In Vitro Cell.Dev.Biol.—Plant (2012) 48:249–258 DOI 10.1007/s11627-012-9435-2
PLANT TISSUE CULTURE
Clonal propagation and production of cichoric acid in three species of Echinaceae Anca-Livia Butiuc-Keul & Laurian Vlase & Cornelia Crăciunaş
Received: 28 July 2011 / Accepted: 6 March 2012 / Published online: 23 March 2012 / Editor: J. Forster # The Society for In Vitro Biology 2012
Abstract In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N6-benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39– 0.73 mg/g dw) were observed in shoots and leaves.
A.-L. Butiuc-Keul (*) : C. Crăciunaş Department of Molecular Biology and Biotechnology, Babeş-Bolyai University, 400084, Cluj-Napoca, Romania e-mail:
[email protected] L. Vlase Faculty of Pharmacy, Department of Pharmaceutical Technology and Biopharmaceutics, University of Medicine and Pharmacy “Iuliu Hatieganu”, 400023, Cluj-Napoca, Romania
Keywords Echinacea . Multiplication . Secondary metabolites . Cichoric acid . Medicinal plant
Introduction Plant species have provided important medicinal remedies for thousands of years. The rapid growth of the marketplace for plant-based health products has been associated with an increased variety of medicinal plant preparations and with a continuous broadening of the categories driven by consumer interest in such products. Even today, the World Health Organization estimates that up to 80 % of people still mainly rely on traditional remedies such as herbs for their medicines (Baum et al. 2006). During the 1990s, popularity of the genus Echinacea Moench (Asteraceae family) as a dietary supplement increased markedly, as the general public learned of its possible efficacy for fighting colds and other illnesses (Baum et al. 2006). Echinacea, also referred to as purple coneflower, is geographically confined to North America and is distributed in dry prairies from Texas to Saskatchewan and from West of the Rocky mountains to Minnesota (Binns et al. 2004). The major producers of Echinacea in Europe are in Germany, Switzerland, The Netherlands, Italy, and Spain (Galambosi 2004). In 1998, Echinacea was the tenth most important medicinal plant sold in Europe with annual sales of about $120 USD million. In North America, Echinacea ranked second in sales in the mainstream market in 2004. Retail sales of Echinacea products are worth more than $158 million annually in the USA, and worldwide, they have been estimated at $1,300 million annually (Blumenthal 2005). Three species of Echinacea are generally used in medicinal plant preparations (Perry et al. 2001): Echinacea purpurea Moench (roots and shoots), Echinacea angustifolia DC (roots), and Echinacea pallida Nutt (roots). Echinacea
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Materials and Methods
E. angustifolia and were immersed in 70 % (v/v) ethanol for 30 s and 15 min immersion in 50 % (v/v) Domestos® (sodium hypochlorite at 5 % v/v). Seeds were collected from field plants from a single batch for each species and were provided by Alexandru Borza Botanical Garden, ClujNapoca, Romania. After sterilization with Domestos solution, seeds were washed three times (5 min) with sterile, distilled water prior to placing on medium consisting of half-strength Murashige and Skoog (MS) salts (Murashige and Skoog 1962), 0.1 g/l myo-inositol, 30 g/l sucrose, and agar 0.8 % w/v (micro-agar, Duchefa, Haarlem, The Netherlands). Culture media were sterilized by autoclaving at 121°C and 103 kPa (1.1 kg cm−2) for 20 min after adjustment of the pH to 5.8 with 1 N KOH. Cultivation was done in 100-ml glass jars containing 15 ml of medium. Cultures were grown for 30 d at constant 22°C, at photoperiod regime of 16 h light (cool-white fluorescent lights, 50 μmol s−1 m−2 photosynthetic photon flux density). Seedlings were placed on MS medium, and after 30 d, 1 cm microcuttings were excised and cultured on MS medium supplemented 1.0 mg/l N6-benzylaminopurine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA) in order to obtain plant material for further experiments. In vitro culture of Echinacea shoots was previously achieved on MS medium supplemented with 1.0 mg/l indole-acetic acid (IAA) and 1.0– 5.0 mg/l BA (Taha et al. 2009). In order to improve the multiplication rate of these species of Echinacea, in our experiment nodal explants of 1 cm obtained on MS medium supplemented with 1.0 mg/l BA and 0.1 mg/l NAA were cultured on MS medium supplemented with indole-3-butyric acid (IBA), NAA, 2-iso-pentenyladenine (2iP), or BA in different concentrations as follows: MS1— without growth regulators; MS2—MS+0.1 mg/l BA+0.1 mg/l NAA; MS3—MS + 1.0 mg/l BA + 0.1 mg/l NAA; MS4—MS + 0.1 mg/l 2iP + 0.1 mg/l IBA; MS5—MS + 1.0 mg/l 2iP+1.0 mg/l IBA; and MS6—MS+2.0 mg/l 2iP+ 0.1 mg/l IBA. Leaf and shoot explants were also placed on MS medium supplemented with 2.0 mg/l 2,4 dichlorophenoxyacetic acid (2,4-D)+0.1 mg/l BA, for callus induction. Cultures were evaluated after 30 d of culture. The number of shoots and roots/explant and shoot length were measured. Ten replicates were used per treatment, and the experiments were repeated twice. After 30 d, the shoots from all 10 replicates grown on MS4 medium (which proved to be superior for multiplication) were collected, and fresh weight (fw) was recorded. Plants were dried at room temperature, and after 1 wk, the dry weight (dw) was also measured. Similar biomass calculations were performed for callus. One-way analysis of variance (ANOVA) and Tukey tests were used to confirm differences between means. MiniTab software was used for statistical analysis.
