An Agrobacterium-mediated transient gene expression system for intact leaves

Abstract

An efficient and reproducible Agrobacterium-mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria, which were pretreated in vir gene-inducing conditions, were infiltrated into complete leaf tissue. Histochemical β-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the induced bacteria entered into the intercellular spaces, thus cnabling T-DNA transfer in all cell layers of the leaf. The protocol was optimized for Phascolus vulgaris leaves, but similar results were obtained with other plant species, such as Phaseolus acutifolius, poplar, and tobacco. A β-glucuronidase chimeric gene interrupted by an intron was used as a marker for histological detection of the sectors.