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3 Bioconfinement of Plants METHODS OF BIOCONFINEMENT Many approaches have been proposed for the biological confinement of plant transgenes (Table 3-1; Daniell, 2002). Some are based on pre- existing agronomic or horticultural methods, others are newly developed, and some are hypothetical. 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. The discussion begins with strategies for blocking sexual and vegetative repro- duction. Other techniques that reduce the spread and persistence of transgenes in wild and cultivated populations of plants are reviewed. The chapter also considers--as best as possible, given the limited data available--the efficacy of those methods at various spatial scales. There is a discussion of whether the methods could affect the populations and ecosystems in which they are deployed. Given that bioconfinement methods are expected to be less than 100% effective, the chapter also asks how to monitor for escape of plant transgenes and whether detection and subsequent culling would be an effec- tive backup to a primary bioconfinement method. Case studies are provided to highlight the bioconfinement issues specific to transgenic trees, turfgrasses and algae. The chapter concludes by asking what consequences might accrue and what mitigation might be necessary if bioconfinement and monitoring of genetically engineered organisms (GEOs) fail. 65

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66 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS TABLE 3-1 Bioconfinement Methods in Plants Major Other Purpose Method Limitations Considerations Confine all gene Sterile triploids Few triploid or sterile Not useful if seed flow via pollen or interspecific hybrid cases apply or production is desired and seeds hybrids are effective Use only male Not feasible if same Not useful if seed or only female species or compatible production is desired plants that can relatives could be propagated cross-pollinate with vegetatively unisexual plants; sex expression can be leaky V-GURTs, V-GURTs under V-GURTs should not such as original development (early); be used in food crops terminator other sterility methods if growers need to require vegetative save seeds propagation Reduce spread V-GURTs with Under development and persistence inducible (early) 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) (early) self-pollination Apomixis Under development Hybrid varieties (asexually (early) would have high yield produced seeds) and breed true; could become invasive

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PLANTS 67 TABLE 3-1 Continued Major Other Purpose Method Limitations Considerations Transgenes Transgenes only Under development Applicable to grafted absent in seeds in rootstocks (early); cannot use scions of certain and pollen transgenic traits in woody species such flowers, fruits, seeds as grapes, fruit trees Transgenes Under development Allows seed excised before (early); very production without reproduction speculative; cannot spread of transgenes use transgenic traits in flowers, fruits, seeds Confine T-GURTs Under development Potentially useful; transgenic traits involving (early); external cues avoids concerns about only (transgenes inducible traits for transgene sterile plants, but can spread) expression might not inactive transgenes be reliable enough for can still spread high efficacy Reduce gene Repressible seed Under development Allows viable seeds to flow to and lethality (early) be produced on same from crop (see Fig. 3-2) cultivar. 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) be leaky Tandem Under development constructs to (early); requires reduce fitness fitness-reducing trait in crop-wild detrimental to wild hybrids and plants but not crop their progeny continued

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68 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS TABLE 3-1 Continued Major Other Purpose Method Limitations Considerations Phenotypic and Domestication Under development; fitness handicaps phenotypes does not prevent to reduce need gene flow for confinement Auxotrophy Under development; (dependence on does not prevent specific nutrients gene flow or growing conditions) Reduce exposure Tissue- and Promoters available, Could alleviate to transgenic organ-specific but greater efficacy the need for products in promoters that needed in many cases; bioconfinement plants limit expression confines transgenic in some cases of transgene traits but not the transgenes; transgenes can spread Minimize or Choice of Economic costs can Often feasible and eliminate alternative be high, especially if highly recommended need for organisms; decision to change when appropriate; bioconfinement choice not to course is made after alternative choices release in field; economic investment should be examined choice not to before GEO is proceed with developed GEO For thorough confinement, pollen dispersal, seed dispersal, and vegetative persistence must be considered. 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. Bananas and seedless grapes are among the sterile food crops that are propagated vegetatively. Many non- sterile cultivated plants are sold as cloned vegetative material, including some varieties of potato, turfgrass, and ornamental plants and poplar trees. Several mechanical, chemical, and genetic methods can be used to block the production of fertile pollen or seeds in those plants. This section reviews genetic approaches that achieve sterility. They include nontransgenic methods

