Methods for genetic transformation of the parasitic weed dodder (Cuscuta trifolii Bab. et Gibs) and for PCR-based detection of early transformation events

The first genetic transformation method has been developed for Cuscuta trifolii, an economically important parasite of alfalfa (Medicago sativa L.). For transgene delivery, Agrobacterium tumefaciens vector was used according to a standard protocol (Method A) and a modified one, which previously resulted in the successful Agrobacterium-mediated transformation of monocot cereals (Method B). In Method B the impacts of physical and biochemical stimulation of the bacterial infection were evaluated on the frequency of transformation such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension into target tissues and the influence of in vitro preinduction of vir genes, respectively. While the physical enhancement of Agrobacterial infection (microwounding, infiltration) had no positive effect on the transformation frequency, the biochemical one (in vitro preinduction of vir genes) resulted in 4-fold increase in comparison with the conventional protocol (Method A). Additionally, a PCR-based technique was developed during the transformation work to monitor early transformation events, using primers designed to amplify specific DNA fragments outside of the T-DNA. Our data confirmed that the presence of a given transgene can be evaluated by using this primer pair as a control. (C) 2002 Elsevier Science B.V. All rights reserved.