Plant material and in vitro culture. In vitro cultures were initiated from seeds of E. purpurea, E. pallida, and
RAPD analysis. Genomic DNA was isolated from leaves using the cetyl trimethylammonium bromide method as
species have been increasingly studied for different applications in biotechnology, especially in vitro culture and preparation of natural extracts (Abbasi et al. 2007). Plant and medical scientists responded to this demand by concentrating their efforts to understand the biology, cultivation, and pharmacology of these plants. Echinacea species, especially E. purpurea contain a variety of chemical compounds (216 different medicinally active compounds) that are structurally diverse (for example, cichoric acid, alkylamides, echinacoside, cinarine, and polysaccharides) and perform multiple activities that are beneficial for health (Murch et al. 2006). Known phenolic compounds in Echinacea species include caffeic acid derivatives such as cichoric acid in E. purpurea and E. pallida, and echinacoside in E. angustifolia (Harborne and Williams 2004). Cichoric acid is the main active compound of Echinacea extract. It is known that natural extracts of Echinacea have significant immunomodulatory, antibacterial, antiviral, antifungal properties (Barrett 2003), and also antioxidant activities (Thygesen et al. 2007). Commercial production of Echinacea has been limited by a range of issues including contamination of plant materials by microorganisms, pollution from the environment, variability of active components, and lack of pure, standardized plant material for biochemical analyses (Raman et al. 2004). To address these issues, in vitro tissue culture techniques for Echinacea represent a desirable alternative to harvesting plants from natural or cultivated habitats. Echinacea species have been regenerated from a range of tissue types varying from in vitro seedlings to mature field-grown plants, with a number of studies having described the biomass production by in vitro culture of Echinacea species (Lata et al. 2004). In addition, several studies have focused on the isolation and characterization of different classes of compounds responsible for the multiple activities of Echinacea extracts (Currier and Miller 2000; Wills and Stuart 2000; Merali et al. 2003). In the present study, an improved method is reported for the in vitro multiplication of E. purpurea, E. pallida, and E. angustifolia. This study is part of a broader research plan aimed at development of alternative methods (such as in vitro culture) for harvesting of plants from natural and cultivated habitats (Butiuc-Keul and Deliu 2001; Butiuc-Keul et al. 2002). In addition to the in vitro multiplication of the three Echinacea species, the amount of cichoric acid in regenerated plantlets and callus tissue was quantified and randomly amplified polymorphic DNA (RAPD) markers were used to assess the genetic variability of the in vitro plantlets and to identify potential somaclonal variants.
CLONAL PROPAGATION AND PRODUCTION OF CICHORIC ACID
described by Doyle and Doyle (1987). DNA mplification by PCR was performed using 10 nucleotides primers (Operon Technologies) and the specific conditions that were previously established in our laboratory (Crăciunaş et al. 2007). Reactions were performed in 0.2-ml Eppendorf tubes containing 2 mM MgCl2, 1 μM of each primer, 200 μM of each dNTP, 1.5 U of Taq (Fermentas), and 25 ng of genomic DNA in a final volume of 25 μl. Steps of the thermocycling reaction conditions were as follows: (1) T095°C for 5 min, (2) T094°C for 45 s, (3) primer annealing at 36°C for 45 s, (4) elongation T072°C for 1.5 min; steps 2–4 were repeated 35 times. A total of 28 random primers were tested, but data are only presented for six primers which generated the best results (Table 1). Amplicons were separated on 1.5 % (w/v) agarose gels and stained with 0.5 μg/ml ethidium bromide. Quantification of cichoric acid. Analysis of cichoric acid from Echinacea extracts was performed using a newly developed procedure of liquid chromatography coupled with mass spectrometry detection. This method provided a highthroughput determination, having a major advantage of rapid analysis (m/z 293+m/z 311, which is specific to cichoric acid. The retention time of cichoric acid was 0.51 min. The concentration of cichoric acid was determined automatically by the instrument data system (QuantAnalysis 1.7 software, Brucker Daltonics, Darmstadt, Germany) using peak area and the external standard method. The calibration curve model was determined by the linear regression, using an 1/Y2 weighting scheme. The lower limit of quantification was established at 0.75 μg/ml. At quantification limit, the method precision (expressed as coefficient of variation, CV %) was 6.8 %, and accuracy (expressed as relative difference between obtained and theoretical concentration, bias %) was 5.3 %, respectively.
