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


CONCLUSIONS
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.
Chen
Xinlu
X
University of Tennessee, Department of Plant Sciences, 2431 Joe Johnson Drive, Knoxville, TN, 37996, USA. jzale@utk.edu.
Equi
Raymie
R
Baxter
Holly
H
Berk
Kyle
K
Han
Jin
J
Agarwal
Sujata
S
Zale
Janice
J
J Biosci Bioeng. 2005 Oct;100(4):391-7
16310727
Plant Physiol. 2002 May;129(1):13-22
12011333
Exp Cell Res. 1968 Apr;50(1):151-8
5650857
Plant Cell Rep. 1998 Apr;17(6-7):482-488
30736623
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8467-71
2554318
Plant Cell Rep. 2009 Jun;28(6):903-13
19308413
Plant Cell Rep. 1996 Nov;16(1-2):12-7
24178645
Plant Physiol. 1997 Nov;115(3):971-980
12223854
Methods Mol Biol. 2009;581:53-9
19768615
Annu Rev Plant Physiol Plant Mol Biol. 1997 Jun;48:297-326
15012265
Plant Cell Rep. 2006 Nov;25(11):1199-204
16773333
Experientia. 1971 Feb 15;27(2):229-30
5544763
Biotechnology (N Y). 1991 Oct;9(10):963-7
1368724
J Biosci Bioeng. 2006 Sep;102(3):162-70
17046528
Plant Cell Rep. 2009 Mar;28(3):387-95
19048258
Biotechnol J. 2008 Mar;3(3):354-9
18064611
Plant Methods. 2009 May 20;5:6
19457242
Plant Biotechnol J. 2008 Sep;6(7):663-78
18498309
Plant J. 1998 Dec;16(6):735-43
10069079