In Vitro Culture of Echinacea Species
Plant tissue culture is the process of producing callus tissue, plant organs, or intact plants using a small piece of a donor plant or even a single cell. This process consists of using an artificial medium and exogenous growth hormones. Successful plant tissue culture is based on the cell theory of Schleiden (1838) and Schwann (1839), and the concept of totipotency (a plant cell is capable of regenerating a whole plant). The portion of plant material used is referred to as the “explant.” The explant may be from any portion of a plant (stem, leaf, nodal segment, etc.). Because this method is essentially a closed system, one may manipulate conditions for specific morphological attributes as well as for an increased yield of a particular chemical constituent. Mass production of clonal plants from a single highly desired plant is often accomplished through in vitro culture. In vitro culture may be used to obtain virus-free plant material by excising only the meristematic dome of a contaminated plant and using it as an explant source. In vitro systems may be used as a model to study various metabolic functions. In addition, transformation experiments are often accomplished in in vitro systems. For example, when products are known to be produced in roots, as is the case with Echinacea spp., hairy root cultures may be initiated using Agrobacterium rhizogenes as the vector.
Advantages of an in vitro system for medicinal plants include: (1) year-round availability of plant materials for extraction of pharmaceuticals produced under controlled conditions; (2) potential regulation of metabolic pathways from which active ingredients or marker compounds are derived; (3) potential genetic modification of cells/tissues to produce specific intermediates or metabolites (); and (4) mass micropropagation of desired plants. Early attempts to produce secondary metabolites were reported in 1960; however, yields were generally low. At times, the secondary product of interest is sequestered in a specific organelle and may not be produced in in vitro cultures. Recent information about signaling mechanisms in plants and the potential control of metabolite production — that is, polysaccharide elicitor from Pseudomonas sp. induced higher levels of rosmarinic acid in oregano shoots callus, elicitor induction of sesequiterpene production in tobacco, and Hyocayamus muticus cell culture — poses potentially interesting possibilities for manipulation of cell cultures for the production of pharmaceutical compounds.
Regeneration of Echinacea Purpurea
Establishing protocols for micropropagation and callus production of Echinacea purpurea L. Moench (purple coneflower) may be a way to recover some of the endangered and overharvested species of Echinacea, as well as a method for providing plant materials for extraction of medicinally important compounds. Regeneration of E. purpurea has been obtained from different explants. Choffe et al. (2000) developed a method for inducing root organogenesis from hypocotyl and cotyledon explants of E. purpurea. Explant material was obtained from 14-day-old seedlings obtained from seeds that had been extensively disinfected prior to germination. Indole acetic acid (IAA) (5 and 15 to 20 μM) and indole butyric acid (IBA) (2.5 to 20 μM) were effective in inducing root formation from either hypocotyl or cotyledon explants. IBA was found to be the most effective auxin tested. The efficiency of auxins for root induction was IBA>IAA>NAA (naphthylene acetic acid). Choffe et al. (2000) obtained regeneration from petiole explants from 2-month-old sterile seedlings cultured on medium with benzylaminopurine (BA) or thidiazuron (TDZ) in combination with IAA. Regeneration was observed either by direct somatic embryo formation on the epidermis of the petiole, or de novo from callus tissues formed in the subepidermal cell layers. Optimal BA level for regeneration from petiole explants was 2.5 μmol/L. When somatic embryos and shoots were separated from the explant tissue and subcultured on basal medium, more than 90% of all regenerates developed into intact plants.
Coker and Camper (2000) obtained callus, shoot, and root formation from hypocotyl explants on media with different hormone combinations. Disinfested E. purpurea achenes were placed in sterile distilled water for 1 to 2 hours to soften the achene prior to inoculation onto potato dextrose agar. Seedlings were harvested at 11 days and hypocotyl sections (4 to 5 mm) served as the explant source. The general medium consisted of Murashige and Skoog Minimal Organics (MSMO), 30 g/L sucrose, and 8 g/L agar at pH 5.7. Hormone regime consisted of 1 to 3 mg/L NAA plus 1 to 2 mg/L kinetin, or 0.5 to 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 to 2.0 mg/L kinetin with subsequent transfer to hormone-free media. All samples were observed for callus, anthocyanin, shoot, and root production. Morphological development of hypocotyl explants varied with hormonal type and concentration. Anthocyanin production was monitored as a visual indicator that secondary metabolism was taking place, but no difference was observed between treatments. Generally, 2,4-D/kinetin combinations produced more callus than explants treated with NAA/kinetin combinations. The percentage of explants forming shoots and roots was higher on media with NAA/kinetin than on media with 2,4-D/kinetin. All combinations of 2,4-D/kinetin treatments induced more callus formation than NAA/kinetin combinations. The greatest number of plantlets was produced with 1 mg/L NAA and 1 mg/L kinetin. Regenerated plants produced flowers similar in color and shape to those of donor plants.
