DESIGNER CONSTRUCTS FOR T-DNA AND DISSOCIATION (Ds) MEDIATED INSERTIONAL MUTAGENESIS IN PLANTS
Andrew L. Eamens, Qian-Hao Zhu, Elizabeth S. Dennis, and Narayana M. Upadhyaya

Following completion of the genome sequencing of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), international focus has now turned to identification of specific functions encoded by each of the predicted ~50,000 plant genes. A genome-wide approach is essential to achieve this mammoth task, which would involve structural similarity studies, expression profiling, production of gene knockouts and gene expression knockouts, and mutant phenotyping. One of the most direct approaches to determine gene function is production of insertion mutations and study of their effects on the plant’s phenotype. In these insertion lines, the inactivated gene contains a known insertion sequence, which simplifies the task of isolating this gene, as it effectively has been "tagged" by the inserted sequence.

The incorporation of reporter gene trapping capacities within the inserted element will, in addition to producing gene knockouts, provide gene expression data as measured by the trap reporter activity^1,2. A gene trap construct contains an intron with multiple splice acceptor sites fused to a promoterless reporter gene to allow for the formation of a functional fusion product when an element inserts within a gene intron to effectively "trap" the endogenous gene. Several groups have used either unidirectional T-DNA or Dissociation (Ds) gene trap constructs to produce rice insertion lines, and gene trapping efficiencies of ~6% have been reported for these constructs³. The efficiency of T-DNA gene traps depends on the frequency of clean T-DNA insertions, i.e., insertions devoid of direct or inverted T-DNA repeats or of the incorporation of vector backbone (VB) sequences derived from outside the T-DNA borders. Such unwanted T-DNA repeats and VB sequences are shown to be present in 30 – 60% of existing insertion lines.

Insertion mutagens used in rice
Rice insertion lines are generated in laboratories world-wide, using retrotransposons (Tos17), T-DNA, iAc/Ds, and En/I as mutagens³. In particular, the two-component iAc/Ds or En/I systems provide the advantage of being able to remobilize the insertional mutagen to produce new insertion lines. We and others have previously shown in rice that the iAc/Ds-based gene and enhancer trap systems yield ~5% unique stable insertion lines in a screening population derived from either crossing of iAc and Ds lines, or from iAc/Ds double transformants. Although variable, on average ~60% of insertions are linked to the Ds launch pad (original location of Ds within the T-DNA). The majority reinsert within 4 cM of either side of the Ds launch pad. We have developed a dual-purpose (T-DNA and Ds) bidirectional T-DNA/Ds gene trap construct suitable for initial T-DNA gene trapping and subsequent Ds-based gene trapping and have tested the construct’s gene trapping efficiency in rice⁴. The increased gene trapping efficiencies of a bidirectional T-DNA gene trap have also been recently reported⁵.

A bidirectional T-DNA/Ds gene trap construct
Our T-DNA/Ds gene trap construct (Fig. 1) contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilisation; promoterless green fluorescent protein (sgfpS65T) and β-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary VB to select against transformants carrying unwanted VB sequences.

A greater than three-fold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3 – 7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of RB flanking sequence tags (FSTs) retained 1– 6 bp of the RB repeat, and 30% of LB FSTs retained 5 – 23 bp of the LB repeat. The remaining FSTs carried deletions of 2 – 84 bp inside the RB or 1 – 97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.

Figure 1.  Salient Features of the biodirectional dual purpose T-DNA/Ds gene trap constructs pEU334BN (GenBank Acc. No.AY488511). This construct contains (from RB) Ds5’ termini fused to a splice acceptor (SA - Arabidopsis GPA1 fourth intron), and the gfp reporter gene (sgfpS65T) with nos terminator as the RB/Ds5’ gene trap. An ampicillin resistance gene (bla) and E. coli bacterial origin of replication (ori) were also included in the same orientation (5’-3’) to act as the plasmid rescue system. In the opposite orientation (3’-5’) a CaMV35S promoter-driven intron-containing hygromycin phosphotransferase (hph) chimeric gene with a nos terminator was included either as a selectable marker following Agrobacterium-mediated transformation or as a Ds tracer following Ds transposition. A nos terminator-containing gus reporter gene (uidA) fused to a second SA, and Ds3’ termini were incorporated immediately inside the T-DNA LB to act as the LB/Ds3’ gene trap. In the modified binary VB (composed of restriction fragments from pCAMBIA1300 and pWBVec8), the Ubi1 promoter-barnase(I)-nosT cassette is proximal to the LB. Cleavage sites of restriction enzymes (XhoI, BglII and NheI) used in the FST rescues are indicated.

Further improvements to the construct design
We have also developed Ds-containing T-DNA constructs with features specifically suited for high efficiency Ds insertional mutagenesis (Upadhyaya et al, to be published). In addition to the features incorporated in the above described construct (bidirectional gene trap, plasmid rescue and barnase gene in the VB), these designer Ds constructs have a herbicide (bar) resistance gene as an initial selection marker or as a Ds reinsertion marker and the hygromycin resistance gene (hph) as a Ds excision marker. Another version of the construct has bar as Ds excision marker and kanamycin resistance gene (nptII) as Ds reinsertion marker or tracer. We are currently producing large numbers of these improved Ds launch pads with clean T-DNA insertion distributed over each of the 12 rice chromosomes. These resources will be made available to the international research community.

By super-infecting callus tissue from the single-copy T-DNA/Ds lines with an Agrobacterium harboring an iAc construct, and containing a visual marker (gfp), we have been able to regenerate, by applying appropriate selection pressure for Ds excision and reinsertion, stable Ds insertion lines at a frequency of ~5% in addition to the expected iAc/Ds double transformants. The progeny of double transformant lines can also be used to yield stable insertion lines by using this labor-reduced visual marker screening followed by herbicide and antibiotic selection.

Prospects
The availability of a large number of clean T-DNA/Ds launch pads throughout the rice genome will facilitate chromosomal region-directed insertion mutagenesis in a high throughput manner by saturating the corresponding genomic region of interest with Ds insertions. A similar approach has already been reported for Arabidopsis⁶. Saturation insertional mutagenesis of the entire rice genome is a huge task and requires serious collaborations among all rice researchers. Availability of such designer Ds launch pads will enable chromosomal region-wise distribution of gene tagging tasks among collaborating laboratories.

1. Sundaresan V et al. (1995). Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev. 9: 1797-1810.

2. Upadhyaya NM et al. (2002). An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice. Functl Plant Biol. 29: 547-559.

3. Hirochika H et al. (2004) Rice mutant resources for gene discovery. Plant Mol. Biol. 54: 325-334.

4. Eamens AL, Blanchard CL, Dennis ES, Upadhyaya NM (2004) A bidirectional gene trap construct for T-DNA and Ds mediated insertional mutagenesis in rice (Oryza sativa L.) Plant Biotechnol. J 2: 367-380.

5. Ryu C-H et al. (2004) Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database. Plant Mol. Biol. 54:489-502.

6. Paul R et al. (2003) A resource of mapped Dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome. Plant Physiol. 132 506–516.

ABERRANT mRNA EXPRESSION OF MAIZE RUST RESISTANCE GENE IN WHEAT AND BARLEY
P Janaki Krishna

Plant resistance (R) genes involved in gene-for-gene interactions with pathogens tend to undergo co-evolutionary arms races in which plant specificity and plant virulence continually adapt in response to each other. Studies in this regard have documented that genes resistant for different plant pathogens—fungi, bacteria, viruses and nematodes—are highly conserved in some motifs among a variety of plant species. Based on these studies, disease resistance genes have been isolated from several plant species. Although originally believed to provide durable resistance, only a few exceptional R genes proved able to control pathogens for an extended period. Though in some cases functional R gene transfer was successful, in other cases it was limited to members of the same family. In other words, sometime host signal transduction molecules are conserved among non-sexually compatible plant species. By exploiting new developments in plant transformation technology, isolated R genes can be transferred from donor species to sexually incompatible recipients. However, the ability of heterologous R genes to recognize pathogen species is still unclear.

