Genetically Engineered Plants In The Environment: Applications And Issues {Manuscript}


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EPA 0823
Genetically Engineered Plants in the Environment-Applications and Issues
Lidia S. Watrud
United States Environmental Protection Agency
National Health and Environmental Effects Research Laboratory
Western Ecology Division •
200 SW 35th Street
Corvallis, OR 97333
%
phone (541) 754-4874
FAX (541) 754-4799
e-mail: watrud.lidia@epamail.epa.gov
Manuscript has been subjected to peer and Agency administrative reviews. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
The information in this document has been funded by the U.S. Environmental
Protection Agency. It has been approved for publication as an EPA document
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ABSTRACT
Almost 20 years have past since the first report of genetic engineering of plants. During
that time, significant technical advances have been made in plant transformation, in gene isolation
and design and in the regulation of gene expression. Increasing numbers of food, fiber and
horticultural species can be engineered with a broad range of engineered traits of potential value
for agricultural, human health and environmental clean up applications. The majority of early
commercial product candidates have been herbaceous crop plants engineered for resistance to
agronomic pests or to herbicides. Field testing of genetically engineered plants has occurred in
numerous countries. However, many unresolved environmental, regulatory, proprietary and
public acceptance issues remain. An overview of the types of engineered plant products that are
being developed is presented. Reported non-target effects of genetically engineered plants on
plant, microbial and invertebrate populations are summarized. Research to assess the potential
long term non-target ecological and health effects of engineered plants is proposed. Technical and
non-technical points to consider in developing and releasing genetically engineered plants are also
discussed.
key words: genetically engineered plants, non-target ecological effects, risk assessment,
transgenic plants
PROTECTED UNDER INTERNATIONAL COPYRIGHT
ALL RIGHTS RESERVED.
NATIONAL TECHNICAL INFORMATION SERVICE
U.S. DEPARTMENT OF COMMERCE

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INTRODUCTION
Selection of natural variants and specific breeding of plants for given agronomic,
horticultural or silvicultural applications are commonly used crop improvement practices. In
conjunction with fertilizers, weed and pest control, plant breeding efforts have been very
successful in producing plants with improved yields and desired crop quality characteristics. For
the most part, planned introductions of herbaceous and woody plants, even in geographies
outside their native ranges, have proceeded without adverse ecological or health effects. Many of
our major crop species, such as wheat, corn, rice, potatoes, and soybeans, have been successfully
introduced world-wide (80). However, numerous escapes from cultivation of non-engineered
agronomic, horticultural and tree species are causing unwanted ecological effects. Examples
include kudzu, johnson grass, purple loosestrife and Melaleuca (64,111, 112, 131, 163, 172.
177). With genetic engineering, traditional breeding barriers between plants can be overcome,
thereby making possible the creation of truly novel plants. Genes from other species or genera
of plants, and even genes from microbes and animals, are being introduced into an increasingly
broad array of herbaceous and woody, food, fiber, ornamental and specialty crop (e. g., nut and
vine) species to create engineered plants having desired characteristics (44,48, 50, 77,126,146).
As these novel plants rapidly progress from laboratory culture to greenhouse and field testing
(86, 149) and into commercial production, tests of their efficacy and yields tend to be well
addressed. However, the degree of evaluation of their potential ecological impacts and human