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PLANTS 69 (triploids); transgenic sterility that is nonreversible; and transgenic approaches that allow for reversible sexual sterility that permits further breeding. The sections that follow discuss options for blocking vegetative spread and for obtaining male sterility. Interspecific Hybrids Interspecific hybrids often exhibit partial or full sterility (e.g., Grant, 1981; Stace, 1975). The sterility of the mule, a horse and donkey hybrid, is well known. 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). In a surprising number of cases, hybrid fitness has been shown to be as high as or higher than that of the parental genotypes (Arnold, 1997; Arnold and Hodges, 1995). For example, Arriola and Ellstrand (1997) compared the fitness of hybrids of Sorghum bicolor (the crop, grain sorghum) and S. halepense (the weed, johnsongrass) and genetically pure S. halepense siblings under field conditions. They report that the hybrids did not significantly differ from the weeds in terms of biomass, tiller number, seed set, or pollen viability. Furthermore, in many species, relatively or fully sterile hybrids reproduce and spread by vegetative reproduction, sometimes even more vigorously than do their sexually fertile relatives (e.g., Ellstrand et al., 1996). It is well known that the fitness of hybrids varies tremendously in different environments (Anderson, 1949; Arnold, 1997). Thus, housing transgenes in interspecific hybrids might afford some moderate bioconfine- ment relative to nonhybrids, but for any given hybrid genotype, male fertility, female fertility, and vegetative reproduction (if appropriate) must be measured in a range of potential field environments to allow an estimate of what amount of bioconfinement might be expected. Strengths In cases where there is complete or near-complete sterility, interspecific hybridity could yield a reasonably easy way to obtain bioconfinement in plants, as in the case of triploid hybrids. As long as sterility is maintained in a variety of environments, the genes of those plants are unlikely to spread through pollen or seed. Weaknesses Sterile interspecific plant hybrids will not be a general solution for plant bioconfinement. Specific hybrids might prove to be very sterile, but it is more likely that interspecific plant hybrids would offer moderate bio- confinement at best and no bioconfinement at all in some cases.

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70 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS Sterile Triploids Breeding methods that disrupt chromosomal pairing during sexual reproduction have been used to create sterile plants. Most plants are chro- mosomally diploid (characterized as 2n). That is, they have two sets of matching homologous chromosomes in their somatic cells. The two sets pair up and separate during the process of gamete formation, and the number of chromosomes is halved for each pollen grain or ovule (those gametes have n chromosomes). The diploid number is restored when the gametes fuse to create a zygote. Organisms with three sets of chromosomes are called triploids (3n). In humans, triploidy is lethal, and it is a rare condition in wild organisms (Chapter 4). It is not uncommon in cultivated plants (Grant, 1981), how- ever, many commercial banana cultivars are triploid and thus seedless (Simmonds, 1995). Spontaneous triploids primarily appear to result from the fusion of a normal gamete (n) with an aberrant unreduced (diploid, 2n) gamete. Spontaneous triploids also can occur from the fusion of a gamete from a diploid species with one from a related tetraploid (4n) species (which produces gametes that bear 2n chromosomes). For example, if a 2n plant is crossed with a 4n plant, all of their progeny would be 3n and would be expected to be sterile. Triploid plants found in the wild typically are par- tially 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) or vegetative reproduction. Triploidy in cultivated plants is main- tained mostly through vegetative propagation. Thus, induction of triploidy (and other odd-numbered chromosome counts) represents a possible option for bioconfinement. Chromosomal situations other than odd ploidy--extra or missing indi- vidual chromosomes (aneuploidy) and translocation heterozygosity--also disrupt gamete formation during meiosis. Although they can cause reduced fertility, they apparently have not been examined for use in bioconfinement. More information on chromosomal variation in plants and its consequences for plant fertility is found in Burnham (1962) and Levin (2002). Strengths If triploidy results in pollen and seed sterility, and if the degree of sterility does not vary from one environment to another, induction of triploidy could be an effective method of bioconfinement. Triploidy induc- tion will be most effective for organisms that do not reproduce asexually, although that complicates options for further breeding and multiplication. Triploidy also can be induced in other transgenic organisms such as fish (Chapter 4).

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PLANTS 71 Weaknesses Much like interspecific hybridity, the efficacy of triploidy induction varies by genotype and environment. Unisexual Plants Lacking Mates Many dioecious (unisexual) plants can be propagated vegetatively, among them holly, kiwi, gingko, avocado and asparagus, such that only one sex is used for genetic engineering. Sex-specific molecular markers can be used to identify male or female plants before massive propagation (e.g., Khadka et al., 2002; Reamon-Buttner, 1998). 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. For example, all-female cultivars of ornamental nonnative plants could be used in this context. However, this method of bioconfinement is unlikely to be practical in most cases. First, the number of species for which the condi- tions would be met (along with sufficient economic advantages) is small. Second, dioecy is known to be quite leaky (Krohne et al., 1980; Poppendieck and Petersen, 1999); seeds could be produced in low frequency by "male" plants, especially in large-scale plantings. Finally, human error could result in mix-ups that allow both sexes to occur in the same population, resulting in a breakdown of bioconfinement. Strengths This method might be desirable if it is used in combination with other confinement approaches in small-scale plantings. Weaknesses This method is unlikely to be reliable, and it applies only to a narrow range of species. Transgenic Sterility Transgenic methods are available for developing plants that abort young flower buds and thus become sterile through ablation. The resulting plants cannot be used for breeding or for multiplication by seed, but this method has been considered for some clonally propagated plants, such as poplar trees. Strauss and colleagues (1995) reviewed the rationale for at- tempting to engineer nonreversible sterility in forest trees. One strategy for creating sterility-causing transgenes that is particularly attractive for peren-

