INFORMATION SYSTEMS FOR BIOTECHNOLOGY


February 2005
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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RELEASE OF LARVICIDAL CRY PROTEINS IN ROOT EXUDATES OF TRANSGENIC BT PLANTS
Deepak Saxena and Guenther Stotzky

Transgenic plants engineered to express larvicidal Cry proteins from Bacillus thuringiensis (Bt) can reduce the use of broad-spectrum chemical insecticides. However, there is some concern that Bt toxins (Cry proteins) released to soil may pose risks to the environment. The toxins could accumulate to concentrations that may constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects, and other animal classes, and may result in the selection and enrichment of toxin-resistant target insects. Conversely, the accumulated toxins could increase the control of target pests. Accumulation is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays, humic substances) and, thereby, are rendered less accessible for microbial degradation but still retain their toxic activity. The toxins produced by B. thuringiensis subsp. kurstaki (Btk; antilepidopteran), subsp. morrisoni strain tenebrionis (Btt; anticoleopteran), and subsp. israelensis (Bti; antidipteran) bound rapidly on these surface-active particles, persisted in soil and water, and remained larvicidal to the tobacco hornworm (Menduca sexta), the Colorado potato beetle (Leptinotarsa decemlineata), and a mosquito (Culex pipiens), respectively, which were used as assay species1.

Although the major introduction of the toxins to soil occurs after harvest of a Bt crop, the Cry1Ab protein was released in root exudates from transgenic Bt corn plants throughout their growth in sterile hydroponic culture and in sterile and nonsterile soil2. The presence of the toxin was demonstrated by SDS-PAGE and confirmed by immunological and larvicidal assays. The toxin from root exudates was also detected in natural soil 180 days (the longest time evaluated) after growth of Bt corn in a plant-growth room, as well as in the field after harvest and after frost from Bt corn plants that had been dead for several months. The Cry1Ab protein was released in root exudates from all 13 Bt corn hybrids, representing three transformation events (Bt11, MON810, and 176) and evaluated in both the plant-growth room and the field3.

To determine whether the release of Cry proteins in root exudates is a common phenomenon in transgenic Bt plants, the release of different Cry proteins (Cry1Ab, Cry3A, and Cry1Ac) in the root exudates of Bt corn (Zea mays L.), and rice (Oryyza sativa L.), potato (Solanum tubersoum L.), and canola (Brassica napus L.), cotton (Gossypium hirsutum L.), and tobacco (Nicotiana tabacum L.), respectively, was evaluated. Soil and hydroponic solution in which Bt corn, rice, or potato had been grown were both immunologically positive for the presence of the Cry proteins and toxic to the larva of M. sexta (corn and rice) and L. decemlineata (potato). No Cry proteins were detected immunologically or by larvicidal assay in any soil or hydroponic solution in which Bt canola, cotton, or tobacco, as well as all near-isogenic non-Bt plant counterparts or no plants, had been grown4. However, the Cry proteins were detected in the tissues of all Bt plants. There were apparent differences in exudation of the proteins (as evaluated immunologically and by mortality and weight of surviving larvae) between plant species: exudates from Bt corn were more larvicidal than those from Bt rice and potato. No green fluorescent protein (GFP) was detected in hydroponic solutions from canola and tobacco genetically modified to express GFP or both GFP and the Cry1Ac protein.

Cry proteins released in root exudates of Bt corn, rice, and potato accumulated in soil and retained larvicidal activity, probably as the result of the binding of the proteins on surface-active particles in soil, which rendered them resistant to rapid biodegradation1,2,3. Although some Cry proteins were probably released from sloughed and damaged root cells of corn, rice, and potato in soil, the major portion was apparently derived from root exudates, as there was no discernable root debris when plants were grown in hydroponic culture, and no Cry proteins from Bt canola, cotton, or tobacco were detected when grown in soil where some damage to roots probably occurred.

In addition to the introduction to soil of Cry proteins in plant biomass and some in pollen, the proteins will also be released in root exudates during the entire growth of some Bt plants. The continual presence of the Cry proteins in soil could improve the control of insect pests, enhance the selection of toxin-resistant target insects, and/or constitute a hazard to nontarget organisms. The Cry1Ab protein released in root exudates and from biomass of Bt corn had no apparent effects on earthworms, nematodes, protozoa, bacteria, and fungi in soil5; it was not taken up by radish, carrot, turnip, and non-Bt corn6; and it did not move far vertically in soil7.