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biosafety may vary, depending in part on the existence and rigor of regulations (104). Typically,
assessment for ecological and human safety consists of short term, single species, toxicity tests
done under laboratory conditions. The objectives of this review are to (a) provide a brief
summary of the major proposed applications of engineered plants, (b) highlight published results
on their potential non-target effects, (c) suggest points to consider in developing and releasing
engineered plants, (d) propose research needed to identify potential long term effects of releasing
engineered plants and (e) briefly discuss potential impacts of regulations, intellectual property
rights and public acceptance on the development, safety evaluation and commercialization of
engineered plant products.
CREATING ENGINEERED PLANTS
Agrobacterium, particle guns and electroporation represent three major, not necessarily
mutually exclusive or equally effective, approaches for introducing genes into plants (48, 50. 77.
126, 146). In spite of the relative ease of transformation of many plant species, some species,
particularly legumes, cereals, and woody species, often remain recalcitrant to transformation
simply by treatment of tissue surfaces, cells or protoplasts with engineered Agrobacterium (106,
126,162). Higher rates of transformation may sometimes be achieved by using physical
approaches such as mechanical energy or electrostatic forces to introduce organisms, plasmids,
transposable elements or nucleic acids containing sequences of interest (24,25,26,68,90,146).
In ballistics-based approaches, gold or other non-biological particles coated with Agrobacterium,
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plasmids or nucleic acid sequences are literally explosively propelled into plant host tissues,
embryos or cells. In electroporation, charge differences are applied to facilitate entrance of
engineered plasmids or nucleic acids into recipient cells or protoplasts. Preliminary estimates of
transformation success may be obtained in each of these systems by using selective marker genes,
typically for antibiotic resistance, which allow transformed survivors to grow on selective media.
Reporter genes such as the GUS, Green Flourescent Protein and lux systems, which produce
visible stains or emit visible or flourescent light respectively, also may be used to quantify gene
expression and study gene regulation in transfomiants (69, 107, 115,128, 151). Various nucleic
acid-based amplification and hybridization methods and protein- based immunological techniques
may additionally or alternatively be used to detect and quantify gene expression, and to select
candidates for advanced testing and breeding efforts (57, 65, 135). Ideally, transformants having
desired expression levels can be regenerated into whole plants and used in subsequent seed
increase and traditional breeding efforts to produce plants which stably express the desired gene
in progeny which are agronomically fit (17, 85). To help protect proprietary interests, additional
engineering steps may be taken to introduce genes for seed sterility. Use of this controversial
seed sterilizing technology to prevent farmers and growers from saving seed for replanting and
breeding purposes respectively, has been termed "terminator" technology in the public press (28,
81,139).
PROPOSED USES OF GENETICALLY ENGINEERED PLANTS
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In addition to numerous journal articles and reviews (19,49,171), various academic, trade
and activist newsletters provide periodic updates on the kinds of engineered plants which are
being developed and field tested (54,66,156).
Three categories which broadly describe the proposed applications of engineered plants
are crop protection, crop quality and specialty uses. The crop protection category includes
plants designed for resistance to insect pests, to plant diseases and to herbicides. Examples of
plants designed for protection against lepidopteran pests such as homworms, ear worms,
budworms and bollworms, include tomatoes, corn and cotton that express pesticidal delta-
endotoxin or cry genes from Bacillus thuringiensis var. kurstaki (B t.k.), (34,47,122). Addition
of genes to express serine protease inhibitors also has been explored as a means of increasing
B.t.k. activity (51,93). Crystal toxin genes from Bacillus thuringiensis var. tenehrionis (B.t.k.).
have been used to confer resistance to a coleopteran insect, the Colorado potato beetle (103).
Disease resistance strategies have focused primarily on control of viral and fungal diseases
of plants. Incorporation of viral coat protein genes, anti-sense or ribozyme sequences has been
explored as means of conferring resistance to viruses such as TMV and ToMV in tobacco and
tomato, CMV in cucurbit crops and tomatoes, and PVX and PVY in potatoes (9, 21, 29, 60, 73,
89,161). To achieve resistance to fungal pathogens, sequences encoding hydrolytic enzymes
such as chitinases, gluconases and phosphatases have been inserted into plants such as tobacco,
potatoes, com and roses (18,96,152,167). Other disease resistance strategies include insertion
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of sequences to express anti-bacterial cysteine-rich peptides such as thionins and defensins (20),
or production of more broadly active ribosome inactivating proteins (92,169). Insertion of
glucose oxidase genes has been postulated to result in increased production of hydrogen peroxide,
which may be directly toxic to some pathogens; the hydrogen peroxide in turn, has been
suggested to activate or enhance the protective response of plant systemic aquired resistance
(SAR) mechanisms (152). Use of genetic engineering to increase production of secondary
metabolites such as chalcones also has been proposed as a mechanism to confer plant disease
resistance (109). Genetically engineered plants are also being developed as tools to better
understand, induce and regulate SAR responses (22, 37).
Mechanisms to confer herbicide resistance include insertion of plant or bacterial genes
which encode enzymes for herbicide inactivation or degradation, or for inactivation of target sites
(27,63,76,120). For example, bromoxynil resistant cotton and canola have been produced by
introducing microbial genes for the enzyme nitrilase, which can degrade bromoxynil (147), and
resistance to the herbicide glufosinate has been achieved by cloning the gene for phosphinothricin
acetyl transferase from an actinomycete into crop plants (63). Introduction of plant or microbial
sequences encoding modified EPSP (5-enolpyruvl-3phosphoshikimate) synthases, has been used
to produce soybean, cotton and canola plants tolerant to the herbicide glyphosate (76).
The major objectives of crop quality improvements are the modification of traits to
enhance nutritional benefits to consumers or economic benefits to growers and food processing
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companies. Approaches to increase levels of the antioxidant, vitamin E, have been described
(113,141). To produce oils more suitable for cosmetic uses or for human consumption
respectively, the degree of saturation of lipids can be increased or decreased using anti-sense
technology (82,83). Anti-sense technology also has potential applications in controlling shelf
life of produce by inhibiting expression of enzymes involved in cell wall degradation. Using
altered plant or microbial genes for enzymes involved in sugar and starch biosynthesis, starch
levels can be increased in corn and potato cultivars used as starch or ethanol sources; they also
can be used to decrease starch and associated oil absorption in potato cultivars used for French
fries (49,140). Genetic engineering may also be used to either increase the gluten content of
wheat flour used in making bread or decrease it in flour used for making pastries (15).
A major proposed specialty application of engineered plants includes production of
pharmaceuticals. Plants have be£n engineered to produce vaccines, antibodies and peptides (e. g.,
enkaphalins and inteferons), for veterinary and human therapeutic uses (10,61, 99,119,144,
' 165, 183). Plants also have been designed to produce industrial enzymes including bacterial
alpha-amylase, which may be useful in food and beverage processing and in stain removal, and to.
produce fungal lignin-peroxidase to degrade wastes from pulp mills (6). They also have been
proposed as production sources for plastics and pigments (70,123,154).
Phytoremediation is attractive as a lower cost, in situ alternative to transporting
contaminated soils for clean up by extraction or incineration methodologies (4,46,134,136).
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Engineering of plants to selectively develop rhizosphere flora capable of degrading specific
xenobiotic compounds has been proposed (117). Pesticide and heavy metal tolerant plants for
use in clean up of polluted soils, for treatment of industrial waste streams and as environmental
biosensors also have been proposed (84,110,166). Mechanisms for phytoremediation include
enzyme secretion by roots, to degrade xenobiotic chemicals in soil, and binding of metals to
introduced metallothioneins or peptides (108,148).
Strategies to produce plants tolerant to natural stressors such as freezing temperatures
and salts, have been described which utilize gene sources ranging from bacteria to fish (7, 55,62,
102,155,181,182). Availability of plants able to grow in physically and chemically demanding
environments could result in a redefinition of current concepts of arable soils. Coupled with
increases in photosynthetic efficiency and modifications in patterns of carbon allocation (75, 95).
improved stress tolerance could lead to the development of plants customized for optimal growth
and yield even in areas which have short growing seasons and suboptimal growing conditions.
POTENTIAL NON-TARGET ECOLOGICAL AND HEALTH EFFECTS OF
ENGINEERED PLANTS
Numerous international symposia have been held to discuss proposed applications and
biosafety considerations for the release of engineered plants (Table 1). In part because of the
newness of the technology, relatively little is available in the peer-reviewed literature on observed
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non-target ecological effects of engineered plants. Most of the ecological risk concerns have
focused on three areas: (a) the potential for gene flow and outcrossing of herbicide resistance
genes, with the resultant development of crop-weed hybrids (b) the development of resistance to
B. t. k delta-endotoxins in lepidopteran insect pest populations and (c) the effects of pesticidal
plants on soil foodweb biota.
Many of the early and continuing concerns regarding the release of engineered plants have
centered on the potential for escape of herbicide resistant plants or their genes, to crop and non-
crop relatives (5, 11, 16,30,31,32, 33,45, 53,58,71,74, 78, 79, 97, 129, 130, 138, 145, 157, '
158,159,179). Publications on gene flow between engineered and non-engineered plants in
greenhouse or field situations are becoming increasingly available (12,23,36,91, 127).
Accordingly, strategies to reduce and manage the risks of gene flow from engineered plants are of
interest, and have been discussed (72, 133). For cotton, soybeans and corn, which at least in the
major growing areas in the continental U.S., do not have closely related wild relatives, outcrossing
has not been a significant concern (145). However in the U.S., where sunflowers, cucurbits and
radish have wild relatives; in Canada and the northern U.S., where canola may coexist with wild
mustards; and in Europe, where wild beets may coexist in proximity to cultivated sugarbeet
crops; herbicide resistant gene flow to wild relatives could result in the creation of crop-weed
hybrids. A recent report suggesting enhanced outcrossing of transgenic plants (12) is of
particular ecological concern; it highlights a need to carefully monitor the outcrossing rates of
genetically engineered plants. Additional factors which need to be looked at in longer term
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studies of potential non-target effects of gene flow from transgenic plants are viable seed
production, spread and persistence of crop-weed hybrids.
Preventing or decreasing resistance development to B. 1.1 delta endotoxins in target and
non-target susceptible lepidopteran insect populations, has received much attention from
entomologists (2,59,67,100,101, 132, 153,164). Recently, a major agricultural biotechnology
company announced that growers should plant areas adjacent to fields planted with insect
resistant engineered com, with non-insecticidal ciiltivars (173). These areas would serve as
refugia, in which target pest populations would not be exposed to the pesticidal proteins.
Entomologists have additionally recommended that "pyramiding", the use of multiple engineered
and non-engineered genes to confer resistance to target pests, should also be considered as part of
an over all strategy to slow down resistance development in target pest populations (132).
Several studies are available on the short term ecological effects of engineered plants
containing insecticidal genes on soil foodweb components. Using a broad array of techniques,
changes in the size and diversity of bacterial, fungal and plant feeding nematode populations and
in soil enzyme actvities, have been found in soil exposed to leaf litter from cotton expressing the
B.t.k. delta-endotoxin gene and potato plants expressing the B.t. t. crystal protein gene (39, 40).
Similarly, soil incorporation of tobacco leaves expressing an insecticidal protease inhibitor,
resulted in changes in soil respiration and in populations of nematodes, protozoans and
microarthopods (42). Using immunological methods, the persistence of B.t.k delta-endotoxin
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and protease inhibitor in engineered leaves of cotton and tobacco respectively, was observed for
several months following incorporation into soil (42,121). Using molecular methods, antibiotic
resistance marker sequences in plasmid DNA and in potato leaf litter, could be detected for
months following incorporation into field soils and soil microcosms, respectively (175,176).
Non-target effects of alfalfa plants designed for industrial enzyme production include changes in
the community composition and substrate utilization patterns of microbial populations,
decreases in plant biomass and changes in nutrient content of both greenhouse grown and field
grown engineered alfalfa plants (35,41,170). Differences have been noted in endophytic and
rhizosphere microbial communities between nonengineered canola cultivars and those engineered
to be herbicide resistant (143). Delays and decreases in arbuscular mycorrhizal infection have
been observed in some tobacco transformants engineered to express phosphatases for fungal
disease resistance (167). In contrast, tobacco engineered for disease resistance with defensin
genes had no inhibitory effect on arbuscular mycorrrhizal infection (13). In alfalfa containing a
fungal lignin-peroxidase gene, a trend toward decreased arbuscular mycorrhizal infection was
observed in plants grcnvn in greenhouses (170). An excellent review is available which
summarizes the potential and reported effects of transgenic plants producing anti-bacterial and
antifungal proteins on saprophytic soil microflora (56). A summary of reported short term non-
target effects of engineered plants expressing traits for insect, disease or herbicide resistance and
for production of specialty chemicals is presented in Table 2.
The possibility exists that non-target or unintended effects may be the result of
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somaclonal variation. For example, in a study of non-transformed cotton plants regenerated from
tissue culture, significant differences in boll and seed number and in fiber quality were attributed
to somaclonal variation (3). It is thus conceivable that some non-target or unintended effects of
engineered plants may not be due to direct or indirect activities of engineered gene products per
se, but to somaclonal variation. Additional possibilities include positional and pleiotropic effects
related to where introduced genes have inserted into the host plant genome. The generally
random nature of gene insertion may result in activation or inactivation of genes having functions
which differ from those of the inserted gene. "Whether introduced genes, somaclonal variation,
positional or pleotropic effects result in unwanted agronomic, health or ecological effects or
potentially yield and crop quality benefits, are areas deserving of careful screening, selection and
monitoring (39,105,180).
Few reports are available in the peer reviewed literature on evaluation of potential effects
of engineered plants on human health (52,114). Development of allergenicity to proteins in
engineered plants is a potential concern, since it may not be apparent with short term, single
acute exposures. Over a long period of cumulative dietary or contact exposure however,
susceptible individuals may develop allergic responses to these proteins. Interest in modifying
seed storage proteins, such as those found in soybeans or Brazil nuts (87) appears to have
waned, in large part due to allergenic concerns (116). Another human health concern which has
been raised and debated, and which is more of a concern in some parts of the world than in
others, is transfer of antibiotic resistance from genes in ingested plant tissues to human gut flora.
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Because of their common use as selective markers to facilitate the detection of transformants.
DNA encoding resistance to antibiotics such as streptomycin and kanamycin, is present in many
engineered plant tissues. Potential ways to minimize concerns about the transfer of antibiotic
resistance genes from plant tissue to intestinal flora are utilization of transformation strategies
which can avoid the use of antibiotic resistance markers, removal of antibiotic resistance genes by
ge : and biochemical means, or determination of acceptable levels of risk.
REGULATORY AND ECONOMIC POINTS TO CONSIDER IN DEVELOPING
GENETICIALLY ENGINEERED PLANTS
It is understandably difficult to propose universally applicable testing requirements for
the broad spectrum of engineered gene and plant possibilities. Arguments for case by case
regulation or no regulation, have thus sometimes been brought forward. Both national and
international efforts continue to harmonize regulations for environmental testing permits and for
commercial use registrations (168). International entities such as the Organization for Economic
and Community Development (OECD), the United Nations Industrial Development
Organization and national Departments, Agencies and Ministries of Agriculture, Food Safety, the
Environment and Forestry of individual countries, may each provide varying degrees of oversight
and regulatory guidance. Based on the nature of specific crops and traits, either engineered plants,
or their active (engineered) ingredients may be regulated. For example, in the U.S., shipment and
field tests of engineered plants are regulated by the U.S. Department of Agriculture (U.S.D.A.),
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the active engineered pesticidal ingredients expressed in engineered pestididal plants are regulated
by the U.S. Environmental Protection Agency (U.S.E.P.A.), and food additives are regulated by
the Food and Dnig Administration (F.D.A.). An overview of the coordination of the various
roles of U.S.D.A., F.D.A. and E.P.A., under their respective statutory authorities (Plant Pest
Act; Federal Insecicide, Fungicide and Rodenticide Act and the Toxic Substances Control Act;
Federal Food, Drug and Cosmetic Act), have recently been summarized (8). In addition to federal
regulations, individual states may have notification, permitting or other regulator)' requirements.
Points that may be useful to consider as engineered plants are developed, tested and considered
for commercialization, are presented in Table 3. These include the market need for that type of
plant product, efficacy, economic returns and short and long term non-target ecological and health
effects.
RESEARCH NEEDS FOR ASSESSMENT OF NON-TARGET ECOLOGICAL EFFECTS
OF ENGINEERED PLANTS
Given the published observations on outcrossing potential of genes to weeds, effects on
the size and diversity of soil foodweb populations and on host plants, a need for longer term
ecological monitoring seems apparent. For example, if plants designed for phytoremediation,
crop protection or specialty chemical production result in accumulation of toxic compounds in
their shoots or rhizospheres, potential impacts on herbivores, pollinators, pathogens, pests,
symbionts, detritovores and saprophytes might be anticipated. Downstream effects on rates of
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litter decomposition and nutrient cycling could also develop. Similarly, escape, persistence and
reproduction of herbicide tolerant plants or crop-weed hybrids, could bring about changes in
plant community composition, much in the same way that exotic weeds have invaded and altered
rangelands, grasslands, wetlands and forests.
The effects of potential changes in agonomic practices necessitated by the use of
engineered crops, also need to be addressed. If the types and rates of agricultural chemical
application for the engineered crop differ from those of non-engineered cultivars, the associated
spectrum of pests and pathogens may change on each. The pest and pathogen spectrum may also
change on adjacent fields of other crops and on non-crop plant species. If modified crop chemical
recommendations are needed for engineered cultivars, the impacts of both crop and chemicals on
subsequent crop rotation and chemical options also will need to be considered.
Reliance on methods used to monitor the fate, transport and persistence of chemicals may
not be sufficient or even appropriate, for novel biologicals produced by some kinds of engineered
plants. Similarly, use of single species, short term test systems may not be appropriate or
sufficient for some types of engineered plant products. Increased attention should be given to
utilizing and developing methods to look at both short and long term soil foodweb, trophic and
community level responses, as alternatives or supplements to single species acute toxicity tests.
Studies are needed to determine if long term dietary or contact exposure to engineered products
may lead to toxicity in wildlife and humans and development of allergenicity to humans.
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Use of modern molecular and immunological methods, often similar or identical to those
used in gene isolation and in quantification of gene expression, may facilitate detection', tracking
and fate of engineered genes and gene products (121,125,142,175,176). Knowledge of the
degree of spread of transgenic genes and genetically engineered plants can then be used to design
and implement control strategies appropriate (i. e., if needed and in accordance with determined
risks), for escaped and persistent genetically engineered crops and crop-weed hybrids. Fig. 1
summarizes the major types of targeted applications of engineered plants; it also highlights areas
where research may be needed to identify and mitigate potential long term non-target ecological
and health effects of genetically engineered plants.
NON-ECOLOGICAL ISSUES REGARDING THE RELEASE AND
COMMERCIALIZATION OF GENETICALLY ENGINEERED PLANTS
It is clear that many different kinds of plants can and have been genetically engineered for
diverse, novel and potentially useful agronomic and specialty chemical applications. Issues that
are unclear and which continue to be debated in the media, court rooms, board rooms and in the
court of public opinion, are who if anyone, should own or manipulate genes or life forms initially
found in nature (38, 150). In many countries, regulatory paths remain unclear, or are not yet in
place. Even as commercial products have begun to be marketed, difficult questions regarding
regulation, ownership and pi'blic acceptance persist (137,139). Cost of access to engineered
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seed and biological, marketing and contractual strategies to limit growers attempts to save and
replant seed progeny, can become challenging public relations issues (81,174). Patents may
barely be granted and licensing agreements signed, before they are challenged in court (1,14, 43,
98,118, 124). Licensing and royalty fees may be paid simply as costs of doing business, to
avoid lengthy and costly legal battles over patent rights.
CONCLUSIONS
Technical advances now permit introduction of genes from diverse sources into a broad
array of herbaceous and woody food and fiber crops. Engineered plants have begun entering
commerce, particularly in the U.S., and are being tested in numerous countries. Gene flow
studies have documented transfer of engineered genes to crop and weed species; soil foodweb
studies have demonstrated effects of several types of engineered plants on microbial and
invertebrate populations in soil. Consequently, there is a need for ecological and health effects
studies to be performed both prior to and after broad scale release. In addition, monitoring and
mitigation plans are needed to help ensure the long term environmental and human safety of
releasing and using engineered plant products.
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REFERENCES
1.	Agris, C. H. 1998. Patenting DNA sequences. Nature Biotechnol. 16:877.
2.	Alstad, D. N., and D. A. Andow. 1995. Managing the evolution of insect resistance to
transgenic plants. Science 268:1894-1895.
3.	Altman, D. W., D. M. Stclly, and D. M. Mitten. 1991. Quantitative trait variation in
phenotypically normal regenerants of cotton. In vitro Cell. Dev. Biol. 3:132-138.
4.	Anderson, T. A., E. A. Guthrie and B. T. Walton. 1993. Bioremediation in the
Rhizosphere. Environmental Sci. & Technol. 27:2630-2636.
5.	Arias, D. M., and L. H. Rleseberg. 1994. Gene flow between cultivated and wild
sunflower. Theoret. & Appl. Genetics 89:655-660.
6.	Austin, S., E. T. Bingham, D. E. Matthews, M. N. Shahan, J. Will, and R. R. Burgess.
1995. Production and field performance of transgenic alfalfa expressing alpha-amylase and
manganese dependent lignin peroxidase. Euphytica 85:381-393.
7.	Baertlein, D. A., S. E. Lindow, N. J. Panopoulos, S. P. Lee, T. H. H. Chen, and M. N.
Mindrinos. 1992. Expression of a bacterial ice nucleation gene in plants, Plant Physiol.
100:1730-1736.
8.	Beach, J. E. 1998. No "killer tomatoes": easing federal regulation of genetically engineered
plants. Food & Drug Law J. 53:181-191.
9.	Beachy, R. N., S. Loesch-Fries, and N. E. Turner. 1990. Coat protein-mediated
resistance against virus infection. Annu. Rev. Phytopathol. 28:451-474.
10.	Benvenuto, E., R. J. Ordas, R. Tavazza, G. Ancora, S. Biocca, A. Cottanea, and P.
19

