Bt corn grows on millions of hectares in the U.S. and elsewhere, but its usefulness will be cut short if pests evolve resistance to Bt toxins. So far, field-evolved resistance to Bt crops has not been reported. Although only one pest (diamondback moth) has evolved resistance to Bt sprays in the field, more than a dozen have evolved resistance to Bt toxins in the laboratory and in greenhouses.

To slow pest resistance to Bt crops, the U.S. Environmental Protection Agency (EPA) has mandated the "high-dose/refuge strategy" requiring farmers to grow non-Bt refuges near Bt crops. The non-Bt refuges are designed to enable survival of susceptible pests. Ideally, rare resistant individuals emerging from Bt corn mate almost exclusively with relatively abundant susceptible individuals emerging from refuges. If resistance is inherited as a recessive trait, the hybrid offspring resulting from matings between resistant and susceptible adults will be killed by the Bt crop. Models predict that the strategy will delay resistance substantially if these assumptions hold, but pollen-mediated gene flow from Bt crops to refuge plants could disrupt this.

Gene flow in plants occurs when pollen from one plant fertilizes another plant, carrying with it the genes from the first plant. We focused on corn in this study, but all genetically engineered crops produce pollen-containing transgenes. If pollen from transgenic plants moves by wind or if insect pollinators fertilize related non-transgenic plants, the transgenes may be integrated into the genome of the offspring, altering the traits of the offspring. Previous concerns about gene flow from transgenic crops have emphasized movement of transgenes to wild relatives of crops, landraces, and organic plantings while effects on pest resistance had been overlooked.

Gene flow to refuges from Bt corn can cause Bt toxin production in seeds (i.e., kernels) but not in vegetative tissues of refuge plants. Such refuge contamination, causing toxin production in corn kernels, could affect caterpillars of pests such as corn earworm (Helicoverpa zea) that eat corn kernels. In addition, during their second or later generations, 20 to 50% of populations of European corn borer, (Ostrinia nubilalis), the main target of Bt corn, also eat kernels.

Corn is primarily wind-pollinated, with approximately 97% outcrossing between plants and fertilization occurring at up to 200 m. To test the hypothesis that gene flow from Bt corn causes Bt toxin production in non-Bt corn refuges, we sampled kernels from ears of non-Bt corn along transects near Bt corn. Tests of non-Bt corn sampled from the field showed that the concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt corn. These results imply that pollen-mediated gene flow from Bt corn caused Bt toxin production in kernels of non-Bt corn refuge plants.

Methods
Six pairs of near-isogenic Bt (4 Bt11 insertion event hybrids, and 2 Mon810 event hybrids) and non-Bt corn hybrids were planted in adjacent plots in the field, with each non-Bt hybrid planted in 36 rows downwind of 8 rows of its Bt counterpart (0.965 m row-spacing). Each commercially available Bt hybrid was produced by crossing a Bt line with a conventional line and backcrossing the resulting Cry1Ab-producing plants with the conventional line for several generations. Each backcrossed Bt line was crossed with a second conventional line to produce hybrid seed for planting that was hemizygous for the cry1Ab gene. Plants grown from this hybrid seed should carry the cry1Ab gene in half of their eggs and pollen. Thus, Bt plants fertilized with Bt pollen are expected to have the cry1Ab gene in 75% of their seeds and pollen (50% hemizygous and 25% homozygous). Ears were harvested from two rows of each Bt subplot and rows 1–4, 8, 16, 24, and 32 of each non-Bt subplot (0.96 to 31 m from the Bt subplot). From each sample, 30 randomly selected kernels were set aside and the remaining kernels ground into a powder.

We used enzyme-linked immunosorbent assay (ELISA) to test for Cry1Ab. To estimate the mean concentration of Cry1Ab in ears of each subplot row, we tested 2 g of each ground kernel sample with ELISA. To estimate the percentage of kernels containing Cry1Ab, 30 seeds from each sample were germinated and leaf punches from each seedling tested for Cry1Ab with ELISA by Mid-West Seed Services (Brookings, South Dakota, USA).

Results
Gene flow from Bt corn to non-Bt corn occurred at up to 31 m, the greatest distance we examined. ELISA tests of non-Bt corn showed that the concentration of Bt toxin Cry1Ab and the percentage of kernels with Cry1Ab decreased with distance from Bt corn (Figs. 1 & 2).