Researchers from CSIRO Plant Industry (Australia), Kansas State University (USA), and the University of Sydney (Australia) studied the function and expression of a maize R gene, Rp1-D, to investigate its potential against related pathogens in transgenic wheat and barley. Though Rp1-D genes confer no detectable resistance phenotype, genes conferring over a dozen different race specificities exist in cereals, including species like wheat and barley. Utilization of maize rust resistance genes in controlling other cereal rusts, particularly wheat and barley, could significantly affect yield and be a boon to farmers. The researchers examined whether a full-length clone (wt-Rp1D) of the Rp1-D rust resistance gene, which was successful in controlling common maize leaf rust caused by Puccinia sorghi, could function in wheat and barley and provide protection against wheat stem rust caused by Puccinia graminis f.sp. tritici and barley leaf rust caused by P. hordei.

Transformation experiments demonstrated unambiguously that Rp1-D encodes a fully functional Rp1-D gene when introduced into maize. However, after transferring this construct into both barley and wheat, no differences were observed in response to the disease between transgenic and nontransgenic lines, either at the macroscopic or microscopic level.

The team designed several experiments to investigate the lack of expression of the Rp1-D gene in wheat and barley. Experiments revealed that: 1) low levels of transcript wt-Rp1D were present in wheat and barley, which resulted in truncated transcripts; and 2) truncated mRNAs are not a consequence of gene function. Molecular analysis of truncated transcription products from barley revealed premature polyadenylation, resulting in transcripts that are unable to encode functional Rp1-D protein. Similarly, RT-PCR analysis confirmed the presence of truncated Rp1-D transcripts in transgenic wheat lines. Further experiments confirmed that truncated transcripts are generated by the endogenous maize Rp1-D gene. Data revealed that prematurely truncated and polyadenylated transcripts are produced by the endogenous Rp1-D gene, and that the point of truncation is variable.

The authors reported that transformation of maize with the Rp1-D gene, which was regulated by either its endogenous promoter or a maize polyubiquitin promoter, conferred race-specific rust resistance in plants expressing this gene. Conversely, in wheat and barley plants, the Rp1-D gene constructs were incapable of recognizing or acting against wheat and barley rust isolates, demonstrating that inferior heterologous host recognition could result in overexpression of the processing signal such that a majority of the transcripts were processed into a truncated form. However, none of the mechanisms accounted for the low transgene expression levels observed in these heterologous cereal hosts, unless aberrant mRNA processing is associated with mRNA instability. This aberrant mRNA processing was unrelated to gene function because an inactive version of the gene also generated such transcripts.

These data demonstrate that resistance gene transfer between species may not be limited only by divergence of signaling effector molecules and pathogen avirulence ligands, but potentially also by more fundamental gene expression and transcript processing limitations and aberrant expression of the Rp1-D transgene in these species. Regardless of the regulatory sequences employed, minimum threshold level of Rp1-D protein has not been produced in these lines. Therefore, the ability of this maize R protein to interact with signaling apparatuses present in these other grass species and its ability to recognize rust pathogens of wheat and barley remains inconclusively tested.

If truncated R proteins are necessary for resistance, premature transcript polyadenylation is a potential mechanism for generating transcripts encoding such proteins and this process presumably would involve specific RNA processing signals. These transcripts may arise as an RNA processing artifact or a post-transcriptional regulatory process that is more apparent for the Rp1-D sequence in barley and wheat than in maize. In conclusion, the maize Rp1-D gene does not provide resistance in wheat and barley for the reasons explained and hence further investigation in this research area is required.

1. Ayliffe MA, Steinau M, Park RF, Rooke L, Pacheco MG, Hulbert SH, Trick HN, & Pryor AJ. (2004) Aberrant mRNA processing of the maize Rp1-D rust resistance gene in wheat and barley. Molecular Plant-Microbe Interactions 17(8): 853-864.

2. Whitham S, McCormick S, & Baker B. (1996) The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc Natl Acad Sci USA 93(16): 8776-8781.