Why Cry proteins were released in root exudates of Bt corn, rice, and potato but not in exudates of Bt canola, cotton, and tobacco is not known. The methods of transformation of the cry genes, somaclonal variation, differences in level of protein expression (although all species had the cauliflower mosaic virus 35S promoter, except rice, which had the ubiquitin promoter from corn), or location of the endoplasmic reticulum relative to the plasma membrane (in corn, this appears to be a close relation) may have been involved1. Although Cry1Ab and Cry1Ac proteins differ in some aspects, the differences are apparently small, as their insect targets are similar and they cross-react with antibodies to each other1. Nevertheless, the differences may be responsible for release of the Cry1Ab protein and the lack of release of the Cry1Ac protein in root exudates. Further studies, especially by plant physiologists and anatomists, are obviously necessary.

The relevance of these observation also requires clarification, especially as at least 26 plant species, including corn, cotton, potato, canola, rice, broccoli, peanut, eggplant, and other crop species, have been modified to express Cry proteins, and 8.1 million hectares of Bt corn or 26% of total corn acreage, 2.4 million hectares of Bt cotton or 45% of total cotton acreage, and 0.02 million hectares of Bt potato or 3.5% of total potato acreage were planted in the United States alone in 20004.

The release of Bt toxins in the root exudates of some plant species, and their persistence in soil, indicate that caution must be exercised before plants and animals genetically modified to express pharmaceuticals (e.g., vaccines, hormones, antibiotics, blood substitutes, enzymes) and other bioactive compounds ("pharms") are introduced to the environment8,9. In contrast to pesticidal transgenic plants, the targets of these compounds, which are seldom found in natural habitat5 (i.e., they are essentially environmental xenobiotics), are human beings and other "higher level" eukaryotes rather than insects, nematodes, and protozoa. Inasmuch as the persistence in and the effects on the environment of these biomolecules have not been studied adequately, their potential hazards are not known and cannot be predicted. As with Bt plants, where only a portion of the plants is harvested and the remainder of the biomass is incorporated into soil wherein the toxins released from disintegrating biomass are rapidly bound on surface-active particles, a substantial portion of the biomass of plant pharms containing the products of introduced novel genes will also be incorporated into soil. With pharms of transgenic animals, the feces, urine, and subsequently even carcasses containing bioactive compounds will eventually reach soil and other natural habitats. If these bioactive compounds (including prions from diseased animal carcasses) bind on surface-active particles—and as many of these compounds are proteinaceous, they most likely will—they may also persist in these habitats. If they retain their bioactivity, they could affect the biology of these habitats. Consequently, before the large-scale use in the field of such plants and animals pharms, the persistence of their products and the potential effects of the products on the inhabitants of soil and other habitats must be thoroughly evaluated.

Acknowledgments

These studies were supported, in part, by grants R826107-01 from the US Environmental Protection Agency, 2003-35107-13776 from the US Department of Agriculture, and N0721 from the New York University Research Challenge Fund. The opinions expressed herein are not necessarily those of the EPA, USDA, or RCF. Seeds of cotton and potato ("eyes") were kindly provided by Monsanto Co., of rice by Drs. I. Altosaar and Q. Shu, and of tobacco and canola by Dr. C. N. Stewart.

Sources

1. Stotzky G (2001) In: D. K. Letourneau, B.E. Burrows (Eds.), Genetically Engineered Organisms: Assessing Environmental and Human Health Effects. CRC Press, Boca Raton. pp. 187-222

2. Saxena D, Flores S, and Stotzky G (1999) Nature 402, 480

3. Saxena D, Flores S, and Stotzky G (2002) Soil Biology & Biochemistry 34, 133-137

4. Saxena D et al. (2004) Plant Physiology and Biochemistry 42, 383-387

5. Saxena D, and Stotzky G (2001) Soil Biology & Biochemistry 33, 1225-1230

6. Saxena D, and Stotzky G (2001) Nature Biotechnology 19, 199

7. Saxena D, Flores S, and Stotzky G (2002) Soil Biology & Biochemistry 34, 111-120

8. Fischer R et al. (2004) Current Opinions in Plant Biology 7, 152-158

9. Daniell H, Streatfield SJ, and Wycoff K (2001) Trends in Plant Science 6, 219-226

Deepak Saxena1 and Guenther Stotzky2
1
College of Dentistry (
ds100@nyu.edu)
2Laboratory of Microbial Ecology (gs5@nyu.edu)
Department of Biology, New York University


FREQUENCY OF POLLEN DRIFT IN GENETICALLY ENGINEERED CORN
B. L. Ma

Cross fertilization and concerns over cross contamination
Corn (Zea mays L.), also called maize, is a monoecious crop with male (staminate inflorescence) and female (pistillate inflorescence) flowers formed in separate parts of the same plant, leading to a high degree of cross-pollination between plants. It is reported that cultivated corn freely crosses with nearly all members of the genus, including several hundred mutants1. The male inflorescence (tassel) of corn can produce considerably more pollen grains than are required for pollination of a single plant. Westgate et al.2 estimated that individual tassels produced 4.5 x 106 pollen grains, and pollen shedding often lasts 5 or 6 days.

Interest in pollen movement has increased recently due to possible contamination of conventional crops from pollen of genetically engineered (GE) genotypes. Under natural conditions, pollen can travel from field to field, but the majority of pollen grains are assumed to fall within the row space, as corn pollen is one of the heaviest and largest (about 90-100 µm in diameter) among wind-dispersed pollen grains1,4. For corn producers, the major issue is that contamination of conventional hybrids by pollen from neighboring GE hybrids will restrict marketing the grain harvested from the contaminated field. Such grain is declared as essentially transgenic and will only be accepted by specific elevators and processors3. Thus, there is an urgent need to understand pollen-mediated gene flow and the minimum distance required to isolate conventional hybrids from neighboring GE cornfields.

The endosperm of corn kernels can be yellow or white, with yellow dominant to white. These colors are easily observable and can be used as markers in studies designed to measure cross-fertilization. It is possible to quantify the extent of out-crossing between genotypes by planting a white-kernel hybrid next to a yellow hybrid and measuring the incidence of yellow kernels on white cobs. Using this production system, we determined: i) the frequency of cross-fertilization of a corn genotype by foreign pollen of neighboring hybrids; and ii) the practical distance from neighboring GE corn fields required to grow non-GE corn.

Site description for field experiment

Field experiments to quantify the level of cross fertilization of white corn by pollen from neighboring Bt yellow corn were planted at three locations in Ottawa, Canada, (45°22’N, 75°43’W) for three growing seasons (2000, 2001, and 2002). All sites were open fields, and in all cases, there were no corn crops, fences, or blocks to stop wind flow within at least 200 m in all directions. At each site, the yellow Bt corn was planted in the center (36 rows of 27 x 27 m) while a white corn hybrid was planted in the surrounding area (Fig. 1).

In this region, the prevailing wind in July and August is generally from the northwest direction. Therefore, white corn planted east and south of the yellow Bt corn was designated as "downwind," while white corn west and north was "upwind." Hybrids and planting dates for each site-year are listed in Table 1.

Assessment of the frequency of cross-fertilization

At maturity, a thorough systematic sampling of white corn was conducted to determine out-crossing of white corn kernels by the yellow Bt pollen (Fig. 2).

Figure 2.
Example of cross-fertilization of white corn by neighboring yellow corn pollen from the first adjoining row up to 19th row from the pollen source as compared to the pure white and yellow corn ears.

In each site, in both downwind and upwind directions, ears of white corn were sampled from rows No.1 (the first row of white corn adjacent to the yellow Bt hybrids), 7, 13, 19, 25, 31, 37, 43, and 48 (37 m) bordering the yellow Bt corn. In the northern and southern ends of yellow Bt corn rows, white corn ear samples were taken from rows 1, 7, 13, 19, 25, and 33 (row number was arbitrarily defined, but plant number was counted always starting from the white kernel plant bordering the yellow corn). In all designated rows, ears from every 10th plant of white corn (i.e., 1st, 11th, 21st, and so on) were collected, marked, and measured. The relative distance to the yellow corn determined the position of all sampled plants in the field. A plant was considered as 0% cross-fertilized if there were no yellow-colored kernels in the sampled ear as well as in the ears of two adjacent plants (e.g., if the target was an 11th plant, plants 10th, 11th and 12th were also field-checked to insure no yellow kernels were present). Within a single ear, the total number of rows, number of kernels per row, and the total number of yellow kernels were counted. Percent out-cross was calculated as the number of yellow kernels divided by the total number of kernels (white+yellow) per ear.

An exponential decline model was used for fitting the cross-fertilization data as a function of distance to the yellow Bt corn pollen:

Y = Y0 e –BX [1]

where Y is cross-fertilization (%), Y0 is cross-fertilization extrapolated to X = 0, B is a shape coefficient, and X is the distance (m) of the sampled ear to the pollen source of the yellow corn.

Results and discussion

Pollen shedding and silking dates, time taken to reach the stages, and CHU accumulated for both yellow and white corn hybrids are presented in Table 1. The hybrid 4521Bt (yellow) at Site #2 (year 2000), V414W (white) at Site #3 (year 2001), and both hybrids at Sites #2 and #3 in 2002 had non-uniform plant growth (uneven plant size) and development (phenological stages) requiring longer periods to complete their flowering (Table 1), and probably asynchronous pollination within the population.

Seasonal weather conditions affected the level of cross-fertilization. The observed maximum out-crossing was over

82%, indicating that, in corn, cross-fertilization between genotypes is more favored than that within the population (>50% kernels are yellow in the white kernel corn). The average level of out-crossing in the first adjacent row was greater in 2000 (18.2%) than in 2001 (12.3%) or 2002 (13.3%). A consistent pattern was also observed in subsequent rows.

The level of cross-fertilizations across site-years fluctuated greatly because of wind speed and directions, but as a rule, the farther away from the yellow Bt pollen source, the lesser was the percent out-crossing (Fig. 2). Overall, maximum cross-fertilization occurred in the first row of the white corn adjacent to the yellow Bt hybrid. The extent of cross-fertilization in the subsequent rows declined exponentially to 0 or near 0% toward the edge of the field. Greater than 1% cross-fertilization was found after the 37th border row (28 m) downwind from the prevailing wind direction or the 13th row (10 m) in the upwind direction in all site-years. In the white corn rows on the straight northern and southern sides of the yellow Bt corn field, a considerable amount of out-crossing (7-15%) was recorded, mainly within 7.4 m (41st plants) from the pollen source, with substantial differences in level of out-crossing among site-years.

The rate of cross-fertilization considering distance to the pollen source was well represented by the exponential decline function (P<0.01), with R2 = 0.64 for downwind and 0.58 for upwind (Fig. 3).

The Eq [1] also fits the data of cross-fertilization in the northern and southern sides of yellow Bt corn. According to Eq [1], the extrapolated zero (or 0.0001%) cross-fertilization would have occurred in the white corn at about 28–30 m downwind or 18–23 m upwind from the pollen source (Fig. 3), suggesting that pollen traveled shorter distances, or cross-fertilization declined more quickly, along the same row direction than along cross row directions. In general, although the model fits the data, the R˛ values in all cases were not very large, indicating factors other than distance, e.g., wind speed and directions during the peak pollination period, also played important roles in the extent of cross-fertilization. The risk of cross-fertilization of white corn (or other non-GE corn) by pollen from neighboring yellow Bt corn was very low beyond the 37th row (28 m) from the source.

Summary and Concluding Remarks

Using yellow kernel corn as a marker of cross-fertilization in white corn is a useful tool to estimate pollen dispersal. The experimental results showed that the majority of corn pollen grains settle close to the source. There was an exponential decrease in pollen dispersal as the distance from pollen source increased. From a practical point of view, and considering differences in planting dates from neighboring cornfields and/or different maturity of two hybrids involved, our data suggest it is possible to produce non-GE corn grains by removing the outside rows of corn plants (about 30 m) adjacent to the GE corn field in concern, if the acceptance level is set at <1% cross-fertilization. Although the chances of cross-fertilization of white corn by pollen from neighboring yellow corn at the distance of 28 m was minimal, this study did not examine the situation in which Bt and non-Bt corn were separated by a non-corn space3.

References

1. Burris JS (2001) Adventitious pollen intrusion into hybrid maize seed production fields. Proc. 56th Annual Corn and Sorghum Research Conference 2001. American Seed Trade Association, Inc. Washington, D.C.

2. Westgate ME, Lizaso J & Batchelor W (2003) Quantitative relationship between pollen-shed density and grain yield in maize. Crop Sci. 43, 934-942

3. Ma BL, Subedi KD & Reid LM (2004) Extent of cross-fertilization in maize by pollens from neighboring transgenic hybrid. Crop Sci. 44, 1273-1282

4. Stewart DW, Ma BL & Dwyer LM (2001) A mathematical model of pollen dispersion in a maize canopy. 2001 Annual Meeting Abstracts, The ASA-CSSA-SSSA Headquarters, Madison, WI.

B. L. Ma
Eastern Cereal and Oilseed Research Centre
Agriculture and Agri-Food Canada
mab@agr.gc.ca


ASSESSING THE BENEFITS AND RISKS OF GE CROPS: Evidence from the Insect Resistant Maize for Africa Project
H De Groote, S Mugo, D Bergvinson, and B Odhiambo

Background
Genetically engineered crops have been highly successful in developed countries, increasing yields and profits without negative health or environmental effects. However, the technology has generally not been well received in Europe, where environmentalists and green activists are worried about irreversible environmental damage. Moreover, European agriculture has a consistent overproduction problem, so yield enhancing technologies are not of critical importance. Expected benefits to the European consumer are also small. Therefore, Europe has accepted the precautionary principle, which imposes very stringent regulations and requirements of risk assessment on GE crops, basically banning them for the time being. In 2004, Europe approved the importation of the first GE maize food, but use of GE maize seed is generally not allowed.

