Biological Confinement of Genetically Engineered Organisms (2004) / Chapter Skim
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3. Bioconfinement of Plants
3. Bioconfinement of Plants
Pages 65-129
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From page 65... ...
In a few cases, there are data that illustrate the efficacy of those approaches; in other cases, the approaches are untested. This chapter reviews and analyzes as many bioconfinement methods for genetically engineered plants as the committee could identify, although the survey is incomplete because new methods are proposed constantly.
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From page 66... ...
of vegetative promoters that propagules kill vegetative tissues Confine pollen Male sterility Available for some Crop requires other only species, could be lost plants as source of in later generations; pollen if seed transgenic methods production is desired could be more durable Transgene in Under development; Possible to obtain chloroplast; not feasible for plants high concentrations maternal with paternal of desired genetically inheritance inheritance of engineered proteins, chloroplast DNA but many traits (most gymnosperms) cannot be conferred by chloroplast genes Cleistogamy Under development Results in (closed flowers)
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From page 67... ...
Seeds sired relatives on other cultivars or wild relatives would not be viable Cross- Under development incompatibility (early) ; speculative Chromosome Under development; Applies only to crops location in possible if relative that are allopolyploids allopolyploids has nonhomologous (wheat, cotton, chromosomes; can canola)
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From page 68... ...
V-GURT, variety genetic use restriction technology; T-GURT, trait genetic use restriction technology. Sterility Because transgene escape by pollen or seeds is not possible for plants that do not produce fertile pollen or seeds, the task of bioconfinement is simplified because it is necessary only to keep track of vegetative dispersal units, such as tillers, rhizomes, and stolons.
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From page 69... ...
In some cases, interspecific hybrids have almost complete male and female sterility. However, most interspecific plant hybrids are not fully sterile (e.g., Stace, 1975)
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From page 70... ...
Triploid plants found in the wild typically are partially or fully sterile with respect to pollen and seed production. Those that are fully sterile persist only if they are capable of asexual seed production (apomixis)
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From page 71... ...
. In fields, bioconfinement could be achieved if such plants are grown in unisexual stands far from conspecifics or wild relatives with which there could be cross-pollination.
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From page 72... ...
. One type of reversible sterility blocks gene flow through pollen and seeds, thereby, for example, preserving a seed company's ownership of transgenic germplasm.
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From page 73... ...
. Several related transgenic sterility methods are in development commercially and by independent researchers, but little has been published about them beyond general descriptions in patent applications (FAO, 2002)
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From page 74... ...
V-GURT methods could be useful for bioconfinement of grasses, trees, and other horticultural species in which it is desirable to strongly limit gene flow. The social, political, and ethical issues attending the use of V-GURTs in food crops will need to be addressed.
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From page 75... ...
could be developed into a transgene bioconfinement method for vegetative propagules. Pontier and colleagues (1999)
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From page 76... ...
This section begins with a discussion of male sterility, which can be achieved through conventional and transgenic approaches. Nontransgenic Male Sterility Male sterility, the inability of a plant to produce fertile pollen, is a useful tool for hybrid breeding and hybrid seed production because selfpollination is prevented.
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From page 77... ...
Thus, genic male sterility systems could be preferable to cytoplasmic systems if pollen-mediated gene flow must be kept to a minimum. There is substantial concern over transgene flow from GEOs to natural populations of related plants via pollen, so the use of male sterility is recommended whenever feasible.
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From page 78... ...
When effective, male sterility can greatly reduce pollenmediated crop-to-crop and crop-to-wild gene flow. Weaknesses Most types of male sterility are leaky, so it will be important to test the reliability of this trait in a representative range of environmental conditions.
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From page 79... ...
. The greater production is possible because chloroplast transgenes are present as multiple gene copies per cell, and they are little affected by pre- or post-transcriptional gene silencing (Heifetz, 2000)
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From page 80... ...
This approach could prevent transgene dispersal in pollen while preventing some of the disadvantages of male sterility, such as loss of pollen for cross-pollination. Weaknesses Technical difficulties have prevented this bioconfinement method from being feasible, and many types of desirable traits cannot be produced by
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From page 81... ...
. Therefore, creating plants with obligate cleistogamy has been mentioned as a possible bioconfinement method (e.g., Lu, 2003)
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From page 82... ...
