INFORMATION SYSTEMS FOR BIOTECHNOLOGY


April 2002

COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


.pdf version
IN THIS ISSUE:
The Maturation of Agricultural Biotechnology Risk Assessment Research
Plant GM Technology Web Site Announced
Emerging Trends: Metabolic Engineering
Nontarget Impacts of Bt Corn: A Risk Assessment
Cloning Updates
Litigation in the Wind


THE MATURATION OF AGRICULTURAL BIOTECHNOLOGY RISK ASSESSMENT RESEARCH

In the midst of the rumble of the Quist and Chapela report1 of introgression of DNA from transgenic corn into Mexican landraces and the subsequent scientific aftershocks,2,3 Dr. Allison Snow, a professor at Ohio State University and plant ecology researcher, recently convened a scientific methods workshop entitled "Ecological and Agronomic Consequences of Gene Flow from Transgenic Crops to Wild Relatives." The workshop proceedings, which contain contributed papers and group report recommendations, can be accessed at http://www.biosci.ohio-state.edu/~lspencer/gene_flow.htm. The workshop was funded by the USDA Biotechnology Risk Assessment Research Grants Program (BRARGP) and held in Columbus, Ohio, on March 5th and 6th, 2002. Contrasting with the controversial report and issue of intraspecific gene flow in corn, the papers presented during the workshop focused on the sustained efforts through several years by many research groups on gene flow from domesticated crops to wild-growing relatives that might be in the same or a related species.

While I have attended many conferences similar to this one since I jumped into the fairly young field of biotech risk assessment research in 1994, I was struck by the growing maturity of the research presented in the workshop. Much of the credit of the notable maturation goes to BRARGP
( http://www.reeusda.gov/crgam/biotechrisk/biotech.htm), which has provided scarce research funds to an ever-increasing community of researchers. Says Dr. Deborah Sheely, the program director of BRARGP, "Over the years, the research supported by USDA's Biotechnology Risk Assessment Program has generated information essential for making science-based regulatory decisions. This kind of information is now more important than ever, and further research on the issues discussed at this workshop will help to facilitate the prudent use of biotechnology in agriculture." There have been some recent developments in the science presented in Columbus that illustrate the field's maturation.

The first development is that several groups are now using actual transgenic plants to study gene flow problems of interest to biotechnology concerns. Since most researchers performing experiments in assessing the risks of transgenic plants do not have the expertise to genetically engineer plants, they have, in the past, often used nontransgenic plants coupled with allozyme or molecular markers to infer how transgenes will move to their intended hosts. This shift from using nontransgenic to transgenic plants is not trivial. As I pointed out in a commentary last year, ecologists do not have access to the most ecologically relevant germplasm, that is, the actual transgenic events that will be someday commercialized.4 In at least one study, academic biologists had collaborated with companies to perform the research—another favorable development in the science.

The second reason that biotech risk research has matured is that there is more dialogue between a number of groups and academic disciplines interested in the ecology of transgenic plants. For instance, to the credit of the conference organizers, of the sixty-six attendees present, there were agricultural scientists (e.g., weed scientists, agronomists, and molecular geneticists), ecologists (e.g., population ecologists, ecological geneticists, and evolutionary ecologists), regulatory officials, industry scientists, government scientists, and even representatives from NGOs such as English Nature and the Union of Concerned Scientists. And dialogue actually occurred. It was heartening to see the free flow of information among various interest groups and disciplines. For example, in many past meetings of this sort there has been noticeable tension between, say, ecologists and industry representatives. It is a tremendous development that people who may have different goals and perspectives would have meaningful discourse about this potentially controversial science.

The first two reasons for increased maturation have given rise to the third: the quality of the science is visibly improving. While industry is now investing small amounts of money into risk assessment research, there is still relatively little research money allocated to BRARGP, the only competitive funding source devoted to biotech risk research in the US. The BRARGP panel has only had about $1.5 M per year to grant for research for a number of years, which is not very much. (There is a chance that program funding for BRARGP will increase significantly in FY 2003 and that the panel may widen the scope of the risk assessment research it funds as well.) So, an increase in the amount of money available for biotech risk research cannot be credited for the noted improvement in quality of research (though more money allocated to BRARGP is not a bad idea in these days of relevant biotech research needs). I suspect that the improved quality of biotech risk assessment research has partially arisen because the panel has funded a few groups for sustained efforts over several funding cycles. A corollary is that the most successful groups have involved multidisciplinary teaming approaches. These facts should encourage USDA BRARGP to perhaps use increased funding (if the increase does indeed occur) to request longer-term research projects as well as requiring interdisciplinary teams for part of the funding.

Breakout groups are always part of workshops, such as the one in Columbus. Most breakout group schemes revolve around commodity or plant species groups. This particular workshop boldly organized the small group discussions around the following five topics: 1) Hybridization and introgression; 2) Fitness of feral crops, crop-wild hybrids, and transgenic wild plants; 3) Effects of introgressed transgenes on plant populations; 4) Effects of introgressed transgenes on the genetic diversity of wild relatives; and 5) Indirect effects of introgressed transgenes on ecological communities. The goal of the group discussions was to determine what information experiments could provide and how experiments should be designed to meet the needs of federal regulators who are charged with ecological biosafety oversight. What are the future research needs? According to Dr. Snow, "the biggest research gaps are understanding whether fitness-related transgenes can affect the population dynamics, competitive ability, geographic distribution, and/or genetic diversity of wild and weedy species."

