Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Stem and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1–3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μM). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA). Echinacea, purple coneflower, is a genus of herbaceous perennials, including nine species primarily indigenous to the Central and Plains States (McGregor, 1968). It has received considerable attention in recent years for its ornamental value in the landscape and in florist bouquets and for its medicinal qualities. This popularity has led to a need for improvements in propagation methods over traditional seed propagation and crown division to facilitate faster multiplication and support rapid availability of elite clonal selections. Several Echinacea species have high economic value. Echinacea purpurea has long E. angustifolia has higher value per kilogram of root. High value of Echinacea has led to species endangerment problems because of harvesting of native colonies for sale to pharmaceutical companies (Foster, 1990). Two of the nine species (E. tennesseensis Small. and E. laevigata Blake.) are on the federal endangered species list or are being considered for this list. Efforts are currently under way to multiply these species for reestablishment in the wild. Ornamental and medicinal value of the midwestern species, E. angustifolia, E. pallida, and E. purpurea, may also lead to their endangerment through overharvesting of native populations. Most Echinacea species are easily seedpropagated following stratification. Seedpropagated plants often will not flower until the second year. Many years are required for the number of generations of self-pollination required to achieve inbred lines, and some species, such as E. angustifolia, are self-incompatible. Inherent heterozygosity and tendency toward interspecific hybridization often require vegetative propagation to maintain superior characteristics of select individuals. Echinacea angustifolia and E. pallida produce thick taproots that can be propagated as root cuttings, while E. purpurea has a fibrous root system and is best vegetatively propagated by crown division or basal stem cuttings. Micropropagation offers improvements over traditional vegetative propagation because of the faster rate of multiplication (Lineberger, 1983), and can also be effective in propagating species that are troublesome to clone by conventional means (Bridgen, 1986). This study was undertaken to investigate micropropagation for rapid multiplication of Echinacea species. Materials and Methods Experiment 1. Isolation of aseptic cultures from seedling and stem explants. Aseptic explant isolation was compared with seed and stem explants. Seeds of E. angustifolia, E. pallida, and E. purpurea ‘Bravado’ were isolated using four methods: 1) intact; 2) after removing the outer seedcoat (pericarp); 3) after removing the pericarp plus the integument layer; and 4) after removing the pericarp plus the integument layer plus the endosperm layer (excised embryos). One-centimeter shoot tip and stem explants were also evaluated. These were collected prior to flower development from actively growing, 2-year-old E. purpurea ‘Bravado’ and E. angustifolia stems that were 15 to 30 cm long. Stock plants were grown in a greenhouse in pasteurized 2 polystyrene : 2 sphagnum peat moss : 1 clay-loam field soil media in 15-cm-diameter, standard, round plastic pots. Plants were watered as needed and fertilized weekly with 300 mg·L 1 20N–8.7P–16.6K soluble Peters brand fertilizer (Grace Sierra Horticultural Products Co., Allentown, Pa.). All explants were treated with a 15% household bleach solution (final concentration was 0.8% sodium hypochlorite) containing 0.1% Tween 20 detergent; they been a popular garden ornamental and was rated one of the top-selling perennials surveyed from 1992 through 1995 (Rhodus, 1995). The cultivar Magnus was voted the perennial plant of the year in 1998 by the Perennial Plant Association. Echinacea purpurea has been a prolific producer of high-quality, longstemmed flowers and was ranked third of 19 species for potential profitability as a fieldgrown florist crop (Starman et al., 1995). In addition to ornamental value, Echinacea is one of the most important medicinal herbs in commerce today (Foster, 1985), and has a long history of medicinal use dating back to the Plains Indian tribes of midwestern America (Foster, 1990; Kindscher, 1989; Shemluck, 1982). Medicinal effectiveness apparently stems from the presence of several endogenous compounds effective in immuno-stimulation (Kindscher, 1989). Use of medicinal Echinacea products is at an all-time high in Europe and North America (Rawls, 1996). Echinacea-based products are the bestselling herbal remedies in the United States, with 9.1% of the market share. Commercial production of Echinacea for medicinal products in the United States and Canada involves E. purpurea (80% of planted area) and E. angustifolia (20% of planted area). However, were submerged in the solution with agitation for 15 min followed by rinsing for 30 s in sterile deionized water. Explants were then placed aseptically on sterile medium containing Murashige and Skoog (MS) (1962) minerals, 0.56 mM myoinositol, 1.20 μM thiamine HCl, 4.44 μM BA, 87.6 mM sucrose, and 0.7% Bacto agar (Difco Laboratories, Detroit). These ingredients were used in all experiments except where