Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules

• We established a novel maize transformation system using ovule as target.
• Several important factors were optimized to standardize the transformation protocol.
• Morphologically normal, fertile and independent transgenic lines were obtained.
• The transformation resulted in stable transgene expression and inheritance.
• We confirmed the maize ovule culture and transformation system is genotype independent.

A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24 h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2–3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly.