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

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 beta-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 beta-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.

Figure 1

Microtitre plate with switchgrass seedlings assayed for GUSPlus activity after treatment with or without thiol compounds. Coculture with (a) Agrobacterium in water and acetosyringone; (b) with Agrobacterium in water, acetosyringone and L-cysteine; (c) with Agrobacterium in water, acetosyringone and dithiothreitol (DTT); and (d) with Agrobacterium in water, acetosyringone, L-cysteine and DTT.

Figure 2

Microtitre plate of seedlings inoculated with Agrobacterium, treated by sonication, needle wounding or mixing by vortex with carborundum and stained for β-glucuronidase (GUS)Plus activity. (a) Control (sonicated) switchgrass seedlings; (b) sonicated seedlings that were needle inoculated; (c) sonicated seedlings that were mixed by vortex with carborundum. All seedlings were assayed for GUSPlus activity.

Figure 3

Microtitre plate comparing switchgrass seedlings after a 3-day inoculation with Agrobacterium harboring two different β-glucuronidase (GUS) plasmids and assayed for GUS activity. Switchgrass seedlings inoculated with Agrobacterium harboring (a) pCambia 1305.1 and (b) pCambia 1305.2.

Figure 4

Duration of β-glucuronidase (GUS) expression after a 3-day cocultivation with Agrobacteriumharboring pCambia 1305.1, pCambia 1305.2 and a control. Seedlings cocultivated with (a, c) pCambia 1305.1 and (b, d) pCambia 1305.2 at 3 and 5 days after cocultivation, respectively, and stained for GUS. (e, f) Control seedlings at 2 and 7 days after cocultivation without Agrobacterium, respectively, and stained for GUS.

Figure 5

MU fluorescence due to β-glucuronidase (GUS) activity in switchgrass seedlings days after a 3-day cocultivation with Agrobacterium harboring pCambia1305.1, pCambia1305.2 and a control. The number of days after cocultivation is shown on the x axis, and the 4-methylumbelliferone (MU) fluorescence per seedling is shown on the y axis. Standard errors are shown for each measurement.