3. Zhang X-C and Gassmann W. (2003) RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames. Plant Cell 15(10): 2333-2342.

Researchers are designing genetically engineered insects to benefit public health, enhance agriculture, and provide novel types of economically useful bugs. These efforts include genetically altering mosquitoes to render the insects incapable of transmitting malaria, engineering a bacterium in the kissing bug’s gut to kill the parasite that causes Chagas’ disease, modifying pink bollworms to carry a gene that would prevent offspring from developing, and genetically engineering honeybees to make them resistant to diseases and parasites. According to the Pew Initiative on Food and Biotechnology, genetically modified (GM) insect projects may advance from confined field trials to full environmental release within three to five years.

In January 2004 the Pew Initiative released its study on scientific and legal issues arising from insect bioengineering. Michael Fernandez, the Initiative’s director of science, explained that his organization published "Bugs in the System?" to jump-start dialogue about the benefits, risks, and regulatory challenges of genetically engineered insects. The Initiative took the initiative by sponsoring a two-day workshop on biotech bugs during September 20-21, 2004, in Washington, D.C. Conference speakers stressed that, while the technology steadily progresses, mechanisms for regulating biotech bugs—at both national and international levels—have failed to keep pace.

The Regulation Gap: United States
Eric N. Olsen, an attorney in the Washington, D.C. office of Patton Boggs, explored how the U.S. regulatory system currently governs GM insects and how the system could be adapted to cover the technology. The U.S. Coordinated Framework for Regulation of Biotechnology directs the Food and Drug Administration, the Department of Agriculture, and the Environmental Protection Agency to regulate biotech products according to their intended use. Olsen explained that the Coordinated Framework does not address GM insects, and that the federal government has done little to indicate if it will regulate bioengineered bugs. Only the USDA, he noted, has asserted authority over some GM insects.

The USDA’s Animal and Plant Health Inspection Service regulates GM insects that are, or could be, plant pests under the authority of the Plant Protection Act. This statute regulates a GM organism if the donor organism, recipient organism, vector, or vector agent is classified as a plant pest. GM plant pests are subject to APHIS review and approval before field trials or environmental release. Olsen suggested that the USDA may find additional authority to regulate GM insects under the Animal Health Protection Act, Honeybee Act, and Virus-Serum-Toxin Act.

The EPA derives a limited authority to regulate GM insects from the Federal Insecticide, Fungicide and Rodenticide Act and the Toxic Substance Control Act. FIFRA gives the power to regulate a substance as a "pesticide" if the substance is intended to prevent, destroy, repel, or mitigate a pest. GM insects engineered to target pests might fall within this definition of pesticide. TSCA covers new chemical materials produced for a commercial purpose. Olsen noted that the EPA has applied TSCA to GM microorganisms, and that the agency claims that TSCA also covers GM insects. Whether the EPA will use TSCA to regulate biotech insects remains to be seen.

Because the FDA regulates food safety, the agency would have authority over food products created by biotech bugs, such as honey from GM honeybees. Olsen also suggested that GM insects designed to improve public health could be regulated by the FDA as drugs or drug delivery devices.

In short, no U.S. statute covers all types of GM insects, and even combined, the statutes leave gaps. For example, TSCA applies to substances produced for a commercial purpose; therefore, it may not apply to GM insects designed to achieve a public health objective. APHIS covers many GM insects, but the agency also lacks clear authority and expertise to protect public health. Olsen argued that FDA’s authority to regulate new animal drugs—broadly defined as substances that alter an animal’s structure or function—could provide the basis for asserting authority over all GM insects. A downside to an FDA-dominated regulation of GM bugs is that the agency closes its regulatory process to public participation. Olsen, like many of the workshop presenters, stressed that agencies need to provide an opportunity for public input before an agency makes a final decision, and that afterwards, an agency should make the basis for the decision public.

While concerns about regulation often focus on the release of GM insects into the environment, Marjorie A. Hoy, of the University of Florida’s Department of Entomology and Nematology, called for the development of uniform guidelines to govern the use of GM insects in the lab. The adequacy of procedures designed to ensure that researchers retain their GM insects in the laboratory, she noted, is currently assessed on a case-by-case basis, primarily by institutional biosafety committees.