; for example, in the case of dandelions, the apomicts are widespread, and the sexual populations are mostly restricted to narrow refugia. Strengths Obligate apomixis with full male sterility will be an effective bioconfinement method only if the confinement goal is to prevent the formation of hybrid progeny.
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From page 83... ...
Weaknesses The technique could not be used for nonwoody species, and it can be applied only for transgenic traits that are expressed in the rootstock or
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From page 84... ...
Artificially Induced Transgene Expression A promising method for reducing the effects of unwanted transgenes is to use a system in which the transgenic trait is activated by an artificial stimulus, such as a chemical spray (Figure 3-1; Daniell, 2002; FAO, 2002)
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From page 85... ...
. Reducing Gene Flow to Crop Relatives Several approaches could be used to restrict the spread of transgenes to sexually compatible wild relatives and cultivars of a crop.
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From page 86... ...
With this system, field-grown commercial plants produce viable seeds when they naturally self-pollinate or cross with each other in the field (Figure 3-2; 25% of the seeds are expected to be inviable because they would be homozygous for the seed-lethal transgene and would lack the repressor transgene)
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From page 87... ...
Concerns about the consequences for nearby relatives of producing dead seeds from the crop are similar to those for terminator and related transgenic sterility methods. Continued use of this system in a single locale could lead to the introgression of the repressor gene into nearby natural populations.
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From page 88... ...
Weaknesses Such bioconfinement methods have not yet been created and tested. Because some cross-incompatibility barriers can be breached by environmental factors, such as high temperature or the presence of pollen from a compatible relative (e.g., Richards, 1997)
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From page 89... ...
As with some of the other proposed bioconfinement methods, this one would work primarily for preventing or reducing introgression from the transgenic organism to wild populations.
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From page 90... ...
created an experimental model system. They transformed tobacco with an herbicide resistance gene linked to a dwarfing gene.
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From page 91... ...
Weaknesses This method depends on using linked alleles that are harmful to wild relatives but neutral or beneficial for crops . Although they will frustrate the evolution of weediness, if introgression of the biocontrol alleles into a very small population did occur by pollen or seed swamping, then depressed fitness of future generations of that population could result, increasing the risk of extirpation of that population, which would be a concern if an endangered wild relative is involved.
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From page 92... ...
By adding a back-up confinement method, such as male sterility, gene flow and persistence of genetically engineered traits in other poplar populations could be so low as to become negligible. Strengths Auxotrophy and other handicap strategies might contribute eventually to integrated confinement methods.
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From page 93... ...
Some of them also could be useful in bioconfinement methods, such as inducible lethality in seeds, which involve targeted blocking of plant growth and development. Green-Specific (Chloroplast-Targeting)
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From page 94... ...
was expressed with the maize streak virus coat protein promoter in transgenic rice, the GUS protein was produced only in the vascular tissues, particularly in phloem-associated tissues (Mazithulela et al., 2000)
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From page 95... ...
. Pollen-Specific Gene Expression The need for pollen-specific transgene expression may be relatively uncommon, unless the transgenic trait is needed specifically in pollen.
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From page 96... ...
If field releases are essential for a given genetically engineered application, some characteristics of the transgene "host" species, such as whether it is traded as a commodity crop, like corn and soybean, could greatly influence whether bioconfinement is needed. Likewise, if all possible plant species that host a given genetically engineered application require strict containment rather than confinement, plans to produce field-released plants should be abandoned.
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From page 97... ...
Moreover, abandonment of a project could prevent some benefits from being realized. The following sections include a discussion of bioconfinement options for genetically engineered trees, and short overviews of related topics in grasses and algae.
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From page 98... ...
Genetic engineering could help restore those species within a manageable period and without the genetic dilution that occurs with sexual hybridization. If the introduction of genetically engineered trees is restricted to areas they once inhabited, growth and eventual spread should remain restricted to their natural ranges.
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From page 99... ...
XMT antisense PQ - Ethylene production reduced (2) ACO or ACS Cranberry IR - Lepidopteran resistant (1)
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From page 100... ...
BAC HT - CBI (8) CBI HT - Glyphosate tolerant (19)
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From page 101... ...
