a high-throughput transient gene expression system for switchgrass (panicum virgatum l.) seedlings柳枝稷的高通量瞬时基因表达系统(黍virgatum l .)幼苗.pdfVIP

Chen et al. Biotechnology for Biofuels 2010, 3:9 /content/3/1/9 R E S E A R C H Open Access Research A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings Xinlu Chen, Raymie Equi, Holly Baxter, Kyle Berk, Jin Han, Sujata Agarwal and Janice Zale* Abstract 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 sho