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development, is disruption of pollen fertility. For example, inactivation of the UDP-
glucose pyrophosphorylase 1 gene for flower development has resulted in a male
sterile phenotype in rice (Woo et al. 2008). Post-zygotic methods may involve
transgene placement in the crop loci that confer lower fitness and competitiveness
to the wild relative and are thus negatively selected in the wild plants (Kwit et al.
2011). It is difficult to achieve with random T-DNA-based transformation, but
transgene insertion at a targeted locus is currently feasible using zinc-finger nucle-
ase technology (Li et al. 2009; Shukla et al. 2009). The creation of selectively
terminable transgenic lines represents another strategy, as demonstrated in rice by
the tagging of a gene of interest with an RNAi cassette that suppresses the bentazon
detoxification gene CYP81A6 (Lin et al. 2008). This has resulted in the creation of
rice that is sensitive to a major herbicide, bentazon. Therefore, any possible hybrids
outside of the field could be controlled by spraying bentazon during the conven-
tional rice weed control process.
Use of antibiotic or herbicide-resistance genes as a simple method to select for a
transformation event is often criticized, although the risk of their horizontal transfer
from engineered plant is essentially negligible (Bennett et al. 2004). The more
realistic threat is, however, the mobilization of genes and elements proximal to the
gene for antibiotic resistance, which is always also the heterologous gene-of-interest.
As genetic determinants of antibiotic resistance are widely distributed in the envi-
ronment, a potential mechanism of horizontal transfer involving homologous recom-
bination exists. The avoidance of antibiotic or herbicide markers is thus encouraged.
For example, the plants harboring antibiotic resistance transgenes are no longer
authorized for application in the EU since 2005 (EU directive 2001/1//EC). In this
context, alternative selection systems are being developed, including positive selec-
tion employing the E. coli pmi encoding a phosphomannose isomerase (Bojsen et al.
1998). Since mannose-6-phosphate formed from mannose in planta is toxic to
glycolysis in plants and Pmi enzyme converts this compound to natural metabolite
fructose-6-phosphate, pmi/mannose system offers benefits of positive selection.
However, the best solution to the selection marker problem is the precise excision
of the marker gene from a chromosome using site-specific recombinases, such as
Cre, FLP, PaA, or PhiC31 (Zuo et al. 2002; Gilbertson 2003; Thomson et al. 2009;
Kempe et al. 2010). This strategy would then render transgenes containing only
those heterologous genes, which are to be employed for the phytoremediation job.
12.3 Molecular Targets to Genetic Manipulation in Plants
Prerequisite to the accumulation of metal in the aboveground tissues is its mobili-
zation from soil, metal uptake and root-to-shoot translocation mechanism, and
competence to detoxify (over)accumulated metal species (Clemens et al. 2002).
Tight control and regulation of accumulation and homeostasis thus evolved in all
plants for essential metals and is of central importance both at organism and cellular
level. Uptake of nonessential metals employs the same mechanisms as adopted by