Agrobacterium tumefaciens-mediated genetic transformation of Eleutherococcus sessiliflorus by phosphinothricin acetyl transferase gene

Abstract

An Agrobacterium-mediated transformation system for Eleutherococcus sessiliflorus was established using uidA gene coding for the β-glucuronidase (GUS) and pat gene that confers resistance to the herbicide Basta® (bialaphos). Either fine embryogenic calluses or early globular embryo clusters were cocultivated with A. tumefaciens for 3d. Transient GUS expression levels were higher in globular embryo clusters than in embryogenic calluses. The high level of transient expression coincided with stable expression of the uidA gene. The frequency of GUS-positive somatic embryos increased following continuous subculture of explants (once every 2 wk) on selection medium containing 200 mgl−1 cefotaxime and 25 mgl−1 kanamycin. After 10 subcultures, more than 95% of somatic embryos were GUS-positive. Cotyledonary embryos were transferred to a medium containing 14.4 μM gibberellic acid to induce germination and plantlet conversion. Transgenic plantlets were confirmed by polymerase chain reaction (PCR) and Southern analysis. Each transgenic line was cloned by induction of embryogenic callus from roots and hypocotyls. Transgenic plants grown in soil medium were resistant to 200 mgl−1 phosphinothricin (Basta®) application.