Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl(-1) kinetin, and 100 mgl(-1) kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl(-1) each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the β-glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl(-1) myo-inositol, 1 mgl(-1) 2,4-D, 0.5 mgl(-1) BA, and 0.5 mgl(-1) kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.
Mature zygotic embryos dissected from ginseng (Panax ginseng C.A. Meyer) seeds were cultured on Murashige and Skoog (MS) medium containing various concentrations of 2,4-D and kinetin. Somatic embryos were induced directly from cotyledonary tissue and from intervening callus. The frequency of somatic embryo induction was up to 55% of zygotic embryo explants. Upon transfer onto half-strength MS medium supplemented with 1 mg BA/liter and 1 mg GA3/liter, most somatic embryos developed into plantlets. More than 50% of the plantlets flowered after 4 weeks of culture, and some developed immature fruits in vitro. These results indicate that adulthood of ginseng root explants is not a prerequisite for flowering of plantlets regenerated through somatic embryogenesis. Chemical names used: (2,4 -dichlorophenoxy) acetic acid (2,4-D); N-(2-furanylmethyl) -1H-purin-6-amine(kinetin); N-(phenylmethyl) -1H-purin-6-amine (BA); gibberellic acid (GA3).
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