Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation

The visual marker GUS has been utilized in this study to understand the Ara bidopsis thaliana vacuum infiltration transformation process by Agrobacteri um tumefaciens. High transformation frequencies of up to 394 transgenic see ds per infiltrated plant were achieved. The results showed that the majorit y of the transgenic seeds from single infiltrated plants were from independ ent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 d ays post-infiltration. In addition, all ovules from a single infiltrated pl ant were examined every other day. GUS expression frequencies of up to 1% o f pollen were observed 3-5 days post-infiltration, whereas frequencies of u p to 6% were detected with ovules of unopened flowers 5-11 days post-infilt ration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathwa y.