ELECTROPORATION OF RAPESEED PROTOPLASTS - TRANSIENT AND STABLE TRANSFORMATION

Protoplasts of Brassica napus hypocotyls were transfected using electr oporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable con ditions for gene transfer. To monitor the introduction of DNA into pro toplasts a plasmid containing the beta-glucuronidase (EC 3.2.1.31), an d the neomycin phosphotransferase (EC 2.7.1.95) genes was used. By usi ng this construct, expression of a screenable marker gene for transien t expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electropo ration conditions resulted in a frequency of 0.1% transiently transfor med protoplasts. Microcalluses were cultured under selective condition s in a bead-type culture system. Resistant callus, with an absolute tr ansformation frequency of 4.9 x 10(-5) and a relative transformation f requency of 0.3% could be achieved. X-ray irradiation of newly electro porated protoplasts did not enhance absolute transformation frequencie s. From some of the resistant calluses, transgenic plants could be reg enerated which were characterized by molecular analysis.