Containing Gene Flow

Several molecular mechanisms have been suggested for containing gene flow (i.e., to prevent gene flow between the phytoremediating species and relatives), especially by pollen, ignoring the other routes of sporophyte propagule (seeds and asexual parts) movement, especially transgenes within the phytoremediating species (i.e., to prevent outflow to related species), or to mitigate the effects of transgene flow once it has occurred [20, 22, 26, 29]. It is more important to prevent gene flow from the phyto-remediating species to outside the contaminated site than to prevent influx into the phytoremediation site, as the phytoremediating species should be most fit to live the contaminated site. Even though the hybrids may be the same in either direction, the likelihood of such a hybrid establishing on a phytoremediation site is minimal.

3.2.1 Containment by targeting genes to a cytoplasmic genome

The most widely discussed containment possibility is to integrate the transgene of choice in the plastid or mitochondrial genomes [30-32]. There are good reasons to engineer phytoremediating genes into chloroplasts besides the presumed biosafety. The chloroplasts are often the targets of environmental contaminants and need protection. Additionally, many genes of value come from bacteria with similar codon usage as chloroplasts. Such genes often need to be re-engineered to plant codon usage before inserting into the nuclear genome [33]. Indeed the bacterial genes merA/merB that convert organomercurials into elemental mercury (which is later volatilised) were successfully introduced into chloroplasts of tobacco [34]. Still, the same genes were active in Arabidopsis when the merB was augmented with a peptide that targeted the gene product into the endoplasmic reticulum, despite the bacterial codon usage differences [35].

The opportunity of gene outflow is limited due to the predominantly maternal inheritance of these genomes in many, but far from all species. This is presently an arduous technology, which so far is limited to a few species. It does not preclude the outside species from pollinating the bioremediating species, and then acting as the recurrent pollen parent, but this is less of a problem on a bioremediation site than off site.

The claim of strict maternal inheritance of plastome-encoded traits [32, 36, 37] was not substantiated. Tobacco [38] and other species [39] often have between a 10-3-10-4 frequency of pollen transfer of plastid inherited traits. Pollen transmission of plastome traits can only be easily detected using both large samples and selectable genetic markers. A large-scale field experiment utilized a Setaria italica (foxtail or birdseed millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant leaf marker) crossed with five different male sterile herbicide susceptible lines. Chloroplast-inherited resistance was pollen transmitted at a frequency of 3 x 10-4in >780,000 hybrid offspring [40]. At this transmission frequency, the probability of transgene movement via plastomic gene flow is orders of magnitude greater than by spontaneous nuclear genome mutations. Thus, chloroplast transformation is probably unacceptable for preventing transgene outflow, unless stacked with additional mechanisms, and as noted above, will not at all impede gene inflow. Maliga [32] discounts the relevance of the findings with tobacco and Setaria as being due to an origin of the plastids from interspecific (closely related) cytoplasmic substitution, where pollen transmission barriers can break down [41]. Setaria viridis, the wild progenitor of Setaria italica is biologically con-specific with it [42]. There are two problems with this denigration of the relevance of pollen movement of plastome encoded genes: 1) it is just such interspecific movement that could be a problem between phytoremediating species and related species; 2) he [32] ignores the discussion in Darmency et al. [39] of cases of intraspecific transmission of plastomic traits by pollen at about the same frequency, within the same species, as reported above between species.

3.2.2 Male sterility coupled with transplastomic traits

A novel additional combination that considerably lowers the risk of plastome gene outflow within a field (but not gene influx from related strains or species) can come from utilizing male sterility with transplastomic traits [40]. Introducing plastome-inherited traits into varieties with complete male sterility would vastly reduce the risk of transgene flow, except in the small isolated areas required for line maintenance. Such a double failsafe containment method might be considered sufficient where there are highly stringent requirements for preventing gene outflow to interbreeding species adjacent to the phytoremediation sites. Plastome-encoded transgenes for non-selectable traits (e.g. for phytoremediation) could be transformed into the chloroplasts together with a trait such as tentoxin or atrazine resistance as a selectable plastome marker. With such mechanisms to further reduce out-crossing risk, plastome transformation can possibly meet the initial expectations.

