Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue

Abstract

The development of a barley (Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation (Ac/Ds) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time‐consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co‐transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uidA reporter gene (Gus), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase‐mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.

Temperature Shift Coordinately Changes the Activity and the Methylation State of Transposon Tam3 in Antirrhinum majus

“…If Tam3 TPase activity is present in a plant cell, the Tam3 element is expected to be excised from the plasmid DNA after integration into the cell. A transient assay for Ac activity used to demonstrate Ds excision after bombardment of transgenic barley (Hordeum vulgare) callus lines containing Ac has been reported (McElroy et al, 1997). Using this assay, we delivered the plasmid pBS18-10E, carrying a 1.6-kb partial Tam3 DNA sequence, into leaf cells through particle bombardment.…”

The hAT family: a versatile transposon group common to plants, fungi, animals, and man

“…This allows selection of plants with transposed and reinserted copies of Ds (Hehl and Baker 1989;Lassner et al 1989;Masterson et al 1989;Balcells et al 1991). The activity of Ac has been demonstrated in many different plants, including barley (McElroy et al 1997), tobacco (Jones et al 1990), tomato (Healy et al 1993), and rice (Izawa et al 1991;Murai et al 1991). For more details see Kunze (1996).…”

Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the Lr21-Mediated Leaf Rust Resistance Pathway

“…Mutagenized diploid wheat collections are not available, and, since wheat is recalcitrant to T-DNA transformation, no T-DNA insertion libraries exist in diploid wheat that could be used to streamline gene isolation. No active wheat transposons have been characterized, and transgenic systems for transposon-based gene tagging are not widely available (McElroy et al, 1997). Despite these obstacles, at least three wheat R-genes, Lr21 (Huang et al, 2003), Lr10 (Feuillet et al, 2003), and Pm3b (Yahiaoui et al, 2004), have been isolated through strategies employing chromosome walking followed by complementation in transgenic plants to confirm gene function.…”