-------
Galeffl. 1991. "Phytoantibodies'Ya general vector for the expression of hemoglobin
domains in transgenic plants. Plant Mol. Biol. 17:865-874.
11.	Bergelson, J. 1994. Changes in fecundity do not predict invasiveness: a model study of
transgenic plants. Ecology 75:249-252.
12.	Bergelson, J., C. B. Purrlngton, and G. Wichmann. 1998. Promiscuity in transgenic
plants. Nature 395:25.
13.	Bianciotto, V., I. Martini, and P. Bonfante. 1998. Arbuscular mycorrhizal interactions
with transgenic plants expressing antifungal proteins, p. 29. In Proceedings of Second
International Conference on Mycorrhiza, July 5-10, 1998, SLU, Uppsala, Sweden.
14.	Bisbee, C. 1998. Patent law and litigation. Genetic Engineering News. 18:11.
15.	BlechI, A. E., H. Q. Le, and O. D. Anderson. 1998. Engineering changes in wheat flour
by genetic transformation. J. Plant Physiol. 152:703-707.
16.	Boudry, P., M. Morchen, P. Sanmitou-Laprade, P. Vernet, and H. Van Dijk. 1993.
The origin and evolution of weed beets: consequences for the breeding and release of
herbicide-resistant transgenic sugar beets. Theoret. & Appl. Genetics 87:471-478.
17.	Brandle, J. E., S. G. McHugh, L. James, H. Labb6, and B. L. Miki. 1995. Instability of
transgene expression in field grown tobacco carrying the csrl-1 gene for sulfonylurea
herbicide resistance. Bio/Technology 13:994-998.
18.	Broglie, K., I. Chet, M. Holliday, R. Cressman, P. Biddle, S. Knowlton, C. J.
-j
Maiivais, and R. Broglie. 1991. Transgenic plants with enhanced resistance to the fungal
pathogen Rhizoctonia solani. Science 254:1194-1197..
20