African countries are caught in a quandary—should they embrace the technology to help feed their hungry people, or rather protect them from potential dangers? Potential advantages of the technology include: increased yield (for the only continent that has benefited little from the Green Revolution), increased food security (for the only region in the world where the percentage of malnourished children is expected to rise during the next 20 years), and a technology easy to disseminate (for a region where extension services have collapsed and liberalization is lagging).

Despite these potential benefits, deployment of GE crops in Africa remains highly controversial. Among the arguments against them are: GE crops would not respond to small farmers’ priorities; their traits would not reply to a particular demand; and seed would be expensive. Another argument alleges that GE technology would only be beneficial to the agro-businesses, which can protect their interests through Intellectual Property Rights (IPR) and ‘terminator’ genes, and make farmers dependent on new varieties while they lose biodiversity of their old ones. Further, GE crops could pose serious risks to the environment through the development of resistance in target insects, gene flow into weeds and local varieties, and from the disruption of non-target organisms. Moreover, African countries might not be sufficiently equipped with the appropriate biosafety regulations to make an informed choice. Finally, it is argued that poor people, if given a choice, would not necessarily opt for GE crops but might prefer other solutions.

We argue that African farmers and consumers have the right to choose their own technologies, based on the best available knowledge1. African scientists need to develop and test GE crops on the alternative precautionary principle, that is, poor farmers and consumers risk being denied a chance to improve their livelihood based on an academic debate in which they cannot participate. On this principle, the Insect Resistant Maize for Africa (IRMA) project was launched in 1999, using both conventional breeding and biotechnology, and combining the best available science, biophysical as well as social. After five years of research in the first phase, it can be shown how most, but not all, concerns against Bt maize can be answered.

Overview of research results

Research shows that demand for Bt maize is likely to be high. Not only is maize the major food crop in Kenya but, after progress in the 1960s and 1970s, maize yields and production have stagnated while production per capita has decreased. While more maize is grown in the high-potential zones, the level of poverty is higher in the low-potential zones (Fig. 1).

During participatory rural appraisals (PRA) with 43 villages, more than 900 farmers explained which varieties they grow and why, and expressed the constraints and pest problems they face. Most farmers grow local varieties, except for in the high-potential zones. The two major criteria for variety selection are early maturity and yield, in addition to three other important traits—tolerance to drought, field pests, and storage pests. The three major constraints to maize production were cash constraints, lack of technical expertise and extension, and problems with maize seed—high cost, poor quality, and low availability. Pest problems are usually found among the top six constraints. The two most important pest problems farmers encounter are stem borers and weevils, which rank in the top three in all agro-ecological zones.

Yield losses due to stem borers were calculated based on farmers’ estimates from a survey of 1400 farmers, and resulted in a first estimate of 12.9%2. These losses were higher in the low-potential zones (15–21%) than in the high-potential zones (10–12%). Next, yield losses were measured in 150 farmers’ fields using a simple experiment comparing protected and unprotected maize, leading to an estimated loss of 13.5%, totaling 0.4 million tons annually, valued at US$ 80 million3 (Fig. 2).

Supplying the Bt technology for Kenyan maize production does not pose major technological problems. IRMA, working within the regulatory system, introduced several samples of maize leaves with different Bt genes (one per plant) for bioassays4. Effective Bt genes were found against all major stem borer species, except for one, Busseola fusca, which dominates in the higher altitudes and is economically more important (Fig. 3). In bioassays of multiple genes per plant, however, higher levels of efficacy were found. These events will now be tested in the recently approved biosafety greenhouse, followed by trials in an open quarantine facility5. Moreover, a review of relevant Intellectual Property Rights, including a Freedom to Operate review, concluded that there are no patents filed in Kenya that would restrict the use of Bt genes in maize. Finally, local seed companies have shown great interest in adopting the technology, as long as the costs are reasonable. The estimated demand and supply were combined in an economic surplus model, which calculated a modest profitability with the currently available Bt genes3. The project would be highly profitable if a gene or combination of genes can be found against B. fusca. More than two thirds of the benefits would go to the consumer through a reduction in prices.

Demand and supply need to find one another through markets, within the regulatory framework. Biosafety guidelines were established and Institutional and National Biosafety Committees set up to implement these. Over the years, these committees have become experienced and efficient in dealing with biosafety applications, partially due to the experience and interaction with IRMA. An analysis of the seed sector found that liberalization has increased the number of companies and varieties dramatically, but overall markets are still dominated by one company and a limited number of varieties, especially in the highlands. Moreover, the amount of improved maize seed sold has not increased over the years.

