Preventing Establishment By Transgenic Mitigation

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If a transgene confers even a small fitness disadvantage, the less fit transgenic volunteers and their own or hybrid progeny should only be able to exist as a very small proportion of the population. Therefore, it should be possible to mitigate volunteer establishment and gene flow by lowering the fitness of transgene recipients below the fitness of competitors, so that the volunteer or hybrid offspring will reproduce with considerably less success than its non-transgenic competitors. A concept of "transgenic mitigation" (TM) was proposed [22], in which mitigator genes are linked or fused to the desired primary transgene. Thus, a transgene with a desired trait is directly linked to a transgene that decreases fitness in volunteers (Fig. 1). TM could also be used as a stand-alone procedure with non-transgenic phytoremediating species to reduce the fitness advantage of hybrids and their rare progeny, and thus substantially reduce the risk of exo-feral hybrid volunteer persistence.

This TM approach is based on the premises that: 1) tandem constructs act as tightly linked genes, and their segregation from each other is exceedingly rare; 2) the gain of function dominant or semi-dominant TM traits chosen are neutral or favourable to phytoremediating species, but deleterious to volunteer progeny and their hybrids due to a negative selection pressure; and 3) individuals bearing even mildly harmful TM traits will be kept at very low frequencies in volunteer/hybrid populations because strong competition with their own wild type or with other species should eliminate even marginally unfit individuals, and prevent them from persisting in the field population [22].

Thus, it was predicted that if the primary gene(s) for phytoremediation advantage being engineered into a phytoremediating species or a crop will not persist in future generations if it is flanked by TM gene(s), such as genes (for crops) encoding dwarfing, strong apical dominance to prevent tillering (in grains) or multi-heading (in crops like sunflowers), determinate growth, non-bolting genes, uniform seed ripening, non-shattering, anti-secondary dormancy. When they are in such a tandem construct, the overall effect would be deleterious to the volunteer progeny and to hybrids. Indeed a

TM gene such as anti-shattering should decrease re-seeding, and thus the number of initial volunteers. With crops or phytoremediating species there is typically a small amount of shattering due to imperfect harvesting equipment, which may leave a few seeds behind. Because the TM genes will reduce the competitive ability of the rare hybrids, they should not be able to compete and persist in easily measurable or biologically significant frequencies in agroecosystems [20, 22].

Once TM genes are isolated, the actual cost of cloning them into TM constructs is minimal, compared to the total time and effort in producing a transgenic phyto-remediating species. The cost is even inconsequential in systems where biolistic co-transformation allows introducing genes into the same site such that the tandem construct is made by the plant.

3.3.1 Demonstration of Transgenic Mitigation in tobacco and oilseed rape

We used tobacco (Nicotiana tabacum) as a model plant to test the TM concept: a tandem construct was made containing an ahasR (acetohydroxy acid synthase) gene for herbicide resistance as the primary desirable gene of choice, and the dwarfing Agai (gibberellic acid-insensitive) truncated gene as a mitigator [23].

Dwarfing would be disadvantageous to the rare weeds introgressing the TM construct, as they could no longer compete, but is desirable in many crops, preventing lodging and producing less stem with more leaves. The dwarf and herbicide resistant TM transgenic hybrid tobacco plants (simulating a TM introgressed hybrid) were more reproductive than the wild type when cultivated alone (without herbicide). They formed many more flowers than the wild type when cultivated by themselves, which is indicative of a higher harvest index. Conversely, the TM transgenics were weak competitors and highly unfit when co-cultivated with the wild type in ecological simulation of competition. The inability to achieve flowering on the TM plants in the competitive situation resulted in zero reproductive fitness of the TM plants grown in an equal mixture with the wild type at typical field spacing of plants resulting from seed rain of volunteer weeds [23].