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72 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS nial plants is to ablate floral tissues by the expression of cytotoxin genes that are fused to developmentally induced promoters expressed in flowers. Promoters from floral-specific genes tend to work well across species. Thus, ablation methods based on these genes probably will not require cloning of new gene homologues from each new transgenic species and genotype. Practical constraints include the requirement for vegetative propagation if complete sterility is engineered and the need for sterility to be highly stable in long-lived species such as trees and perennial grasses. Strauss and col- leagues (1995) suggest that long-term stability could require suppression of more than one floral gene or use of more than one genetic mechanism for sterility. A shortcoming of nonreversible sterility is that it precludes options for further breeding and seed production within the genetically engineered line that could be needed in the future. For trees or other perennials that do not flower in the first 510 years--the breeding period is longer than the generation of new transformants--that limitation might not be a major concern because new transformants could be made within the same period. The engineering of sterility by ablation can be conducted as the last step in the improvement process after breeding or genetic engineering for other traits has been accomplished. The preablation, fertile versions of the lines would still be available for use in breeding or seed production. Reversible Transgenic Sterility Plants that are permanently sterile, such as those described above, constitute an evolutionary dead-end. Researchers have proposed various transgenic methods by which sterility can be gained or lost by design (Fig- ure 3-1; Daniell, 2002). One type of reversible sterility blocks gene flow through pollen and seeds, thereby, for example, preserving a seed company's ownership of transgenic germplasm. With this method, transgenes that confer desirable traits are linked to transgenes that cause sterility, and the two are inherited together. Because this strategy restricts access to fertile plants, it is known as variety genetic use restriction technology (V-GURT). Trait genetic use restriction technologies (T-GURTs) induce transgenic traits in fertile plants by means of a specific stimulus, such as a chemical spray. The term GURT has gained wide use in scientific and policy discussions (e.g., FAO, 2002), but this report focuses on bioconfinement uses of GURTs and related techniques, keeping in mind that incentives for developing those methods are often based on proprietary commercial goals. One of the first V-GURTs was the so-called terminator technology protection system in which transgenic plants produced dead seeds. V-GURTs have not yet been used in any deregulated or commercialized crops, but, the terminator technology patent application was extremely

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PLANTS 73 Fertile V-GURTs: Dead seeds; plants original "Terminator" Sterile Fertile seeds; plants but only during breeding; dead seeds on field plants T-GURTs: Fertile Fertile seeds; plants transgenes expressed only when induced Fertile seeds; on same GE crop variety: dead seeds on other plants FIGURE 3-1 Proposed transgenic bioconfinement methods in plants. V-GURT, variety genetic use restriction technology; T-GURT, trait genetic use restriction technology. controversial, especially in developing countries. The V-GURT approach induces seeds that grow into plants that produce nonviable offspring when they are cultivated in farmers' fields. Induction can occur by soaking the seed source in a solution that induces a promoter, setting the stage for late- acting lethality in ripened seeds (Figure 3-1, V-GURT example 1). In field- grown plants, a promoter that is expressed late in seed development acti- vates a lethal gene that renders the seeds unviable but still fully formed, which is important if the seed is to be sold for food, feed, or other uses. However, seeds in the original seed lot that are not induced properly can develop into fertile plants rather than sterile ones. Such incomplete sterility seems quite likely, based on the status of the technology (Daniell, 2002), and other V-GURTs are likely to be more effective. To avoid the problem of incomplete induction of sterility, plants could be engineered with sterility as the default condition, and breeders could use a stimulus to induce a pro- moter to render them fertile (Figure 3-1; adapted from FAO, 2002). Several related transgenic sterility methods are in development com- mercially and by independent researchers, but little has been published about them beyond general descriptions in patent applications (FAO, 2002). One exception is the research published by a group that developed a method called "recoverable block of function" (Kuvshinov et al., 2001), which consists of a DNA sequence element (a "blocker") that interrupts a specific molecular or physiological function in the host plant, leading to death of

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74 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS the host plant or its seeds. A second DNA sequence element (for "recovery") restores the blocked function in the host plant. The blocker and the recovery sequences are physically linked to the transgene of interest in one construct so that they integrate into the genome together and remain united during sexual reproduction. The recovery function is designed to be activated by exogenous chemical or physical treatment. Thus, the dispersal of pollen or seeds with the recoverable block of function construct would result in progeny that would die or be unable to reproduce because the recovery function would be inactive. The work is still in the early stages, and it might or might not reach commercial development. Sterility systems for genetically engineered plants have been criticized because they would prevent growers from saving seed and having the option of using transgenes to improve local varieties. If implemented widely, V-GURTs such as the terminator technology would force growers to buy new seed each year to benefit from modern varieties. Many growers do buy new, certified seed each year, to save time and obtain a high-quality product that is free of contaminating pathogens and weed seeds. Many food crops and annual ornamental plants are sold as F1 hybrids, among them corn, sunflower, and petunias. Seeds from those plants can be saved but they do not "breed true," so new seeds must be purchased each year. The socio- economic issues surrounding V-GURTs and other sterility methods are discussed in Chapter 1. Environmental effects of the methods are discussed later in this chapter. 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. Strengths Reversible sterility methods could become very useful for bioconfine- ment because they could be used to block the dispersal of pollen and seeds that bear unwanted transgenes. Weaknesses The effectiveness of those novel methods has not been determined nor has their acceptability to consumers. The efficacy of reversible sterility could be diminished by gene silencing or recombination events that cause the sterility construct to become dissociated from the transgenes that require confinement. Research is needed to develop appropriate inducible pro- moters. Public access to data on the efficacy of transgenic reversible sterility, including long-term studies of transgene stability, will be essential. The technology should not be used in food crops for which growers need to save

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PLANTS 75 seeds for future planting or breeding. Possible environmental concerns should be evaluated on a case-by-case basis and are discussed later in this chapter. V-GURTs will not prevent clonal propagation of many plants, such as some species of grasses, shrubs, and trees. Mortality of Vegetative Propagules Vegetative spread, both natural and human-mediated, is common in perennial species. Vegetative clones of semidomesticated and nondomesticated grasses, trees, and shrubs can spread over large areas and survive for decades as new ramets are produced and old ones die off. Some plants--especially species that occur along river margins and shorelines--also have vegetative parts that break off and disperse. Many perennial crops, horticultural plants, and woody species can be multiplied and distributed by rooting clonal segments of the plant and meristematic tissue. Depending on the plant's growth habit and ability to be cloned, strategies for minimizing vegetative propagation could be an essential component of bioconfinement. The ability to propagate plants vegetatively is often desirable for commercial produc- tion, but in wild species, this trait often is associated with enhanced com- petitive ability. Transgenic methods can be used to restrict the spread of vegetative propagules, such as tillers, rhizomes, and root suckers. Given that it will rarely be practical to breed plants that have lost this ability, one of the few options for bioconfinement of vegetative parts is to use a GURT that is induced to kill the plant at some point in its development before it is cloned or propagated (FAO, 2002). Many inducible promoters could be used, including those triggered by chemical applications or winter conditions. Programmed cell death (PCD) is a normal part of development, and, when it is better understood, that response to stress in plants as well as animals (Zhivotovsky, 2002) could be developed into a transgene bio- confinement method for vegetative propagules. Pontier and colleagues (1999) observed that a senescence-like process is triggered during the for- mation of necrotic lesions in disease-resistant plants. They suggested that cells committed to die in resistant plants during this hypersensitive response (HR) to pathogens might release a signal that induces senescence in neigh- boring cells. The signaling pathway responsible for PCD and HR involves changes in the antioxidant systems that are activated by nitric oxide and reactive oxygen species (De Pinto et al., 2002). AtMYB30, transcriptional regulation gene, has been identified as a positive regulator of the hyper- sensitive cell death program in plants in response to pathogen attack (Vailleau et al., 2002). Several lesion mimic mutants have been isolated in Arabidopsis and in other plants that display accelerated HR (Jambunathan et al., 2001). Lesion mimics also can be generated in plants by various

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PLANTS 119 FIGURE 3-3 A wild hybrid, F. arundinacea and L. multiflorum Lam. cross-pollination of nontransgenic creeping bentgrass plants at a distance of 8,000 m (Ellstrand and Hoffman, 1990). Turfgrasses have small pollen that can blow great distances. Normally, the two factors of distance and wind direction are considered to predict the distance that pollens can travel (Giddings, 2000; Giddings et al., 1997b). However, other factors, such as speed and wind turbulence--especially if "whirl winds" are present--are important in the unintended deposition of pollen in other fields. Other factors include relative humidity and temperature (Wipff and Fricker, 2001). Because there are no models to predict those factors, an old method of exponential power function (Bateman, 1947) can be used to predict turf- grass pollen disposition (Wipff and Fricker, 2001). Wipff and Fricker (2001) 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. bentgrass seed is produced. Pollen movement was determined by placing transects of nontransgenic creeping bentgrass around a nursery of 286 plants genetically engineered for tolerance to the herbicide glufosinate. In 1998, transgenic turfgrass pollen grains were observed to travel 1,066.8 m along southwest transects and 1,309.4 m along northeast transects from the

