A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings
© Chen et al; licensee BioMed Central Ltd. 2010
Received: 27 October 2009
Accepted: 7 May 2010
Published: 7 May 2010
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.
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.
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.
Perennial lowland switchgrass (Panicum virgatum L.) was chosen by Oak Ridge National Lab as a herbaceous biofuel crop of choice in 1991 because of its relatively high biomass yields in a number of replicated trials across seven states in the USA . Switchgrass can be propagated by seed, survives drought better than Miscanthus , and has the ability to grow on marginal land with low fertility requirements, increasing its attractiveness in southeast USA.
Most of the economically important monocots have been relatively recalcitrant to genetic transformation compared with some dicots . Transient gene expression [4, 5] and stable genetic transformation of embryogenic calli in switchgrass have been reported [6–8]. In planta transformation of germinating cereal seedlings has been demonstrated after needle inoculation [9, 10], and after shoot excision with no callus phase .
Several wounding treatments and additives have been shown to increase Agrobacterium-mediated transient gene expression and stable genetic transformation. Thiol compounds [12, 13], sonication-assisted Agrobacterium transformation (SAAT) , a combination of SAAT and vacuum infiltration , heat and separation by centrifugation , surfactants , and mixing by vortex with carborundum  have been applied to a variety of explants of different species in an effort to increase transient gene expression and hence stable transformation of plants.
Transient gene expression systems are ideal for testing and comparing genetic constructs; however, increases in transient gene expression does not have a definite correlation with an increase in the production of stable transformants. Alpeter et al. concluded in 1996 that transient gene expression was not correlated with stable transformation in wheat . However, in other studies, increased numbers of transgenic wheat and corn were regenerated from dissected explants after optimization of transient expression from reporter genes [17, 19]. Transformation efficiencies of soybean and Ohio buckeye were also increased after optimization of transient expression .
In this paper, we describe optimization experiments and wounding treatments that significantly increased transient expression of a commercial reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS) gene) in germinating switchgrass seedlings. The optimization experiments determined the most favorable Agrobacterium strain and acetosyringone concentrations. Wounding treatments such as sonication, mixing by vortex with carborundum, vacuum infiltration, needle wounding, separation by centrifugation, heat treatments, and additives such as L-cysteine, dithiothreitol (DTT), acetosyringone and surfactants were systematically tested in an effort to determine which treatment or combination of treatments increased transient GUS expression and the likelihood of producing stable transformants in switchgrass.
Results and discussion
Comparisons of different Agrobacterium strains and acetosyringone concentrations
Four different Agrobacterium strains (AGL1, C58, GV3101 and EHA105) were tested for their ability to deliver transfer (T)-DNA to dehusked, 3-day-old switchgrass seedlings at various acetosyringone concentrations (0, 50, 100 and 200 μM). All seedlings were treated with one of four Agrobacterium strains, sonicated for 1 minute, incubated for 30 minutes and then cocultivated with or without different concentrations of acetosyringone for 3 days. Seedlings were inoculated with each strain, which harbored the vector pCambia1305.2, and after cocultivation for 3 days, the number of GUS-positive seedlings was assessed. pCambia1305.2 carries the CaMV35S promoter:GRP signal peptide: catalase intron: GUSPlus: nos terminator . The GUSPlus gene was originally isolated from a Staphylococcus species and is more stable at higher temperatures and in fixatives than the β-glucuronidase gene cloned from Escherichia coli . The microbial glycine-rich signal peptide (GRP) from the lac Z alpha fragment permits secretion of GUS from the cytoplast into the apoplast .
Comparison of GUSPlus expression in switchgrass seedlings induced by four Agrobacterium strains, at different acetosyringone concentrations.
Mean ± SD*
9.0 ± 3.0a
5.0 ± 1.2b
1.5 ± 0.3c
0.5 ± 0.3c
The effects of various treatments and additives on GUSPlus expression
A comparison of thiol compounds and their effect on transient gene expression of GUSPlus.