Fig. 1. Mean (± S.E.) concentration of Bt toxin Cry1Ab in kernels of Bt and non-Bt corn ears as a function of distance between Bt and non-Bt plots. Row distance = 0.97 m. Rows -1 and -3 were Bt corn approximately 2 and 4 m from the non-Bt corn, whereas, rows 1 to 32 were non-Bt corn approximately 1 to 32 m from Bt corn.

The mean concentration of Cry1Ab in kernels of Bt hybrids was 260 ± 19 ng/g (Fig. 1). The concentration of Bt toxin in non-Bt corn was highest within 2 m of Bt corn, decreasing rapidly with distance. The maximum Bt toxin concentration in kernels of non-Bt corn was 140 ng/g in refuge plants at about 1 m distance from Bt plants. This maximum toxin concentration in non-Bt corn was 45% of the mean concentration in adjacent Bt plants. At 31 m from Bt corn, the mean concentration of Cry1Ab in kernels of non-Bt corn was 4.4 ± 1.2 ng/g, which is 1.7% of the concentration in the kernels of adjacent Bt plants.

Fig. 2. Mean (± S.E.) percentage of Cry1Ab corn kernels in Bt and non-Bt corn ears as a function of distance between Bt and non-Bt plots. Rows labeled as in Fig. 1.

In Bt corn, the mean percentage of kernels with Cry1Ab was 74.5%, which is consistent with the 75% expected from hemizygous parents (Fig. 2). The maximum percentage of kernels of non-Bt corn with Cry1Ab was 60%, which occurred at 1 m from Bt corn. In proportion to the mean of 75% Bt kernels for the ears of adjacent Bt corn, the maximum of 60% Bt kernels in non-Bt corn was 0.8 (60% divided by 75%).

Discussion
The movement of Bt genes into non-Bt refuge plants has important implications for resistance management. If some target pests are killed by Bt toxins in refuge plants near Bt corn, refuges will produce fewer susceptible insects than expected. If so, the value of refuges for delaying resistance will be diminished. Further, a mosaic of kernels with and without Bt toxin in various proportions could cause some pests to eat a mixture of Bt kernels and non-Bt kernels in the same ear. This in turn might expose pests to a wide range of doses, rather than the uniform high dose envisioned in the ideal application of the high-dose/refuge strategy. Exposure to intermediate doses might favor survival of hybrid pests produced by matings between resistant and susceptible parents, which would undermine the strategy. Even in uniform plantings of Bt corn, toxin levels vary among leaves, silks, shanks, and kernels of a single plant, as well as among plants and through time. This variation calls into question the assumption that pests always receive a high toxin dose when eating Bt plants. Nonetheless, the additional variation in toxin concentration introduced by gene flow from Bt corn to nearby non-Bt corn could reduce the effectiveness of the refuge strategy.

The extent of Bt gene flow into refuges depends on many factors, including refuge size, shape, distance from the Bt crop, wind speed and direction, and similarity in maturation times between Bt and non-Bt hybrids. Thus, various physical, ecological, and molecular methods of gene containment could reduce contamination of refuge plants by gene flow from Bt crops. In our experiment, non-Bt corn was planted downwind from Bt corn. Planting non-Bt corn upwind of Bt corn would reduce gene flow into refuges, but increased gene flow in the opposite direction could produce Bt corn with intermediate toxin concentrations. This type of gene flow appears to be a less serious concern because vegetative tissues of Bt corn as well as some portions of all the kernels such as the seed coat would produce Bt toxin regardless of such gene flow.

Although movement of Bt genes into refuges has received little attention previously, the potential for movement of corn transgenes has been recognized in other contexts. In particular, the U.S. EPA mandates that experimental Bt corn hybrids must be planted more than 200 m from other corn to limit gene flow. Paradoxically, EPA guidelines for Bt corn resistance management suggest that non-Bt corn refuges in Bt corn fields can be as narrow as 4 m and that they must be planted within 800 m of Bt corn, preferably 400 m, to increase mating between insects from Bt corn and refuges. Revised guidelines should consider the effects of gene flow between Bt and non-Bt plants as well as gene flow between resistant and susceptible insects. In particular, the minimum width of non-Bt corn refuges should be increased to at least 30 m.