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120 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS nursery. In 1999, transgenic pollen traveled 331.5 m to the southwest, 575.1 m to northeast, 262.4 m to the northwest, and 331.5 m to the southeast from the nursery. The experiments resulted in the introgression of the bar gene from creeping bentgrass into A. canina, A. capillaris, A. castellana, A. gigantea, and A. pallens species. Turfgrasses can vegetatively multiply easily and effectively by rhizomes and stolons. Those underground parts often are translocated by machinery. Birds and mammals also facilitate the dispersal of turfgrass because they feed and forage in and around turfgrass stands for seeds and insects. Grass seeds are ingested and excreted or carried on fur or feathers for deposition elsewhere. For all of the reasons discussed above, transgenic turfgrasses, perhaps especially creeping bentgrass, can be considered potentially difficult to con- fine (Box 3-3). It also must be recognized that bentgrass is a commercially important turfgrass because of its extensive use in golf courses: More than 65% of the transgenic field test permits issued have been for bentgrass (Table 3-3). Bioconfinement Methods for Transgenic Turfgrasses Each bioconfinement technique discussed above could be used in future transgenic turfgrass products. The possibilities include chloroplast trans- BOX 3-3 Turfgrass Might be Difficult to Confine Transgenic turfgrasses carry a particularly high risk of escape for two reasons: Turfgrasses are perennial, so they have many seasons in which to spread through pollen and seeds, and they form unintended hybrids (which themselves would be long-lived) easily. Turfgrasses are open-pollinated plants with a very high cross- ability, primarily with species that are aggressive weeds. Most turfgrasses have many species that outcross heavily among themselves (Giddings et al., 1997a) and even among different turfgrass genera. For example, in nature, Agrostis spp. (bentgrass) cross-breeds with members of the Polypogon genus; and it is believed that Agrostis parlatorei Breistr and A. moldavica Dobrescu and A. moldavica Beldie are derived from multiple cross-hybridization between A. casstellana and P. veridis (Wipff and Fricker, 2001). Also, there are several examples of anthropogenic hybrids between ryegrass (Lolium spp.) and Fescue (Festuca spp.) genera. Figure 3-3 shows a wild hybrid between tall fescue (F. arundinacea) and annual ryegrass (L. multiflorum Lam) 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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PLANTS 121 genesis, tissue- and organ-specific gene expression, male sterility, apomixis, terminator gene technology, gene silencing, suicide genes, ablation, exci- sion, and inducible promoters. However, few bioconfinement techniques have been reported for turfgrasses, in part because little funding has been available for basic research. A significant increase in support will be needed to promote development of an adequate arsenal of bioconfinement tech- niques for the safe use of transgenic turfgrasses. It should be noted that some transgenes could have beneficial effects, should they transfer to other grasses through pollen flow or by other means. Many people suffer from ryegrass pollen allergies, and ryegrass was recently genetically engineered with an antisense-mediated silencing of the gene (lot p5) that encodes the rye pollen allergen. The lot p5 gene antisense construct was expressed in ryegrass under regulation of a pollen-specific promoter. The pollen from those transgenic plants showed low IgE antibody-binding capacity of pollen extract as compared with control pollen, meaning that the pollen of the genetically modified ryegrass could contain minimal amounts of allergen or none at all (Bahalla et al., 1999). This could be of great benefit to allergy sufferers. TRANSGENIC ALGAE Microscopic and macroscopic algae are a diverse group of organisms that are taxonomically distinct from plants. Microalgae are discussed along with bacteria and other microbes in Chapter 5. Commercial production of macroalgae is an important sector of aquaculture, especially in Asia. Seaweeds, such as Laminaria, Porphyra, Undaria, and Graciliaria, are grown for food and food additives, including polysaccharides such as carageenan (Renn, 1997). Commercial transgenic macroalgae have not been developed, in part because of technical obstacles, but there is increasing interest in using them to enhance fuel, polysaccharide, fish feed, and phar- maceutical production and in environmental bioremediation (Minocha 2003; Stevens and Purton, 1997). As with grasses and trees, some commer- cially grown algae have tremendous potential to disperse and persist in natural habitats. Some algae are considered invasive because they out-compete native species and dominate marine ecosystems when introduced to new areas (Occhipinti-Ambrogi and Savini, 2003). Because algae often are cultured outside their native ranges, some nontransgenic species have been managed using bioconfinement methods. For example, a "biological design" method has been used in Maine to confine nonengineered nori (Porphyra spp.). An introduced species of nori (P. umbilicalis) is cultivated commercially on rafts that float in coastal waters where a closely related native species of nori also occurs. Concerns were raised that the introduced species would

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122 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS become invasive and harm native populations by hybridization or competi- tion. However, extensive field studies documented that, under ambient conditions, the introduced species was not invasive and did not reproduce, most likely because of its poor survival in winter (Levine et al., 2001). Thus, this nonnative nori appears to be biologically confined, as long as its repro- ductive capacity continues to be inhibited by local conditions. Other bioconfinement methods would be needed for genetically engi- neered algae that can survive and spread in natural habitats near aquacul- ture 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. Macroalgae are plastic in growth form. They often have complex life histo- ries that involve multiple reproductive pathways, including parthenogenesis and vegetatively dispersed propagules. Researchers do not fully understand sex determination, reproduction, or other aspects of the life history of many species; in some cases, they have not even identified which life stage is reproductive. Therefore, any efforts to study and then biologically confine transgenic algae will have to proceed on a case-by-case basis. EFFECTIVENESS AT DIFFERENT SPATIAL AND TEMPORAL SCALES Most of the bioconfinement methods discussed here are equivalent to natural mechanisms of reproductive isolation that act to maintain species barriers. In plants, the leakiness of those species boundaries is well known (Arnold, 1997; Grant, 1981; Levin, 1978). Within species, distinctive breed- ing systems such as dioecy (male or female plants) and self-incompatibility also are known to be leaky (e.g., Lloyd, 2000; Poppendieck and Petersen, 1999). Moreover, experience suggests that sterility is rarely absolute. Thus, in most circumstances, single-method efforts at bioconfinement are likely to be less than 100% effective in preventing the escape of transgenes, espe- cially if large numbers of plants are involved. The same could be true of multiple-method bioconfinement efforts if there is a chance that individual methods could fail. Unless a bioconfinement method is 100% effective in preventing the movement of seed, pollen, spores, and vegetative propagules, its efficacy generally would vary considerably over different spatial and temporal scales. Spatial Scale Bioconfinement generally will work best for small numbers of plants that are physically isolated (on the order of kilometers at least) from other

