SOMATIC EMBRYOGENESIS OF PRUNUS-SUBHIRTELLA AUTUMNO-ROSA AND REGENERATION OF TRANSGENIC PLANTS AFTER AGROBACTERIUM-MEDIATED TRANSFORMATION

Embryogenic lines of Prunus subhirtella autumno rosa were established on a modified MS medium supplemented with 1 mg/l NAA, 0.06 mg/l IBA an d 0.04 mg/l BA from petioles of axenically grown shoots of adult origi n. To induce normal development of plantlets we compared a range of ap proaches on solid culture media as well as in suspension cultures incl uding treatments with ABA, GA3, zeatin, darkness, and cold. A series o f experiments were conducted to follow the temporal pattern of somatic embryo development. Separation of embryos at different stages of deve lopment was carried out by sieving the suspension cultures through nyl on nets. While the embryogenic masses were used for further subculture s, well formed embryos were used for germination experiments. Transfor med Prunus subhirtella plants were regenerated from somatic embryos by inoculating an embryogenic callus with Agrobacterium strain LBA 4404 containing the beta-glucuronidase (GUS) gene on plasmid pBinGUSint. Se veral putative transformed embryogenic calli were selected for continu ed proliferation on kanamycin containing media. Finally transgenic pla nts were regenerated on shoot multiplication medium containing kanamyc in. Embryos and plants were shown to express the GUS gene by histochem ical assays and northern blot analysis. With a yield of 110 transgenic lines from a single transformation experiment this approach appears i deal for the study of the influence on level of expression caused by d ifferent copy number, site of insertion etc. This will be helpful in e stablishing parameters according to which the best transgenic line for a chosen purpose should be selected.