References
1. Chilcutt CF & Tabashnik BE. (2004) Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic corn. Proceedings of the National Academy of Sciences USA 101: 7526-7529.

2. Gould F. (1998) Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annual Review of Entomology 43: 701-726.

3. Shelton, AM, Nyrop JP, Seaman A & Foster RE. (1982) Distribution of European corn borer (Lepidoptera: Pyralidae) egg masses and larvae on sweet corn in New York. Environmental Entomology 15: 501-506.

4. Tabashnik BE. (1994). Evolution of resistance to Bacillus thuringiensis. Annual Review of Entomology 39, 47-79.

Two major research directions have been established since the original work demonstrating the functional expression of antibodies in plants¹. The first was using plants as bio-reactors for large-scale therapeutic antibody production; the second, expressing antibodies to affect physiological processes of the plant by a method termed immunomodulation². We have recently expanded the immunomodulation potential for plant-expressed Abs by successfully testing the hypothesis that an Ab with specific affinity for a herbicide can confer resistance when expressed transgenically in planta³. For this proof of concept model system, we chose an Ab against the auxinic herbicide picloram and tobacco as the transgenic host because tobacco is highly susceptible to the effects of this herbicide⁴.

One hundred primary transgenic Nicotiana tabacum plants were produced using a binary expression construct and Agrobacterium-mediated plant transformation with kanamycin selection. The binary construct was designed to express a single-chain Fv (scFv) recombinant Ab format derived from the gene sequence of a previously characterized anti-picloram monoclonal Ab⁵. The scFv was placed under control of a tobacco constitutive promoter, tCUP, and expression was targeted to the endoplasmic reticulum by the C-terminal addition of a KDEL motif and an N-terminal secretion signal. Also included in the design of the scFv was the addition of both a c-MYC epitope tag and a polyhistidine (6xHis) tag for detection and quantification of plant-expressed anti-picloram scFv. Ninety-nine of the 100 primary transgenic plants tested positive for functional anti-picloram scFv and were rank-ordered as determined by indirect picloram enzyme-linked immunosorbant assay (ELISA). Competitive indirect ELISA demonstrated the specificity of anti-picloram scFv for picloram, as this free antigen inhibited the indirect ELISA with an I.C. 50 of 150 ppb; two structural herbicidal analogues, clopyralid and triclopyr, did not inhibit this indirect competitive ELISA.

Seven independent primary transgenic (T₀) plants of varying anti-picloram scFv expression, as well as a non-specific scFv control, were selfed and T₁ seeds collected for picloram resistance bioassay studies. T₁ seeds were germinated on agar medium containing picloram over a concentration range of 10^-11 to 10^-4 M to determine an effective dose for demonstrating picloram resistance among experimental plants compared with controls. As illustrated in Figure 1, anti-picloram scFv-expressing seedlings on 10^-8 M picloram demonstrate visibly better growth characteristics than transgenic control seedlings, which developed typical auxinic herbicide effects such as epinasty, hypertrophy, and a reduction of true leaf development. Four weeks post-plating, six seedlings from 10^-8 M picloram plates were transplanted into soil lacking picloram and allowed to grow for two more weeks, after which total leaf areas and shoot fresh weights were determined. When compared to the control, six of the seven seedling sets had significantly different shoot weights and leaf areas at the 0.01 probability level, while the seventh was significantly different at the 0.05 probability level.

Figure 1. Effect of 10 nM picloram on control and anti-picloram scFv-expressing tobacco seedlings (T1 transgenic #21, 3 independent T-DNA insertions). The photograph was taken 4 weeks after sowing surface-sterilized seeds on MS plus sucrose agar medium containing 10 nM picloram.

One of our highest anti-picloram scFv expressing lines, T₁ #21, and the nonspecific scFv-expressing control line were selected on kanamycin plates for a field spray simulation bioassay. These were subsequently propagated in 10 cm pots and grown to the six-leaf stage of development when they were sprayed with 1 mL of either 0, 1, 10 or 100 µM solutions of picloram, which are equivalent to agronomic doses of 0, 0.05, 0.5 and 5.0 g ai/ha, respectively. Experiments were performed in quadruplicate and sampled after 4 weeks post-spraying. The 1 µM picloram treatment had little or no effect on either control or experimental plants. Both control and experimental plants of the 100 µM picloram treatment were severely affected; three of the four control plants died, whereas only one of the four anti-picloram scFv plants died. All survivors at this concentration were severely stunted. The most significant phenotypic differences were observed with the 10 µM picloram treatment (Fig. 2), where the growth rate of T₁ #21 plants were similar to untreated plants, whereas control plants were stunted and had obvious picloram injury at this dose. Additionally, control and anti-picloram scFv-expressing plants treated with 10 µM picloram were allowed to grow to the flower stage of development. Control plants did not develop primary meristems; however, they did develop secondary meristems that produced small flowers and seeds that did not mature. Anti-picloram scFv-expressing experimental T₁ #21 plants produced normal flowers from primary meristems with viable seeds that were indistinguishable from untreated wild type plants.

