Plant Cell Reports

Plant Cell Reports (1991) 10:85-89

9 Springer-Verlag1991

Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea M . Trypsteen 1, M . Van Lijsebettens 2, R. Van Severen 1, and M. Van Montagu z Laboratory of Pharmacognosy and Phytochemistry, State University of Ghent, Harelbekest:raat 72, B-9000 Gent, Belgium z Laboratory of Genetics, State University of Ghent, Ledeganckstraat 35, B-9000 Gent, Belgium Received November 26, 1990/Revised version received February 25, 1991 - Communicated by W. Bary S u m m a r y . Echinacea purpurea seedlings were inoculated with several Agrobacterium rhizogenes strains in order to obtain hairy roots. Infection with A. rhizogenes strains LMG63 and LMG150 resulted in callus formation. Upon infection with strains ATCC 15834 and R1601 hairy roots were obtained. Opine detection confirmed transformation of E. purpurea. Comparative H P L C fingerprint analysis o f the alkamides from natural plant source, control tissues, and transformed callus and roots indicated that transformed callus and hairy roots might be a promising source for continuous and standardized production of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and related amides.

Key words:Agrobactetqum rhizogenes/alkamides/Eehinaceapurpurea/ transformation Abbreviations: HPLC, high-pressure liquid chromatography; MS, Murashige and Skoog culture medium

are valuable plant products because of their pungent and local anaesthetic properties, insecticidal and higher cited biological activities (Greger 1984, 1988). Plant extracts are a major source for the purification of pharmaceuticals and fine chemicals. Alternative to field production of plants, in vitro culture techniques of plant cells and organs have been developed to obtain a more standardized way for secondary metabolite production (Hamill et al. 1987; Flores et al. 1987). A promising in vitro source are the genetically transformed "hairy" roots produced by plant tissue after infection with the soil bacterium Agrobacterium rhizogenes (for review, see Gelvin 1990). Recently fast-growing hairy root cultures were established to use as a continuous source for a standardized production of valuable secondary metabolites in several plant species (Signs and Flores 1990). This paper reports on transformation of Echinacea purpurea by Agrobacterium rhizogenes and establishment of hairy root and callus lines. The profile of the different alkamides in control tissues, transformed calli, and hairy roots is compared through H P L C fingerprint analysis.

Introduction

Materials and methods

Echinacea purpurea (Asteraceae) root extracts and tinctures are frequently used in pharmacy and medicine for their immuno-modulating properties (Hahn and Mayer 1984; Bauer and Wagner 1988; Hudson and Towers 1988). Besides high-molecular-weight compounds such as polysaccharides and glycoproteins, low-molecular-weight components such as alkamides and cichoric acid derivatives contribute to the cited biological activity (Bauer and Wagner 1988). Furthermore the dodeca-2E,4E, 8Z, 10E/Ztetraenoie acid isobutylamide is an effective inhibitor of the lipoxygenase enzyme (Wagner 1989). Alkamides, commonly present in plant species belonging to the tribes Anthemideae and Heliantheae o f the Asteraceae family

Bacterial Strains and Plant Material. Agrobacterium rhizogenes mannopine-type strains LMG63 and LMG150 were obtained from the Laboratory of Microbiology (P,ijksuniversiteit Gent, Belgium). Agropine-typestrains ATCC 15834and R1601 were kindlyprovided by Plant Genetic Systems N.V. (Gent, Belgium) and M. Gordon, State University of Washington (Seattle, USA), respectively. R1601 contains a pRiA4-derived plasmid (Pythoud et al. 1987). All strains were grown at 28~ on solid YEB medium (Vervliet et al. 1975). Strain RI601 was grown on the same medium containing 100/zg/ml kanamycin and 100 #g/ml ampicillin. Seeds orE. put~ourea were obtained from Biohorma (Elburg, The Netherlands). After immersingin alcohol for 30 seconds, the seeds were surface-sterilized for 15 minutes in a solution containing 5.0% NaOCI and O.1% Tween 20. Seeds were rinsed 5 times in sterile distilled water, blotted dry, and aseptically germinated on solid B5 medium without hormones (Gamborg et al. 1968) at 24~ 160 #mol/m2/see

Offprint requests to: M. Van Lijsebettens

86 white light intensity with a 16-hour day/8-hour night rhythm. The same growth conditions were used for tissue culture procedures.

