Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum)

Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta β-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable gene expression in switchgrass leaves. Assays on extracts from leaves were also performed to measure expression levels; however, these assays showed low expression levels, which may have been due to low recombinant protein recovery and decomposition in the leaf. Bead beating was successful for wounding the plant surface, but did not improve the consistency of expression based on histochemical staining observations. Surfactant was necessary for improving contact between the leaf surface and Agrobacterium suspension and consistently improved expression when vacuum application level was low (25 kPa). Increasing vacuum application from 25 to 5 kPa improved expression only when surfactant concentration was low. When a suspension of A. tumefaciens containing 1000 ppm Break-Thru surfactant was added to harvested leaves and 25 kPa vacuum applied, a fairly uniform expression was visualized across the leaf surface within 2-3 days of incubation, suggesting that agroinfiltration is a rapid tool for examining expression of transgenes in switchgrass leaves.