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PLANTS 123 populations of the same species or from compatible relatives. Relatively small plant populations tend to be gene flow sinks rather than gene flow sources. All other things being equal, when population sizes vary, gene flow tends to be asymmetric: There is more flow from large populations into small ones than the other way around (Handel, 1983; Levin and Kerster, 1975). Thus, if a bioconfined crop were planted in the midst of other varieties of the same species (e.g., maize grown in Iowa), the percentage of efficacy of less-than-perfect bioconfinement would be expected to drop radically as the number of bioconfined plants increased from dozens to thousands. First, the chance of genetic changes that "disarm" confinement traits, such as mutations that silence transgenic sterility systems, increases with population size. Second, larger populations are more likely to disperse pollen, seeds, or vegetative propagules than are small populations (e.g., Handel, 1983; Levin and Kerster, 1975), and this could compromise back- up strategies such as physical isolation of the bioconfined crop. 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 rela- tionships 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 commer- cially. The high economic value of those crops and the requirement to segregate them from related crops or wild species will mandate their culti- vation in small or isolated populations. However, most plants grown for other uses are likely to be cultivated on a much larger scale. If, for example, bioconfinement is desired for corn or tobacco varieties that produce indus- trial chemicals, some of those crops could be grown on thousands of acres with millions of plants at each site and millions of other, nontransgenic, plants growing nearby. Another aspect of spatial scale is the number of populations that will be cultivated and the number of regions in which the crop can be grown. Local varieties of corn and soybean are grown over vast areas in the United States; fruit orchards and vineyards tend to be smaller and more regional. Major commodity crops that constitute the basis of industrialized agriculture could pose the greatest challenges for bioconfinement because they are grown on an enormous scale. Likewise, forage crops planted on rangeland occupy vast geographic areas, especially in the western states. Even highly managed tree plantations and golf courses represent large populations, each of which consists of thousands or millions of individual plants. When bioconfined plants are grown in many regions, there is a greater chance that they will be planted in the proximity of sexually compatible cultivars or wild relatives. This magnifies the chances of unwanted effects should bioconfinement break down.