The International Regulation Gap
A system for regulating GM insects at the international level does not exist. Yet Mark Mansour, a partner in Morgan Lewis’ Washington, D.C. office, noted several international agreements and groups that have laid the potential groundwork for regulation: the Cartagena Protocol on Biosafety, the International Plant Protection Convention, and the Codex Alimentarius Commission Ad Hoc Biotechnology Task Force.

The Cartagena Protocol on Biosafety applies to the transboundary movement and use of living modified organisms that may have adverse effects on biological diversity and human health. Mansour suggested that the Protocol may provide a template for the international regulation of biotech insects. He warned, however, that this approach has several drawbacks: the United States is not a signatory to the agreement, and the vague Precautionary Principle inspires the Protocol’s decision-making processes. The Precautionary Principle can come into play when policymakers must decide whether to adopt new technology if the technology may harm the environment. A significant problem in implementing the Precautionary Principle as a policy tool arises from the extreme variability in its interpretation with approaches ranging from risk averse to risk-taking.

The International Plant Protection Convention aims to prevent the spread and introduction of plant pests and to promote appropriate measures for controlling plant pests. The treaty is governed by the Interim Commission on Phytosanitary Measures, which adopts international standards. Mansour proposed that these standards may extend to biotech insects considered plant pests, but probably will not cover insects targeted for human and animal diseases.

Established by the United Nations’ World Health Organization and the Food and Agricultural Organization in 1962, the Codex Alimentarius Commission aspires to protect consumer health and ensure fair trade practices by establishing standards and principles for use in international food trade. The organization’s first task force on biotechnology completed its work in 2003, and a new biotech task force will begin work next year. The 2005 Task Force may examine the development and regulation of transgenic animals, which could provide a model for regulating biotech insects.

In Mansour’s view, successful international regulation of GM insects will depend largely on the progress of debates about ethical aspects of the technology, the interpretation of biosafety protocol and other international agreements, and developments in the regulation of biotech bugs in the United States and European Union. International cooperation will be needed to ensure establishment of a science-based regulatory system.

The Need for Public Participation and Transparent Decision-Making
GM insects pose novel regulatory issues: engineered insects can scatter throughout their surroundings from their point of release, and many programs will require GM insects to persist in nature. These attributes can produce risks to public health, agriculture, and the environment. During the conference, the Union of Concerned Scientists’ Margaret Mellon asserted that scientists must take the initiative to obtain funding for studying risk assessment of GM insect projects.

Refined risk assessment techniques should provide a foundation for regulating GM insects, but more will be needed to gain public confidence. Paul B. Thompson, from Michigan State University’s department of philosophy, warned against the allure of a strictly risk-benefit style of decision-making that excludes public participation. Public trust in transgenic insect technology will not be won simply by explaining how potential benefits exceed possible risk. Similarly, Fred L. Gould, professor of entomology at North Carolina State University, declared that the development of meaningful and broadly acceptable regulations requires the inclusion of environmental groups and concerned citizens into discussions about proposed research projects. Hoy echoed these sentiments, noting that an estimation of a risk—that is, an estimate that a hazard will occur—is a scientific question free of policy implications. On the other hand, the acceptability of risk is a political question, one affected by public concerns.

On Oct 6, 2004, Monsanto posted a net loss of $42M for the fourth quarter, spurring a 3.2% single day drop in share price¹. Continued erosion of sales, down 3% from a year earlier, has increased expectations for the agrochemical giant’s newest product: low linolenic VISTIVE™ soybeans.