Participants of the workshop also learned of two international opportunities to further the dialogue of transgene flow and introgression. The first is the 7th International Symposium on the Biosafety of Genetically Modified Organisms to be held in Beijing, October 2002 (http://www.worldbiosafety.net). The second is "Introgression from GMOs into Wild Relatives and their Consequences" to be held in Amsterdam in early 2003, which should be the European version of the Columbus workshop (contact Detlef Bartsch: bartsch@rwth-aachen.de). It will be interesting to observe the development of maturity of the field of biotechnology risk assessment research in Europe.

In light of the upcoming international meetings, it is critical for stakeholders in the US to more seriously consider international teaming in biotech risk research and international cooperation in funding such research. As we have witnessed recently, biotech developments in the US do not remain contained within our borders. It might be that the successful importation and exportation of crops and foods will soon depend on importing and exporting collaborative grants and research expertise, especially in biosafety. That would be a mature step indeed.

Sources

1. Quist D and Chapela IH. 2001. Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature 414: 541-543.

2. Mann CC. 2002. Has GM corn `invaded' Mexico? Science 295: 1617-1618.

3. Conko G and Prakash CS. Report of transgenes in Mexican corn called into question. ISB News Report, March 2002, 3-5. ( http://www.isb.vt.edu/news/2002/news02.mar.html#mar0202 )

4. Stewart CN Jr and Wheaton SK. 2001. GM crop data—agronomy and ecology in tandem. Nature Biotechnology 19: 3.

C. Neal Stewart, Jr.
UNC-Greensboro
cnstewart1@juno.com


PLANT GM TECHNOLOGY WEB SITE ANNOUNCED

The entrenched debate on the applications of recombinant DNA technology to crop plants has persuaded the editors of the The Plant Journal to organize a Web site resource featuring a special `rolling' issue on "Plant GM Technology," which they say will offer a "science-based analysis of the issues surrounding transgenic crop plants." Access to the online articles on the Web is free to everyone (http://www.blackwell-science.com/tpj/gm). The philosophy of the Journal in organizing this site is to "undertake a holistic view of the issues involved and to provide an independent and authoritative resource of world-class academic information that will facilitate an informed debate ... " on GM technology and its application to crop plants.

The premier issue contains a major authoritative review on plant GM technology in relation to food and food safety issues. The review panel was chaired by Harry Kuiper, Head of the Department of Food Safety and Health at RIKILT (the State Institute for Quality Control of Agricultural Products in The Netherlands). In addition, according to the Web site, a "major review on GM plants in relation to the environment is also in the pipeline, prepared by a group of scientists under the chairmanship of Tony Conner from the New Zealand Institute for Crop and Food Research."

Complementing these major reviews will be case studies, which will target specific issues. The first case studies to appear include a discussion of the development and use of Bt cotton, authored by Frederick J. Perlak and colleagues of the Monsanto Company, and an interpretation of the monarch butterfly controversy co-written by Anthony Shelton and Mark Sears. In the future, links will be added to include Web sites and articles prepared by organizations such as the Royal Society, United Nations, and Rockefeller Foundation. These articles are intended to comprise committee reports that are written by specialists who can present perspectives on "plant GM technology and its field applications from many different geographical locations and many different academic and cultural backgrounds."

Source

Bowles D and Klee H. 2001. Special Issues on Plant GM Technology. The Plant Journal. http://www.blackwell-science.com/tpj/gm

Ruth Irwin
Information Systems for Biotechnology
Virginia Tech
rirwin@vt.edu



EMERGING TRENDS: METABOLIC ENGINEERING

The second wave of genetically modified plants is nigh. These organisms address consumer demand on two fronts. They deal with concerns regarding safety and genetic pollution by using gene containment technologies, by not including DNA from viruses, pathogens, or bacteria, and by passing the stringent regulatory hurdles now in place for genetically modified organisms. They also deal with concerns of consumer participation in that they provide benefits to the end-user, the customer, rather than solely to suppliers, distributors, and transnational conglomerates. They are more sophisticated in both their science and marketing, and it is unfortunate that they have arrived after "industry-benefit" products such as late-ripening tomatoes and pesticide-resistant corn.

Metabolic engineering is the in vivo manipulation of biochemistry to produce non-protein products or to alter cellular properties. The products may be native or novel, and the tools used are usually those of genetic engineering. The non-protein products may be alkaloids such as quinine, lipids such as long-chain polyunsaturated fatty acids, polyterpenes such as rubber, structural components such as lignin, osmoprotectants such as glycine betaine, aroma compounds such as S-linalool in tomatoes, pigments such as blue delphinidin in flowers, vitamins such as folic acid, biodegradable plastics such as polyhydroxyalkanoates, and more. Metabolic engineering is a key part of the second wave of plant genetic engineering products.

