Agrobacterium-Mediated Transformation in Prunus Species

摘要

In recent years our laboratory has worked on transformation of Prunus species mediated by Agrobacterium tumefaciens in order to obtain plant resistance to sharka disease. In plum, kanamycin selection applied either immediately or with a one-month delay both produced clones with stably integrated transgenes (Damiano et al., 2007a; Ilardi et al., 2007). These clones, derived from hypocotyl sections of seeds of 'Stanley' and rootstock 'St. Julien', infected with strain EHA101 carrying the plasmid pGA482GG/PPV-CP-33, after three years of in vitro culture, contain the NPTII, GUS and PPV coat protein genes, as confirmed by PCR. Some of the transgenic clones obtained in 2001, derived from 'Stanley', grown in the greenhouse since 2003, express histochemical GUS reaction in the floral organs and pollen grains. In apricot, 'Boccuccia Spinosa' and 'Monaco Bello', antibiotic selection was applied during the multiplication phase due to the high sensitivity of hypocotyl sections and embryo apices to kanamycin (Damiano et al., 2007b). PCR initially confirmed integration of transgenes in some clones, but after six months NPTII, GUS and PPV coat protein genes were no longer detected. Difficulties have been encountered in regeneration of peach: thus far no transformation events have occurred. These results suggest that species and antibiotic selection affect the efficiency of transformation and the stability of the transgenic plants. Regarding the use of the reporter gene, though generally unaccepted for the field release, it could be a useful tool for monitoring pollen spread. Concerning virus resistance strategy, the full coat protein viral gene was integrated in the transgenic lines, but the aim will be to induce resistance by RNA interference, preventing exogenous protein expression.