From the data above it is clear that transgenic mitigation should be advantageous to a phytoremediation species growing alone, while disadvantageous to a hybrid with it living in the competitive environment of the phytoremediation site, or off site. If a rare pollen grain bearing tandem transgenic traits bypasses containment, it must compete with multitudes of wild type pollen to produce a hybrid. Its rare progeny must then compete with more fit wild type cohorts during self-thinning and establishment. Even a small degree of unfitness encoded in the TM construct would bring about the elimination of the vast majority of progeny in all future generations, as long as the primary gene provides no selective advantage that counterbalances the unfitness of the linked TM gene. Most phytoremediating genes have a drag, not an increased fitness off the phytoremediation site. We have inserted the same construct into oilseed rape and have tested the selfed progeny, as well as hybrids with the weed Brassica campestris

Fig. 1. Transgenic Mitigation to prevent establishment of (A) volunteers and (B) hybrids between phytoremediation species and relatives. The phytoremediation species bears desirable transgenes coupled in tandem with transgenes encoding traits that are neutral or positive for the phytoremediating species, but render volunteers or hybrids unfit to compete outside of cultivation. Source: From ref. [1], with permission of Springer Verlag.

Fig. 1. Transgenic Mitigation to prevent establishment of (A) volunteers and (B) hybrids between phytoremediation species and relatives. The phytoremediation species bears desirable transgenes coupled in tandem with transgenes encoding traits that are neutral or positive for the phytoremediating species, but render volunteers or hybrids unfit to compete outside of cultivation. Source: From ref. [1], with permission of Springer Verlag.

x B. rapa. When cultivated alone, the dwarf transgenic oilseed rape grew at almost the same rate as the transgenic (Fig. 2A), but produced twice as much seed as the non-transgenic isoline (Fig. 2C). When the TM transgenic oilseed rape plants were co-cultivated in competition with the wild type, they were unable to grow normally (Fig. 2B), and hardly set seed (Fig. 2C) because they were so unfit to reproduce.

Cultivated alone In competition

Cultivated alone In competition

Fig. 2. Suppression of B. growth and C. seed yield of TM (transgenic mitigator) bearing oilseed rape plants carrying a dwarfing gene in tandem with a herbicide resistance gene (closed symbols and bars) when in competition with non-transgenic plants (open symbols and bars)), and A. near-normal growth of the transgenics and C. much higher seed yield of the transgenics when cultivated separately without herbicide at 3 cm spacing in a biocontainment screenhouse. (Unpublished data: Al-Ahmad and Gressel, 2005).

Fig. 2. Suppression of B. growth and C. seed yield of TM (transgenic mitigator) bearing oilseed rape plants carrying a dwarfing gene in tandem with a herbicide resistance gene (closed symbols and bars) when in competition with non-transgenic plants (open symbols and bars)), and A. near-normal growth of the transgenics and C. much higher seed yield of the transgenics when cultivated separately without herbicide at 3 cm spacing in a biocontainment screenhouse. (Unpublished data: Al-Ahmad and Gressel, 2005).

The rare hybrid offspring from escaped pollen bearing transgenic mitigator genes would not pose a dire threat, especially to wild species outside fields, as the amount of pollen reaching the pristine wild environment would only be at a minuscule fraction of the pollen from the wild type. This is dependent on the distance, source size, and on fertility barriers. Large-scale cultivation creates large pollen sources, and in theory a wild population having its niche on "the edge of agriculture" with coincident pollen shed could be swamped. There has been pollen flow, but no swamping with native DNA of wheat sporadically appearing in a ruderal Aegilops sp. [21]. Presently, there are no well documented cases where fertility barriers do not prevent more than the formation of a few infertile hybrids near the borders, as well as the rare introgressions, as have been happening for time immemorial. Any unfit hybrids and their rare backcross offspring containing transgenes linked to TM genes should still be eliminated. Further large-scale field studies will be needed with crop/weed pairs to continue to evaluate the positive implications of risk mitigation.

3.3.2 Risk that introgression of TM traits will affect relatives of the phytoremediating species

A model by Haygood et al. [56] claims to "prove" the premise that "demographic swamping" by transgenes would cause "migrational meltdown" of wild species related to the crop or phytoremediating species, especially if the introgressed genes confer unfitness. This proposition that recurrent gene flow from crops or phytoremediating species, even TM gene flow, could affect wild relatives deserves some discussion, as it negates the concept of transgenic mitigation.

They claim that their model demonstrates that recurrent gene flow from transgenic crops or phytoremediating species with less fit genes will cause wild populations to shrink. Firstly, conventional crops already belie this possibility. There are few if any major domesticated crops that are fit to live in a wild ecosystem, so their normal genes should confer a modicum of unfitness. Such crop x wild hybrids continually form, yet no evidence is presented that demographic swamping has occurred due to recurrent gene flow from the crops or phytoremediating species, nor could we locate any published data to that effect. Indeed, considerable evidence has been presented that many crops exist near their wild or weedy progenitors, without causing the extinction of the progenitors, despite gene flow.

