Saccharomyces cerevisiae, and R/RS from Zygosaccharomyces rouxii [reviewed by
Ebinuma et al. (2001)]. The target sequences are placed around the genes targeted for
excision followed by the introduction of the recombinase in a second round of transform-
ation (Dale and Ow 1991). Again, this approach suffers from the criticism that it is
restricted to seed-propagated plants to segregate the recombinase gene from the gene
of interest. This has been partially overcome by the introduction of the recombinase
by transient expression. Although excision occurs at a lower frequency, the recombinase
gene is not integrated into the genome. Another promising approach incorporated the
gene of interest along with the selectable marker genes and recombinase gene on one
vector (Fig. 9.13). This strategy overcomes many of the earlier limitations by using an
inducible promoter to express the recombinase, resulting in the autoexcision of the
recombinase and the selectable marker genes simultaneously (Zuo et al. 2001;
Ebinuma et al. 2001). This approach eliminates the need for successive rounds of trans-
formation or crossing and minimizes the period of exposure of plants to the action of
recombinases. Prolonged exposure to recombinases is a concern as unpredictable del-
etions in the genome may occur due to the action of the recombinases on cryptic
target sites. Although the extent of such deletions is uncertain in the nuclear genome,
examples have been reported in the plastid genome with Cre. With time the excision
systems and gene regulatory systems will improve, and the technology is likely to be
refined to practical levels. The strategies discussed above as well as other methods for
marker gene removal in plants have been reviewed by Darbani et al. (2007).
9.7 Conclusions
It is difficult to imagine plant biotechnology without marker genes. As we have seen, effec-
tive marker genes have demonstrated little to no effect on the plant except their intended
effect. Nonetheless, marker genes have been somewhat controversial, especially antibiotic
resistance genes, because of the concern about horizontal gene transfer (HGT). HGT is the
movement of DNA from one species to an unrelated species—in this case from transgenic
plants to bacteria. In the event that an antibiotic resistance gene were to be horizontally
transferred to bacteria, some people worry that new antibiotic resistance problems could
be created that could harm human or ecosystem health. Even though HGT has not been
demonstrated from transgenic plants to bacteria in a realistic experimental system, it has
affected the politics of regulation and the perception of transgenic plants, which will be
covered in later chapters.
9.7. CONCLUSIONS 237