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Background  Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass ( Panicum virgatum  L.) has previously been reported after cocultivation of explants with  Agrobacterium  and biolistics of embryogenic calli. Experiments to increase transient gene expression  in planta  may lead to stable transformation methods with increased efficiency.    Results  A high-throughput  Agrobacterium -mediated transient gene expression system has been developed for  in planta  inoculation of germinating switchgrass seedlings. Four different  Agrobacterium  strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS) gene). The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG) assays showed a peak of β-glucuronidase (GUS) enzyme activity 3 days after cocultivation with  Agrobacterium  harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with  Agrobacterium  harboring pCambia1305.1.    Conclusion   Agrobacterium  strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo) compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock. The addition of thiol compounds such as L-cysteine and dithiothreitol in combination with acetosyringone during cocultivation also increased transient expression. The combination of multiple wounding treatments along with the addition of thiol compounds during cocultivation increased transient expression levels from 6% to 54%. There were differences in temporal GUS expression induced by pCambia1305.1 and pCambia1305.2.

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings