Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted ?-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.

Agrobacterium-mediated transformation of American ginseng with a rice chitinase gene

“…We also tested cotyledons for their response to transformation, but unlike the results reported for Korean ginseng (Lee et al 1995), the rate of callusing frequency was only around 0.5% (data not shown). The pre-culture of explants on tissue culture medium prior to infection by Agrobacterium has been reported to enhance transformation frequency (Sunilkumar et al 1999).…”

Rapid induction of Agrobacterium tumefaciens-mediated transgenic roots directly from adventitious roots in Panax ginseng

“…This danger is avoided by the use of constructs with an intron sequence inserted within the coding sequence of GUS gene (Ohta et al 1990). Although pBI121 vector did not contain intron in GUS gene, false GUS-positive activity by X-gluc assay was not observed during genetic transformation in Panax ginseng (Lee et al 1995), indicating the GUS false positive result dose not occur or negligible in Panax ginseng.…”

“…The first attempt for the production of transgenic ginseng plant was reported by Lee et al (1995). They introduced the b-glucuronidase (GUS) gene into cotyledon explants by A. tumefaciens LBA4404 and transgenic plants were obtained by somatic embryogenesis.…”

Agrobacterium rhizogenes-Mediated Transformation of Plants

“…A method to avoid the regeneration of chimerie plants might be to regenerate them from hairy root -derived protoplasts (Lee et al 1995;Sevon et al 1995).…”