Figure 2. Figure 2. Differential susceptibility of control and anti-picloram scFv-expressing tobacco plants to a 10 μM dose of picloram. T1 #21 seedlings and control seedlings were selected on kanamycin plates and transplanted to soil. Experimental and control plantlets were sprayed once with a 10 μM solutions of picloram in 1ml volumes at the four-leaf stage of development and the response comparison recorded 6 weeks post spraying.

Although picloram is no longer considered an important agronomic herbicide, we have successfully used this proof of concept model system to test the hypothesis that antibodies with specific xenobiotic affinities are able to generate xenobiotic resistance when expressed in plants. Our results support the concept that affinity-adsorption occurs in planta, thereby resulting in the sequestration of the xenobiotic to levels below that of a physiologically toxic threshold dose. However, as with many proofs of concept technologies, improvements will have to be made to commercialize this Ab technology because our highest picloram resistance, 0.5 g ai/ha, is below application rates of herbicides in use today, 5 to 1000 g ai/ha. This could be done by taking advantage of the fact that Ab-antigen interactions rely on the quantity of Ab being expressed, as well as the affinity of the antibody-antigen interaction. Therefore, anti-herbicide immunomodulation technology could become applicable to all types of herbicides, as both in planta Ab expression and Ab affinity is improved through the use of recombinant Ab technologies such as affinity maturation procedures. For example, a 10-fold affinity improvement of our anti-picloram scFv for picloram would likely result in resistance levels as high as 5 g ai/ha. Therefore, the first successful demonstration of this technology for commercial field dose applications will likely come from plants protected with Abs against low-use-rate herbicides, such as acetolactate synthase inhibitors, which are applied at rates as low as 5 g ai/ha. Alternatively, the development of catalytic Abs⁶ specific for xenobiotic detoxification would overcome the obvious limitation of this 1:1 stoichiometric neutralization approach and may allow for Ab-based resistance to higher use rate herbicides.

In addition to generating xenobiotic resistant crops for weed control, the results of this proof of concept study suggest other novel generic uses for anti-xenobiotic Ab expression in plants. For example, Abs expressed in plants could provide a seemingly unlimited choice for generating novel selectable markers for use in plant biotechnology by expressing Abs to different xenobiotics in plants. Our results also suggest the potential for a generic bioremediation strategy by expressing anti-xenobiotic Abs against organic environmental contaminants, such as PCPs, PCBs, residual herbicides, and even chemical warfare agents. If the Ab were catalytic, the contaminant could be detoxified in planta; alternatively, non-catalytic antibodies could provide a sink for the adsorption of the compound from the soil with little or no effect on the plant.

References
1. Hiatt A, Cafferkey R & Bowdish K. (1989) Production of antibodies in transgenic plants. Nature 342: 76-8.

2. Conrad U & Manteuffel R. (2001) Immunomodulation of phytohormones and functional proteins in plant cells. Trends Plant Sci. 6: 399-402.

3. Almquist KC et al. (2004) Immunomodulation confers herbicide resistance in plants. Plant Biotechnology Journal 2: 189-197.

4. Cox C. (1998) Herbicide Fact Sheet: Picloram. Journal of Pesticide Reform 18: 13-20.

5. Yau K et al. (1998) Bacterial expression and characterization of a picloram-specific recombinant Fab for residue analysis. J. Agric. Food Chem. 46: 4457-63.