There are other mundane yet fatal flaws in their model based on shaky premises and assumptions not borne out by plant biology. Three problematic issues that seem to invalidate the relevance of their model for the vast majority of conceivable crop or phytoremediating species/wild species systems, are discussed below:

• to get the level of swamping that they [56] discuss, the wild relative and the phytoremediating species would have to live in the same ecosystem. There are typically geographic separations between phytoremediation ecosystems and wild ecosystems, with the extent of pollen flow decreasing exponentially with distance between them - usually to a low asymptote due to wind currents or insects not fully following simple physics. There should always be far more wild pollen in the wild ecosystems, so hybridisation events in the wild from crop pollen will be rare, even with masses of pollen occurring within the agroecosystem. Thus their basic assumption of transgenic pollen swamping wild type pollen in the wild is invalid. Indeed, even when they assume an enormous 10% of hybridisations in the wild each generation coming from transgenic pollen, according to their model it will take about 20 generations of recurrent pollination for the unfit allele to become fixed in half the population, and 50 generations for an unfit gene to asymptotically reach 80% of the population. As discussed below, their other assumptions leading to these numbers are also off target, so it should actually take much longer;

• they assume synchronous flowering, no self-fertilization, and no genetic or other barriers to cross-fertilization; indeed, this negates the definition of speciation. It is exceedingly rare for pollen from one species to fertilize another species without any genetic barrier in the wild relative. Of the species mentioned in [57], this might only occur with con-specific wild sunflowers, which might fit this criterion, but even in this case there are genomic deterrents to introgression (as reviewed in [26]. The flow of genes between con-specific rice and red (weedy)-rice does not fit their assumptions because they are cleistogamous, predominantly self-fertilizing before the flowers open, and the amount of outcrossing possible is very low. Of course weedy rice is not a wild species (by definition), so it too is not really relevant to their case. There are fertilization barriers of different chromosome numbers, non-homology etc, which limit fertilization of wild relatives of oilseed rape and wheat, so they are outside the models;

• their models assume animal-type replacement rates - a few progeny per mating, where lower fitness can indeed become fixed. Most wild relatives of phytoremediating herbaceous or tree species produce copious amounts of seed to replace parents. Hundreds to thousands typically germinate in the area occupied by a parent and the process of self-thinning is ferociously competitive, eliminating less fit individuals. Our experimental data show that at realistic seed output and seeding rates, unfit individuals are eliminated or remain at a low frequency, just as unfit mutations are maintained in populations at some low frequency (the relative fitness multiplied by the mutation frequency).

Their conclusion that "the most striking implication of this model is the possibility of thresholds and hysteresis, such that a small increase in (unfit gene) immigration can lead to fixation of a disfavoured crop allele " [56] flies in the face of evolutionary evidence, and decades of classic and contemporary field data showing that only near-neutral genes exist in pockets of the evolutionary landscape of plants, and blatantly unfit plant genes are not known to exist in such pockets unless all the fit genes are somehow removed. Just as endogenous unfavored gene mutations exist in the wild at a frequency lower than the mutation rate, transgenes from phytoremediating species that have a fitness penalty will exist in the wild at a rate lower than the immigration rate. As discussed above, the immigration rate to the wild is perforce very low. Unfit genes are eliminated from populations of plants that produce large numbers of seeds, whereas the genes could be fixed in populations of animals with few progeny. When a model contradicts reams of data, it is more likely than not that the model is invalid.

Haygood et al. [56] further contend that their model would work if the phytoremediating species were heterozygous for the unfit gene (and many transgenic hybrids have the transgene in a single parent and are thus hemizygous). The data in Fig. 2 clearly demonstrate that when even half of the backcross progeny contain a TM construct, they cannot compete with their non-transgenic sibs, let alone the wild type. Part of the problem may be that Haygood et al. [56] (p. 1880 column 2) "assume (that) the number of plants surviving to maturity does not vary from one generation to the next", a questionable assumption for unfit phenotypes when they must compete with fit cohorts and other species.