Two genes are often better than one
The second wave is late because it is a more ambitious, technically challenging task to modify physiology and biochemistry than to overproduce a protein such as Bt or spider silk monomer. Often, several novel or altered genes must be introduced (the process is often termed `gene stacking'), or several changes made to existing metabolism. The first wave of modified plants used single-gene single-enzyme manipulations, but these are often unpredictable in their effectiveness due to the multilevel regulation of cellular metabolism. A multidimensional network of equilibria rich in feedbacks, fail-safes, redundant backups, and antagonistic pathways is unchanged by most single gene additions or deletions.1 Variation in the wild already includes a lot that can be achieved by such manipulations, though single-gene manipulations have of course been successful, as demonstrated by the publications arising from yeast and Arabidopsis single-gene knockout populations and the raft of publications on the manipulation of lipid metabolism. However, little has been done where the biochemical pathways concerned are obscure, branched, or essential.

This delimitation of possibilities via single-gene alterations is in part due to the nonrandom sampling of genomes that has taken place in the past decade. The gene resources available for the first wave of genetically enhanced plants have been predominantly EST datasets, which are rich in genes involved in the biochemical labor but not in those which supervise, manage, and control.2 An example is transcription factors, which control and coordinate the expression of numerous other genes involved in a common biochemical goal. Transcription factors offer the possibility of powerful single-gene manipulations leading to metabolic engineering, for example by turning on or off whole suites of biochemical reactions, yet genes for these proteins are largely missing from EST datasets. After the publication of the Arabidopsis genome sequence, it was discovered that greater than 5% of genes in this plant were transcription factors and only 10% of those had been characterized. Mendel Biotechnology (Hayward, California) is a company founded solely to assess the function of each of these genes and develop products;3 there is clearly value in single-gene manipulations of the right variety. Meanwhile, plant metabolic engineering has had significant successes, for example in biodegradable plastics and of course vitamin A in Golden Rice.4

Control is everything
Where multiple genetic elements are required for a desired phenotype, an important prerequisite is often to switch them on simultaneously and to a similar degree, yet coordinate expression of more than one gene has proved difficult to achieve. Bacterial polycistronic constructs, in which one promoter drives transcription of numerous genes, do not function in plants. Two types of strategies are employed when introducing multiple genetic modifications: simultaneous and sequential transformation. In the former, several genes are collected into one transforming molecule and introduced, or they may be simultaneously inserted while on different transforming molecules. In sequential transformation, a transgenic plant is re-transformed with a second gene, or two lineages receive one transgene each and their progeny are crossed. All of these approaches suffer from two related major drawbacks. Firstly, there are a limited number of well-characterized, useful gene control elements (i.e., promoters) and only a handful of marker genes with useful characteristics and without patent ownership constraints.5 Gene stacking using multiple or large constructs therefore quickly runs out of options, since any repetition will trigger a viral defense mechanism that switches off the transgenes at some point in the next few generations. The second drawback is that the viral defense mechanism limiting gene stacking also prevents the use of identical promoters and therefore never guarantees coordinate expression. Each approach also suffers unique problems, e.g., retransformation can cause many other changes due to the two cell culture phases, and all add considerably to the research and development costs. In `golden rice,' no attempt was made to coordinate the expression of the genes and all were constitutively overexpressed; the need for the public to see genetic modification used responsibly and with precision and predictability will probably prejudice against such methods in the future.

More advanced methods of coordinate expression have been tried. Bicistronic (two genes from one promoter) constructs have been shown to work where the promoter is bi-directional or the expressed DNA is short. Genes can be linked `in frame' so that their protein products are joined at a cleavable site. More recently, Mlynárová et al. (2002) showed that a DNA element identified from a chicken gene can coordinate the expression of two genes driven by different promoters.6 The transforming construct contained the two genes and their promoters, flanked by chicken lysozyme A element. In tobacco, as in chicken, the flanking DNA elements appear to proffer the genes for expression on an easily accessible DNA loop by binding to the structural proteins supporting the chromosome. Another recent successful and clever approach aimed to reduce the effect of three genes involved in lignin production in tobacco by exploiting the aforementioned viral defense mechanism. Abbott et al. (2002) were able to switch off three genes using a single artificial chimerical gene composed of parts of all three, since, to switch genes off, the defense system requires only part of the gene.7

Different regulatory problems
Across the world, legislation is evolving to deal with the issues surrounding genetic engineering. Like most legislation, this has been reactive and subjective rather than anticipatory and objective. Biotechnology is a fast-moving field; today's technical advances result in tomorrow's genetically modified products, which are eventually regulated by next month's laws. Metabolic engineering poses new problems for legislation designed to react to the existence of the first wave of transgenic organisms.

In many cases, the plant or a relative already manufactures the product. Much legislation and recycled paper has been expended attempting to prevent the escape of genes from transgenic plants that might confer pesticide resistance to weedy relatives. While this unlikely event is indeed a concern with some credibility (though not much), the same rules surely do not apply if the gene concerned is a species-specific switch intended to increase the levels of certain flavor compounds in tomatoes. Not all genetic modifications are created equal, and the merits of each, as well as the financial penalty on developers as they attempt to get their product to market, should be concomitant with the scientifically valid dangers posed and the benefits offered. Likewise, multiple, dispersed genetic alterations such as those arising from sequential transformations require a different approach. Each alteration may make only a small contribution to the transphenotype, or a small step towards it. The transfer of the trait from the host species to a weedy relative is therefore many times less likely. While the movement of one of a suite of genetic elements to another lineage is undesirable and methods to minimize this should be used, triffids will not result. DNA constructs used in metabolic engineering that contain many genes collected together (simultaneous transformation) are of course horizontally transmissible as `trait islands,' but the traits conferred still offer no advantage to any would-be superweed. It should be noted that the distinctions described in this paragraph offer little solace to those whose view is that a plant with two genetic modifications is merely twice as polluted and twice as likely to pollute.