The PRAs also showed that farmers often recycle seed, including hybrids, and that they mark selected plants for this purpose. A study of the credit sector showed that formal agricultural credit has basically collapsed and has been replaced by small, informal finance groups. Farmers who have access to this type of credit use half of it for agriculture, which allows them to double their use of improved maize seed. Regular discussions with farmers, consumers, and institutions during annual stakeholders meetings, group discussions, and other fora reveal that farmers are generally very enthusiastic about Bt maize, while scientists, consumers, and the general audience are cautiously optimistic.

During a survey in Nairobi, few consumers objected to the use of GE crops for food, although they have concerns about risks for environment and biodiversity. Interestingly, upon learning that the Bt gene is dominant (and can therefore be recycled) farmers requested that the project also consider transformation of their local varieties.

Farm surveys showed that most areas have enough alternative hosts that form natural refugia and prevent the build-up of resistance against the toxins. No relatives of maize exist in Africa, so the gene cannot cross into weeds. Farm surveys and PRAs also indicate that biodiversity does not decrease with agricultural intensification. Although the number of local varieties does decrease with intensification, the total number of varieties does not. In the high-potential areas, farmers typically use more varieties than in the low-potential areas, so that their biodiversity indices are higher.

Conclusions

The results of the different studies show how most objections to Bt maize cannot be substantiated. First, it is indispensable to work with Bt maize and introduce it in an experimental setting so that farmers, consumers, and policy makers can make informed decisions. These results indicate that Bt maize responds to an important constraint and that farmers are very interested. Consumers are likely to benefit too, and they do not express strong objections. The poorer farmers in the low-potential areas will benefit relatively more, since they have relatively higher losses, and poor consumers will benefit relatively more since they spend proportionately more of their income on maize. Bt maize is likely to be commercialized by local companies, since there are no restrictive IPRs involved, and thus extra costs will be low. Because the Bt genes are dominant, farmers will not become dependent on the seed industry since they can recycle their seed. Their recycling methods, moreover, are likely to select for the Bt gene and, over time, incorporate the gene into local varieties.

However, local varieties are likely to become contaminated, and this process could be irreversible. IRMA has taken samples of all local varieties in the different zones to deposit in the National Genebank. Further, natural refugia might be insufficient in certain areas. This could be countered by pyramiding several Bt genes in appropriate varieties or mixing seed with sufficient amounts of non-Bt maize. The study of the effects of Bt maize on non-target organisms has not yet been initiated, but identification of these organisms has started and comparative studies will start immediately with field trials.

Acknowledgements

The financial support of the Syngenta Foundation for Sustainable Agriculture is highly appreciated. The views expressed in this paper are the authors’ and do not necessarily reflect the policies, or opinions of their respective institutions.

References

1. De Groote H, Mugo S, Bergvinson D, Owuor G & Odhiambo B (2004) Debunking the myths of GM crops for Africa: the case of Bt maize in Kenya. Paper presented at the American Agricultural Economics Association’s Annual Meeting, Denver, Colorado, August 1-4, 2004

2. De Groote H (2002) Maize Yield Losses from Stemborers in Kenya. Insect Science and its Applications 22, 89-96

3. De Groote H, Overholt W, Ouma JO, & Mugo S (2003) Assessing the impact of Bt maize in Kenya using a GIS model. Paper presented at the Conference of the International Association of Agricultural Economics, Durban (South Africa), August 2003

4. Mugo S, Taracha C, Bergvinson D, Odhiambo B, Songa J, Hoisington D, McLean S, Ngatia I, & Gethi M (2004a) Screening cry proteins produced by Bt maize leaves for activity against Kenyan maize stem borers." Paper presented to the 7th Eastern and Southern Africa Regional Maize Conference and Symposium on Low N and Drought Tolerance in Maize, Kenya, 2002. In Friesen, D. and A. F. E. Palmer (eds.). Integrated Approaches to Higher Maize Productivity in the New Millenium. Proceedings of the 7th Eastern and Southern Africa Regional Maize Conference, Nairobi, Kenya, 11 - 15 February 2002. Mexico, D. F.: CIMMYT, pp. 102-105

5. Mugo S, De Groote H, Songa J, Mulaa M, Odhiambo B, Taracha C, Bergvinson D, Hoisington D, & Gethi M (2004b) Advances in Developing Insect Resistant Maize Varieties for Kenya within the Insect Resistant Maize For Africa (IRMA) Project. In Friesn and A. F. E. Palmer (eds.). Integrated Approaches to Higher Maize Productivity in the New Millenium. Proceedings of the 7th Eastern and Southern Africa Regional Maize Conference, Nairobi, Kenya, 11 - 15 February 2002. Mexico, D. F.: CIMMYT, pp. 31-37

Hugo De Groote
h.degroote@cgiar.org
Stephen Mugo
S.mugo@cgiar.org
CIMMYT



PATENT CHALLENGES TO AGBIOTECH TECHNOLOGIES IN 2004
Phillip B. C. Jones

The classic techniques appeared in the scientific literature 10 to 20 years ago: Agrobacterium-mediated transformation of plants, producing transgenic plants that express Bacillus thuringiensis (Bt) toxin for insect protection, and engineering glyphosate-tolerant plants that express a mutant 5-enolpyruvylshikimate-3-phosphate synthase. Despite their age, conflicts over the rights to use these methods remain vigorous.

