Page 249 - Plant-Based Remediation Processes
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242                                                         P. Kotrba

            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
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