-------
19.	Brown," K. S. 1996. Life on the molecular farm. Bioscience 46:80-83.
20.	Carmona, M. J., A. Molina, J. A. Fernandez* J. J. Lopez-Fando, and F. Garcla-
Olmedo. 1993. Expression of the a-thionin gene from barley in tobacco confers enhanced
resistance to bacterial pathogens. Plant J. 3:457-462.
21.	Cech,T. R. 1987. The chemistry of self-splicing RNA and RNA enzymes. Science
236:1532-1539.
22.	Chamnongpol, S., H. Willekens, C. Langebartels, M. Van Montagu, D. Inz6, and W.
Van Camp. 1996. Transgenic tobacco with a reduced catalase activity develops necrotic
lesions and induces pathogenesis-related expression under high light. Plant J. 10:491-503.
23.	Chfevre, A.-M., F. Eber, A. Baranger, and M. Renard. 1997. Gene flow from transgenic
crops. Nature 389:924.
24.	Christou, P. 1995. Strategies for variety-independent genetic transformation of important
cereals, legumes and woody species utilizing particle bombardment. Euphytica 85:13-27.
25.	Christou, P., D. E. McCabe, and W. F. Swain. 1988. Stable transformation of soybean
callus by DNA-coated gold particles. Plant Physiol. 87:671-674.
26.	Christou, P., T. L. Ford, and M. Kofron. 1991. Production of transgenic rice (Oryza
sativa L.) plants from agronomically important indica and japonica varieties via electronic
discharge particle acceleration of exogenous DNA into immature zygotic embryos.
Bio/Technology 9:957-962.
27.	Comai, L., D. Facciotti, W. R. Hiatt, G. Thompson, R. Rose, and D. Stalker. 1985.
Expression in plants of a mutant aro A gene from Salmonella typhimurium confers
21

-------
tolerance to glyphosate. Nature 317:741-745.
28.	Crouch, M. L. 1998. How the terminator terminates: an explanation for the non-scientist
of a remarkable patent for killing second generation seeds of crop plants. The Edmonds
Institute. 10 p.
29.	Cuozzo, M., K. M. O'Connell, W. Kaniewski, R. X. Fang, N. H. Chua, and N. E.
Turner. 1988. Viral protection in transgenic tobacco plants expressing the cucumber
mosiac virus coat protein or its antisense RNA. Bio/Technology 6:549-554,557.
30.	Dale, P. J. 1994. The impact of hybrids between genetically modified crop plants and
their related species: general considerations. Mol. Ecol. 3:31-36.
31.	Dale, P. J. 1997. Potential impacts from the release of transgenic plants into the
environment. Acta Physiol. Plant. 19:595-600.
32.	Dale, P. J., and J. A. Irwin. 1995. The release of transgenic plants from containment, and
the move towards their widespread use in agriculture. Euphytica 85:425-431.
33.	Darmency, H. 1994. The impact of hybrids'between genetically modified crop species
and their related species: introgression and weediness. Mol. Ecol. 3:37-40.
34.	Delannay, X., B. J. LaVallee, R. K. Proksch, R. L. Fuchs, S. R. Sims, J. J. Augustine,
J. G. Layton, and D. A. Fischhoff. 1989. Field performance of transgenic tomato plants
expressing the Bacillus thuringiensis var. kurstaki insect control protein. Bio/Technology
7:1265-1269.
35.	Di Giovanni, G. D., L. S. Watrud, R. J. Seidler and F. Widmer. 1999. Comparison of
parental and transgenic alfalfa rhizosphere bacterial communities using Biolog GN metabolic
22

-------
fingerprinting and enterobacterial repetitive intergeneric consensus sequence - PCR (ERIC-
PCR). Microb.Ecol. 37:129-139.
36.	Dietz-Pfellstetter, A., and M. Klrchner. 1998. Analysis of gene inheritance and
expression in hybrids between transgenic sugar beet and wild beets. Mol. Ecol.
7:1693-1700.
37.	Doerner, P. W., B. Sterner, J. Schmid, R. A. Dixon, and C. J. Lamb. 1990. Plant
defense gene promoter-reporter gene fusions in transgenic plants: tools for identification of
novel inducers. Bio/Technology 8:845-848.-
38.	Doll, J. J. 1998. The patenting of DNA. Science 280:689-690.
39.	Donegan, K. K., C. J. Palm, V. J. Fieland, L. A. Porteous, L. M. Ganlo, D. L. Schaller,
L. Q. Bucao, and R. J. Seidler. 1995. Changes in levels, species and DNA fingerprints of
soil microorganisms associated with cotton expressing the Bacillus thuringiensis var.
kurstaki endotoxin. Appl. Soil Ecol. 2:111-124.
40.	Donegan, K. K., D. L. Schaller, J. K. Stone, L. M. Ganio, G. Reed, P. B. Hamm, and
R. J. Seidler. 1996. Microbial populations, fungal species diversity and plant pathogen
levels in field plots of potato plants expressing the Bacillus thuringiensis var. tenebrionis
endotoxin. Transgenic Res. 5:25-35.
41.	Donegan, K. K., R. J. Seidler, J. D. Doyle, L. A. Porteous, G. Di Giovanni, F.
Widmer, and L. S. Watrud. 1999. A field study with genetically engineered alfalfa
inoculated with recombinant Sinorhizobium meliloti'. effects on the soil ecosystem.
23

-------
Submitted for publication.
42.	Donegan, K. K., R. J. Seldler, V. J. Fieland, D. L. Schallcr, C. J. Palm, L. M. Ganio,
D. M. Cardwell, and Y. Stelnberger. 1997. Decomposition of genetically engineered
tobacco under field conditions: persistence of the proteinase inhibitor I product and effects
on soil microbial respiration and protozoa, nematode and microarthropod populations. J.
Appl. Ecol. 34:767-777.
43.	Dove, A. 1998. Botanical gardens cope with bioprospecting loophole. Science 281:1273.
44.	Dunwell, J. M. 199S. Novel food products from genetically modified crop plants:
methods and future prospects. Intern. J. Food Sci. & Techn. 33:205-213.
45.	Ellstrand, N. C. and C. A. Hoffman. 1990. Hybridization as an avenue of escape for
engineered genes. Bioscience 40:438-442.
46.	Entry, J. A., L. S. Watrud, R. S. Manasse, and N. C. Vance. 1997. Phyroremediation
and reclamation of soils contaminated with radionuclides, p. 229-306. In Phytoremediation
of Soil and Water Contaminants. ACS Symposium Series 664.
47.	Fischhoff, D. A., K. S. Bowdish, F. J. Perlak, P. G. Marrone, S. M. McCormick, J. G.
Niedermeyer, D. A. Dean, K. Kusano-Kretzmer, E. J. Mayer, D. E. Rochester, S. G.
Rogers and R. T. Fraley. 1988. Insect tolerant transgenic tomato plants. Bio/Technology
5:807-813.
48.	Fraley, R. T., S. G. Rogers, R. B. Horsch, P. Sanders, J. Flick, S. Adams, M. Bittner,
L. Brand, C. Fink, J. Fry, G. Gallup!, S. Goldberg, N. Hoffman, and S. Woo. 1983.
Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. U.S.A. 80:4803-4807.
24