Patent Rights to Agrobacterium Technology
On February 23, 2004, Syngenta International AG (Basel, Switzerland) and Monsanto Company (St. Louis, Missouri) announced an agreement in which the companies cross-license proprietary Agrobacterium-mediated transformation technology. The agreement resolved a patent interference proceeding in the U.S. Patent and Trademark Office (PTO) involving transgenic broad leaf crops.

The Monsanto-Syngenta deal also resolved a lawsuit that had been pending in the U.S. District Court for the District of Delaware. Syngenta had filed the case in 2002, alleging that Monsanto and Delta and Pine Land infringed U.S. Patent No. 6,051,757, which covers methods of transferring genes into dicotyledonous plants using Agrobacterium-based vectors. On the day that the companies announced their new agreement, the Delaware district court dismissed the patent infringement case.

Monsanto continues to build its store of Agrobacterium-related patent rights. In October, the company announced the PTO’s decision that Monsanto’s scientists had invented Agrobacterium transformation methods in dicot plants before the Max Planck Institute and other parties. The decision ended a 12-year patent interference dispute.

Toxic Battles Over Bacillus thuringiensis-producing Plants
On August 13, 2004, the San Diego federal district court held that Dow Chemical Company subsidiary Mycogen was the first to invent the Bt-based insect resistance trait Cry1F. This Bt toxin protein is expressed in Dow AgroSciences’ Herculex™ insect protection for corn and WideStrike™ protection for cotton.

A company that has the rights to a species of Bt toxin protein can still be subject to another company’s broad patent rights on Bt toxin technology. Expansive patent coverage in the Bt toxin arena are at stake in a patent infringement case initiated by Syngenta on July 25, 2002. Syngenta had filed a lawsuit claiming that Monsanto, DeKalb Genetics (a subsidiary of Monsanto), Pioneer Hi-Bred International, Inc. (a subsidiary of E. I. DuPont de Nemours), Dow AgroSciences LLC (Indianapolis, Indiana), and Mycogen Seeds infringe at least one of U.S. Patent Nos. 6,075,185, 6,320,100, and 6,403,865. These patents include claims to synthetic Bt genes designed for increased expression in corn and claims to transgenic corn plants resistant to insects. Among the allegedly infringing products were Monsanto’s YieldGard® and Dow AgroSciences’ Herculex™.

On November 29, 2004—the first day of the trial—Syngenta announced that it had reached an agreement with Pioneer Hi-Bred International. Pioneer would receive a license to Syngenta’s patents covering insect-resistant corn traits.

During December, Judge Sue L. Robinson held that Dow AgroSciences and Monsanto had not infringed two of Syngenta’s patents (6,075,185 and 6,320,100) as a matter of law. These patents focus on methods for optimizing codons for more efficient expression of Bt insecticidal proteins in corn. The defendants’ products use different codons. A jury then decided that the remaining patent—U.S. Patent No. 6,403,865—is invalid because Syngenta did not invent the claimed invention. Syngenta announced its intent to appeal both verdicts.

A handful of Bt toxin patent cases may be resolved in 2005. On April 13, 2004, the PTO issued Syngenta’s U.S. Patent No. 6,720,488, which includes claims to transgenic maize seeds that contain a Bt toxin gene. The next day, the company filed a complaint in the Delaware district court against Monsanto et al. alleging infringement. Syngenta requested the court combine this complaint with the other Bt toxin litigation, but in August, the court denied consolidation. This case may be decided this year.

In February of 2004, the PTO published its decision on an interference proceeding to determine who had invented methods of designing synthetic Bt toxin genes for efficient expression in plants. The PTO decided that Monsanto’s inventors had beat Mycogen Plant Science’s inventors, and the agency eliminated 12 of 14 claims in Mycogen’s U.S. Patent No. 5,380,831. In a patent infringement case decided several weeks later, the Court of Appeals for the Federal Circuit limited the scope of the remaining two claims to a particular Bt toxin gene disclosed in the patent.

