Agrobacterium-mediated transformation of Eupatorium adenophorum

Abstract

A simple and efficient protocol for Agrobacterium tumefaciens-mediated genetic transformation of Eupatorium adenophorum has been developed. Stem segments were pre-cultured on Murashige and Skoog (MS) medium for 24 h and then co-cultured with A. tumefaciens strain LBA4404 harboring the pBI121 plasmid on the same medium in darkness for 3 days. After co-cultivation, explants were transferred to MS medium supplemented with 1.0 mg l -1 naphthaleneacetic acid (NAA), 1.0 mg l -1 6-benzyladenine (BA), 150 mg l -1 kanamycin (Kan), and 200 mg l -1 cefotaxime (Cef), and incubated at 25 ± 1C under a 16-h photoperiod. Callus formation was induced for 3-4 weeks. Induced calluses were transferred to fresh selection medium and subcultured every 15 days. After 6-8 weeks, regenerated plantlets from embryogenic callus could be observed. Buds of 1 cm in length were transferred to half-strength MS medium containing 150 mg l -1 Kan and 200 mg l -1 Cef, and rooted plantlets were obtained after 4 weeks. Putative transgenic plants were confirmed using GUS histochemical assay, polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR. A transfor- mation efficiency of 2% was obtained.