Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Sulfonamide resistance is conferred by the <it>sul</it>I gene found on many <it>Enterobacteriaceae </it>R plasmids and Tn21 type transposons. The <it>sul</it>I gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or <it>Arabidopsis </it>using <it>sul</it>I as a selectable marker generates sulfadiazine-resistant plants. Typically <it>sul</it>I-based selection of transgenic plants is performed on tissue culture media under sterile conditions.</p> <p>Findings</p> <p>A set of novel binary vectors containing a <it>sul</it>I selectable marker expression cassette were constructed and used to generate transgenic <it>Arabidopsis</it>. We demonstrate that the <it>sul</it>I 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 <it>Arabidopsis </it>without use of tissue culture was developed.</p> <p>Conclusion</p> <p>Novel <it>sul</it>I-containing <it>Agrobacterium </it>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.</p

Generation and Characterization of the Western Regional Research Center Brachypodium T-DNA Insertional Mutant Collection

<div><p>The model grass <i>Brachypodium distachyon</i> (<i>Brachypodium</i>) is an excellent system for studying the basic biology underlying traits relevant to the use of grasses as food, forage and energy crops. To add to the growing collection of <i>Brachypodium</i> resources available to plant scientists, we further optimized our <i>Agrobacterium tumefaciens</i>-mediated high-efficiency transformation method and generated 8,491 <i>Brachypodium</i> T-DNA lines. We used inverse PCR to sequence the DNA flanking the insertion sites in the mutants. Using these flanking sequence tags (FSTs) we were able to assign 7,389 FSTs from 4,402 T-DNA mutants to 5,285 specific insertion sites (ISs) in the <i>Brachypodium</i> genome. More than 29% of the assigned ISs are supported by multiple FSTs. T-DNA insertions span the entire genome with an average of 19.3 insertions/Mb. The distribution of T-DNA insertions is non-uniform with a larger number of insertions at the distal ends compared to the centromeric regions of the chromosomes. Insertions are correlated with genic regions, but are biased toward UTRs and non-coding regions within 1 kb of genes over exons and intron regions. More than 1,300 unique genes have been tagged in this population. Information about the Western Regional Research Center <i>Brachypodium</i> insertional mutant population is available on a searchable website (<a href="http://brachypodium.pw.usda.gov" target="_blank">http://brachypodium.pw.usda.gov</a>) designed to provide researchers with a means to order T-DNA lines with mutations in genes of interest.</p></div

Distribution of insertion sites between genic and intergenic regions.

<p>A comparison of the observed and expected percentages of T-DNA insertions is illustrated for each insertion class. Insertions in UTRs and within 1 kb of a gene are observed to be substantially higher than expected. Intergenic insertions >1 kb from a gene are lower than expected.</p

Chromosome distribution of FSTs, ISs, and genes.

<p>Chromosome distribution of FSTs, ISs, and genes.</p

Evaluation of transposon tagging in <i>Brachypodium</i>.

<p><b>A.</b> Diagram of the T-DNA region of Ac-DsATag-Bar_gosGFP. The mobile element is depicted on the upper line. Note that when the mobile element excises the empty donor site (EDS) can be amplified by the flanking PCR primers, solid black arrows. <b>B.</b> Examples of transgenic plants created using Ac-DsATag-Bar_gosGFP. Most plants containing the transposable elements died before flowering or failed to set seed. <b>C.</b> PCR using primers that flank the Ds mobile element. An EDS produces a band while an intact donor site will not produce a band. Note that five of eight lines contain an EDS indicating that Ds was excised. The hygromycin resistance gene was amplified from all lines indicating that they contained at least part of the T-DNA. <b>D.</b> Examples of transgenic plants created using the pdSpm-R tagging construct and the control vector pOL001. Most plants containing the transposable elements died when very small. <b>E.</b> Calluses transformed with the pdSpm-R tagging construct were fluorescent green due to the expression of GFP contained on the T-DNA. By contrast, calluses transformed with a control plasmid, pOL001, were not fluorescent. For both constructs, pictures on the left are taken under UV light through a GFP filter and the pictures on the right are under white light.</p

Flanking sequence tags (FSTs) generated for T-DNA lines.

<p>Flanking sequence tags (FSTs) generated for T-DNA lines.</p

Sequences and locations of primers used for Inverse PCR.

<p>Sequences and locations of primers used for Inverse PCR.</p

T-DNA constructs evaluated for transformation efficiency.

<p>T-DNA constructs evaluated for transformation efficiency.</p