Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants

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Abstract
Maize Type II callus tissue was used as the plant material for genetic transformation via electroporation. Plasmid DNA containing a selectable marker gene (either neomycin phosphotransferase (npt-II) or phosphinothricin acetyl transferase (bar)), and a screenable marker gene (gus A) was incubated with the tissue prior to electroporation. Electroporated callus tissue was placed on selection medium containing kanamycin sulfate or Bast™. No kanamycin resistant colonies were recovered whereas four independent Basta™ resistant callus isolates were recovered from a total of 544 cuvettes electroporated. After 8 to 16 weeks on the Basta™ containing medium, selected calli were isolated and maintained in individual selection plates for 4 to 6 weeks until sufficient tissue accumulated. Enzyme assays and DNA analyses were performed to verify the transformation events. Several plants were regenerated from individual callus isolates. The plants derived from one callus isolate were male sterile while those derived from the other isolates were both male and female fertile. Most plants showed Basta™ resistance. DNA analyses confirmed the presence of the introduced bar gene(s) in the primary regenerants and their progeny. The integration patterns of the inserted DNA appeared to be complex.