-------
49.	Fraley, R. 1992. Sustaining the supply. Bio/Technology 10:40-43.
50.	Fraley, R. T., S. G. Rogers, and R. B. Horsch. 1986. Genetic transformation in higher
plants. Crit. Rev. Plant Sci. 4:1-46.
51.	Fuchs, R. L. 1990. Potentiation of Bacillus thuringiensis insecticidal activity by serine
protease inhibitors. J. Agric. Food Chem. 38:1145-1152.
52.	Fuchs, R. L., and J. D. Astwood. 1996. Allergenicity assessment of foods derived from
genetically modified plants. Food Technol. February:83-88.
53.	Gates, P. 1995. The environmental impact of genetically engineered crops. Biotechnology
and Genetic Engineering Reviews 13:181-195.
54.	Genetic Engineering News, Mary Liebert Publ., New York, NY.
55.	Georges, F., M. Saleem, and A. J. Cutler. 1990. Design and cloning of a synthetic gene
for the flounder antifreeze protein and its expression in plant cells. Gene 91:159-165.
56.	Glandorf, D. C. M., P. A. H. M. Bakker, and L. C. Van Loon. 1997. Influence of the
production of antibacterial and antifungal proteins by transgenic plants on the saprophytic
soil microflora. Acta Bot. Neerl. 46:85-104.
57.	Glick, B. R. and J. J. Pasternak (eds.). 1994. Molecular Biotechnology - Principles and
Applications of Recombinant DNA. ASM Press, Washington, DC.r500 p.
58.	Gliddon, A. 1994. The impact of hybrids between geneticaly modified crop plants and
their related species: biological models and theoretical perspectives. Mol. Ecol. 3:41-44.
59.	Gould, F., A. Martinez-Ramirez, A. Anderson, J. Ferre, F. J. Silva and W. J. Moar.
1992. Broad spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens.
25

-------
Proc. of the Natl. Acad, of Sci. (USA) 89:7986-7990.
60.	Hemenway, C., R. X. Fang, J. J. Kanlewski, N. H. Chua, and N. E. Turner. 1988.
Analysis of the mechanism of protection in transgenic plants expressing the potato virus X
coat protein or its antisense RNA EMBO 17:1273-1280.
61.	Hiatt, A., R. Cafferkey, and K. Bowdish. 1989. Production of antibodies in transgenic
plants. Nature 342:76-78.
62.	Hightower, R., C. Baden, E. Penzes, P. Lund, and P. Dunsmuir. 1991. Expression of
antifreeze protein in transgenic plants. PlarltMol. Biol. 17:1013-1021.
63.	Hinchee, M. A. WM S. R. Padgette, G. M. Kishore, X. Delannay, and R. T. Fraley.
1993. Herbicide-tolerant crops, p. 243-263. In Transgenic Plants: Engineering and
Utilization, Vol. 1, Academic Press, New York.
64.	Holdgate, M. W. 1986. Summary and conclusions: characteristics and consequences of
biological invasions. Philos. Trans. R. Soc. of London B314:733-742.
65.	Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.). 1990. PCR Protocols
- A Guide to Methods and Applications. Academic Press, Inc., San Diego. 482 p.
66.	ISB News Report. Information Systems for Biotechnology, Virginia Polytechnic Institute
and State University.
67.	Ives, A. R. 1996. Evolution of insect resistance to Bacillus thuringiensis-Xrzx\sfoTT(\z&
plants. Science 273:1412-1413.
68.	Jahhe, A., D. Becker, and H. L6rz. 1995. Genetic engineering of cereal crop plants: a
review. Euphytica 85:35-44.
26

-------
69.	Jeffersftn, R. A. 1989. The GUS reporter gene system. Nature 342:837-838.
70.	John, M. E., and G. Keller. 1996. Metabolic pathway engineering in cotton:
biosynthesis of polyhydroxybutyrate in fiber cells. Proc. Natl. Acad. Sci. (USA)
93:12768-12773.
71.	Kareiva, P., R. Manasse, and W. Morris. 1991. Using models to integrate data from field
trials and estimate risks of gene escape and gene spread, p. 31-42. In D. R. MacKenzie and
S. C. Henry (ed.), Proceedings of Kiawah Island Conference, South Carolina, USA 27-30
November 1990.
72.	Kareiva, P., W. Morris and C. M. Jacobi. 1994. Studying and managing the risk of
cross-fertilization between transgenic crops and wild relatives. Mol. Ecol. 3:15-22.
73.	Kavanagh, T. A., and C. Spillane. 1995. Strategies for c ngineering virus resistance in
transgenic plants. Euphytica 85:149-158.
74.	Keeler, K. 1989. Can genetically engineered weeds become crops? Bio/Technology
7:1134-1139.
75.	Kehr, J., F. Hustiak, C. Walz, L. Willmitzcr, and J. Fisahn. 1998. Transgenic plants
changed in carbon allocation pattern display a shift in diurnal growth pattern. Plant J.
16:497-503.
76.	Kishore, G. M. and D. Shah. 1991. Glyphosate-tolerant 5-enolpyruvyl 3-
phosphoshikimate synthase. BiotechnoL Adv. 9:89.
77.	Klein, T. M., M. Fromm, A. Weissinger, D. Tomes, S. SchaiT, M. Sletten, and J. C.
Sanford. 1988. Transfer of foreign genes into maize cells with high-velocity
27

-------
microprojectiles, Proc. Natl. Acad. Sci. U.S.A. 85:4305-4309.
78.	Klinger, T., D. R. Elam, and N. C. Ellstrand. 1991. Radish as a model system for the
study of engineered gene escape rates via crop-weed mating. Conserv. Biol. 5:531-535.
79.	Klinger, T. and N. C. Ellstrand. 1994. Engineered genes in wild populations: fitness of
weed-crop hybrids of Raphanus sativus. Ecol. Appl. 4:117-120.
80.	Kloppenburg, J. R. 1988. First the Seed--the Political Economy of Plant Biotechnology
1492-2000. Cambridge University Press, London. 349 p.
81.	Kluger,J. 1999. The suicide seeds. Time 153:44-45.
82.	Knauf, V. C. 1987. The application of genetic engineering to oilseed crops. Trends
Biotechnol. 5:40-47.
83.	Knutzon, D. S., G. A. Thompson, S. E. Radke, W. B. Johnson, V. C. Knauf, and J. C.
Kridl. 1992. Modification of Brassica seed oil by antisense expression of a stearoyl-acyl
carrier protein desaturase gene. Proc. Natl. Acad. Sci. U.S.A. 89:2624-2628.
84.	Kovalchuk, I., O. Kovalchuk, A. Arkhipov, and B. Hohn. 1998. Transgenic plants are
sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nature
Biotechnology 16:1054-1059.
85.	Koziel, M. G., G. L. Beland, C. Bowman, N. B. Carozzi, R. Crenshaw, L. Crossland,
J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Launis, K. Lewis, D. Maddox, K.
McPherson, E. Meghjl, M. R. Merlin, R. Rhodes, G. W. Warren, M. Wright and S. V.
Evola. 1993. Field performance of elite transgenic maize plants expressing an insecticidal
protein derived from Bacillus thuringiensis. Bio/Technology 11:194-200.
28

-------
86.	Krattiger, A. F. 1994. The field testing and commercialization of genetically modified
plants: A review of worldwide data (1986 to 1993/94). p. 247-266. In A.F. Krattiger, and
A. Rosemarin (ed.), Biosafety for a Sustainable Agriculture: Sharing Biotechnology
Regulatory Experiences of the Western Hemisphere. ISAAA, Ithaca and Stockholm.
87.	Kriz, A. L. and B. A. Larkins. 1991. Biotechnology of seed crops: genetic engineering of
seed storage proteins. HortScience 26:1036-1041.
88.	Laplerre, C., B. Pollet, M. Petit-Conil, G. Toval, J. Romero, G. Pilate, J.-C. Lepl6, W.
Boerjan, V. Ferret, V. De Nadai, and L. Jouanin. 1999. Structural alterations of lignins
in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-
methyltransferase activity have an opposite impact on the efficiency of industrial kraft
pulping. Plant Physiol. 119:153-163.
89.	Lawson, C., W. KaniewskJ, L. Haley, R. Rozman, C. Newell, P. Sanders, and N. E.
Turner. 1990. Engineering resistance to mixed virus infection in a commercial potato
cultivar.resistance to potato virus X and potato virus Y in transgenic Russet BuTbank.
Bio/Technology 8:127-134.
90.	Lebel, E. G., J. Masson, A. Bogucki, and J. Paszkowski. 1995. Transposable elements
as plant transformation vectors for long stretches of foreign DNA. Theor. Appl. Genet.
91:899-906.
91.	Lefol, E., V. Danielou, H. Damnay, M. C. Kerlan, P. Valee, A. Chevre, and M.
Renard. 1991. Escape of engineered genes from rapeseed to wild Brassiceae. Proc.
Brighton Crop Protection Conf. Weeds 3:1049-1056. ¦
29