otherwise stated. Cultures were incubated in 40 μmol·m·s continuous coolwhite fluorescent light at 25 °C. Cultures were evaluated 4 weeks after initiation and data were taken on percent contamination and germination, and number of shoots produced (shoot count). A completely random design was used with four individual cultures (one explant per culture) representing one replicate and three replicates for each treatment– species combination. All data were analyzed by analysis of variance. Analysis of aseptic explant isolation and establishment data was performed on two subsets of the entire data set. The first analysis included only treatments involving seed explants and data on contamination, germination, and shoot count. The second analysis included treatments involving both seed and vegetative explants and data on contamination and shoot count. Experiment 2. Cytokinin influence on shoot formation. Initially, shoot formation in response to a wide range of concentrations of BA was evaluated using excised embryos of E. angustifolia, E. pallida, and E. purpurea ‘Bravado’. Embryos were disinfested and placed on media as described above but modified to include 0.45, 4.45, and 44.5 μM BA. Cultures were evaluated after 4 weeks of incubation as described above for shoot count and percent adventitious shoots (shoots not arising from nodes). A randomized complete-block design was used. Shoot formation in response to a lower cytokinin range was evaluated using the same procedure but with 0, 0.09, 0.9, and 8.9 μM BA. A completely random design was used. Shoot number, leaf length, and root number were recorded after 4 weeks of culture. Experiment 3. Mineral composition and concentration influence on shoot formation. Shoot formation in response to mineral composition and concentration was evaluated with excised embryos of E. angustifolia, E. pallida, and E. purpurea ‘Bravado’. Explants were placed on the medium described above, in which mineral composition was modified to include MS minerals or Woody Plant Medium (WPM) (Lloyd and McCown, 1980) minerals at normal concentration or at one-half normal concentration. Cultures were arranged in a completely random design. Shoot number, leaf length, and root number were recorded after 4 weeks of culture. Experiment 4. Shoot formation response to subculture frequency. Influence of subculture frequency on shoot formation was investigated with established (seed explants isolated and subcultured nine times at 4-week intervals) E. angustifolia shoot cultures. Established cultures were divided and single shoots placed on fresh medium using MS minerals Table 1. Influence of seed covering removal and species on contamination, germination, and shoot count from newly isolated seed explants of three Echinacea species. Seed covering Contamination Germination Shoots layers removed Species (%) (%) per explant 0 E. angustifolia 72 a E. pallida 11 b–d 50 n 0.5 n E. purpurea ‘Bravado’ 39 b 1 E. angustifolia 39 b E. pallida 33 bc 73 m 0.8 mn E. purpurea ‘Bravado’ 19 b–d 2 E. angustifolia 0 d E. pallida 0 d 97 l 1.0 m E. purpurea ‘Bravado’ 8 cd 3 E. angustifolia 0 d E. pallida 0 d 100 l 1.5 l E. purpurea ‘Bravado’ 0 d ANOVA Source of variation df Mean square Species (S) 2 864.32 198.79 0.06 Seed covering removal (SC) 3 3686.48 4930.25 1.45 S × SC 6 772.22 437.99 0.18 Error 24 235.38 335.86 0.11 Mean separation within columns by Duncan’s multiple range test, P ≤ 0.05. NS, *, Nonsignificant or significant at P ≤ 0.05 or 0.001, respectively. Table 2. Influence of explant source and species on contamination and shoot count from newly isolated cultures of two Echinacea species. Contamination Shoots Explant source Species (%) per explant Intact seed E. angustifolia 72 b 0.4 n E. purpurea ‘Bravado’ 39 c Seed minus one covering layer E. angustifolia 39 c 0.9 m E. purpurea ‘Bravado’ 19 cd Seed minus two covering layers E. angustifolia 0 d 1.1 lm E. purpurea ‘Bravado’ 8 d Seed minus three covering layers E. angustifolia 0 d 1.4 l E. purpurea ‘Bravado’ 0 d Shoot tip E. angustifolia 100 a 0.0 o E. purpurea ‘Bravado’ 100 a Stem section E. angustifolia 38 c 1.3 l E. purpurea ‘Bravado’ 11 d ANOVA Source of variation df Mean square Species (S) 1 1304.41 0.03 Seed covering removal (SC) 5 8346.10 1.73 S × SC 5 438.54 0.12 Error 24 3610.56 0.11 Mean separation within columns by Duncan’s multiple range test, P ≤ 0.05 NS, *, **, Nonsignificant or significant at P ≤ 0.05, 0.01, or 0.001, respectively. and BA at 0.9 μM. Cultures were subdivided and transferred to fresh medium at 2-, 3-, and 4-week intervals. Cultures were arranged in a completely random design. Data were recorded for shoots per subculture and total shoots produced after 12 weeks. Experiment 5. Auxin, light, and temperature influence on rooting. Influence of auxin, light, and temperature on root formation from established shoot cultures (after more than 15 subcultures) of E. angustifolia, E. pallida, and E. purpurea ‘Bravado’ was investigated using a two-phase rooting system. Individual shoots were excised from proliferating shoot cultures and placed in sterile medium containing 43.8 mM sucrose, 1.95 mM MES [2(N-morpholino) ethanesulfonic acid], 7 g·L Difco Bactoagar, and 0, 1.5, and 15.0 μM IBA. Media pH was adjusted to 5.5. Shoots were incubated at 21 or 30 °C in the dark or under 40 μmol·m·s 1 continuous cool-white fluorescent light. Data were taken on root count and percent shoots with roots.