained from calli and cultured on Gamborg’s B5 medium supplemented with 3% sucrose (w/v) and 2,4-D (9 µM). Other culture conditions were the same as applied for shoot cultures in B5 standard medium.
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Figure 1. Effect of cellular organization level on furanocoumarins production.
phytohormones: Control (without hormone), D50 (50 µM of 2,4-D), B50 (50 µM of BAP), K50 (50 µM of kinetin), DB50 (50 µM of 2,4-D and 50 µM of BAP) and KD50 (50 µM of 2,4-D and 50 µM of kinetin). 2,4-D was purchased from Fluka Chemie AG (Switzerland), kinetin and BAP from Sigma (St Louis, USA). Carbon source experiments Micropropagated shoots were cultivated on Gamborg’s B5 medium supplemented with kinetin (2 mg l−1 ) and 2,4-D (2 mg l−1 ). Three sugars (sucrose, glucose and fructose) were tested at the concentration of 30 g l−1 and the biomass productions were compared after 26 days. A second assay was led with sucrose. The effects of four concentrations (5, 10, 30 and 50 g l−1 ) on shoot growth and furanocoumarin production were studied during a growth cycle over 26 days period (biomass measurement every 3 days). For the two assays the other culture conditions were the same as applied for standard medium shoot culture. Elicitation assay Fungus culture. Verticillium dahliae culture was initiated on Potato Dextrose Agar medium (DIFCO Laboratories). Elicitor preparation and elicitor experiments. Elicitor homogenization was performed with 7 days-old Verticillium dahliae cultures (6.2 g in 100 ml of ultra
pure water). This solution was autoclaved (120 ◦ C, 1 bars for 20 min) and added to micropropagation medium (2 ml per flask) after 14 days of culture. Effect of light Shoot cultures were grown on Gamborg’s medium supplemented with sucrose 3% (w/v), 2,4-D (2 mg l−1 ) and kinetin (2 mg l−1 ). One treatment was cultivated on darkness and another under light (145 µmol m−2 s−1 ).
Results and discussions Choice of in vitro plant material Four types of plant materials (entire plants, micropropagated shoots, calli and cell suspensions) were studied. Different cellular organizations were represented in this experiment. The undifferentiated cells corresponded to cell suspensions and calli. Differentiated tissues consisted of micropropagated shoots and entire plants. The concentrations of four linear furanocoumarins (psoralen, 5-methoxypsoralen, 8-methoxypsoralen and 5,8-dimethoxypsoralen) were studied (Figure 1). For the four molecules, significantly higher concentrations were found in micropropagated shoots of Ruta graveolens (ANOVA, p