-------
92.	Logemann, J., G. Jack, H. Tommerup, J. Mundy, and J. Schell. 1992. Expression of a
barley ribosome-inactivating protein leads to increased fungal protection in transgenic to-
bacco plants. Bio/Technology 10:305-308.
93.	Macintosh, S. C., G. M. Klshore, F. J. Ferlak, P. G. Marrone, T. B. Stone, S. R.
Sims, and R.L. Fuchs. 1990. Potentiation of Bacillus thuringiensis insecticidal activity
by serine protease inhibitors. J. Agric. Food Chem. 38:1145-1152.
94.	MacKenzie, D. 1990. Jumping genes confound German scientists. New Sci. 128:18.
95.	Mann, C. C. 1999. Genetic engineers aim to soup up crop photosynthesis. Science
283:314-316.'
96.	Marchant, R., M. R. Davey, J. A. Lucas, C. J. Lamb, R. A. Dixon, and J. B. Power.
1998. Expression of a chitinase transgene in rose (Rosa hyhbrida L.) reduces development
of blackspot disease (Diplocarpon rosae Wolf). Mol. Breeding 4:187-194.
97.	Marshall, G. 1998. Herbicide-tolerant crops-real farmer opportunity or potential
environmental problem? Pestic. Sci. 52:394-402.
98.	Mascarenhas, D. 1998. Negotiating the maze ofbiotech "tool patents." Nature
Biotechnology 16:1371-1372.
99.	Mason, H. S., T. A. Haq, J. D. Clements, and C. J. Arntzen. 1998. Edible vaccine
protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a
synthetic LT-B gene. Vaccine 16:1336-1343.
100.	McGaughey, W. H. and R. W. Beeman. 1988. Resistance to Bacillus thuringiensis in
colonies of Indian meal moth and almond moth (Lepidoptera: Pyralidae). J. Econ. Entom.
30

-------
81:28-33.
101.	McGaughey, W. H. and M. E. Whalon. 1992. Managing insect resistance to Bacillus
thuringiensis toxins. Science 258:1451-1455.
102.	McKersle, B. D., J. Murnaghan, and S. R. Bowley. 1997. Manipulating freezing
tolerance in transgenic plants. Acta Physiologiae Plantarum 19:485-495.
103.	McPherson, S. A., F. J. Perlak, R. L. Fuchs, P. G. Marrone, P. B. Lavrik, and D. A.
Fischhoff. 1988. Characterization of the coleopteran-specific protein gene of Bacillus
thuringiensis var. tenebrionis. Bio/Technology 6:61-66.
104.	Mellon, M. and J. Rosier. 1995. Transgenic crops: USDA data on small-scale tests
contribute little to commercial risk assessment. Bio/Technology 13:96.
105.	Meredith Jr., W. R. 1995. Strengths and limitations of conventional and transgenic
breeding. 1995 Proceedings Beltwide Cotton Conferences, San Antonio, TX, USA, January
4-7,1995 1:166-168.
106.	Michler, C. H. 1991. Biotechnology of woody environmental crops. HortScience
26:1042-1044.
107.	Millar, A. J., S. R. Short, N. H. Chua, and S. A. Kay. 1992. A novel circadian
phenotype based on firefly luciferase expression in transgenic plants. The Plant Cell
4:1075-1087.
108.	Misra, S. and L. Gedamu. 1989. Heavy metal tolerant transgenic Brassica napus and
Nicotiana tabacum L. Theor. Appl. Genet. 78:161-168.
109.	Moffat, A. S. 1992. Improving plant disease resistance. Science 257:482-483.
31

-------
110.	Monciardinl, P., D. Fodini, and N. Mai*m!roli. 1998. Exotic gene expression in
transgenic plants as a tool for monitoring environmental pollution. Chemosphere
37:2761-2772.
111.	Mooney, H., S. Hamburg, and J. Drake. 1986. The invasions of plants and animals into
California. In J. A. Mooney and J. A. Drake (ed.). Ecology of Biological Invasions of
North America and Hawaii. Springer-Verlag, New York. 250 p
112.	Morganthaler, E. 1993. What's Florida to do with an explosion of Melaleuca trees? Wall
Street Journal, February 8.1993.
113.	MuIIineaux, P. M., and G. P. Creissen. 1996. Opportunities for the genetic
manipulation of antioxidants in plant foods. Bioactive Components of Food (Biochemical
Society Transactions) 24: 829-835.
114.	Nida, D. L., S. Patzer, P. Harvey, R. Stipanovic, R. Wood, and R. L. Fuchs. 1996.
Glyphosate-tolerant cotton: the composition of the cottonseed is equivalent to that of
conventional cottonseed. J. Agric. Food Chem. 44:1967-1974.
115.	Niedz, R. P., M. R. Sussman, and J. S. Satterlee. 1995. Green fluorescent protein - an
in vivo reporter of plant gene expression. Plant Cell Rep.l4:403-406!
116.	Nordlee, J. A., S. L. Taylor, J. A. Townsend, L. A. Thomas, and R. K. Bush. 1996.
Identification of a Brazil-nut allergen in transgenic soybeans. N. Engl. J. Med. 334:688-
692.
117.	O'Connell, K. P., R. M. Goodman, and J. Handelsman. 1996. Engineering the
rhizosphere: expressing a bias. Tibtech 14:83-88.
32

-------
118.	Ono, R. D. (ed.). 1991. The Business of Biotechnology - From the Bench to the Street.
384 p.
119.	Owen, M. R. L., A. Gandecha, B. Cockburn, and G. C. Whitelom. 1992. The
expression of antibodies in plants. Chem. Ind. (London) 11:406-408.
120.	Padgette, S. R., G. della-Cioppa, D. M. Shah, R. T. Fraley, and G. M. Kishore. 1989.
Selective herbicide tolerance through protein engineering, p. 441-476. In Cell Culture and
Somatic Cell Genetics of Plants, Academic Press, New York.
121.	Palm, C. J., D. L. Schaller, K. K. Donegan, and R. J. Seidler. 1996. Persistence in soil
of transgenic plant produced Bacillus thuringiensis var. kursiaki d-endotoxin. Can. J.
Microbiol. 42:1258-1262.
122.	Perlak, F. J., R. W. Deaton, T. A. Armstrong, R. L. Fuchs, S. R. Sims, J. T.
Greenplate, and D. A. Fischhoff. 1990. Insect resistant cotton plants. Bio/Technology
8:939-943.
123.	Poirier, Y., D. E. Dennis, K. Klomparens, and C. Somerville. 1992.
Polyhydroxybutyrate, a biodegradable thermoplastic produced in transgenic plants. Science
256:520-523.
124.	Pollan, M. 1998. Playing God in the Garden. New York Times Mag. October 25:44.
125.	Porteous, L. A., R. J. Seidler, and L. S. Watrud. 1997. An improved method of
purifying DNA from soil for polymerase chain reaction amplification and molecular ecology
applications. Mol. Ecol. 6:787-791.
126.	Potrykus, I. 1990. Gene transfer to cereals: an assessment. Bio/Technology 8:535-542.
33

-------
127.	Purrington, C. B. and J. Bergelson. 1995. Assessing weediness of transgenic crops:
industry plays plant ecologist. Trends in Ecol. & Evol. 10:340-342.
128.	Quaedvlieg, N. E. M., H. R. M. Schlaman, P. C. Admtraal, S. E. Wijtlng, J.
Stougaard, and H. P. Spaink. 1998. Fusions between green fluorescent protein and
B-glucuronidase as sensitive and vital Afunctional reporters in plants. Plant Mol. Biol.
38:861-873.
129.	Raybould, A. F. and A. J. Gray. 1993. Genetically modified crops and hybridization with
wild relatives: a UK perspective. J. Appl. Ecol. 30:199-219.
130.	Raybould, A. F. and A. J. Gray. 1994. Will hybrids of genetically modified crops invade
natural communities? Trends in Ecol. & Evol. 9:85-89.
131.	Rissler, J. and M. Mellon. 1993. Perils amidst the promise, ecological risks of transgenic
crops in a global market. Union of Concerned Scientists, Cambridge, MA. 92 p.
132.	Roush, R. T. 1998. Two-toxin strategies for management of insecticidal transgenic crops:
can pyramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. B.
353:1777-1786.
133.	Ruesink, J. L., I. M. Parker, M. J. Groom and P. M. Kareiva. 1995. Reducing the risks
of non-indigenous species introductions. Bioscience 45:465-477.
134.	Salt, D. E., M. Blaylock, N. P. B. Kumar, V. Dushenkov, B. D. Ensley, I. Chet, andl.
Raskin. 1995. Phytoremediation: a Novel Strategy for the Removal of Toxic Metals From
the Environment Using Plants. Biotechnology 13:468-474.
135.	Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). 1989. Molecular Cloning - A
34