Harbage (2001) established a micropropagation method for E. purpurea, E. angustifolia, and E. pallida. Removal of seed coverings enabled production of contaminant-free cultures of E. purpurea and E. angustifolia, but not E. pallida. Shoot-tip explants were contaminated in all cases. Generally, shoot formation increased with BA concentration (0.45 to 4.45 μM) for all three species. Rooting was affected by species but not by light, temperature, or IBA concentration. Rooting could be induced in BA-free medium without auxin addition.
Liquid Cell Culture
Liquid cell cultures can be derived from callus or directly from explant material. Establishment usually involves placing an explant on solidified nutrient medium supplemented with growth hormones for initial callus production. Explants form callus that is subsequently subcultured to increase mass and then transferred to liquid nutrient medium. Alternately, explant tissue (leaf, root, stem) can be placed in liquid nutrient medium to obtain a cell suspension. Liquid cell cultures provide a source of material for various metabolic studies or for transformation studies. Growth hormones in the medium can influence secondary product formation; for example, 2,4-D addition to the medium may accelerate callus formation, suppress subsequent morphogenesis, and prevent secondary product formation. Lower 2,4-D concentrations in combination with other hormones often favor secondary product formation.
Protocols were established in the late 1980s for liquid cell cultures of E. purpurea (). Luettig et al. (1989) used the supernatants from liquid cell culture of E. purpurea to obtain highly purified arabinogalactans. The arabinogalactans were then used to measure macrophage activation in a mice assay. Wagner et al. (1988) studied immunologically active polysaccharides of E. purpurea from cell cultures. Leaf and stem explants were cultured in Linsmaier/Skoog medium with 2,4-D. Polysaccharides were extracted after a 14- to 21-day period and were then used in immunological assays. Three polysaccharides, two neutral furogalactoxy-glucans, and an acidic arabinogalactan were detected and shown to be immunologically active. Proksch and Wagner (1987) concluded that the polysaccharides obtained with liquid cell cultures were structurally different from those found in intact E. purpurea plants. Similarly, Schollhorn et al. (1993) used cell suspension studies to provide polysaccharides for immunochemical investigations. In this study, a polyclonal IgG-antibody produced from rabbits was used to study the relationship between the polysaccharide structure and binding. Schollhorn et al. (1993) determined a high degree of structural similarity between the acidic arabinorhamnogalactan from the plant material and the acidic arabinorhamnogalactan from the cell suspension. Like Proksch and Wagner (1987), Schollhorn et al. (1993) determined that the acidic heteroxylan and fucogalactoxyloglucan were structurally different between the plants and the cell suspension cultures.
Hairy root cultures can be induced in tissue cultures using inoculation with strains of Agro-bacterium rhizogenes. Hairy root cultures can provide a source for the standardized production of secondary metabolites in several plant species. Trypsteen et al. (1991) transformed E. purpurea with several strains of A. rhizogenes. Two strains produced callus while the other two strains resulted in formation of hairy roots. Callus and hairy roots produced on the plants were analyzed as a possible source of isobutylamides. Opine detection confirmed successful transformation. High performance liquid chromatography (HPLC) alkamide patterns for control and transformed tissues indicated the following: similar levels in control and transformed callus to that in root tissue and some differences in selected peak intensities; levels in transformed and control root tissue were similar with slight differences in selected peak intensities.
In Vitro Culture of Echinacea Species: Summary
Echinacea species have been successfully regenerated from several explants, hypocotyl, cotyledon, petiole, and shoot-tips. Explants successfully used for liquid cell-suspension cultures include callus tissue, leaf, and stem. Root tissue has been successfully transformed by Agrobacterium to produce hairy root cultures and callus. Potential uses for cultured material includes year-round plant availability, material for mass production of specific clonal lines or repopulation of endangered species, a source of virus-free plant material, as well as a source of material for transformation experiments. Cultured material may also be used as a model system for studying metabolic functions as well as to manipulate metabolic pathways and the production of metabolites. This cultured material may be used for mass production of secondary products μ.e., arabinorhamnogalactan , heteroxylan, and fucogalactoxyloglucan ).
Pamela S. Coker and N. Dwight Camper “In Vitro Culture of Echinacea Species” (2004)