Rapid and efficient Agrobacterium-mediated transformation of Panax ginseng by plasmolyzing pre-treatment of cotyledons

A rapid and efficient genetic transformation of Panax ginseng cotyledon explants following a plasmolyzing pre-treatment was investigated. When cotyledon explants of P. ginseng were pre-treated with 1.0 M sucrose, transient expression of the β-glucuronidase (GUS) gene was strongly enhanced following co-cultivation with Agrobacterium tumefaciens harboring the GUS gene. This enhanced expression coincided with a high frequency of stable transformation (three times higher than non-treatment). Blue-colored cells (indicative of the presence of the GUS gene) were detected over large areas of cotyledons pre-treated with sucrose. In contrast, when the plasmolyzing pre-treatment was not applied, GUS-positive cells were observed to be scattered on the cotyledons. Somatic embryos developed directly on cotyledon surfaces without intervening callus formation within 2 weeks and matured to the cotyledonary stage after about 7 weeks. Cotyledonary embryos regenerated into small transgenic plantlets on medium supplemented with gibberellic acid within 1 month. Thus, about 3 months of culture was required for small transgenic ginseng plantlets to be obtained. The presence of transformed ginseng plantlets was confirmed using the staining reaction of X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and by the polymerase chain reaction.