-------
Laboratory Manual. Cold Spring Harbor Laboratory Press.
136.	Schnoor, J. L., L. A. Light, S. C. McCutcheon, N. L. Wolfe and L. H. Carreira. 1995.
Phytoremediation of organic and nutrient contaminants. Environ. Sci. & Technol. 29:318-
323.
137.	Schartz, D. A. 1999. Biodiversity inventory stirs debate over ownership of organisms.
Environ. Sci. & Technol. News: January 1:13A.
138.	Seidler, R. J. and M. Levin. 1994. Potential ecological and non-target effects of
transgenic plant gene products on agriculture, silviculture and natural ecosystems: general
introduction. Mol. Ecol. 3:1-3.
139.	Service, R. F. 1998. Seed-sterilizing'terminator technology'sows discord. Science
282:850-851.
140.	Shewmaker, C. K. and D. M. Stalker. 1992. Modifying starch biosynthesis with
transgenes in potatoes. Plant Physiol. 100:1083-1086.
141.	Shintani, D., and D. DellaPenna. 1998. Elevating the vitamin E content of plants
through metabolic engineering. Science 282:2098-2100.
142.	Shirai, N., K. Momma, S. Ozawa, W. Hashimoto, M. Kito, S. Utsumi, and K. Murata.
1998. Safety assessment of genetically engineered food: detection and monitoring of
glyphosate-tolerant soybeans. Biosci. Biotechnol. Biochem. 62:1461-1464.
143.	Siciliano, S. D., C. M. Theoret, J. R. de Freltas, P. J. Hucl, and J. J. Germida. 1998.
Differences in the microbial communities associated with the roots of different cultivars of
canola and wheat. Can. J. Microbiol. 44:844-851.
35

-------
144.	Smith, M. D. 1996. Antibody production in plants. Biotcchnol. Adv. 14:267-281.
145.	Snow, A. A., and P. Morin-Palma. 1997. Commercialization of transgenic plants:
potential ecological risks. Bioscience 47:86-96.
146.	Southgate, E. M., M. R. Davey, J. B. Power, and R. Marchant. 1995. Factors affecting
the genetic engineering of plants by microprojectile bombardment. Biotechnol. Adv.
13:631-651.
147.	Stalker, D. M., K. E. McBride, and D. Malyj. 1988. Herbicide resistance in transgenic
plants expressing a bacterial detoxification gene. Science 242:419-422.
148.	Steffens, J. C. 1990. The heavy metal binding peptides of plants. Annu. Rev. Plant
Pi.ysiol. Plant Mol. Biol. 41:533-575.
149.	Stone, R. 1994. Large plots are next test for transgenic crop safety. Science
266:1472-1473.
150.	Stuber, C. W., G. H. Helchel, and D. E. Kissel (ed). 1989. Intellectual property rights
associated with plants. American Society of Agronomy Special Publication Number 52,
Madison, WI. 206 p.
151.	Suter-Crazzolara, C., M. Klemm, and B. Reiss. 1995. Reporter genes. Methods in Cell
Biol. 50:425-438.
152.	Swords, K. M. M., J. Liang, and D. M. Shah. 1997. Novel approaches to engineering
disease resistance in crops. In J. K. Setlow (ed.). Genetic Engineering, Vol. 19:1-13. Plenum
Press, New York.
153.	Tabashnik, B. E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. of
36

-------
Entomol.39:47-79.
154.	Tanaka, Y., S. Tsuda, and T. Kusumi. 1998. Metabolic engineering to modify flower
color. Plant Cell Physiol. 39:1119-1126.
155.	Tarczynski, M. C., R. G. Jensen, and H. J. Bohnert. 1993. Stress protection of
transgenic tobacco by production of the osmolyte mannitol. Science 259:508-510.
156.	The Gene Exchange. Published quarterly by the Union of Concerned Scientists.
157.	Tiedje, J. M., R. K. Cofovell, Y. L. Grossman, R. E. Hodson, R. E. Lenski, R. N. Mack
and P. J. Regal. 1989. The Planned Introduction of Genetically Engineered
Organisms:Ecological Considerations and Recommendations. Ecology 70:298-315.
158.	Till-Bottraud, I., X. Rebould, P. Brabant, M. Lefranc, B. Rherissi, F. Vederl and H.
Darmency. 1992. Outcrossing and hybridization in wild and cultivated foxtail millets:
.consequences for the release of transgenic crops. Theoret. & Appl. Genetics 83:940-946.
159.	Timmons, A. M., E. T. O'Brien, Y. M. Charters, S. J. Dubbels, and M. J. Wilkinson.
1995. Assessing the risks of wind pollination from fields of genetically modified Brassica
napus ssp. oleifera. Euphytica 85:417-423.
160.	Tuominen, H., F. Sitbon, C. Jacobsson, G. Sandberg, O. Olsson, and B. Sundberg.
1995. Altered growth and wood characteristics in transgenic hybrid aspen expressing
Agrobacterium tumefaciens T-DNA indoleacetic acid-biosynthetic genes. Plant Physiol.
109:1179-1189.
161.	Turner, N. E., K. M. O'Connell, R. S. Nelson, P. R. Sanders, R. N. Beachy, R. T.
Fraley, and D. M. Shah. 1987. Expression of alfalfa mosaic virus coat protein confers
37

-------
cross-protection in transgenic tobacco and tomato plants. Embo J. 6:1181-1188.
162.	Tzfira, T., A. Zuker, and A. Altman. 1998. Forest-tree biotechnology: genetic
transformation and its application to future forests. Tibtech 16:439-446.
163.	U.S. Congress, Office of Technology Assessment. 1993. Harmful Non-Indigenous
Species in the United States, Washington, D.C. 57 p.
164.	U.S. Department of Agriculture. 1992. Scientific evaluation of the potential for pest
resistance to the Bacillus thuringiensis (Bt) delta-endotoxins. A conference to explore
resistance management strategies, Beltsville, MD, January 21-23,1992. 19 p.
165.	Vandekerckhove, J., J. VanDamme, M. Lijsebettens, J. Botterman, M. DeBlock, M.
Vanderviele, A. DeClerce, J. Lemans, M. Montagu, and E. Krebbers. 1989.
Enkephalins produced in transgenic plants using modified 2S seed storage proteins.
Bio/Technology 7:929-982.
166.	Vickers, K. M., and P. G. Lemaux. 1998. Biotechnology and the environment: challenges
and opportunities. HortScience 33:609-614.
167.	Vierheilig, H., M. Alt, J. Lange, M. Gut-Rella, A. Wiemken and T. Boiler. 1995.
Colonization of transgenic tobacco constitutively expressing pathogenesis-related proteins
by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Appl. & Environ.
Microbiol. 8:3031-3034.
168.	Virgin, I., and R. J. Frederick. 1995. The impact of international harmonisation on
adoption of biosafety regulations. African Crop Sci. J. 3:387-394.
169.	Wang, P., O. Zoubenko, and N. E. Turner. 1998. Reduced toxicity and broad spectrum
38

-------
resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral
protein II. Plant Mol. Biol. 38:957-964.
170.	Watrud, L. S., G. Di Giovanni, C. Coleman, and T. Shiroyama. 1998. Responses of
parental and transgenic genotypes of alfalfa (Medicago saiiva L.) to mycorrhizal
inoculation, p. 183. In Proceedings of Second International Conference on Mycorrhiza,
July 5-10, 1998, SLU, Uppsala Sweden.
171.	Watrud, L. S., S. G. Metz and D. A.. Fischhoff. 1996. Engineued plants in the
environment, p. 165-189. In E. Israeli and M. Levin (ed.). Engineered Organisms in
Environmental Settings. Biotechnological and Agricultural Applications. CRC Press, Boca
Raton, FL.
172.	Watrud, L. S., and R. J. Seidler. 1998. Nontarget ecological effects of plant, microbial,
and chemical introductions to terrestrial systems. In P.M. Huang (ed.). Soil Chemistry and
Ecosystem Health. Special Publication no. 52:313-340. Soil Science Society of America,
Madison, WI.
173.	Weiss, R. 1999a. Com seed producers move to avert pesticide resistance. The Washington
Post: January 9,1999, p. A4.
174.	Weiss, R. 1999b. Seeds of discord. The Washington Post, February 3, 1999, p. A01.
175.	Widmer, F., R. J. Seidler, K. K. Donegan, and G. L. Reed. 1997. Quantification of
transgenic plant marker gene persistence in the field. Mol. Ecol. 6:1-7.
176.	Widmer, F., R. J. Seidler, and L. S. Watrud. 1996. Sensitive detection of transgenic
plant marker gene persistence in soil microcosms. Mol. Ecol. 5:603-613.
39