Phenotype Key: AP, agronomic properties; BR, bacterial resistance; FR, fungal resistance; HT, herbicide tolerant; IR, insect resistant; MG, marker gene; NR, nematode resistance; OO, other; PN, plant nutrition; PR, bioremediation; PQ, product quality; SM, selectable marker; VR, virus resistance. Gene Key: 4CL, 4-Coumarate:CoA ligase antisense gene from poplar; ACO, ACC oxidase antisense from coffee; ACOp, ACC oxidase antisense from Prunus; ACS, ACC synthase antisense from coffee; ACSp, ACC synthase antisense from papaya; AHAS, acetohydroxyacid synthase; ALS, acetolactate synthase; Att-E, attacin gene from Hyalophora cecropia; BAC, bacteropsin gene from Halobacterium halobium; BARNASE, barnase gene; barstar, barstar gene from Bacillus amyloliquefaciens; C4H, Cinnamate 4-hydroxylase gene from Populus tremuloides; CAT, chloramphenicol acetyltransferase gene from E
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From page 102... ...
coli -glucuronidase gene; HYR, hygromycin phosphotransferase gene; IAAm, IAA monooxygenase gene; LEC, lectin genes from barley, rubber tree, and/or stinging nettle; LFY, leafy homeotic regulatory gene from Arabidopsis thaliana; LGB, lignan biosynthesis protein gene from pea; LYZ, lysozyme gene from cow; magainin, magainin gene from Xeanopus laevis; MIR, mercuric ion reductase from E coli ; MS1, male sterility protein gene from Populus trichocarpa; NPTII, neomycin phosphotransferase gene from E
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From page 103... ...
After transformants are selected in which the transgene has stably integrated, loss of the phenotype can occur subsequently, because of the loss of expression of the transgene -- by gene silencing. In the Arabidopsis thaliana model system, transgene inactivation has been correlated with multiple copies of the transgene, with the presence of vector backbone sequences, with DNA methylation, and with transgene position in a genome (De Buck et al., 2001; De Wilde et al., 2001; Meza et al., 2002)
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From page 104... ...
However, breeding for resistance traits has not been widely attempted for forest trees in the past because of the inherent difficulties in protracted multigenerational breeding. Thus, traditional knowledge does not exist to evaluate whether gene flow from resistant genotypes in managed stands to wild stands will disrupt existing food chains or have other significant nontarget effects in subsequent generations.
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From page 105... ...
If the transgene were to provide a substantial competitive advantage, even severe restriction of transgene flow might be insufficient to prevent colonization after escape. Such extreme cases are trait and species specific and should be identified during risk analysis in the planning or development phases.
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From page 106... ...
Transgene flow could be substantial if genetically engineered trees are permitted to reach sexual maturity and flower within the natural geographic range of wild relatives. The extent of hybridization
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From page 107... ...
In commercial poplar plantations, studies involving nontransgenic DNA markers have shown unexpectedly low levels of gene flow to nearby wild populations, despite the potential for extensive gene flow (Slavov et al., 2002)
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From page 108... ...
Within natural forest ecosystems, however, they might not be. Secondary Phenotypic Effects of Transgenesis As in nontransgenic methods of genetic modification, the process of inserting transgenes into plant cells and regenerating whole plants from those cells can result in different types of unintended phenotypic effects.
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From page 109... ...
The total lignincellulose mass in the genetically engineered trees was essentially unchanged. Furthermore, leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both in the cells and in whole plants in the transgenic lines.
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From page 110... ...
To investigate transgene silencing in a tree system, Kumar and Fladung (2001) analyzed aspen (Populus tremula L.)
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From page 111... ...
Stable suppression of fertility could require targeting of multiple floral genes or the combined use of several genetic mechanisms for inducing sterility and other bioconfinement methods. Engineering of complete sterility or male sterile lines could help to achieve gene confinement, and it could stimulate faster wood production, reduce the production of allergenic pollen, and (in the case of male sterility)
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From page 112... ...
is an engineered form of auxotrophy that could reduce gene flow from transgenic trees to wild relatives. The RBF system is superior to single-gene-mutation auxotrophs because hybrids between the transgenic plants carrying the RBF and the wild relatives would die or be unable to reproduce because of the blocking construct.
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From page 113... ...
Outlook for Bioconfinement of Transgenes in Trees Safe and effective bioconfinement methods should lead to greater acceptance of transgenic trees by the general public and to increased opportunities for the creation and deployment of genetically engineered trees by researchers in the public and private sectors. Given the number of options available and the frequency with which new approaches are being reported, there is every reason to believe that effective bioconfinement methods will
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From page 114... ...
