High-efficiency transformation of Lycium barbarum mediated by Agrobacterium tumefaciens and transgenic plant regeneration via somatic embryogenesis

We have developed a reliable and high-frequency system of transformation and regeneration via somatic embryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-beta-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50 mg l(-1) kanamycin (Km), approximately 60% of the explants produced kanamycin-resistant (KMR) calli. After three subcultures on the same selective medium, KmR calli were transferred onto SE induction medium containing 25 mg l(-1) Km, where they produced numerous somatic embryos (166 g(-1) fresh weight callus) that subsequently developed into KMR plantlets. Compared with the untransformed control calli, the transformed calli maintained their higher frequency of. SE up to 16 months after transformation. GUS staining and polymerase chain reaction and Southern blot analyses confirmed the integration of the T-DNA into the plant genome.