3.2.3 Genetic use restriction technologies and recoverable block of function

Other molecular approaches suggested for transgene containment include: seed sterility, utilizing the genetic use restriction technologies (GURT) ('terminator gene') [43, 44], and recoverable block of function (RBF) [45] to prevent transgene flow. Such proposed technologies control both the gene influx of exo-ferality and endo-feral volunteer seed dispersal, but theoretically if the controlling element of the transgene is silenced, expression would occur, rendering a critical defect in principle and practice. The frequency of loss of such controlling elements is yet unclear, as there have been no large-scale field trials to test this.

3.2.4 Repressible seed lethal technologies

An impractical technology has been proposed to use a "repressible seed-lethal system" [46]. The seed-lethal trait and its repressor must be simultaneously inserted at the same locus on homologous chromosomes in the hybrid used for phytoremediation (in our specific case), to prevent recombination (crossing over), a technology that is not yet workable in plants. The hemizygote transgenic seed lethal parent of the hybrid cannot reproduce by itself, as its seeds are not viable. If the hybrid could be made, half the progeny would not carry the seed lethal trait (or the trait of interest linked to it) and they would have to be culled, which would not be easy without a marker gene. A containment technology should leave no viable volunteers with the transgene, but this complex technology would kill only 25% of the progeny and 50% would be like the hybrid parents and 25% would contain just the repressor. Thus, the repressor can cross from the volunteers to related weeds, and so can the trait of choice linked with the lethal, and viable hybrid plants could form. The death of a quarter of the seeds in all future generations is inconsequential to plants that copiously produce seed, as long as the transgenic trait provides some selective advantage.

In summary, none of the above containment mechanisms is absolute, but the risk could be reduced by stacking a combination of containment mechanisms, compounding the infrequency of gene introgression. Still, even at very low frequencies of gene transfer, once gene transfer occurs, the new bearer of the transgene could disperse throughout the population if it has just a small fitness advantage.

3.2.5 Transient transgenics

It is possible to insert certain phytoremediation traits encoding transgenes on RNA viruses or in endomycorrhizae that are expressed in the plant, but are not carried through meiosis into reproductive cells, and thus there will be no gene flow via seeds or pollen. Attempts had been made to use endophytes to carry useful genes into plants by pressure-infiltrating the endophytes into seeds [47, 48]. The advantage of the technology was that it was not variety specific, such that indigenous species or varieties can be used. There are endophytes that naturally participate in phytoremediation processes, e.g. the Methylobacterium sp. that inhabits poplars and degrades explosives [49]. Genes from this or similar species can and have been engineered into other endophytic bacteria, with quite promising results [50, 51].

The same or other infection procedures could be used to introduce phytoremediation traits by disarmed plant disease viruses as the vector. The possibility that such a procedure might work was borne out in many cases with dicots showing that they express virus-encoded genes, e.g. [52]. It was possible to infect Arabidopsis with tobacco etch virus carrying the bar gene; the gene was fully expressed in the plants [53].

Cucurbits artificially infected with an attenuated zucchini yellow mosaic potyvirus containing the same transgene s were resistant in the field [54]. An NTPII carrying wheat streak mosaic virus was used to infect various grains, and the gene was expressed (immunologically) [55]. The virus carrying the genes was expressed in the roots following leaf infection, though not in all tissues.

Considerable technological obstacles of infection of the phytoremediating species will have to be worked out. While no gene flow from the plants is expected, endophytic bacteria are prone to horizontal gene transfer among themselves, an issue which bacterial biosafety experts will have to consider. There are biosafety issues relating to the mode of disarming to be considered, and it must be demonstrated that there is no gene introgression from the virus to the plant chromosomes, as well as no-extra-nuclear transmission of the virus through ovules or pollen in very large numbers of individuals. It is necessary to transfect the phytoremediating species every generation, which may be easier with perennials, such as poplar, and it may be more cumbersome with annual species.

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