In summary, where might their model have some validity? Even though, despite their claims, the model has limited validity for the "wild" ecosystems, the model might be valid for a few weeds (not wild species) related to phytoremediating species. Weeds are man-made domesticated species (of a sort), and they are dependent on human controlled agro-ecosystems. These systems change continuously, which leads to continual shifting weed populations with an ever-changing composition. Over time new species invade, and old species go extinct, adapt, or are once more confined to their original natural environments. This is the nature of agriculture itself. It is likely that weeds that are evolutionarily threatened by the flow of unfit genes would evolve exclusionary mechanisms that block extinction;, e.g., they could evolve a shift to predominant self-fertilization that would protect them from transgenic pollen bearing unfit genes. The model of Haygood et al. [56] may be right for certain animal systems but irrelevant for the vast majority of plant systems. They fail to mention specific plant systems where their model might be valid. Indeed, the species that naturally phytoremediate mine sites

(for the last 2000 years in the case of Roman sites) are so unfit to compete off of mine sites that the heavy metal resistant genes are not found in the same species of windpollinated grasses a few cm from the edge of mine tailings [58]. Some pollen flowed, but the hybrid offspring cannot compete with wild-type offspring.

3.3.3 Following transgene flow to volunteers and feral forms

Using the various containment and mitigation strategies it should be possible to keep transgene "leaks" below risk thresholds, which have to be specified by science-based regulators on a case-to-case basis. As the numbers of transgenic species being released is increasing, and the problems of monitoring for such genes increases geometrically, we suggested that a uniform biobarcodeTM system be used, where a small piece of non-coding DNA having uniform recognition sites are at the ends (for single PCR primer pair amplification) with an assigned variable region in between. Thus, PCR-automated sequencing could be used to determine the origin of "leaks", contamination, liability, as well as intellectual property violations [59].

4. Special transgenic mitigation genes for phytoremediation

As more genes become isolated and their properties elucidated, it appears that many might be specifically utilizable to contain and mitigate gene flow in plants used for phytoremediation. Some genes that can be used for containment might be better used for mitigation. For example, various Populus species have been genetically engineered and field-tested out of doors for heavy metal tolerance or for metabolising halogenated hydrocarbons, as well as male sterility, and lack of fertility [60], but necessarily linked in tandem, so the traits can segregate. Male sterility and lack of fertility can prevent gene outflow, albeit typically leaky. Thus, some pollen bearing the phytoremediation traits can escape to the wild, and some pollen from the wild can fertilize the few flowers appearing on a tree. In the case of vegetatively propagated species such as poplars, male sterility can be coupled with female sterility, which will prevent pollen from nearby related species from effectively pollinating the phytoremediating poplar. Additionally, floral ablation can be used (no pollination in either direction) can be used, as described in a review of the earlier literature [61]. A presently used cytotoxin gene under the control, of a PTD flower promoter imparts "high levels" of floral ablation in poplar, a species commonly used for phytoremediation [62], with complete loss of flower buds in some lines tested in the greenhouse, in plants also engineered for early flowering. Whether they are leaky and allow some flowering as plants mature is being tested in field trials now in progress (S.H. Strauss, Oregon State Univ., pers. comm. 2004). If the infertility is not 100% and the genes are just used for containment, i.e., not engineered in a tandem construct with the phytoremediation genes, the infertility genes can segregate from the phytoremediation genes in further generations, giving fertile plants with the phytoremediation traits. If the same infertility genes are engineered in a tandem construct or in such a way that they will be linked in planta (as happens with most biolistic co-transformants), the two sets of traits will remain linked, and the rare escapee bearing infertility and phytoremediation will remain "mitigated", i.e., in a perennially low proportion of the population.

Some traits are appropriate containing/mitigating both tree, shrub, and herbaceous phytoremediating plants, for example: the overexpression of a cytokinin oxidase [63], which reduces the levels of isopentenyl and zeatin type cytokinins. This in turn leads to phenotypes with far reduced shoot systems (unfitness to compete) but with faster growing more extensive root systems [64], all the better for extracting toxic wastes.

Irreversible sterility is best for trees and shrubs that can be vegetatively propagated, reversible male sterility is better for herbaceous species, as it allows seed production, as described below.

