Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation AciDs plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences signifcantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.Arabidopsis thaliana is an excellent model plant for classical and molecular genetic studies. Current efforts toward characterizing the genome of Arabidopsis include restriction fragment length polymorphism analysis (1, 2), production and physical mapping of yeast artificial chromosome libraries (3-5), and Agrobacterium tumefaciens T-DNA insertional mutagenesis (6-8). An alternative means of isolating genes, which has been effective in many other model systems, is the use of transposable elements. In this paper, we report the development of a two-element Activator/Dissociation (Ac/ Ds) transposable element system suitable for gene tagging in Arabidopsis.The Ac/Ds transposable element system was originally discovered in maize by McClintock (9). It has since been shown that Ac transposes in Nicotiana tabacum (10) and numerous other dicotyledonous plant species (for review, see ref. 11). Its behavior in heterologous plant species has been most intensively studied in tobacco (10, 12-21), tomato (22-24), and A. thaliana (25-28). Many transposition-related properties associated with Ac activity in maize are also found in heterologous systems. For instance, the 4.6-kilobase (kb) autonomous element forms 8-base-pair duplications upon insertion (10), is capable of mobilizing receptor Ds elements (12,13,25,29), and preferentially transposes to nearby locations (16,28).To develop an efficient transposon tagging system for Arabidopsis and other plants, we have constructed a twoelement Ac/Ds system containing plant selectable markers designed to monitor the presence of Ac, Ds, and Ds-related excision events (25,30). A marked Ds element (DsDHFR) was constructed by inserting a plant selectable marker cassette, including a dihydrofolate reductase (DHFR) gene under the control of a cauliflower mosaic virus (CaMV) 35S promoter, into the central region of a Ds element. We have previously shown that the DsDHFR element transposes in tobacco and Arabidopsis, and the DHFR cassette does not decrease the frequency of Ds transposition (25). Furthermore, it was determined that approximately two-thirds of excised DsDHFR elements reinserted in transgenic tobacco protoplasts while the oth...
Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.
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