For the future
There are exciting possibilities and near-horizon products from plant metabolic engineering. For example, recent publications have covered increasing essential oil production, decreasing lignin deposition, stimulating the bioconversion of secondary metabolites to medicinally important alkaloids, introducing processing steps leading to the production of industrial feedstock chemicals, and improving tomato flavor.8 Interest is high in the industry—those involved include not only the established agribiotech companies such as Monsanto and Syngenta, but food commodity producers such as Nestlé and Fonterra, along with university research groups around the world. The biotechnology revolution is poised to deliver. The public and legislators have heard this before and remember the mistakes of the past from the manipulation of the human food chain without consumer benefit to the promised-but-still-pending panaceas of the human genome. The second wave of agricultural biotechnology, metabolic engineering, will demonstrate why this industry is in existence. It is time to make good on some promises.

Sources

1. ISB News Report, July 2001.
(
http://www.isb.vt.edu/news/prep/news01.jul.html#jul0102 )

2. ISB News Report, December 2001.
( http://www.isb.vt.edu/news/2001/news01.dec.html#dec0101 )

3. Riechmann JL, et al. 2000. Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science: 290(5499): 2105-2110.

4. See Bohmert, et al., 2000, Planta 211: 841-5 for biodegradable plastics via one large transforming molecule containing four genes, and Ye et al., 2000, Science 287: 303-5 for `golden rice' via multiple simultaneous transformations.

5. ISB News Report, August 2001.
( http://www.isb.vt.edu/news/2001/news01.aug.html#aug0103 )

6. Mlynárová L, et al. 2002. Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco. Genetics 160: 727-40.

7. Abbott JC, et al. 2002. Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco. Plant Physiology 128: 844-53.

8. See Mahmoud and Croteau, 2001, PNAS 98: 8915-20 for essential oils (although they have yet to combine the two effects they describe for optimum production); Abbott et al. cite above for lignin; Van der Fits and Memelink, 2000, Science 289: 295-7 for alkaloids via transcription factor manipulation; Cahoon et al. 2002, Plant Physiology 128: 615-24 for an industrial feedstock chemical via a key single gene; Wang et al. 2001, Phytochemistry 58: 227-32 for tomato flavor via a key single gene.

Zac Hanley and Kieran Elborough
Consultants in Plant Biotechnology
New Zealand
http://www.greengenz.com



NONTARGET IMPACTS OF Bt CORN: A RISK ASSESSMENT

One of the components of sustainable agriculture is to make prudent use of chemical insecticides and biopesticides. Bacillus thuringiensis (Bt), a common soil organism known for its ability to kill the larvae of certain moths, is one example of a frequently used biopesticide. In dried form, Bt was registered and approved as an insecticide in 1961. It is generally regarded as safe for humans and the natural enemies of many crop pests.

Among the list of Bt-containing crop plants currently cultivated across the globe, GM corn ranks second in abundance. Several seed companies, including Dekalb, Monsanto, Ciba (now Syngenta), and Mycogen (now Dow), have developed corn events that express a Bt toxin active against the European corn borer. However, the reports that Bt corn pollen may harm monarch butterfly larvae1 and that DNA from genetically engineered corn may have introgressed into local landrace maize varieties in Mexico2 have escalated the call for more detailed studies on nontarget impacts of Bt corn. It should be noted that, in these reports, perceived risks of biotechnology are not weighed against benefits, at least in the short term, partly because the benefits are not yet fully known as this new technology is increasingly applied around the world.

Reports of harm to monarch butterfly larvae feeding on milkweed grown adjacent to Bt corn fields prompted the formation, in early 2000, of a consortium of research teams to investigate the threat. The researchers, consisting of scientists from government, universities, industry, and environmental groups, published the results of this two-year collaboration as a series of reports in the Proceedings of the National Academy of Sciences in October 2001 (http://www.pnas.org). The researchers report the results of extensive studies on the effects of Bt corn pollen on nontarget organisms, primarily monarch butterfly larvae and, in one case, black swallowtail caterpillars.

In one study,3 the research team conducted field experiments to study the effect of Novartis event 176 Bt corn pollen on nearby nontarget lepidopterans, namely monarch butterfly and black swallowtail caterpillars. They reported a very low survival rate, only 7%, among the monarch larvae population; however, this survival rate was not due to Bt pollen exposure, as the rate did not vary as a function of distance from the transgenic corn. (Note that with only 22 of 600 monarch larvae surviving, the statistical power of this result was extremely low.) Likewise, the mortality rate of the black swallowtail caterpillars did not vary as a function of distance; however, the overall survival rate for swallowtails was much greater than that of the monarch. The team also reported that, despite five rainfall events, the presence of the 176 pollen exerted a sublethal affect on swallowtail caterpillars. In this case, the sublethal affect was negatively impacted by proximity to the transgenic corn: those larvae located 7 m from the corn were three times larger than those closest to the corn field. In laboratory bioassays, the authors noted that the LD50 for black swallowtail caterpillars is not, to a large extent, different from that reported for monarch larvae.