This will only occur if there is acknowledgment by industry that bioconfinement of transgenes is necessary and beneficial; if the public accepts that there can be an acceptable risk; and if more public funding becomes available for the discovery, development, and appropriate testing of bioconfinement methods in trees. The likelihood of public acceptance of nominal risk associated with the growth of trees under bio- and physical confinement could be improved by the use of transgenes derived from the same or similar tree species and by the development and adoption of methods for monitoring wild populations for entry of transgenes from genetically engineered tree plantations.
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From page 115... ...
. Genetically Engineered Turfgrasses Since 1993, the Animal and Plant Health Inspection Service has issued more than 200 permits for small field tests of transgenic turfgrass species in the United States (Table 3-3; Wipff and Fricker, 2001)
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From page 116... ...
CBI AP - Growth rate altered & CBI HT - Glyphosate tolerant (6) FR - Rhizoctonia solani resistant (2)
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From page 117... ...
coli -glucuronidase gene; HYR, hygromycin phosphotransferase gene; IMT, inositol methyl transferase; LEA, late embryogenesis abundant protein gene from barley; Lsd (Sac) , levansucrase gene from Bt; Npr1, nonexpressor of pathogenesis-related gene from Arabidopsis thaliana; NPTII, neomycin phosphotransferase gene from E
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From page 118... ...
. Potential for Gene Flow Grasses that are cultivated for turf, forage, and ornamental uses pose several challenges for bioconfinement because of their capacity for outcrossing, hybridization, and vegetative propagation.
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From page 119... ...
measured gene flow from herbicide-resistant transgenic creeping bentgrass into wild relatives. The primary objectives of the study were to investigate intra- and interspecific gene flow of transgenic creeping bentgrass in the Willamette Valley of Oregon, where nearly all U.S.
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From page 120... ...
developed by Tim Phillip at the University of Kentucky. More intensive bioconfinement methods, such as the use of plastid transgenesis and male sterility are needed in genetically engineered turfgrass production.
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From page 121... ...
. Because algae often are cultured outside their native ranges, some nontransgenic species have been managed using bioconfinement methods.
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From page 122... ...
Other bioconfinement methods would be needed for genetically engineered algae that can survive and spread in natural habitats near aquaculture facilities. There is no feasible method of inducing sterility in algae, and the lack of basic understanding of the biology of reproduction in most algae is a major obstacle to developing a feasible method in the near future.
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From page 123... ...
Although most of the data that associate population size and gene flow come from the literature on pollen flow, there is every reason to assume that similar relationships would occur for the dispersal of seed and vegetative propagules. Small populations could be common for a few types of transgenic crops -- such as pharmaceutical-producing plants -- that are grown commercially.
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From page 124... ...
If a failed bioconfinement method can be recognized by distinctive phenotypic traits, such as the presence of flowers in otherwise sterile plant varieties, it might be possible to cull abnormal plants in small fields. That practice is used in certified seed production programs, where inspectors go through the fields to remove or cut off any "off-type" plants that do not conform to desired phenotypic standards.
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From page 125... ...
This method could be used on a case-by-case basis, but if the bioconfinement method failed it might lead to the unwanted spread of herbicide resistance as well as to the spread of the bioconfined transgene. However, in short-term, small-scale experiments, herbicide resistance could be a useful marker for testing the efficacy of new bioconfinement methods before they are used on a commercial scale.
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From page 126... ...
Given enough resources for statistically meaningful sampling efforts, it might be possible to detect failed bioconfinement, but there is still the problem of detecting failure early enough to mitigate or eradicate unwanted plants. If those plants reproduce and spread, either by further cultivation or by naturally occurring gene flow, subsequent efforts to stop the process could be futile.
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From page 127... ...
Two types of effects are discussed: those in which the confinement method functions as intended, and those that result from an unintended breakdown. For bioconfinement methods that rely on complete sterility, unwanted ecological or evolutionary effects are likely to be negligible if the method functions properly.
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From page 128... ...
Large population size is common for most wild relatives of crop species. Male sterility is a bioconfinement method that sometimes is misunderstood to be a danger to wild populations.
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From page 129... ...
It also is useful to consider possible consequences when bioconfinement methods do not function properly, for example because of gene silencing or recombination that disconnects linked transgenes (Box 3-1)
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