Abstract
BackgroundSulfonamide resistance is conferred by the sulI gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. The sulI gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or Arabidopsis using sulI as a selectable marker generates sulfadiazine-resistant plants. Typically sulI-based selection of transgenic plants is performed on tissue culture media under sterile conditions.FindingsA set of novel binary vectors containing a sulI selectable marker expression cassette were constructed and used to generate transgenic Arabidopsis. We demonstrate that the sulI selectable marker can be utilized for direct selection of plants grown in soil with a simple foliar spray application procedure. A highly effective and inexpensive high throughput screening strategy to identify transgenic Arabidopsis without use of tissue culture was developed.ConclusionNovel sulI-containing Agrobacterium binary vectors designed to over-express a gene of interest or to characterize a test promoter in transgenic plants have been constructed. These new vector tools combined with the various beneficial attributes of sulfonamide selection and the simple foliar screening strategy provide an advantageous alternative for plant biotechnology researchers. The set of binary vectors is freely available upon request.
Highlights
Sulfonamide resistance is conferred by the sulfonamide resistance gene (sulI) gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons
Further analysis has demonstrated that the nos promoter, in contrast, does not alter the expression conferred by nearby promoters [27,28] and was chosen as the promoter to control sulI expression in pSUNG
We speculate that this is likely due to the nos promoter driving lower levels of sulI expression in the transgenic plants than is typically observed for the double enhanced CaMV 35S promoter. We believe this is a likely explanation, other potential causes for this difference are possible. These results demonstrate that repeated foliar application of the sulfadiazine/silwet solution does not cause tissue damage to sulI resistant plants and can be successfully used to identify transgenic Arabidopsis
Summary
Sulfonamide resistance is conferred by the sulI gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. Transformation of tobacco, potato or Arabidopsis using sulI as a selectable marker generates sulfadiazine-resistant plants. Used selectable marker genes for plant transformation include the neomycin phosphotransferase II gene (nptII) [4,5,6] that confers resistance to aminoglycoside antibiotics such as kanamycin, neomycin, and G418, as well as the hygromycin phosphotransferase II gene (hptII) [7] and bialaphos resistance gene (bar) [8] that confer resistance to hygromycin and the herbicide glufosinate, respectively. A lesser known and less frequently utilized plant selectable marker is the sulfonamide resistance gene (sulI), which confers resistance to sulfadiazine and other sulfonamide chemicals [9]. SulI is used less often than nptII, hptII, and bar due to its infrequent inclusion as a marker in the commonly used plant transformation vectors. Sulfonamides exhibit substantial potency as a plant selection agent; e.g. 5 mg/L sulfadiazine is sufficient for Arabidopsis selection [12]
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