Results Shoot induction and multiplication. In vitro cultures were established by aseptic germination of seeds on half-strength MS medium. The rate of seed germination was 80–90 % (data not shown). Previous studies revealed a high level of infection of seeds with microorganisms (Mechanda et al. 2003), and several researchers have proposed removal of the seed coat layers to prevent contamination (Harbage 2001). However, a sequential process of surface sterilization proved to be highly efficient in ensuring visually contamination-free seed germination. Surface sterilization of seeds was also accomplished by other authors (Taha et al. 2009). Based on ANOVA analysis and the Tukey test, culture media was shown to significantly influence both shoot multiplication and root induction rates in all three species of Echinacea. The best media for shoot multiplication were MS4 for E. purpurea, for which on average 7.1 shoots/nodal explant and 4.6 roots/nodal explant were obtained, and MS5 for E. pallida (5.4 shoots/nodal explant) and E. angustifolia (3.9 shoots/nodal explant). The highest numbers of shoots/ nodal explant were obtained in E. purpurea, then E. pallida, and E. angustifolia. MS4 medium was also superior for root induction in E. purpurea (4.6 roots/nodal explant) and E. pallida (4.5 roots/nodal explant). In E. angustifolia, the
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Table 2. Influence of culture medium on shoot multiplication and elongation of the three species of Echinacea after 30 d Species
Culture medium
E. purpurea
MS1 MS2
1.4±0.52 a 1.5±0.53 a
MS3 MS4 MS5 MS6
2.6±0.7 b
2.5±0.14 d
2.8±0.92 g
1.5±0.18 i
MS1 MS2
1.0±0 a 1.3±0.48 a
3.4±0.59 f 3.6±0.38 f
1.7±0.67 g 2.3±0.82 g
1.9±0.27 jk 1.6±0.4 i, j
MS3
2.5±0.71 b
2.5±0.32 de
2.4±0.7 g
1.4±0.18 i
MS4 MS5
4.8±0.79 c 5.4±0.84 c
2.7±0.32 e 2.1±0.23 d
4.5±1.08 h 1.7±0.67 g
2.4±0.15 l 2.2±0.23 kl
MS6
3.2±0.63 b
2.2±0.26 d
2.3±0.82 g
1.3±0.21 i
MS1 MS2
1.0±0 a 1.4±0.52 b
3.9±0.5 h 3.4±0.25 g
3.3±0.82 ij 3.8±0.79 j
3.8±0.29 o 3.3±0.2 n
MS3
3.6±0.7 c
3.2±0.22 fg
3.3±0.67 ij
1.9±0.16 k
MS4 MS5
3.7±0.67 c 3.9±0.74 c
2.9±0.28 ef 2.6±0.23 d e
3.3±0.67 ij 2.7±0.82 i
2.6±0.18 m 3.4±0.18 n
MS6
3.8±0.79 c
2.4±0.19 d
2.6±0.7 i
2.3±0.24 l
E. pallida
E. angustifolia
Number of plantlets/explant
Length of plantlets (cm)
Number of roots/explant
Length of roots (cm)
4.1±0.54 e 4.2±0.57 e
1.9±0.57 fg 2.2±0.79 fg
2.0±0.23 j 2.0±0.28 j
3.1±0.88 b
2.6±0.29 d
1.7±0.67 f
1.4±0.18 i
7.1±0.99 c 2.6±0.7 b
2.5±0.17 d 2.4±0.17 d
4.6±0.84 h 2.2±0.92 fg
2.4±0.16 k 1.3±0.16 i
Nodal explants used for this experiment were generated from shoots produced on the same respective media (1.0 mg/l BA+0.1 mg/l NAA). Values represent means±SD, n010. Values with different letters within a column are significantly different (p
