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Electroporation of rapeseed protoplasts – transient and stable transformation
Author(s) -
Bergman Per,
Glimelius Kristina
Publication year - 1993
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1993.tb01378.x
Subject(s) - protoplast , electroporation , transformation (genetics) , plasmid , biology , callus , microbiology and biotechnology , neomycin , reporter gene , gene , gene expression , botany , biochemistry , antibiotics
Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β‐glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead‐type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10 −5 and a relative transformation frequency of 0.3% could be achieved. X‐ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.