Mean ± SE†
7.0 ± 0.6b
H2O, cysteine, AS
8.7 ± 2.3b
H2O, DTT, AS
9.7 ± 2.2b
H2O, cysteine, DTT, AS
15.7 ± 1.9a
A comparison of mixing by vortex with carborundum, needle wounding and sonication on GUSPlus expression.
Mean ± SE†
9.3 ± 1.2c
21.0 ± 2.1a
16.0 ± 2.3b
A comparison of vacuum infiltration and separation by centrifugation on GUSPlus expression in sonicated switchgrass seedlings.
Mean ± SE†
Control-1 (no vacuum; no separation by centrifugation)
4.2 ± 0.6b
Control-2 (no vacuum; separation by centrifugation)
10.8 ± 2.6a
Vacuum (1 minute); separation by centrifugation
10.8 ± 1.6a
Vacuum (2 minutes); separation by centrifugation
11.0 ± 2.1a
Vacuum (4 minutes); separation by centrifugation
12.2 ± 3.2a
Vacuum (8 minutes); separation by centrifugation
12.5 ± 0.5a
Vacuum (16 minutes); separation by centrifugation
13.8 ± 1.4a
A comparison of two different surfactants, at five concentrations, on transient gene expression of GUSPlus.
GUS- positive seedlings, n†
GUS- positive seedlings, %
GUS- positive seedlings, n†
GUS- positive seedlings, %
GUS- positive seedlings, mean ± SE
22.0 ± 0.8a
23.7 ± 0.5a
26.9 ± 1.4a
19.3 ± 1.6b
4.4 ± 0.8c
The effect of heat shock on transient expression of GUSPlus.
Mean ± SE†
22.0 ± 1.2b
27.0 ± 0.6a
26.0 ± 0.6a
25.3 ± 0.9a
25.7 ± 0.9a
GUS expression comparisons between pCambia 1305.1 and 1305.2
Comparison of GUSPlus activity between pCambia 1305.1 and 1305.2 after 3 days of cocultivation.
Mean ± SE†
12.0 ± 1.1a
19.0 ± 2.6b
These experiments optimized the treatments, additives and Agrobacterium strains for transient gene expression in switchgrass. The Agrobacterium strain AGL1 was most able to infect 3-day-old Alamo switchgrass seedlings in the presence of 100 μM acetosyringone. Double or triple wounding treatments resulted in the highest levels of transient GUSPlus expression. The treatments that significantly increased GUS expression were sonication, needle wounding, mixing by vortex with carborundum, separation by centrifugation and heat shock. Mixing by vortex with carborundum and separation by centrifugation appeared to have the greatest effect on GUS expression in switchgrass. The addition of L-cysteine and DTT during cocultivation also significantly increased GUS expression. Transient gene expression was increased from 6% in untreated seedlings to 54% after the application of a sequential set of treatments and additives. The GUS genes encoded on the two pCambia plasmids, 1305.1 and 1305.2, had different temporal expression patterns in switchgrass seedlings, and histochemical and MUG assays suggest that the enzyme activity of 1305.1 accumulates in the cytosol over a longer period than that of 1305.2.
Plant material and preparation
Alamo seeds, purchased from the Bamert Seed Company in Muleshoe, TX, were dehusked by soaking in 60% H2SO4 for 30 minutes with shaking, and then washed six times in sterile distilled water with shaking, 5 minutes per wash. The seeds were sterilized in 250 ml of 100% commercial bleach (6% NaClO) with 0.05% of Tween-20 for 30 minutes with shaking and washed six times with sterile distilled water with shaking, 5 minutes per wash. Sterilized seeds were plated on seed germination medium composed of MS salts , supplemented with Gamborg B5 vitamins , 2% sucrose, 0.3% Gelrite (Research Products International, Mt. Prospect, IL), pH 5.8 and maintained at 24 ± 2°C in the dark.
