Mass Propagation of Echinacea angustifolia: A Protocol Refinement using Shoot Encapsulation and Temporary Immersion Liquid System

Tissue culture of Echinacea angustifolia allows for selection and rapid multiplication of genetically superior clones. In this study, temporary immersion liquid bioreactor (RITA system), synthetic seed technology and semi-solid culture conditions were evaluated for shoot multiplication efficiency. After 25 days of culture, the highest multiplication rate was observed in explants cultured in temporary immersion liquid system. Previous reports on micropropagation of E. angustifolia have had low success rate in rooting. Shoots were rooted under autotrophic conditions on sugar-free semi-solid media supplemented with 2.5 μM IBA and using synthetic seed technology by shoot encapsulation in calcium alginate gel cultured on MS medium supplemented with 2.6 μM NAA. Rooting response was noticed in three weeks with a success rate of 83%. Arbuscular mycorrhiza infection helped the acclimatization survival rate of rooted plantlets. INTRODUCTION Echinacea is one of the most popular herbal supplements among the general public in the United States and the popularity of this medicinal plant is due to its use in the treatment of infections and colds, based on its immune-stimulant and antiinflammatory effects (Giles et al., 2000). The current sales of Echinacea are in $39 million per year according to Blumenthal (2002) market report. Several clinical studies on the therapeutic use of Echinacea have demonstrated the benefits on recovery and prevention of infections (Giles et al., 2000 and Lindenmuth and Lindenmuth, 2000), but other studies have failed to show efficacy (Mark et al., 2001 and Vonau et al., 2001). Lack of product standardization and consistency within protocols are possible reasons for these inconsistencies in clinical studies (Melchart and Linde, 1999). For commercial preparations of Echinacea, roots and foliage of three different species (Echinacea purpurea, Echinacea angustifolia and Echinacea pallida) have been wild crafted or cultivated. Thus, authenticity and genetic identity are important issues that may affect quality, safety and efficacy of the final products. Variation among the final product formulations (encapsulated powder, expressed juices and tinctures) may lead to different chemical compositions, which may also contribute to these inconsistencies. As the popularity of Echinacea as a dietary supplement depends on quality of the products, selection of superior genotypes based on biological activity is of interest. Developing a mass propagation protocol to rapidly increase superiors E. angustifolia genotypes is the objective of this study. Choffe et al (2000) have reported a successful micropropagation protocol of E. purpurea but rooting has been a challenge for mass propagation of E. angustifolia (Harbage, 2001). Many plant species produced in nutrientrich substrate under sterile conditions fail to survive the transference to soil conditions. Inoculation by arbuscular mycorrhizal fungi (AMF) has aided the survival and growth of these species during plantlet acclimatization (Azcon-Aguilar et al.1996, Salamanca et al. 1992). Further improvements in the existing E. angustifolia micropropagation protocol are needed including optimization of the in vitro microenvironment and the survival rate during acclimatization. In this study, we are attempting to improve the culture conditions Proc. XXVI IHC – Future for Medicinal and Aromatic Plants Eds. L.E. Craker et al. Acta Hort. 629, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 410 by using temporary immersion liquid system and acclimatization of plantlets by mycorrhization, refining E. angustifolia protocol of mass propagation. MATERIALS AND METHODS Plant Material All experiments were conducted with shoot cultures obtained from germinated seeds using hypocotyl as an explant. The North Central Regional Plant Introduction Station (NCRPIS) in Ames, Iowa provided seeds of E. angustifolia (PI 312814 accession). Prior to inoculation on the germination media, seeds were surface disinfected by washing with 1% NaOCl (20% v/v bleach) and 0.1% Tween 20 for 10 minutes, followed by washing three times in sterile distilled water (5 minutes per wash). Aseptic seedlings were initiated on agar (0.8%) solidified half strength MS medium (Murashige and Skoog, 1962) without sucrose using ventilated closures. Cultures were incubated at 25 ± 2° C, 16h photoperiod under fluorescent light with a photon flux of approximately 52μmol. m.s. After 10 days, 0.5-1.0 cm long hypocotyl were taken as explants for the initiation of shoot cultures. Shoot cultures were obtained on MS half strength (MS/2) macronutrients medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with 2.2 μM of 6 Benzyladenine (BA) per liter, adjusted to pH 5.7. The same multiplication medium was used in the three cultured systems described below. Shoot Multiplication Echinacea shoots were transferred into three different culturing systems for shoot multiplication: temporary immersion system (RITA bioreactor), encapsulated shoots using synthetic seed technology and semi-solid culture conditions. For the temporary immersion, 15 explants were inoculated on the upper compartment of each RITA system in each of three bioreactors. Each replicate consisted of three explants removed at 5-day interval per RITA system during the growing period of 25 days. For synthetic seed technology, each explant was encapsulated in 5% sodium alginate and 50 mM CaCl2 producing individual beads. Three individually encapsulated shoots were cultured per vessel on semi-solid MS media supplemented with 2.2 μM BA. A total of 15 vessels composed the encapsulated shoot treatment with three replicates. Same arrangement was used for the cultures on semi-solid media supplemented with 0.8% (w/v) agar. Rooting Shoots were transferred onto rooting media maintaining under the same conditions as described. Treatments were replicated six times in rooting, each RITAsystem contained 15 shoots per bioreactor. For the synthetic seed technology, three encapsulated shoots were inoculated per vessel. Three shoots per vessel were also transferred to semisolid culture condition. An additional treatment was tested for rooting under autotrophic condition using semi-solid sugar free media with vented closures. In rooting E. angustifolia under these different culture conditions, both MS and MS/2 macronutrients supplemented with three different auxins; 2.6 μM NAA (αnaphthalene acetic acid), 2.5 μM IBA (indole-3-butyric acid) and 2.8 μM IAA (indole-3acetic acid) were tested. Rooted and un-rooted plantlets were rinsed with sterile water to remove residual rooting medium and transferred to planting tray kits with 6 cells and a plastic dome and based tray marketed by Wal-Mart Stores, Inc. for acclimatization to soil. Fungal Inoculums Inoculums of Glomus mosseae (Nicol and Gerd) Gerd and Trappe, Gigaspora ramisporophora Spain, Sieverding and Schenck, Scutellospora fulgida Koske and Walker, Entrophospora colombiana Spain and Schenk were provided by Dr. Gisela Cuenca of Instituto Venezolano de Investigaciones Científicas, Caracas Venezuela.