Inoculation and ~ssue Culture. Infections were done on etiolated seedlings germinated for 10 to 14 days in the dark. Hypocotyls were inoculated several times with a sterile needle which had been touched to the surface o f a fresh Agrobacterium culture. Control plantlets were wounded with a sterile needle. The hypocotyl bases of the inoculated seedlings were placed on hormone-free B5 medium containing 1% (w/v) agar and grown in the dark. Callus produced at the infected sites was excised and placed on solid MS medium (Murashige and Skoog 1962) containing300 #g/mlcefotaximum(Claforan| #g/ml 6-benzylaminopurine and grown at low light intensity. After ten weeks of culture, it was necessary to add naphthalene acetic acid at 0.05 #g/ml concentration to maintain further growth. Hairy root tips produced at the infeeted wound sites were excised and placed on solid hormone-free MS medium containing 300 #g/ml cefotaximum and kept in the dark. For roots produced upon infection with strain R1601 kanamycin sulphate was added to the medium in a concentration of 50/zg/lnl. All explants were subcultured every 14 days and after 6 subcultures cefotaximum was omitted.

Opine Detection. Opines (mannopine and agropine) in bacteria-free transformed callus and hairy roots were extracted and anaiyzed by paper electrophoresis and silver staining according to the method o f Petit et al. (1983). After hot-acid extraction of callus tissue (500 mg fresh weight) and hairy roots (100 mg fresh weight), 5 to i0 #1 of the extracts were spotted on Whatman 3MM paper and electrophoresed at 15 V/em for 30 minutes in a buffer o f formic acid, acetic acid, distilled water (3:6:91, v/v/v;pH 1.9). After drying overnight, the electrophoretograms were stained with silver nitrate (Trevelyan et al. 1950), fixed with a sodium thiosulfate solution (10%, v:v in water) and washed with tap water. Non-transformed tissues were treated in parallel as controls. Analysis o f Alkamides. Bacteria-free callus and hairy roots were taken for alkamide extraction 6 to 8 months after initiation. They were harvested at the end of a 14-day subculture period, in the late log phase o f their growth curve. Control roots were taken from 4-year-old soil-grown plants. Callus tissue ( 1 - 2 g ) and hairy roots (100-500 mg) were freeze-dried, powdered, and exhaustively extracted by shaking with

n-hexane; 1% (v:v) and 10 % (v:v) methanol, respectively, was added to facilitate the extraction. After filtration solvents were evaporated to dryness in a Buchi rotavapor at 40~ and the residue was dissolved in 1 ml ethanol. HPLC analysis of the hexane extracts was performed with an ISCO HPLC equipment (HPLC pump model 2350, gradient programmer model 2360, V4 UV-detector, controlled by the ISCO Chemresearch data management system version 2.4). Five to ten microliters of each extract was injected into a Rosil C18 HL (RSL-ALLTECH; Eke, Belgium) column (3 #m, 15 cm x 4.6 m internal diameter) and eluted with an acetonitrile:water gradient (40-80% acetonltrile, linear, 30 minutes). The flow rate was 1 .O ml/minute and the detection wavelength was 254 nm as described by Bauer and Remiger (1989). UV spectra were recorded with a diode array spectrophotometer (HP 8452A) coupled on-line.