6. Wentworth P & Janda KD. (2001) Catalytic antibodies: structure and function. Cell Biochem. Biophys. 35: 63-87.

TRANSGENIC PLANTS PRODUCE OMEGA-3 AND OMEGA-6 FATTY ACIDS
Baoxiu Qi

Recently, the importance of fish and fish oils in the diet has become significantly more recognized. It is known that they play very important roles in human health and nutrition and are important building blocks in neonatal retinal and brain development, as well as being important factors in cardiovascular health and disease prevention^1-2. The active ingredients in fish and fish oils are omega-3 very long chain polyunsaturated fatty acids (VLCPUFAs), such as eicosapentaenoic acid (EPA, C20:5 ω3) and docosahexaenoic acid (DHA, C22:6 ω3). The realization of their importance started in the 1970s from the epidemiological finding that Greenland Eskimos only had one-eighth of the fatal coronary heart disease of Eskimos who lived in Denmark. The reason is that the Greenland Eskimos have a traditional `marine' diet rich in very long chain ω3 PUFAs. The diet of most modern societies is nowadays relatively low in ω3 PUFAs with a concomitant increased level of ω6 PUFAs intake, largely resulting from a preference for plant-seed oils and food products from intensively bred animals. In addition, some communities do not have access to fish supplies, due to geographic or economic reasons; some people are allergic to fish, others choose not to eat fish because of their vegetarian life style. Global fish stocks are declining due to general overfishing and overly efficient fishing methods, and the oils derived from fish are sometimes contaminated with a range of pollutants, heavy metals, and toxins. Therefore, alternative sources of these VLCPUFAs are clearly desirable, and the concept of obtaining them from higher plants in commercial and sustainable quantities is particularly attractive. However, no oil-seed species produces such products naturally, so there is considerable interest recently in genetically engineering the capacity to synthesize these fatty acids in agronomically viable oil-seed species.

Humans can synthesize omega-6 and omega-3 VLCPUFAs from the so-called essential fatty acids, linoleic acid (LA, C18:2 ω6) and α-linolenic acid (ALA, C18:3 ω3) by a Δ⁶-desaturation pathway in which the characteristic first step is the Δ⁶-desaturation of LA and ALA to yield γ-linolenic acid (GLA, C18:3 ω6) and stearidonic acid (STA, C18:4 ω3), respectively. Further fatty acid elongation and desaturation steps give rise to arachidonic acid (AA, C20:4 ω6) and EPA. DHA is the product of further elongation and desaturation of EPA. However, these pathways are very inefficient, and to obtain these VLCPUFAs directly from the diet is considered necessary.

An alternative pathway for the biosynthesis of AA and EPA operates in some organisms. Here, LA and ALA are first elongated specifically to eicosadienoic acid (EDA, C20:2 ω6) and eicosatrienoic acid (EtrA, C20:3 ω3), respectively. Subsequent Δ⁸ and Δ⁵ desaturation of these products yields AA and EPA. We recently reported the reconstitution of these Δ⁸-desaturation pathways for VLCPUFA synthesis in Arabidopsis thaliana, and the accumulation of appreciable quantities of AA and EPA in the transgenic plants³ by sequential transfer and expression of three genes encoding a Δ⁹-specific elongating activity from Isochrysis galbana (IgASE1)⁴, a Δ⁸-desaturase from Euglena gracilis (EuΔ8)⁵, and a Δ⁵-desaturase from Mortierella alpina (MortΔ5)⁶, respectively. For expression in Arabidopsis, the coding regions of these three genes were placed in CaMV 35S promoter-nos terminator expression cassettes of the plant binary vectors pCB302-1, pBECKS, and pCAMBIA 1300, respectively. Arabidopsis thaliana ecotype Columbia 4 was subjected to Agro-bacterium-mediated transformation via floral dipping. Basta herbicide, kanamycin, and hygromycin were used to select single, double, and triple transformants, respectively. Homozygous plants with a single copy of the transgenes were selected by two rounds of self-fertilization after each transformation and subjected to further transformation with the subsequent gene. Total fatty acids in the leaf tissues of wild type, single transgenic plants expressing IgASE1, double transgenic plants expressing both IgASE1 and EuΔ8, and triple transgenic plants expressing IgASE1, EuΔ8, as well as MortΔ5 were extracted, methylated, and analyzed by gas chromatography (GC).

