Submitted to: Transgenic Research
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/1/2006
Publication Date: 4/1/2007
Citation: Bregitzer, P.P., Cooper, L.D., Hayes, P.M., Lemaux, P.G., Singh, J., Sturbaum, A.K. 2007. Viability and bar expression are negatively correlated in oregon wolfe barley dominant hybrids. Transgenic Research. Plant Biotechnology Journal v 5 p 381-388 Interpretive Summary: The production of transgenic cereals generally involves the introduction of DNA into, and plant regeneration from, individual cells that grow as a mass (called "callus") of cells. Many of these cells will be capable of regenerating into plants, but only a small fraction will be transgenic. To avoid the task of hunting for that rare transgenic plant among hundreds or thousands of non-transgenic plants, selectable markers are employed. A selectable marker is essentially another gene that encodes a protein that imparts a special characteristic that enables separation of the non-transgenic cells from transgenic cells. Selectable markers are most often antibiotic or herbicide resistance genes. For barley and other cereals, the herbicide resistance gene bar has been very useful, not only because it helps identify transgenic plants but because it has not been seen to cause any harm to the plants nor interact substantially with normal plant processes. However, in our work with transgenic barley plants, we found one type of barley--a specialized stock used for research purposes called Oregon Wolfe Barley Dominant (OWBD)--that does not tolerate the expression of the bar gene. Introduction of this gene into OWBD plants makes them very unhealthy, and they often die. Problems of this type have never been observed in a number of other studies of transgenic cereals containing this gene. This research raises questions: have we not looked closely enough to detect problems--perhaps more minor than those seen in this study--in the previous studies? Or have we just discovered an unusual case of genetic incompatibility that has no relevance to the more widespread use of this selectable marker? The present study examined barley other than OWBD but finds no evidence of problems, suggesting that the problems seen in OWBD may have only limited consequences.
Technical Abstract: The expression level of the selectable marker bar, which encodes phosphinothricin acetyl transferase (PAT), was negatively correlated with the viability of barley hybrids between 20 Golden Promise-derived transgenic lines (Ds-bar lines) and a specialized marker stock, Oregon Wolfe Barley Dominant (OWBD). Each Ds-bar line was homozygous for a single modified maize Ds element that encoded bar and which had been delivered via transposition to a different genomic location in each line. All Ds-bar lines were viable and morphologically similar. Only 4 of the 20 hybrid populations were viable. The remaining populations died as seedlings, or survived to the tillering stage but died before producing seed. It was hypothesized that the OWBD genome could not tolerate high levels of phosphinothricin acetyl transferase (PAT), the protein encoded by bar; that the Ds-bar lines had variable and heritable levels of bar expression; and that inviable hybrids had high levels of bar expression. Three lines of evidence support this hypothesis. First, unrelated transgenic lines that had been previously characterized as having variable and heritable levels of bar expression were crossed to OWBD; the viability of these hybrids was negatively correlated with bar expression in their transgenic parent. Second, ELISA and qRT-PCR data showed variable bar expression among Ds-bar lines that was negatively correlated with the viability of their hybrids with OWBD. Finally, ELISA and qRT-PCR data showed that the relative levels of bar expression in Ds-bar lines were heritable in hybrid progeny derived from crosses to barley genotypes other than OWBD. All hybrids were viable in the latter crosses, suggesting a peculiar sensitivity of OWBD to PAT.