Results and discussion One month after bacterial infection two different responses could be observed at the Echinacea hypocotyls depending on the strain used. Inoculation with Agrobacterium rhizogenes strains LMG63 and LMG150 resulted in green, compact, tumor-like callus formation at the wound sites (Fig. 1A). This callus was made bacteria-free and maintained in tissue culture as described in Materials and methods. Strains ATCC 15834 and R1601 induced slight callus formation from which hairy roots emerged (Fig. 1B). These roots continued to grow when excised and transferred to MS medium without hormones (Materials and methods) in contrast to control roots that did not proliferate under these tissue culture conditions (Figs. 1C and 1D). The putative transformed "hairy" roots did not produce an excessive number of root hairs but they did show the typical morphological characteristics such as fast growth, branching, and negative geotropism. Some of these roots produced callus and shoots spontaneously (Fig. 1E). Wounded sites of control seedlings did not produce any callus or root growth.

Fig. 1. E. purpurea response toA. rhizogenes infection. A. Tumorous callus growth on etlolated hypocotyI upon infection with LMG63 or LMG150. B. Hairy root formation on etiolated hypocotyl upon infection with ATCC 15834 or R1601. C. Hairy root growth in tissue culture on hormone-free MS medium; arrows indicate the start point from which new root tissue is formed after a 2-week subculture period. D. Control roots do not grow under the same conditions. E. Spontaneous callus and shoot formation on some transformed roots.

87 Table 1. EfficiencyofA. rhizogenes infection on E. purpurea seedlings Bacterial straina

Efficiene~ of infection"

Type of responsec callus roots

LMG63 LMG150 ATCC 15834 R1601 -

30/35 18/25 12/30 6/15 0/20

+++ ++ + + -

+++ +++ -

a See Materials and methods; b number of seedlings with response upon infection on total of infected seedlings; c - , no response; +, + +, + + +, increasing response intensity.

Information on response frequencies of infected seedlings is given in Table 1. Upon infection with strains LMG63 and L M G 1 5 0 at least 70% o f the inoculated sites produced callus. Infection with strains ATCC 15834 and R1601 led to the formation of hairy roots in 40% o f the cases. Successful infection responses were only obtained on hypocotyls o f young, etiolated seedlings, no response was obtained if older plants were used. In many plant species it is well known that the type and age o f the explant is critical for good response to Agrobacterium infections (Rech et al. 1989). To confirm that the callus and root proliferations were genetically and stably transformed, the presence o f opines was measured in these tissues. Opine synthesis is encoded by the T R - D N A , a piece of the bacterial Ri plasmid that is transferred to and integrated into the plant cell genome during Agrobacterium infection (Willmitzer et al. 1982). These compounds are not synthesized in uninfected plant tissues. Measurements were done on bacteria-free calli and roots, excised from the inoculation sites and cultured for several months in tissue culture (Figs. 2A and 2B; Materials and methods). Figure 2A shows that the L M G 6 3 - and LMG150-induced callus lines examined contained mannopine. This compound was absent in control callus. A T C C 15834- and R1601-induced hairy roots contained mannopine and agropine as expected; these were absent in control root extracts (Fig. 2B). From this analysis it is clear that we succeeded in A. rhizogenes transformation o f E. purpurea, hereby extending the list o f medicinally important species o f the Asteraceae family that are susceptible to A. rhizogenes infection (Mugnier 1988; Signs and Flores 1990). The H P L C alkamide pattern was determined in control and transformed callus and root lines as explained in Materials and methods and is presented in Figures 3 and 4. The major alkamides in E. purpurea root extracts identified through H P L C by Bauer and Remiger (1989) were further characterized using UV, NMR, and MS spectra (Bauer et al. 1988a); they showed that additional UV spectra recorded on-line during H P L C analysis were sufficient to distinguish the various structural types of the