Agrobacterium strains, plasmid and bacterial induction
Four Agrobacterium strains, AGL1 , EHA105 , GV3101  and C58  were evaluated for their ability to infect germinating switchgrass seeds. AGL1 and EHA105 are hypervirulent strains [27, 28]. All Agrobacterium strains harbored pCambia1305.1 or pCambia 1305.2 http://www.cambia.org/daisy/cambia/585.html . These plasmids are the same except the pCambia1305.2 carries the GRP signal peptide sequence which permits secretion of GUSPlus enzyme from the cell. Both carry the catalase intron:GUS sequence to prevent expression of GUS genes in Agrobacterium and the hygromycin gene as a plant selectable marker. Four different concentrations of acetosyringone (0, 50, 100, 200 μM) were tested to determine which induced the virulence of Agrobacterium to the greatest extent as determined by GUS staining.
The Agrobacterium was grown in liquid YEP medium (10 g l-1 Bacto Peptone, 10 g l-1 yeast extract, 5 g/l NaCl, pH7.0) overnight and separated by separation by centrifugation the next morning at 1376× g at room temperature (22 ± 4°C) for 10 minutes. The bacterial pellet was gently resuspended in liquid resuspension solution (0.1× MS, 1× vitaminB5, 3% sucrose, 1.2 g/l 2-(N-Morpholino)ethanesulfonic acid (MES), pH 5.4) and diluted to OD600 = 1.0, then 1 M acetosyringone (Acrose Organics, Morris Plains, NJ, USA) dissolved in dimethyl sulfoxide, and added to a final concentration of 100 μM for a 3-hour induction period (henceforth termed the Agrobacterium resuspension solution).
Treatments and Agrobacterium inoculation
To test which strain of Agrobacterium induced the greatest GUSPlus staining, four Agrobacterium strains harboring pCambia1305.2 were used to inoculate 3-day-old switchgrass seedlings. In some treatments, the junction area between the root and shoot were pierced two or three times with a sterile needle that had been dipped in Agrobacterium resuspension solution under a dissecting microscope (Stereomaster, Fisher Scientific, Pittsburgh, PA, USA). After wounding, the entire seedlings were placed in Agrobacterium resuspension solution, incubated for 30 minutes and placed onto sterile 8.4 cm filter paper in a 100 × 15 mm Petri dish (BD Biosciences, Franklin lakes, NJ, USA) wetted with either 1.6 ml of sterile H2O and 100 μM acetosyringone or 1.6 ml of a solution comprising H2O, DTT (154 mg/l; ), L-cysteine (400 mg/l; [11, 12]) and acetosyringone (100 μM), incubated at room temperature in the dark for 3 days of cocultivation. Three replicates, with 50 seedlings per replicate were tested for each strain and at each acetosyringone concentration.
For the sonication treatments, 10 seedlings were placed in sterile tubes to which 500 μl of Agrobacterium resuspension solution was added, and the tubes were placed in a sonicator (Branson 1210; Fisher Scientific, Atlanta, GA, USA) for various durations (0, 0.5, 1, 2, 4 and 8 minutes), after incubation with Agrobacterium resuspension s olution for 30 minutes. The seedlings were spread onto sterile 8.4 cm filter paper in a 100 × 15 mm Petri dish; the paper was wetted with either 1.6 ml of sterile H2O and 100 μM acetosyringone, or sterile H2O, DTT (154 mg/l; ), L-cysteine (400 mg/l; [12, 13]) and acetosyringone (100 μM), and were cocultured for 3 days at room temperature in the dark. There were three replicates, with 50 seedlings per replicate.
Vortex-carborundum-sonication treatments consisted of placing 50 seedlings in 50 ml tubes (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) to which 5 ml of Agrobacterium resuspension solution and 1 ml of 0.1% carborundum solution (w/v) (Fisher Scientific, Atlanta, GA, USA; ) were added. The tubes were then mixed by vortex at 4000 rpm for 2 minutes. After the carborundum treatment, the seedlings were placed in 1.5 ml microcentrifuge tubes (10 seedlings per tube), to which 500 μl of the Agrobacterium resuspension solution was added, and the tubes were sonicated for 1 minute. The sonicated seedlings were incubated for 30 minutes at room temperature, separated by centrifugation at 2400 g, for 1 minute, spread onto filter paper and cocultivated with Agrobacterium as described previously. There were three replicates, with 50 seedlings per replicate.