A few months later, Monsanto claimed that the PTO’s interference decision prompted Dow AgroScience to dismiss with prejudice a Bt toxin patent lawsuit that Mycogen had filed in 1995. But the company has not given up. Mycogen filed a complaint for "patent interference dissatisfaction" in an Indiana federal district court, requesting a reversal of the PTO’s inventorship decision.

Another Bt toxin case began when Monsanto filed an action in a Missouri district court seeking a declaratory judgment that its Bt toxin-expressing transgenic corn does not infringe four patents owned by Aventis CropScience, a predecessor of Bayer BioScience. The patents concern methods for expressing a truncated version of a Bt insecticidal protein. Although Monsanto scored a victory in the district court, Bayer won on appeal. The appellate court bounced the case back to the lower court for further proceedings.

Resisting Competitors for the Glyphosate-tolerant Trait
On May 12, 2004, Syngenta announced that it had acquired from Bayer CropScience the rights to GA21, a glyphosate tolerance technology in corn. The technology enables farmers to control weeds in corn with post emergence applications of glyphosate herbicide. Syngenta said that it will offer the technology—renamed "Agrisure GT Advantage" for 2005 and beyond—through the Garst brand and through licenses with other seed companies.

On the same day, Monsanto announced that it had filed a lawsuit in the Delaware district court against Syngenta. In the complaint, Monsanto alleged that Syngenta has infringed Monsanto’s Patent No. 4,940,835 by attempting to make and use glyphosate-resistant genes and crops, and by conspiring with others to make glyphosate-resistant crops. The company requested damages for past infringement and a permanent injunction against Syngenta to prevent further patent infringement.

The lawsuit may have its origins in an October 2003 agreement between Bayer CropScience AG and Monsanto, which resolved long-standing patent disputes between the companies. As part of the agreement, Monsanto and Bayer cross-licensed enabling technology for certain herbicide-tolerant crops. Monsanto asserts that the deal had not included a license for using Monsanto’s technology in corn, and therefore, Syngenta could not have purchased rights to this use of the technology from Bayer CropScience.

In July, Monsanto filed another lawsuit against Syngenta—this time, in an Illinois federal district court. The company alleged that Syngenta infringed patents held by Monsanto’s DeKalb Genetics Corp. Monsanto asked the court to prevent Syngenta from developing, using, and selling corn seed carrying the glyphosate-tolerant trait. Monsanto claimed that Syngenta infringed patents issued to DeKalb Genetics Corporation.

On the same day, Monsanto amended a breach-of-contract case filed in a St. Louis County court. The company originally filed the lawsuit on May 10, 2004, and had sought to limit a soybean license granted to Ciba-Geigy Corp., one of Syngenta’s corporate predecessors. Monsanto now asked the court to terminate the license that allows Syngenta to sell soybeans engineered to be resistant to Roundup® herbicide.

When Monsanto had filed its May 12 patent infringement suit against Syngenta, David Jones, Syngenta’s head of business development, had dismissed the lawsuit as "a flagrant attempt to intimidate customers and restrict choice in the market." Building on this theme, Syngenta filed an antitrust lawsuit against Monsanto in Delaware district court one day after Monsanto had amended its breach-of-contract claims. Syngenta alleged that Monsanto has engaged in a pattern of illegal and improper activities to maintain its monopoly in key corn traits in the United States. The company asserted that Monsanto’s illegal activities included attempts to prohibit Syngenta from competing with GA21 technology, and attempts to force seed companies to stop producing GA21 seeds, to destroy GA21 inventories, and to adhere to exclusive dealing contracts. Monsanto spokeswoman Lori Fisher said that the antitrust lawsuit appeared to be designed to divert attention from the lawsuits that Monsanto had filed.

Although this flush of litigation suggests that Monsanto considers GA21 technology to be the flagship of its line, this is not the case. Monsanto has phased out GA21 technology in the marketplace in favor of a glyphosate-tolerant event called "NK603."

Selected References

Anonymous (2004) Syngenta refutes Monsanto lawsuit as totally without merit. May 13, 2004. Available at: http://www.syngenta.com

Anonymous (2004) Monsanto wins key patent dispute regarding dicot plant transformation. PR Newswire. October 5, 2004

Sinclair N (2004) Syngenta sues Monsanto alleging GM corn seed monopoly. Chemical News & Intelligence. July 28, 2004

Suhr J (2004) Rivals Monsanto, Syngenta sue each other. Associated Press Online. July 28, 2004

Syngenta Seeds, Inc. v. Monsanto Company et al., Civ No. 02-1326 (D. Del., dismissed February 23, 2004)

Syngenta Seeds, Inc. v. Monsanto Company et al., Civ No. 02-1331 (D. Del. 2004)

Phillip B. C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org




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