In another study,4 researchers examined the densities of monarchs from May through August in four different breeding ranges of North America and overlapped those densities with the time of corn pollen shed to assess the likelihood of monarch exposure to Bt corn pollen. Habitats were chosen in areas where breeding monarchs and host milkweed plants are typically found—corn fields, corn field edges, other agricultural fields, and non agricultural areas. Monarchs were found on milkweeds in cornfields as well as in non-agricultural areas throughout the summer breeding season; the greatest overlap between anthesis and monarch presence occurred in the northern part of the breeding range due to early pollen shed in the south. Using Iowa as an example, the authors estimated that approximately 3% of monarchs would be exposed to Bt pollen over the course of a summer. The authors also noted that due to the preponderance of agricultural land in the Midwestern United States, the majority of monarchs, by a factor of 73 to 78 times, originate in agricultural habitats. Consequently, the team suggested that farming practices affecting milkweed abundance, such as tillage methods, herbicide use, and cropping choices, have a large impact on monarch abundance, regardless of any possible impact of transgenic crops. Nevertheless, the impact of Bt pollen landing on milkweeds must be included in risk assessment studies.

That conclusion was reiterated by another study5 that investigated the factors affecting the density of corn pollen deposited on milkweeds in and near cornfields in five geographically distant localities. As would be expected, pollen density was highest inside the cornfield and gradually decreased from field edge outward. Other factors affecting pollen deposition included leaf characteristics, leaf position on plant, plant position in corn canopy, wind direction, and, most importantly, rainfall, as a single rain event can remove between 54-86% of pollen from leaves. The highest-positioned milkweed leaves, where the young larvae usually feed, had on average less than half of the pollen density of middle leaves, which had more pollen than lower leaves. The authors suggested that leaf orientation on milkweed—upper leaves are upright, middle are horizontal, and lower leaves tend to point down—may be influencing the amount of pollen washed away. In addition, only milkweed located on the downwind side of fields received appreciable pollen. The researchers concluded that the impact of Bt pollen on monarch populations ultimately depends on the toxicity of the pollen and its density in relation to morbidity threshold levels, and the likelihood that larvae will be present at the time of anthesis.

In a companion paper to this study,6researchers reported on the relative sensitivities of monarch larvae to Bt proteins and pollen applied artificially in the laboratory to leaf discs of common milkweed. Studies were carried out using four Bt toxins, Cry1Ab, Cry1Ac, Cry9C, and Cry1F, representing the proteins expressed in various existing (commercial and noncommercial) corn hybrids. The authors used three methods of delivering the Bt toxin: 1) purified toxin incorporated into an artificial diet; 2) field-collected pollen applied directly to discs; and 3) pollen contaminated with corn tassel material applied to discs. The results indicated that sensitivity to Bt toxin depends on the age of the larvae, amount of nonpollen material contaminating the sample, and the particular transgenic event used. Except for pollen from the Cry1Ab event 176 hybrid and non-sifted (contaminated) pollen, larvae survival was not significantly affected by pollen densities of less than 1000 grains/cm2. By comparison, the previous study5 reported that average pollen densities in the field range from 10 to 425 grains/cm2. The authors noted that event 176, a hybrid that expresses the highest level of Bt toxin, currently represents less than 2% of transgenic corn planted and is being phased out of commercial distribution.

In a fifth PNAS study, researchers reported on field tests intended to determine the effects of Bt pollen on monarch butterfly.7 The goal was to expose larvae to Bt and non-Bt pollen under conditions reflecting the natural rates of pollen deposition and of toxin degradation within pollen. For the event 176 test plot located in Maryland, there was a significant difference in the survival rate of larvae feeding on leaves outside the field (63%) versus those inside (25.1%). They determined that growth and survival of first instars were detrimentally affected with a minimum of only 67 pollen grains/cm2 of event 176. In contrast, there was no statistical difference in survival for larvae feeding inside versus outside the field for both the Bt11 and non-transgenic hybrids. Pollen grain densities on leaves inside the field ranged from 504 to 586 grains/cm2 inside and 18 to 22 grains/cm2 outside the fields. These results are in harmony with laboratory studies indicating that Bt11 pollen densities of less than 1000 grains/cm2 are not toxic to first instar monarchs. A similar lack of adverse effect was also documented for the Mon810 event, which expresses very low levels of Cry1Ab toxin in comparison to event 176. The authors also noted the importance of comparing risks of Bt corn to other agricultural practices used to control lepidopteran pests. On three separate occasions, they sprayed non-Bt plots with lambda-cyhalothrin insecticide (Warrier 1E, Syngenta Crop Protection) and, not surprisingly, within one hour, recorded 91-100% and 21-45% mortality of larvae inside and outside the plots, respectively. The researchers suggested further studies to assess potential sublethal effects of longer-term Bt toxin exposures on larvae and called for studies with larger sample sizes and much higher doses to examine possible lifetime and reproductive impacts.

