Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Abstract

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.