Transformation of cherry:: Prunus cerasus L. 'Montmorency' and Prunus cerasus x P canescen 'Gisela 6' mediated by Agrobacterium tumefaciens and a two-step selection system

A two-step selection method was established to improve Agrobacterium tumefaciens-mediated stable transformation of sour cherry Prunus cerasus L. `Montmorency' and rootstock P. cerasus x P. canescens 'Gisela 6'. Whole leaf explants, each with four cuts perpendicular through the midrib, were co-cultivated with EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted B-glucuronidase (GUS) reporter gene (gusA). Selection and regeneration of transformed cells and shoots of both cultivars was carried out on selection media starting with 25 mg L-1 kanamycin (Km) for 4 weeks followed by 50 mg L-1 Km for 8 weeks. Compared with a one-step selection using 50 mg L-1 Km, the two-step selection method minimized necrosis of inoculated leaf explants, and the transformation frequency was increased from 3.1% to 7.6% for `Montmorency' and from 3.3% to 9.7% for 'Gisela 6', respectively. Southern analyses of the GUS- and PCR-positive transformants, 3 for `Montmorency' and 2 for 'Gisela 6', confirmed stable integration of the transgenes.