Authors of the final study reported in PNAS8 made a collaborative effort to conduct a formal risk assessment of the impact of Bt corn on populations of monarch butterfly. They used a well-documented, standardized approach to risk assessment that necessitated a consideration of both the expression of toxicity and the likelihood of exposure. Drawing on the results of the previous four studies,4-7 the authors first characterized the toxicity of Bt corn pollen and then quantified risk by examining monarch exposure according to phenological overlap between anthesis and monarchs, spatial overlap between milkweed hosts and cornfields, and pollen densities. Using this formalized approach, the authors determined that a potential hazard to monarchs exists only with use of the high-expressing 176 event, which, as stated above, is being phased out of the market. They therefore concluded that Bt corn pollen from current commercial hybrids has a negligible impact on monarch butterflies; and, while acknowledging that sublethal effects have not yet been thoroughly examined, they concluded that if found, the sublethal effects would have little overall impact because exposure of larvae to Bt pollen is low. This research substantiated an earlier US Environmental Protection Agency conclusion that the impact of Bt corn pollen on monarch butterfly is negligible due to limited environmental exposure.

In summary, except for event 176 Bt corn hybrids expressing Cry1Ab protein, Bt corn pollen has negligible nontarget impacts. The above studies demonstrate the complex and comprehensive nature of assessing nontarget impacts of Bt corn. A number of factors impact nontarget effects in real field situations: agricultural practices; prevalence of host weeds; occurrence of rain events; timing of anthesis; pollen density and toxicity; and larval stage. In view of these variables, risks associated with GM crops cannot be generalized based on sporadic studies. Consequently, these studies represent a concerted effort to study the impact of GM crops on monarch butterfly based on sound science and proven methods of risk assessment, and exemplify an approach that is a laudable model for future endeavors.

Sources

1. Losey JE, et al. 1999. Transgenic pollen harms monarch larvae. Nature (London) 399: 214.

2. Quist D and Chapela IH. 2001. Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature 414: 541-543.

3. Zangerl AR, et al. 2001. Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions. PNAS 98: 11908-11912.

4. Oberhauser KS, et al. 2001. Temporal and spatial overlap between monarch larvae and corn pollen. PNAS 98: 11913-11918.

5. Pleasants JM, et al. 2001. Corn pollen deposition on milkweeds in and near cornfields. PNAS 98: 11919-11924.

6. Hellmich RL, et al. 2001. Monarch larvae sensitivity to Bacillus thuringiensis-purified proteins and pollen. PNAS 98: 11925-11930.

7. Stanley-Horn DE, et al. 2001. Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies. PNAS 98: 11931-11936.

8. Sears MK, et al. 2001. Impact of Bt corn pollen on monarch butterfly populations: A risk assessment. PNAS 98: 11937-11942.

Ruth Irwin
ISB
Virginia Tech
rirwin@vt.edu
P. Janaki Krishna
IPE, Biotechnology Unit
Hyderabad, India
jankrisp@yahoo.com


CLONING UPDATES

These pigs are knock-outs
Xenotransplantation involves the transfer of cells, tissues, or organs between species. The host immune system, however, will attack and destroy these foreign tissues. As a step towards the development of organ transplants between pigs and humans, two research groups recently reported the production of cloned pigs lacking the alpha-1,3-galactosyltransferase gene. A team consisting of scientists from the University of Missouri, Immerge BioTherapeutics Inc., and Kangwon National University was the first to report their findings in the February 8, 2002, issue of Science, while the other group from PPL Therapeutics Inc. reported their results in the March 2002 issue of Nature Biotechnology.

Galactose alpha-1,3 galactose is a major cell surface antigen on pig cells that causes hyperacute rejection in humans following pig-to-human tissue/organ transplants. Humans lack galactose alpha-1,3 galactose on the surface of their cells because they lack the enzyme alpha-1,3-galactosyltransferase (alpha-GT). Thus one approach that has been proposed to overcome this immunological barrier to pig-to-human transplants is the development of a pig that lacks the alpha-GT gene.

Using gene-targeting technology, both groups successfully "knocked-out" or inactivated one copy of the alpha-GT gene in porcine fetal fibroblasts. Cells with a mutation in one copy of the alpha-GT gene were isolated, verified by PCR and/or Southern blot, and then used as nuclear transfer donors to generate cloned piglets. The University of Missouri group produced six live pigs, of which one died shortly after delivery and a second died 17 days after birth, whereas the PPL Therapeutics group produced five live pigs. All surviving pigs appear to be healthy or suffer from only minor abnormalities.

Because the existing cloned pigs still retain one wild type copy of the alpha-GT gene, the next step for both groups is to generate a pig with both copies of the alpha-GT gene inactivated. Two approaches can be used to achieve this goal. The first involves natural mating of the current alpha-GT knock-out pigs to generate a homozygous knock-out pig. The other method requires a second round of gene targeting and nuclear cloning starting with fetal fibroblasts derived from pigs with one copy of alpha-GT already inactivated. Given the time needed for pigs to attain sexual maturity, the in vitro knock-out/nuclear transfer approach would seem to be the faster alternative. At present, the precise effect of knocking out both copies of the alpha-GT gene in a pig is not known, although studies in mice have shown that mice lacking both copies of the alpha-GT gene are viable.