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124 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS Temporal Scale In the same vein, the efficacy of bioconfinement should decrease as temporal scale increases. The longer a population is in place, the greater the chance that bioconfinement will erode, and the more opportunities the population will have to disperse pollen, seed, and vegetative propagules. Perennials are long-lived by definition, but even annual plants can occur in long-lasting populations. Indeed, if some small amount of viable seed is released undetected into the soil, that seed bank can grow considerably over a series of years. Environmental conditions also vary from one year to the next, and the efficacy of bioconfinement varies under different environ- mental conditions; opportunities for failure increase over time. Perennials such as turfgrasses and trees can behave very differently from annual crops. Where annuals grow, flower, set seed, and die within a single year, perennials are heterogeneous. Depending on the species, they might or might not flower within a year of germinating. Some species do not flower for many years. Some perennial species live a few years; others (including some grasses and trees) can live for hundreds or even thousands of years. Many perennials (especially grasses) reproduce vegetatively, many do not. Each combination of species-specific temporal patterns will have a different influence on bioconfinement strategies. A perennial in which flowering is delayed for many years and in which vegetative reproduction does not occur will be relatively easy to confine, especially if plants are harvested thoroughly before they flower. At the other extreme, a perennial that creates vegetative propagules regularly, flowers at an early age, and continues to flower every year could be structured to produce so many progeny by seed, pollen, and propagule that finding an effective bioconfine- ment strategy could be a significant challenge. MONITORING AND MANAGING CONFINEMENT FAILURE The degree to which failed confinement can be monitored and managed depends on whether the GEOs are easily detected, the scale at which they are released into the environment, and their subsequent population dynamics and the degree to which they can hybridize with related species. Early detection of failed methods is important, especially if the confined transgenes are likely to spread, but this might be possible only for small- scale plantings of some crops. 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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PLANTS 125 However, failures of many bioconfinement methods will be much more difficult to detect. Elaborate experiments would be needed to identify the proper functioning of a repressible seed-lethal transgene. And most bio- confined plants will be grown on such large areas of land that repeated, comprehensive inspections would be impractical. For large-scale releases, it is important to have easily recognized diag- nostic features that allow the detection of failed confinement. In some cases, genetically based color traits, such as red kernels in corn, could be used to identify a particular transgene, assuming that the color trait stays tightly linked to the confined transgene. Distinctive phenotypes have been bred into some conventional crops, such as oilseed and "confectionary" sunflower, which have black seeds instead of striped seeds, respectively. Experimental lines of transgenic rice that have vitamin A precursors pro- duce recognizable yellow grains, hence the name "golden rice" (Ye et al., 2000). An advantage of visually distinctive traits is that they are easy to identify with minimal expertise. However, a disadvantage is that they could be unreliable because of phenotypic plasticity, variable gene expression, or recombination that separates the genetic marker from the bioconfined transgene. Transgenic methods could be used to introduce general or specific markers for the purpose of monitoring bioconfined transgenes. A general method could be to add a gene that expresses GFP, although that requires examining the plants in the dark with ultraviolet light--a technique with obvious limitations (Leffel et al., 1997). Another option is to assay for specific novel proteins in leaves or seeds using rapid enzyme-linked immuno- sorbent assays (ELISAs) that are similar to those at work in home test kits for pregnancy. Several companies market kits for detecting commonly used transgenes, such as antibiotic resistance proteins, that are often used as markers in genetically engineered plants. The kits are simple to use on leaf samples in the field, but false-negative results are common (Ilardi and Barba, 2001), and the cost of large-scale testing can be prohibitive. In some cases, transgenic resistance to a particular herbicide could be inserted in the same construct as a bioconfined transgene to monitor for possible failure. Seed lots could be sampled and screened for the presence of rare, unexpected transgenes by applying the herbicide to large numbers of plants grown in field experiments (e.g., Scheffler et al., 1993). Herbicide- resistant survivors could be analyzed further to confirm the presence of the unwanted transgene. 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 resis- tance 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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126 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS In the future, unique DNA fingerprints could be linked to bioconfined transgenes to function as "bio-barcodes" TM (Gressel, 2002). Those markers also could be useful for identifying nonconfined transgenes for labeling, but they require more elaborate and expensive laboratory techniques than are needed for the phenotypic traits mentioned above. Broothaerts and col- leagues (2001) described a multiplex polymerase chain reaction (PCR) tech- nique that simultaneously demonstrates the presence of a transgene sequence and an endogenous gene using a single reaction. Common transgene-specific primers were used in combination with conserved primers for polymorphic endogenous genes. The polymorphisms detected for the endogenous genes permitted the host plant's genotype to be determined, and they confirmed that the PCR had worked properly. The authors proposed the technology for use in protection against mislabeling of cultivars during subculturing and other laboratory and greenhouse operations, as well as for screening for transformants in the production of new transgene lines. The approach also would be useful in identifying cases of transgene escape into other culti- vars or genotypes of the same species and their sexually compatible wild relatives. Greater attention to the need for monitoring could lead to new and more effective approaches. For example, there is much interest in develop- ing a "synthetic nose" remote sensing system that could identify portions of an agricultural field that are under attack by insects. This method would detect and profile volatile emissions from the plants (www.aginfo.psu.edu/ News/march03/sentinel.html). Such devices are being developed for national defense and agronomic uses. Expression of transgenes for insect resistance also gives the genetically engineered plants a profile of volatile emissions that is different from that of wild-type plants of the same genotype, so it is possible that such transgene constructs could be detectable. Remote detec- tion systems could be used to survey large natural areas for transgene or plant escapes at some point in the future, but that possibility is still quite speculative. 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. Therefore, plants that are judged to be serious enough risks should not be released because bioconfinement is always expected to be imperfect. Population, Community, and Ecosystem Effects Bioconfinement has rarely been used for cultivated plants, yet several

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PLANTS 127 new methods could become available within the next 510 years. Given the diversity of methods that are under development (Table 3-1), it is difficult to project environmental effects. Here, a few examples can be used to illustrate possible direct and indirect consequences of future bioconfinement strategies. 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. For example, when a fully sterile crop or crop-wild hybrid produces no pollen, no viable seeds, and does not reproduce vegeta- tively, the transgene will not spread. Under what conditions could this pose a problem? A possible source of food for insects or wildlife could disappear if seed crops are eliminated through bioconfinement, although the ramifica- tions could be relatively unimportant in some circumstances. For example, if vast tracts of planted, seed-producing trees, such as Douglas fir, were replaced with sterile trees, animal populations that depend on the seed source could be harmed. Whether that would threaten ecologically, eco- nomically, or socially important species would require further, case-by-case investigation. Another hypothetical effect of transgenic sterility might occur if pollen from a crop with seed-specific sterility inundates small populations of wild relatives growing nearby. With extensive immigration of sterility-causing genes, the wild plants' seed production could be reduced (seeds sired by the transgenic pollen would be dead). Under some circumstances, this effect of "usurping" ovules and interfering with seed production might cause the native populations to shrink. However, few examples involving endangered wild relatives of crops have been identified (Hancock, 2003). Sexually com- patible taxa that occur near crops often are weedy or colonizing species for which small population size is not a concern. If bioconfinement were indi- rectly responsible for greater contact between the crop and the wild rela- tive, a possible case of unintended consequence could be argued. Moreover, if a crop's wild relatives are an important source of germplasm for further breeding, as is the case for perennial wild rice (Oryza rufipogon) in South- east Asia (Lu et al., 2003), extra precautions might be needed to ensure that gene flow from a V-GURT does not exacerbate the erosion of valuable genetic diversity. A more far-reaching fear among some members of the public is that sterility genes could spread throughout natural populations of wild rela- tives in a silenced (inactive) condition and later be reactivated, leading to massive die-off in populations of sexually compatible crop relatives. It is difficult to conceive of specific mechanisms that would support this

