Investigation On Somatic Hybridizatzon In Apple

On purpose to establish a regeneration system for the production of somatic hybrids ill apple we used the methods of symmetric and asymmetric protoplast fusion. Several parameters using polyethylenglycol (PEG) mediated fusion were tested. As source material two different transgenic rootstocks clones, one transgenic scion, one nontransformed rootstock and two scion clones were taken. The transgenic clones used were obtained by A. tumefaciens transformation and expressed the two marker genes npt II and GUS. For the protoplast fusion experiments isolation and cultivation occurred were performed according to Huancaruna Perales and Schieder (1993). For asymmetric hybridization, donor protoplasts of transgenic genotypes were additionally irradiated at 1000-1250 Gy before mixing. Parameters as PEG concentration and incubation time, among others, were tested. For shoot induction protocalli were transferred to shoot regeneration medium containing NAA, BAP and TDZ. The kanamycin selection of the regenerated protocalli from fusion experiments started after four weeks in protoplast liquid medium or after six weeks on shoot regeneration medium. Whereas, protocalli for the control experiments were cultivated on antibiotic free medium. The hybrid character of the regenerated protocalli and shoots were analyzed by RAPD/PCR. Until now some somatic hybrids could be identified.