For vacuum infiltration treatments, 3-day-old seedlings were placed in sterile 1.5 ml microcentrifuge tubes (10 seedlings per tube) to which 500 μl of Agrobacterium resuspension solution was added, and the tubes were placed in a sonicator (Branson 1210; Fisher Scientific) for 1 minute. The tubes were then placed in a vacuum chamber under vacuum (610 mm of Hg) for 0, 1, 2, 4, 8 and 16 minutes. This was followed by incubation with Agrobacterium resuspension s olution for 30 minutes and separation by centrifugation (1 minute at 2400 g). The 3-day cocultivation was the same as described above.
Heat-shock treatments were applied by inserting the samples (10 seedlings per microcentrifuge tube in 500 μl Agrobacterium resuspension solution) into a heating block at various temperatures (25, 37, 40, 43, 47 degrees Celsius) for 2 minutes, then placing the tubes into a sonicator for 1 minute (as previously described), followed by incubation for 30 minutes, and separation by centrifugation at 2400 for 1 minute. The 3-day cocultivation was as described above, using three replicates, with 50 seedlings per replicate.
For surfactant treatments, two surfactants (Li700 (Loveland Products Inc., Greeley, Colorado, USA) and Silwet L-77 (Lehle Seeds, Round Rock, TX, USA)) were compared at five concentrations (0, 0.01, 0.02, 0.04 and 0.1% v/v). The surfactants were added to the Agrobacterium resuspension solution in which the seedlings were placed (each tube contained 500 μl of solution per 10 seedlings). The samples were sonicated for 1 minute, incubated for 30 minutes and separated by centrifugation at 2400 for 1 minute. The 3-day cocultivation was as described above, using three replicates, with 50 seedlings per replicate.
Histochemical and fluorometric assay of GUS expression
GUS staining was performed according to Jefferson et al.  with some modifications. The seedlings were incubated overnight at 37°C in a solution containing 50 mM sodium phosphate buffer (pH 7.0), 2 mM EDTA, 0.12% Triton, 0.4 mM ferrocyanide, 0.4 mM ferricyanide, 1.0 mM 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium salt (X-Gluc) (Gold Biotechnolgy, St. Louis, MO, USA) and 20% methanol anhydrate. Seedlings with GUS foci > 2 mm were counted as positive.
Fluorometric MUG assays were performed according to Jefferson et al.  with some modifications. Shoots of 50 seedlings per treatment were excised, placed in 1.5 ml microcentrifuge tubes, and homogenized with a small mortar and pestle and liquid nitrogen. After homogenization, 250 μl of extraction buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% SDS, 10 mM β-mercaptoethanol) was added. The pellet was resuspended by mixing by vortex and separated by centrifugation at 10,000 at 40°C. The supernatant was removed, frozen in liquid nitrogen, and stored at - 80°C. Aliquots of the supernatant (25 μl) were added to 1 ml GUS assay buffer (2 mM 4-methylumbelliferyl-beta-D glucuronide (Sigma, St. Louis, MO, USA) and 10 mM β-mercaptoethanol in extraction buffer), incubated at 37°C for 5, 35 and 95 minutes. Samples (200 μl) of this reaction were mixed with 800 μl stop solution (0.2 M Na2CO3), and the fluorescence measure with excitation at 365 nm and emission at 455 nm in a FLx 800 fluorescent microplate reader (BioTek Instruments, Winooski, VT). Protein content was measured at 595 nm using a commercial kit (Quick Start Bradford Protein Assay Kit; Bio-Rad, Hercules, CA, USA). Three replicates of 50 seedlings per replicate were measured at each time interval.
The number of seedlings expressing GUS foci ≥ 2 mm was counted and analyzed using one-way or two-way ANOVA for a fixed effects model (Minitab. 15 software). If the counts were small (<10), tended toward a Poisson distribution and did not satisfy normality, the data were transformed using the square root transformation, and ANOVAs were performed on transformed data . Mean comparisons were performed using The Tukey multiple comparisons at the 5% level (Minitab. 15). Replicates were performed in time, using different solutions, and the entire series of experiments were repeated with different seed lots and sometimes different operators, but gave similar results.
XC, KB, RE, JH and HB were partially or fully supported by the BioEnergy Science Center (BESC). The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
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