Efficient transformation of scutellar tissue of immature maize embryos

Abstract

  An efficient transformation system for maize was established by improving transformation conditions for the particle bombardment of the scutellar tissue of immature embryos. Particle bombardment was carried out using constructs containing the pat gene as the selection marker and a PDS 1000/He gun (Biorad). Transformation parameters, such as the amount of gold particles used per bombardment, particle velocity, preculture time of the scutellum prior to bombardment and osmotic treatment of the target tissue before and after bombardment, were analysed. Fertile transgenic regenerants of the maize inbred lines H99, A188 and Pa91 and the crosses A188×H99 and Pa91×H99 were selected on Basta-containing medium. The transformation frequency was between 2% and 4%. A total of 29 transgenic plant lines was obtained and verified with Southern blot analysis. All of the transgenic plants were fertile and set seeds. The R1 progeny of single plants was analysed. A Mendelian segregation of the transgenes was observed for all of the transformants tested. For 1 candidate, stable inheritance and stable expression of the transgenes were followed up to the R₄ generation.