Multiplication and germination of somatic embryos of american ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets

Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μμM αα-naphthaleneacetic acid (NAA) and 9 μμM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2––100%%, while somatic embryos were produced from these calluses at a frequency of 25––87.5%% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μμM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1%% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μμM gibberellic acid and 5 μμM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.