Figure 1. GC profiles of Arabidopsis leaf fatty acid methyl esters extracted from (a) an untransformed plant; (b), a single-transgenic plant expressing the I. galbana Δ9-elongating activity IgASE1; (c), a double transgenic plant expressing IgASE1 and the E. gracilis Δ8-desaturase (EuΔ8); (d) a triple transgenic plant expressing IgASE1, EuΔ8 and the M. alpina Δ5-desaturase (MortΔ5). The peaks in the boxed region are designated 1 to 8 in the order of increasing retention time; peaks 4 and 8 are 20:4 and 20:5, respectively.

The GC profile of total fatty acids extracted from leaves of wild type and transgenic plants is shown in Figure 1. LA and ALA, the substrates for the Isochrysis C18-Δ⁹-elongating activity IgASE1, are major fatty acids in the leaves of wild type plants (Fig. 1a). Two additional fatty acid species are apparent in the single transgenic plants expressing IgASE1 (Fig. 1b). These were identified as EDA and EtrA. C20:2 and C20:3 accumulated to 8.4 mol% and 10.4 mol% of total fatty acids, representing conversions of 51% and 22% of their C18 substrates, respectively. Two additional peaks are apparent in the GC profile of the leaf fatty acids of the double transgenic line expressing both IgASE1 and EuΔ8 (Fig. 1c) compared to its single transgenic parent (Fig. 1b). These were identified as dihomo-γ-linolenic acid (DGLA, C20:3 ω6) and eicosatetraenoic acid (ETA, C20:4 ω3), respectively. DGLA and ETA accounted for 8.7 and 6.5 mol% of the total fatty acids and represented a conversion of 88% and 63% of their respective substrates. The total C20 PUFA content in this double transgenic line was ca. 20 mol% of the total fatty acids. Four other fatty acid peaks are apparent in the triple-transgenic plants expressing IgASE1, EuΔ8, and MortΔ5 compared to the double transgenic plants (Fig. 1d). Two of these, peak 4 and 8, correspond to AA and EPA, the expected Δ⁵-desaturation products of DGLA and ETA, respectively. The yields of AA and EPA were 6.6 and 3.0 mol% of the total fatty acids, representing 84% conversion of substrate DGLA and 71% of ETA. Small amounts (3.2 mol% of total FAs) of two other C20 fatty acids were also present and these were identified as sciadonic acid, C20:3 Δ^5,11,14, and juniperonic acid, C20:4 Δ^5,11,14,17. The total C20 PUFA content was ca. 22 mol% of total fatty acids. All transgenic plants were phenotypically identical to wild type, which implies that production of significant amounts of C20 PUFAs did not affect the plants' normal development.

Our results demonstrate that both the ω3 Δ⁸- and ω6 Δ⁸-desaturation biosynthetic pathways for VLCPUFA production can operate in higher plants, yielding the potentially valuable products AA and EPA in appreciable amounts (6.6% and 3%, respectively). The key step would appear to be the C18-Δ⁹-PUFA-specific elongating activity IgASE1 from Isochrysis, which specifically catalyses the elongation of the essential C18-Δ⁹-PUFAs, LA and ALA. These fatty acids are in abundance in green vegetative tissues of higher plants, and in Arabidopsis their elongation resulted in appreciable levels of EDA (20:2ω6) and EtrA (20:3ω3). The efficiency of this alternative pathway may be because the first step is catalyzed by elongase, which uses LA-CoA and ALA-CoA as substrates, and these have previously been shown to be present in Arabidopsis leaf tissue. Moreover, the subsequent two desaturation steps can occur while the acyl groups remain on the phosphotidylcholine substrate. In contrast, recent attempts to reconstitute the conventional ω6 and ω3 Δ⁶-desaturation pathways in yeast met with limited success⁷. To explain their relatively low yield of AA in yeast, Domergue et al.⁷ argued that insufficient GLA substrate was made available from the site of Δ⁶-desaturation to the acyl-CoA pool for elongation, and that this explained a similar poor activity of the reconstituted conventional pathway in transgenic plants.

The results reported here demonstrate that "pathway engineering" for the viable production of VLCPUFAs in plants is possible. In order to exploit these observations more fully it will be necessary to express the genes in question in a seed-specific manner and in a lipid background rich in the fatty acid substrates that are specific for the elongase component, IgASE1. To this end, oilseed species such as soy and linseed should provide appropriate experimental material for further study. Our data indicate that the use of these alternative Δ⁸-desaturation pathways is likely to be more appropriate for genetic modification of oilseeds than the previously described conventional Δ⁶-desaturase/elongase route. The triple-transgenic plants accumulate both AA and EPA in amounts that suggest that, if their incorporation into seed storage oils can be achieved, the production of these fatty acids in oil seed crops could become an economically viable proposition.