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128 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS hypothesis, but further study should be considered for transgenic sterility methods. Other bioconfinement methods are intended to reduce the fitness of offspring from the crop or its crop-wild hybrids. Multiple scenarios for the fate of such fitness-decreasing transgenes should be considered to evaluate the effects of this process. First, if gene flow is extensive enough or the recipient population is small enough, deleterious transgenes could become fixed in feral or hybrid populations, perhaps leading to reduced popula- tions. This type of "demographic swamping" could occur along contact zones between the crop and its wild relatives (e.g., Haygood et al., 2003). Lower fitness that is shared by all members of small populations along the contact zone could cause the population to shrink and perhaps disappear. A second and perhaps more likely scenario is that fitness-reducing transgenes would be purged by natural selection, a process that is likely to occur with many types of "domestication" crop genes that enter wild or weedy popu- lations. Purging is expected to occur in populations for which gene flow is relatively low and the effective population size of wild relatives is larger than about 100 individuals. Large population size is common for most wild relatives of crop species. Male sterility is a bioconfinement method that sometimes is misunder- stood to be a danger to wild populations. Nontransgenic cytoplasmic male sterility has been used for decades to obtain hybrid seed in crops such as sunflower, canola, and sorghum (but not corn, for which mechanical de- tasseling is the commonly used method). Male sterility generally does not "breed true" or persist because of the large numbers of fertility-restoring genes that are found in cultivated and wild relatives of the crop (Besnard, 2000; Jan, 2000; Ohkawa, 1984; Yamagishi, 1998). In the future, new types of transgenic male sterility could come into common use for hybrid seed production in a wider variety of crops. Thus, male-sterile plants could be grown on much larger lands than at present, and it is possible that sterility would be passed on to plant offspring. If so, it is not expected that wild relatives of a crop would be harmed because fitness-reducing traits are quickly purged from large, interbreeding populations. It is also important to consider the possible indirect effects of various bioconfinement methods. For example, how would a bioconfinement method affect populations of nontarget organisms, such as pollinators and other beneficial insects? Could the method harm animals at higher trophic levels in food webs because their prey are adversely affected? Could a novel trait like apomixis allow a vigorous cultivar to establish feral populations that invade natural areas? Also, would the method facilitate the cultivation of novel crops that produce unhealthy residues or facilitate environmentally damaging agricultural practices? How would those effects compare with existing problems caused by conventional agriculture? There is no reason to

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PLANTS 129 expect unwanted effects as a general feature of bioconfinement, but any large-scale release of novel GEOs should be accompanied by careful risk assessment. To thoroughly evaluate new methods it is necessary to examine anticipated benefits as well as possible risks of specific cases. 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). The ecological and evolutionary consequences of failed methods will depend on the char- acteristics of the transgenic plant, the environment in which it occurs, and the effectiveness of physical confinement. Failure of confinement methods-- biological and otherwise--that are used to prevent pharmaceutical proteins in a commodity crop like maize from entering the food supply could lead to huge socioeconomic damage and unwanted effects on human health and nontarget organisms. Likewise, if bioconfinement fails to prevent the spread of an invasive horticultural variety, economic and environmental damage could be extensive. If bioconfinement is used with low-risk GEOs, however, the consequences of failure should be negligible. In general, the reason for investing in bioconfinement in the first place is usually strong enough to indicate the potential seriousness of the consequences of failure. Specific consequences of bioconfinement failure will depend on the type and the scale of the damage, as is discussed in Chapter 2, reflecting the "hazard exposure" equation used in academic discussions of risk assess- ment (see also Figure 2-1). In some cases exposure could be very small (e.g., Slavov et al., 2002, model on gene flow from poplar). However, in complex and constantly evolving ecological systems, the probability of exposure and the risk of harm from such exposure can be difficult to quantify empirically. Also, public perception of risk often is based on other, less tangible criteria. A basic tenet of this report is that bioconfinement is likely to fail to some extent, even when multiple methods are used to safeguard against failure.