-------
177.	Williams, M. C. 1980. Purposefully introduced plants that have become noxious or
poisonous weeds. Weed Science 28:300-305.
178.	Williamson, M. H. and K. C. Brown. 1986. The analysis and modelling of British
invasions. Philos. Trans. R. Soc. London B314:505-522.
179.	Williamson, M., J. Perrlns, and A. Fitter. 1990. Releasing genetically engineered plants:
present proposals and possible hazards. Trends Ecol. Evol. 5:417-419.
180.	Wilson, F. D., H. M. Flint, W. R. Deaton, and R. E. Buehler. 1994. Yield, yield
components, and fiber properties of insect-resistant cotton lines containing a Bacillus
thuringiensis toxin gene. Crop Sci. 34:38-41.
181.	Winicov, I. 1998. New molecular approaches to improving salt tolerance in crop plants.
Annals of Botany 82:703-710.
182.	Winicov, I. and D. R. Bastola. 1997. Salt tolerance in crop plants: new approaches
through tissue culture and gene regulation. Acta Physiol. Plant. 19:435-449.
183.	Zhu, Z., X. Li, and Y. R. Sun. 1991. Expression of human alpha-interferon gene in
transgenic rice plants. Physiol. Plant. 82:A31.
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TABLE 1. Examples of meetings on biosafety and risk assessment of engineered plants
Meeting/Title
Location/Year
Held
Editor/Publishcr/Datc
1st International Symposium on the Bio-
safety Results of Field Tests of Genetically
Modified Plants and Microorganisms
Kiawah Island,
SC, 1990
MacKenzie, D.R., Henry, S.C. (eds.).
Agricultural Research Institute,
1991.
Pcsticidal Transgenic Plants: Product
Development, Risk Assessment and Data
Needs
Annapolis,
MD, 1990
US EPA, Office of Pesticide
Programs, 1991
Workshop on Safeguards for Planned
Introduction of Transgenic Oilseed
Ithaca, NY,
1990
USDA, Animal and Plant Health
Inspection Service, 1990
Symposium on Ecological Implications of
Transgenic Plant Release
College Park,
MD, 1992
Lev'.n, M. and R.J. Seidler (eds.),
Blackwell Scientific Publ., Oxford,
UK. Molecular Ecol. 3:1-90, 1994
2nd International Symposium on the Bio-
safety Results of Field Tests of Genetically
Modified Plants and Microorganisms
Goslar,
Germany,
1992
Casper, R., Landsmann, J. (eds.).
Biologische Bundcsanstalt fur
Land-und Forstwirtschaft, 1992
Toward Enhanced and Sustainable
Agricultural Productivity in the 2000's:
Breeding Research and Biotechnology
Taipei,
Taiwan, 1993
Academia Sinica, Nankang,
Taichung District Agricultural
Improvement Station, 1994
3rd International Symposium on the Bio-
safety Results of Field Tests of Genetically
Modified Plants and Microorganisms
Monterey,
CA, 1994
Jones, D. D. (ed.), University of
California, 1994
OECD Workshop on Ecological
Implications of Transgenic Crop Plants
Containing Bacillus thuringiensis Toxin
Genes
Queenstown,
New Zealand,
1994
Hokkanen, H.M.T. (ed.). University
of Helsinki, Finland, 1994
Herbicide-resistant Crops: a Bitter or Better
Harvest?
Memphis, TN,
1995
Southern Weed Science Society,
Champaign, IL, 1995
Dialogue on Risk Assessment of Transgenic
Plants: Scientific, Technological and
Societal Perspectives
Dornach,
Switzerland,
1997
Heaf, D. (coordinator),
Ifene, UK, 1997
4th International Symposium on the Bio-
safety Results of Field Tests of Genetically
Modified Plants and Microorganisms
Tsukuba-
machi, Japan,
1997
Matsui, S., Miyasaki, S., Kasamo, K.
(eds.). Japan International
Research Center for Agricultural
Sciences, 1997
Virus-resistant Transgenic Plants: Potential
Ecological Impact
Godollo,
Hungary, 1997
Tepfer, M. (ed.), Springer Verlag,
Berlin, Germany, 1997
5th International Symposium on The
Biosafety Results of Field Tests of
Genetically Modified Plants and
Microorganisms
Braunschweig,
Germany,
1998
Biologische Bundesanstalt fur
Land-und Forstwirtschaft

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TABLE 2. ' Examples of non-target and unintended effects of engineered plants
Trait
Plant
Effects
References
Insect resistance
cotton
and
potato
Changes in size and diversity
of soil microbial, nematode
and microarthopod
populations; changes in soil
enzyme activity
Donegan et al., 1995
Donegan et al., 1996

tobacco
Changes in soil respiration;
changes in size and diversity of
protozoa, nematode and
microarthopod populations
Donegan et al., 1997
Disease Resistance
tobacco
Decrease and delay in
arbuscular mycorrhizal
infection
Vierhelig et al., 1995
Herbicide Resistance
Arabidopsis
Gene outcrossing
Bergelson et al., 1998

beets
Gene outcrossing
Dietz-Pfeilstetter and
Kirchner, 1998

canola
Gene outcrossing
Ch£vre et al., 1997;
Lefol et al., 1991;
Purrington &
Bergelson, 1995

canola
Change in endophytic and
rhizosphere microbial
populations
Siciliano et al., 1998
Specialty Uses:
Lignin-Peroxidase
alfalfa
Changes in rhizosphere and
soil microbial populations
Di Giovanni et al.,
1999;
Donegan el al., 1999

alfalfa
Reduced shoot biomass and
changes in shoot
macronutrient content
Donegan et al, 1999

alfalfa
Reduced shoot biomass;
changes in macronutrient and
micronutrient content;
decreased mycorrhizal
infection
Watrud et al., 1998
Auxin, Enzymes
aspen
Altered wood anatomy and
shoot growth; change in lignin
structure
Tuominen et al., 1995;
Lapierrt et al., 1999
Pigments
petunia
Loss of color
Mackenzie, 1990

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TABLE 3. Points to consider in developing genetically engineered plants
Criteria
Questions
Market Need/Fit
Are effective products currently available
Technical Feasiblity
Are transformation systems and genes available
for crop and trait of interest
Efficacy
Will it work better, faster, more safely than
existing products
Agronomic Impacts
Will herbicide, insecticide and fungicide
recommendations for current crop differ from
recommendations for non-engineered cultivars
Will modified chemical recommendations affect
future crop rotations and chemical selections
Economics
Who owns gene sources and modified genes
Do farmers have rights to save seeds
What are anticipated returns to developers and
growers
Ecological and Health Effects
Are there potential adverse effects to crop and
non-crop plants
Are there potential adverse effects to humans,
wildlife, beneficial microbes and invertebrates
Mitigation
Are monitoring and control methods available
Regulations
Is a regulatory framework in place; are
regulatory requirements known
Public Acceptance
1
Is proposed product perceived to be beneficial,
safe, ethical

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Potential Invertebrate and
Vertebrate Non-Target Effects
Resistance Development
in Pest Populations
Changes in Populations of
Herbivores, Pollinators
and Detritovores
Toxicity to Wildlife and Toxicity
and Allergenictty to Humans
Technical Feasiblitv


Targeted Commercial Applications
Specialty Uses
Crop Quality
Potential Plant and Microbial
Non-Taraet Effects
Gene Outcrossing: Weediness of
Crop/Weed Hybrids; Changes in
Plant Community Composition
Changes in Community Composition
of Saprophytic, Pathogenic and
Symbiotic Plant and Soil Microbial
Populations
Changes in Nutrient Composition,
Rates of Litter Decomposition and
Nutrient Cycling

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FIQ. 1. Rationale and commercial applications for genetically engineered plants are
represented by the supports and central target areas, respectively, Arrows Identify areas of
concern where research Is needed to Identify and mitigate potential short-term and long-term
non-target and unintended ecological and health effects of genetically engineered plants.

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