Health concerns
The long-term health effects of cloning have always been of concern. In the March 2002 issue of Nature Genetics, a Japanese research group examined the lifespan of mice cloned from somatic cells. Twelve cloned mice were generated following nuclear transfer of immature Sertoli cells. The weight gain of these cloned mice did not differ from control mice produced by natural matings. Serum biochemical analysis of the cloned mice, performed at three and 14 months of age, revealed that levels of lactate dehydrogenase and ammonia were significantly higher in the cloned mice—a finding that is symptomatic of liver damage.

At 311 days after birth, the first cloned mouse died. By 800 days, 10 of the 12 cloned mice had died, while only one control mouse had died. Necropsy analysis of six cloned mice revealed severe pneumonia (6/6), necrosis in the liver (4/6), and tumors (1/6). These results suggest that the cloned mice died as a result of malfunctions in the liver and/or lung.

Twinning isn't everything
Recently, cats have joined sheep, mice, cattle, goats, and pigs as animals cloned by nuclear transfer. Researchers at Texas A&M's College of Veterinary Medicine reported the first successful cloning of a household pet. Their first attempt using fibroblasts from adult mucosa resulted in a single conceptus that died in utero at 44 days of gestation.
a. Nuclear-donor adult female cat
b. Cloned kitten with surrogate mother
(Used with permission: Nature. http://www.nature.com)

The second attempt using cumulus cells resulted in a single healthy cloned kitten that was delivered by Caesarean section. The efficiency of cat cloning was comparable to that for other cloned mammals: one live birth from 87 transferred embryos. Although the kitten was shown by genotype analysis to be derived from the donor cumulus cells, the kitten's coat color patterning was not identical to the donor mother. This phenotypic difference is due to the fact that the pattern of pigmentation of multicolored animals is a result of both genetic and non-genetic (developmental) factors.

Advances in cloning technology are moving at a rapid pace. The long-term effects of cloning, however, still remain to be determined. Furthermore, it is clear that both genetic and non-genetic factors shape an individual so that a clone will never be an exact copy of the donor.

Sources

1. Lai L, et al. 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295: 1089-1092.

2. Dai Y, et al. 2002. Targeted disruption of the alpha-1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnology 20: 251-255.

3. Ogonuki, N, et al. 2002. Early death of mice cloned from somatic cells. Nature Genetics 30: 253-254.

4. Shin T, et al. 2002. A cat cloned by nuclear transplantation. Nature 415: 859.

Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu



LITIGATION IN THE WIND

According to Darwin's theory, change has fueled the engine of evolution. Today, change is the spark that sets off lawsuits. The introduction of genetically modified (GM) crops has kindled its share of litigation, typically in the form of farmers and farm interest groups versus agbiotech companies. However, farmers who grow GM crops may not be immune from a lawsuit.

Farmers vs. agbiotech companies
In the fall of 2000, remnants of the GM corn StarLink were found in the human food supply. StarLink expresses Cry9C, an insecticidal protein that had not been approved for human consumption by the US Environmental Protection Agency and by various agencies outside the US. The discovery of StarLink "contamination" severely affected domestic and foreign markets for US corn products, which, in turn, spawned at least nine class action lawsuits in six states against Aventis CropScience USA LP (Research Triangle Park, North Carolina), the company that commercialized StarLink.

Last year, for example, a class action suit was filed in a Wisconsin federal district court on behalf of farmers who claim that they have lost money due to the depression in prices after StarLink was found to have entered the food supply (Southview Farms v. Aventis CropScience USA Holding, Inc.). Another class action suit, Mulholland v. Aventis Crop Science USA, was filed on behalf of farmers who did not grow StarLink. Here, the plaintiffs claimed that Aventis failed to take the appropriate measures to prevent the GM corn from entering the human food supply, and that, as a result, the plaintiffs lost significant domestic and foreign markets. The Mulholland complaint includes allegations of public nuisance, consumer fraud, deceptive business practices, and negligence. In Mudd v. Aventis Crop Science USA, non-StarLink growers filed a class action suit based on negligence and strict liability claims.

Concerns about GM crops also provoked the recent filing of a class action lawsuit against agbiotech companies in Canada. In this case, two farmers who specialize in organic produce initiated the lawsuit to recover compensatory damages for revenue lost by contamination of organic canola crops. The plaintiffs also requested an injunction to stop field trials of Monsanto's Roundup Ready wheat.

Larry Hoffman and Dale Beaudoin, two organic farmers in Saskatchewan, filed a statement of claim in the Court of Queen's Bench, seeking the class action lawsuit against Monsanto Canada, Inc. (Misssissauga, Ontario) and Aventis CropScience Canada Holding Inc. (Regina, Saskatchewan). They assert that the companies have ruined the province's organic canola market and must be prevented from doing the same to the organic wheat market. According to the complaint, Monsanto's Roundup Ready canola or Aventis CropScience's Liberty Link canola has been found growing on land for which it was not intended, and consequently, few, if any, seed suppliers will certify their seeds as organic. The farmers allege that the two companies are responsible for any GM contamination on the grounds of negligence, nuisance, trespass, pollution under the Saskatchewan Environmental Management Protection Act, and failure to conduct an environmental assessment. Estimates run to millions of dollars in damages for the loss of canola as an organic crop
in Saskatchewan.