4.1. CONTAINMENT/MITIGATION FOR HERBACEOUS PHYTOREMEDIATION AGENTS

Mitigating genes should easily prevent or delay flowering in rosette type herbaceous species such as the Brassica spp. that are two phase species, where the vegetative material is harvested, and flowering (bolting) is detrimental. This could easily be effected by preventing gibberellic acid biosynthesis [65], either in a TM construct and/or by permanent mutation of the kaurene oxidase gene using a chimeraplastic gene conversion system [66], a system that as yet is hard to use in plants. Kaurene oxidase suppression would require the use of gibberellic acid to 'force' flowering for seed production. There should be a concomitant biosafety requirement that seed production areas be far removed from areas where weedy or other feral or wild relatives grow to prevent pollen transfer.

Delaying of bolting and flowering by using a different transgene has recently been demonstrated. Curtis et al. [67] engineered a fragment of the GIGANTEA gene, the gene encoding a protein that is part of the photoperiod recognition system, into radish using an antisense approach. Bolting was considerably delayed, and thus seed production could come about without reversal mechanisms if seed producers waited long enough. If despite all isolation distances, a TM construct or a mutant in a seed production area introgresses with a wild species, the progeny will also be delayed, i.e., the transgenic hybrid would be non-competitive with cohorts.

4.2. SPECIAL CONTAINMENT/MITIGATION GENES FOR PHYTORE-MEDIATING TREES

In forestry, the possibility of gene flow is especially problematic as the duration until long-term implications of gene movement become apparent can be longer than human lifetimes. The introgression of traits from these species to wild populations has been extensively discussed by [20, 68] and thus containment/mitigation requirements should be stringent. Some phytoremediating species such as the poplars are vegetatively propagated and thus flowers and seeds are not important - indeed may provide a metabolic/genetic drag. Such phytoremediating trees can be vegetatively propagated, and if sterile, besides possibly higher yield and biosafety, allergy-causing pollen clouds and messy fruits would be prevented. An ideal gene for doing this is barnase under the

T29 tapetum-specific promoter [69]. The ribonuclease is only produced in the tapetum and prevents pollen formation with no other ill effects.

If one has an important phytoremediating species in which transgenics are exceedingly worthwhile, yet the risks of cultivation too great, one could envisage using a pollen sterility system coupled with flower drop, as described above and the crop could be propagated by artificial seed, e.g., artificially encased somatic embryos produced in mechanized tissue culture systems. As noted above, such genes are being tested [60], but whether in tandem with phytoremediation traits, or separate is not clear.

Poplar height is under control of gibberellic acid, just as it is with herbaceous species [70]. The GAI and related dwarfism genes are thus being tested in poplar to ascertain whether the shorter, fatter trees concept cited will grow any faster and be less competitive under competition. So far a field trial has been growing for one year and the researches at Oregon State University have many short, fattish trees (size varies from 1/3 to 2m)...but it will take several more years to ascertain the capacity to mitigate (Steven Strauss, personal communication, 2004). They believe that better genes or more specific promoters may be needed to really make the concept work. The professional foresters are quite sceptical, given that tall and straight trees is what they have been taught to seek all their careers (Steven Strauss, personal communication, 2004).

Another approach by scientists at Oji Paper Company in Japan for an analogous situation has been announced (in a news release) [71]. They engineered Eucalyptus to withstand very acid soils, and graft non-transgenic rapidly growing Eucalyptus on the transgenic acid-tolerant rootstock. There can be no transgene flow from these plants, unless suckers or shoots form on the rootstocks. Similar grafting approaches could be used with many bioremediating tree species.

5. Concluding remarks

Systems exist that can theoretically preclude a phytoremediating species from becoming established outside the contaminated area being treated, whether by containing gene flow or by preventing the establishment of hybrids by mitigation. There is evidence that some of these systems are efficient in crops, and there is no reason they could not be used in phytoremediating species, where a risk of transgene flow is perceived. Thus, if a risk of establishment is discerned using the enabling decision tree proved above, such a risk should not preclude developing transgenic phytoremediation species - it should stimulate the imagination to devise and test systems to deal with the potential problems.

Acknowledgements

The second and last authors' research on transgenic mitigation was supported by the Levin Foundation, by INCO-DC contract no. ERB IC18 CT 98 0391, and by a bequest from Israel and Diana Safer. This chapter is a heavily augmented and updated version of Gressel and Al-Ahmad [1], and updated portions from that review are included with the permission of the Springer Verlag.

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