Abstract
Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.
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This work was supported by the Brain Korea 21, Biogreen 21 (20080401034001), and National Research Laboratory Programs and by grants from the Plant Signaling Network Research Center, the Korea Science and Engineering Foundation (2007-03415), and from the Agricultural R & D Promotion Center (309017-5), Korea Ministry for Food, Agriculture, Forestry and Fisheries.
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Communicated by J. R. Liu.
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Kim, M.J., Baek, K. & Park, CM. Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis . Plant Cell Rep 28, 1159–1167 (2009). https://doi.org/10.1007/s00299-009-0717-z
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DOI: https://doi.org/10.1007/s00299-009-0717-z