And what about the farmers who decide to produce GM crops designed by an agbiotech company?


Farmer vs. farmer
Farmers who grow GM crops might find themselves as defendants in a lawsuit filed by neighbors who complain about crop contamination. For instance, plaintiffs might allege that pollen from the defendant's GM crops drifted over a property line (via wind, insects, etc.) and contaminated their non-GM crops.

Commentators have suggested that the plaintiffs of such lawsuits might assert claims of trespass to land, private nuisance, negligence, or strict liability. A claim of trespass to land can arise when someone crosses the legal boundary of another's land or causes something to cross that boundary. A private nuisance is often described as something that decreases the value of an individual's property or interferes with their use or enjoyment of the property. For a claim of negligence, a plaintiff must establish the existence of a duty owed by the defendant to the plaintiff, a breach of that duty, and an injury proximately resulting from the breach of duty. In contrast, strict liability is a type of liability without fault in which a person engages in an "abnormally dangerous" activity. Factors that a court may consider in determining whether an activity is abnormally dangerous include: whether the activity involves a high degree of risk of harm; whether the gravity of the harm that may result from the activity is likely to be great; whether the activity carries risk that cannot be eliminated by the exercise of reasonable care; whether the activity is a matter of common usage; whether the activity is inappropriate to the place where it is carried out; and the value of the activity to the community.

With regard to strict liability, commentators suggest that a court may compare a genetic contamination case to a pesticide drift case, such as the 1977 Washington State supreme court case, Langan v. Valicopters, Inc. (88 Wn.2d 855). In this case, the State supreme court affirmed an award of damages to an organic farmer who sued a crop duster in strict liability for the crop duster's use of a chemical pesticide on the organic farmer's land. Although this is characterized as a pesticide "drift" case, the Defendant had sprayed pesticide while he was flying over the Plaintiff's land in a helicopter. The pesticide did not simply float from one property to another. Courts will have to decide whether a short-term chemical drift is really analogous to the type of long-term process that would be required for genetic drift, and whether growing GM crops is the type of "abnormally dangerous activity" that is covered by strict liability. Determinations of trespass to land, private nuisance, negligence, and strict liability have nuances that can vary from state to state.

Dealing with uncertainty
How can a farmer who grows GM crops manage the risk of a potential crop contamination lawsuit? Last November, a conference was held in Minneapolis to consider strategies for the co-existence of GM, non-GM, and organic crop production. Participants included representatives of the USDA, agbiotech companies, and academia. One recommendation from the Minneapolis meeting was to define legal responsibilities for compromised crop production. It would be helpful to establish an acceptable standard of behavior for a farmer who grows GM crops, and to identify the duty owed by that farmer to a neighbor who grows non-GM crops. Setting such a standard should provide more certainty in determining whether crop contamination was due to negligence.

Another recommendation was to establish a pilot program for an indemnity fund to reimburse losses caused by genetic contamination of non-GM and organic corn by GM corn. Many existing insurance policies do not cover pollution-related damages, and insurers may argue that pollen drift is a type of pollution. An alternative recommendation of the Minneapolis conference participants was to modify federal crop insurance programs to provide cross-contamination coverage. Farmers could also ask agbiotech companies that sell GM seed to indemnify them against liability in the event of a lawsuit.

New state laws might provide relief for certain types of GM crop-related lawsuits. Last year, at least four states considered the liability problem. The Massachusetts legislature, for example, had a bill (1789; "An Act Relative to the Liability for Genetically Engineered Food") that would shift liability to agbiotech companies. According to the legislation, a person (i.e., a natural person or business) who genetically engineers an organism for use as food shall be strictly liable for damages caused by the use of the product on the condition that the harm was not the result of another person violating reasonable safety precautions that were outlined in a signed agreement by both persons. The damages include loss of price due to crop contamination. Taking a different approach, the House and Senate of South Dakota passed a resolution urging Congress to create legislation that places all liability for damages caused by GM seeds on the companies that develop and manufacture the seeds. Currently, however, Congress is not considering this type of legislation.

Selected References

1. Hoffman and Beaudoin v. Monsanto Canada, Inc. and Aventis CropScience Canada Holding Inc. A copy of the Statement of Claim is available at the Saskatchewan Organic Directorate website (http://www.saskorganic.com ).

2. Iowa State University. 2001. Strategies for the coexistence of GMO, non-GMO, and organic crop production (meeting summary). (December). Available at Iowa State University's Biotechnology Home Page
( http://www.biotech.iastate.edu/publications/IFAFS/coexistence.html ).

3. McInnis D. 2002. As more farmers plant GMO crops, legal issues multiply. (February 1). Available at the Checkbiotech.org website (http://www.checkbiotech.org).

4. Moeller DR. 2001. GMO liability threats for farmers. (November). Available at the website of The Institute for Agriculture and Trade Policy (http://www.iatp.org ).

5. Kades D. 2001. Lawsuit filed over genetically modified corn. Wisconsin State Journal D12 (February 17).

6. State Legislative Activity in 2001 Related to Agricultural Biotechnology. (2002). Available from the website of the Pew Initiative on Food and Biotechnology (http://pewagbiotech.org )

Phillip B. C. Jones, PhD., J.D.
Seattle, Washington
phillipjones5939@msn.com





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