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 and 0.04 mg/l BA from petioles of axenically grown shoots of adult origin. To induce normal development of plantlets we compared a range of approaches on solid culture media as well as in suspension cultures including treatments with ABA, GA3, zeatin, darkness, and cold. A series of experiments were conducted to follow the temporal pattern of somatic embryo development. Separation of embryos at different stages of development was carried out by sieving the suspension cultures through nylon nets. While the embryogenic masses were used for further subcultures, well formed embryos were used for germination experiments. Transformed 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. Several putative transformed embryogenic calli were selected for continued proliferation on kanamycin containing media. Finally transgenic plants were regenerated on shoot multiplication medium containing kanamycin. Embryos and plants were shown to express the GUS gene by histochemical assays and northern blot analysis. With a yield of 110 transgenic lines from a single transformation experiment this approach appears ideal for the study of the influence on level of expression caused by different copy number, site of insertion etc. This will be helpful in establishing parameters according to which the best transgenic line for a chosen purpose should be selected.