Eucalyptus: Agrobacterium protocols

Using Eucalyptus camaldulensis as a model system, we describe here a basic Agrobacterium-mediated genetic transformation protocol through organogenesis for the production of transgenic plants. Hypocotyl segments or cotyledon pieces from in vitro seedlings are used as starting materials. The explants are inoculated and cocultivated with a disarmed, binary strain of A. tumefaciens CIB542 harboring a mini Ti plasmid, pBI121. A modified Gamborg’s B5 medium is used as the basal culture medium throughout stages of co-cultivation, callus induction and shoot regeneration. The incorporation of neomycin phosphotransferase II (NPT II) and β–glucuronidase (GUS) genes into the plant nuclear genome are primarily verified by histochemical analysis and polymerase chain reaction (PCR). Modifications of this protocol to use in mature tissues derived from elite trees and other Eucalyptus species are also described.