Fig. 2. A. Detection of mannopine in bacteria-free transformed callus extracts. NS, neutral sugars; lane 1, mannopine standard (Man); lane 2, non-transformed control callus; lanes 3 and 4, LMG63-transformed callus tissue; lane 5, LMG150-transformed callus tissue. B. Detection ofagropine in bacteria-free hairy root extracts. NS, neutral sugars; lane 1, agropine standard (Aglr); lanes 2 and 3, ATCC 15834-transformed hairy roots; lane 4, R1601-transformed hairy roots; lane 5, nontransformed control roots.

major alkamides. Previously, we have isolated and identified the major alkamides represented by peaks A, C, E, H, and I (Figs. 3 and 4) (Trypsteen et al. 1989). These were used as reference compounds in pilot TLC and H P L C analysis to determine their presence in extracts of transformed and control calli or roots in this study. Retention times and on-line recorded UV spectra were used to identify the main isobutylamides. The isobutylamide pattern found in transformed and control callus extracts was similar to the one o f the standard root extract (Fig. 3). However, the relative intensity of the peaks differed and some additional unidentified peaks (X) could be observed. The dodeca-2E,4E, 8 Z, 10E/Z-tetraenoic acid isobutylamide concentration, the major compound in all extracts, was much higher in callus lines than in control

88

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roots (Fig. 3, peak H). Its concentration in natural roots was 0.03% + 0.01 (mg/100 mg dry weight) comparable to the values determined by Bauer and Remiger (1989). In contrast, control calli contained 0.19% _+0.01 and transformed calli 0.36 % + 0.02 of the dodeca-2E,4E, 8Z, 10E/Z-tetraenoic acid isobutylamide (Fig. 3) which indicates a significantly higher production of this compound in callus tissue. The HPLC fingerprints of transformed and nontransformed root extracts were similar; slightly different relative peak intensities could be noticed (Fig. 4). However, the amount of the dodeca2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamide was approximately the same in transformed as in control roots, 0.028% + 0.005 and0.03% ___ 0.01, respectively.

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Fig. 3. HPLC pattern of alkamides in hexane extracts of E. purpurea callus lines. (1) Transformed callus. (2) Untransformed callus. (3) Control root given as standard. HPLC was performed as explained in Materials and methods. Peak intensities are comparable among each pattern. A, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide; B, undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide; C, dodeca2E,4Z-diene-8,10-diynoic acid isobutylamide; D, undeca-2E,4Zdiene-8,10-diynoic acid 2-methylbutylamide; E, dodeca-2E,4E,10Etrien-8-ynoic acid isobutylamide; F, trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide; G, dodeca-2E,4Z-diene-8,10-diynoie acid-2-methylbutylamide; H-I, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide; J, dodeca-2E,4E,SZ-trienoic acid isobutylamide; K, dodeca-2E,4E-dienoic acid isobutylamide; X, unidentified compound.

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Fig. 4. HPLC pattern of alkamides in extracts ofE. purpurea root lines. (1) Transformed hairy root. (2) Control root. Peak intensities are comparable between the two analyses. Nomination of the peaks as in Figure 3.

89 In this study we established conditions for A. rhizogenes infection on sterile E. purpurea plantlets and for subsequent aseptic tissue culture of transformed callus and hairy root lines. This is an important step to make this species accessible for further biochemical and molecular research. Furthermore, the data showed that transformed callus and hairy root lines could be promising sources for a constant and standardized production of the dodeca-2E,4E,SZ,10E/Z-tetraenoic acid isobutylamide and related alkamides. These compounds are gaining interest in the development of new drugs to treat rheumatic diseases or to treat diseases where stimulation of the unspecific enhancement of the immune system is desired (Bauer et al. 1988b; Wagner 1989). Further investigations have to be carried out to improve the production to commercially interesting levels. Acknowledgements. We thank M. De Cock, K. Spruyt, S. Van Gijsegem, and V. Vermaercke for help in preparing the manuscript. MT and RVS are grateful to Biohorma (The Netherlands) for supplying seeds orE. purpurea. Purified agropine was kindly provided by J. Tempg (Gif sur Yvette, France).

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