We have successfully reconstituted the Δ⁸-desaturation pathways for both ω6 and ω3 VLCPUFA biosynthesis in a higher plant. This has been achieved by sequential transfer and expression of three genes in the model plant A. thaliana. The accumulation of AA and EPA in substantial quantities is a significant breakthrough in the search for alternative sustainable sources of these health promoting very long chain polyunsaturated fatty acids.

References
1. Crawford M. (2000) Placental delivery of arachidonic acid and docosahexaenoic acids: implication for the lipid nutrition of preterm infants. Am. J. Clin. Nutr. 71: 275S-284S.

2. Thies F et al. (2003) Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomized controlled trial. The Lancet 361: 477-485.

3. Qi B et al. (2004) Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nature Biotechnol. 22: 739-745.

4. Qi B et al. (2002) Identification of a cDNA encoding a novel C18-Δ⁹ polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett. 510: 159-165.

5. Wallis JG and Browse J. (1999) The Δ8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch. Biochem. Biophys. 365: 307-316.

6. Michaelson LV et al. (1998) Isolation of a Δ⁵-fatty acid desaturase gene from Mortierella alpina. J. Biol. Chem. 273: 19055-19059.

7. Domergue F et al. (2003) Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast. J. Biol. Chem. 278: 35115-35126.

The Federal Court of Canada decided against Schmeiser, and the farmer appealed to the Federal Court of Appeal. After wrestling with Schmeiser's 17 issues for reconsideration, the appellate court affirmed the trial court's verdict. Schmeiser appealed again.

The Supreme Court agreed to consider the case and heard arguments last January. On May 21, the Court published its decision; Monsanto won. For the most part, at least.

Five of the nine justices affirmed the lower court's decision that Schmeiser had infringed Monsanto's patent. First, the majority decided that Monsanto's patent is valid. It is true that Canadian law does not allow for the patenting of transgenic plants, they explained, but Monsanto's patent claims cover genes and modified cells that make up the plant. The claims do not literally cover the modified plant.

If so, then how did Schmeiser use Monsanto's invention by growing transgenic plants? The majority found that the farmer had saved and planted seed, and then harvested and sold plants that contained patented cells and genes. According to case law, a patentee can establish infringement by showing that a defendant's business activity involves an object that includes a patented component. Monsanto had established infringement by showing that Schmeiser had cultivated Roundup Ready canola as part of his business operations. The Court explained that infringement of the Monsanto patent does not require use of the patented gene or cells in isolation, nor does not it require that Schmeiser must have applied Roundup herbicide to aid cultivation.

The Court then considered whether Monsanto should receive the $20,000 patent infringement award conferred by the trial court and affirmed by the appellate court. Canadian patent law provides two types of remedy for infringement: damages or an accounting of profits. Damages represent the patentee's loss, which may include lost profits from sales or lost royalty payments. Monsanto had decided to seek an accounting of profits, which required the courts to calculate the proceeds earned by the infringer.

However, the Supreme Court could find no causal connection between the patented invention and Schmeiser's profits generated by growing Roundup Ready canola. The farmer had sold the Roundup Ready canola for feed and obtained no premium for the fact that the canola was anything but a conventional crop. Schmeiser also had not gained any agricultural advantage from the herbicide resistant nature of the canola, because he had not sprayed with Roundup to reduce weeds. Since the farmer's profits arose solely from qualities of the crop that could not be attributed to the invention, he earned no profit from the invention. Accordingly, the Court set aside Monsanto's award for account of profit.

The trial court had also awarded Monsanto litigation costs, potentially in the neighborhood of $140,000. But the Supreme Court decided that each party must bear its own costs throughout the litigation.

Four justices dissented from the holding that Schmeiser had infringed the patent despite the Court's own ruling that plants are unpatentable. They reasoned that Monsanto's patent claims are valid only if they do not extend patent protection to the transgenic plant itself.

The Schmeiser case vindicated Monsanto's legal rights, even though the company did not collect the monetary award that it had sought. A recent U.S. case yielded a similar outcome.

