Production of four commercially cultivated 'Echinacea' species by different methods of 'in vitro' regeneration

Efficient 'in vitro' procedures for mass propagation of four commercially important 'Echinacea' species have been developed. Plants of 'E. angustifolia', 'E. pallida', 'E. paradoxa' and 'E. purpurea' were regenerated by three methods, namely axillary bud proliferation, adventitious shoot formation and somatic embryogenesis. Shoot tips obtained from 'in vitro' germinated seedlings, adventitious shoots or somatic embryo-derived plantlets, when cultured on Murashige and Skoog medium enriched with 1 μM 6-benzylaminopurine, 2 μM kinetin, 0.5 μM indole-3-butyric acid and 4 mg⁻¹ paclobutrazol multiplied three-fold within 3-4 weeks in culture. Incorporation of paclobutrazol in the shoot multiplication medium was necessary to recover healthy and robust shoots suitable for rooting. Direct, high-frequency shoot formation on intact leaves of shoots grown on 6-benzylaminopurine and kinetin-supplemented media, an unusual and novel observation made in this study, occurred in all the species studied. Rooting of 'in vitro' developed shoots was achieved relatively easily with Murashige and Skoog basal medium rather than with auxin-enriched media. Culturing of hypocotyl explants on medium containing 3,6-dichloro-o-anisic acid (commonly known as dicamba), or 2,4-dichlorophenoxyacetic acid, resulted in direct somatic embryogenesis in all the species examined. The presence of cytokinin was required for somatic embryo germination, but further development of germinated somatic embryos into normal plantlets occurred in Murashige and Skoog medium. We conclude that the procedures described here could be used for rapid propagation as well as genetic transformation of commerically cultivated 'Echinacea' species.