Enhancement of plant performance and biochemical constituents in field and hairy root production of Echinacea

Echinacea is a commercially important source of natural products and phytopharmaceuticals. Echinacea products currently constitute a significant portion of the rapidly growing, multi-billion dollar natural products market. This popularity has led to the expansion of commercial cultivation of Echinacea and the need to find alternative methods to meet the increasing demands of the phytopharmaceutical market. Two methods of producing biochemical constituents in commercial use include extraction from field-grown plants and in vitro hairy root production;Echinacea seeds in the wild typically show a high degree of dormancy. In order to enhance Echinacea seed germination for field production, alternative methods to break seed dormancy were evaluated for the three most important medicinal species of Echinacea, E. angustifolia DC., E. purpurea (L.) Moench, and E. pallida (Nutt.) Nutt. The effects of seed source and production system on growth, yield and biochemical content were evaluated. Cold-moist stratification for 4 wk under 16-24 hr light conditions was determined to be the most effective non-chemical method to break Echinacea seed dormancy and increase germination rates;In the field production studies, where screened cages were evaluated against open field conditions, screened cages were associated with enhanced post-transplant establishment during the first year of growth. Because of the requirement for organic seeds in certified organic production, we evaluated an organic Echinacea seed source and found sufficient yield from this stock. Uniform recommendations, across the three Echinacea species, could not be determined because of numerous significant interactions between production system and seed source, however. Echinacea pallida root yields were equivalent to E. purpurea yields after three growing seasons, and yields were enhanced under screened cages. Echinacea plants in the open field were more affected by aster yellows disease than were those under screened cages. Thus, in areas of aster yellows incidence, satisfactory Echinacea root yields can be obtained under screened cages using organic seeds;Because of difficulties in biosynthesis and in planta variation in levels of bioactive compounds found in Echinacea, application of in vitro techniques in the production of medicinal plants can provide an alternate source of plant material for extraction of active ingredients. Hairy roots of Echinacea may represent an alternative to the production of secondary metabolites, such as alkamides, the most important bioactive compounds produced by Echinacea roots. Hairy roots were induced in the three species of Echinacea by transformation with Agrobacterium rhizogenes and A. tumefaciens containing the rol ABC genes. Plant growth regulators, such as indole butyric acid (IBA), and chemical elicitors, such as jasmonic acid (JA), were evaluated for their effects on growth and production of alkamides in hairy root cultures. Hairy roots induced by the A. tumefaciens strain were more amenable to culturing in liquid medium. Production of alkamides 3, 4, 8, and 9 was increased with the A. tumefaciens (pPZPROL) strain. Growing media supplemented with 3% sucrose provided optimal conditions for hairy root growth. When IBA was added to the growing medium, hairy roots increased their growth rate up to fourteen-fold compared to regular media. Production of Echinacea hairy root cultures was optimized by growing cultures in the dark and using one-half-strength Gamborg\u27s B5 medium supplemented with up to 1 mgL-1 of IBA, 3% of sucrose, and 40 muM of JA. In conclusion, Echinacea biochemical constituents, of interest to the phytopharmaceutical market, can be produced by field-grown Echinacea roots or by hairy root cultures