Mississippi farmer Homan McFarling purchased Monsanto Company's Roundup Ready soybean seeds in 1997 and 1998, signing a Technology Agreement that required him to use the seeds for planting a commercial crop in a single season. The Agreement also prohibited him from saving any crop produced from the seed for replanting. Nevertheless, McFarling saved 1500 bushels of patented soybeans from his harvest in one season, and planted them the next. He repeated this practice during the following year and promised to do so again.

In January 2000, Monsanto sued McFarling in the Eastern District of Missouri, alleging patent infringement and breach of contract. The court prohibited the farmer from using seed that he had collected from crops grown with the patented soybean seed. McFarling appealed to the Court of Appeals for the Federal Circuit, but lost.

Back in district court, Monsanto moved for summary judgement on the patent infringement and breach of the Technology Agreement claims. The company also sought damages.

The Agreement includes a liquidated damages clause that calculates breach of contract damages at 120 times the technology licensing fee. Monsanto argued that the liquidated damages clause should be interpreted to generate an amount of 120 times the technology fee times the number of bags of seed replanted by McFarling. The court decided that this sum would result in a penalty of 120 times the actual damages. Penalty clauses are illegal in Missouri, so the court settled on an amount of $780,000, or 120 times 1000 bags purchased by McFarling times the $6.50 technology licensing fee. McFarling appealed.

On April 9, the Federal Circuit affirmed the district court's decision that McFarling had breached the contract, but vacated the judgment on damages. In the court's view, the Technology Agreement provision that applies a 120 multiplier to the technology fee is an unenforceable and invalid penalty clause. This means that Monsanto's recovery is limited to actual damages. The Federal Circuit sent the case back to the district court to compute actual damages based on the number of bags of seed that McFarling saved and replanted. The amount will probably be a bit less than $780,000.

An announcement made by Syngenta on May 12 triggered yet another patent battle over herbicide resistance technology. The Swiss company publicized its purchase of rights to "GA21" glyphosate tolerance technology in corn from Bayer CropScience, and proclaimed its intention to market GA21 corn in the United States. Taking advantage of the time difference, St. Louis-based Monsanto Company made its own May 12 announcement: it filed a lawsuit in the Delaware district court against Syngenta.

Monsanto alleges that Syngenta's GA21 glyphosate-tolerant corn infringes Patent No. 4,940,835, which covers genes and vectors conferring glyphosate resistance in plants. The company seeks a permanent injunction against Syngenta to prevent commercial sale or distribution of GA21 corn in the United States. David Jones, Syngenta's Head of Business Development, dismissed the lawsuit as "a flagrant attempt to intimidate customers and restrict choice in the market."

The Bt Goes On
The Bacillus thuringiensis toxin patent wars are alive and well. In February the U.S. Patent and Trademark Office (PTO) published its decision on an interference proceeding to determine who had invented methods of designing synthetic Bt toxin genes for expression in plants. Monsanto's inventors had beat Mycogen Plant Science's inventors, the PTO decided. Consequently, the PTO 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 Federal Circuit limited the scope of the remaining two claims to a particular Bt toxin gene disclosed in the patent.

On May 13, Monsanto announced that Dow AgroScience, which acquired Mycogen, dismissed with prejudice a lawsuit that Mycogen had filed in 1995. Monsanto claimed that the PTO's interference conclusion had prompted Dow's decision. But Dow has not given up on the Bt toxin patent. Mycogen filed a complaint for "patent interference dissatisfaction" in an Indiana federal district court on March 29. The company requests the court to reverse the PTO's inventorship decision.

In another Bt toxin case, 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. Monsanto took the position that the patentee's inequitable conduct bars Bayer from enforcing the patents. The district court granted Monsanto's summary judgment motions, holding the four patents unenforceable because of inequitable conduct during patent prosecution. Bayer appealed.

The Federal Circuit reversed the summary judgment on March 20 and sent the case back to the district court for further proceedings. The Federal Circuit decided, among other things, that the lower court had improperly granted summary judgment on Monsanto's inequitable conduct claim, because there was a factual dispute about whether the patentee's statements on the ease of expressing truncated Bt toxin genes in plants were false or misleading, and had been made to deceive the patent examiner.

The International Biotechnology Symposium is held every four years and sponsored by the International Union of Pure and Applied Chemistry (IUPAC). The Symposium will cover the latest developments in:

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