A Troubled Horizon
Since 2000 when U.S. patent protection expired for its flagship product, Round-Up^®, Monsanto has been struggling to keep market share and stay in the black². Despite increased sales in its growing trait business, which partially offset losses in the herbicide arena, a year ago the company posted significant losses—$188M, or $0.72 per share. This year, despite near perfect global farming weather, Monsanto has been unable to stem the tide of falling sales and prices for its Round-Up brand herbicide, a situation exacerbated by global glyphosate (active ingredient in Round-Up) dumping by Chinese manufacturers. U.S. prices for Round-Up are now predicted to hit $11-$13 per gallon, well below Monsanto’s expectations, and market share to fall near 65%, similar to what Monsanto sees in countries where generic glyphosate has been available for years.

Monsanto has been further hit by the inability to collect royalties on pirated soy seeds in Brazil, Paraguay, and Argentina—globally the top three soybean exporters after the United States. Until very recently, genetically modified crops were illegal in Brazil and are still illegal in Paraguay, although farmers are thought to have been planting genetically modified soy for the last six years³. Monsanto’s Round-Up Ready^® soy seeds, genetically modified to withstand Round-Up herbicide, are especially popular because of the reduced time and expense required for their cultivation. Monsanto, as well as producers subject to patent laws who must pay licensing fees to access the technology, believe strongly that everyone who benefits from proprietary technology should have an obligation to pay for it. Licensed producers bear that burden but unlicensed producers do not. Progress was made earlier this year when farmers in Brazil’s Rio Grande do Sul region—where experts estimate close to 90% of the soy is genetically modified—finally agreed to pay a technology fee of $3.50 a ton to Monsanto for use of its seeds. More importantly, the company’s aggressive lobbying paid off when the Brazilian government passed an executive order allowing farmers to plant genetically modified soybeans. Although the bill will give Monsanto legal standing to enforce collection of royalties on unlicensed use of its seeds in the entire country, it will not, however, sanction the sale of genetically modified soybeans

Monsanto faces a more difficult situation in Paraguay. Although 40 – 50% of soy is believed to be genetically modified, Rosa Oviedo, member of Paraguay’s biosafety commission, comments that, "Monsanto has no right to charge royalties. As of now, none of its varieties is legally sanctioned." A bill has been introduced that would legalize biotech crops, but continued peasant and farmer protests against the bill have delayed its passage indefinitely.

Unlike its neighbors, Argentina’s government has allowed Monsanto to build royalties into the price of its seeds. However, the country has been unable to collect these royalties effectively, because pirated Monsanto seeds are widely traded on the black market. In response, Monsanto stopped selling soy seeds in Argentina in 2003. It is also threatening to collect royalties on soy shipments from Argentina to countries where its seeds are patented, if they are found to carry unlicensed Monsanto products.

Other risks to Monsanto include rising oil prices, which could affect its chemical business, and a limited supply of its Posilac™ bovine growth hormone, where unresolved quality control issues are now expected to extend well into 2005. Syngenta’s pending anti-trust lawsuit, filed in July, 2004, which challenges that Monsanto has illegally monopolized key corn traits in the United States, poses another risk to the company.

Promises of Growth
Despite depressed earnings, losses to share price, and a troubled horizon, Monsanto has promised investors a share price growth of 10 – 18% in 2005 and another 10% in 2006¹—to be driven primarily by its growing genomics business and higher prices for soybean traits. However, stock analysts give mixed reviews of the firm’s prospects, many citing concerns that the company is overvalued by Wall Street, and that long-term growth will be below average. Overall, recommendations to investors range from strong sell to strong buy. According to eight independent equity research firms consulted, opinion on Monsanto is split evenly between three recommendations to buy, two to hold, and three to sell. Yet, there is consensus among analysts that makes one point clear: Monsanto’s future will be critically dependent on the success of developing its genetically modified seeds and traits business.

Increasing global sales of cotton, corn, and soybean products is the cornerstone of Monsanto’s growth plan. Sales potential has been boosted by the recent decision of the U.S. Patent and Trademark office that Monsanto was the first company to develop Agrobacterium transformation in dicot plants such as cotton⁴. The decision ends a twelve-year patent dispute between Monsanto and the Max Planck Institute (The Netherlands) and will allow Monsanto to collect fees from companies using the technology to introduce characteristics into dicot plants as well as providing patent protection for its Bollgard^® brand insect-protected cotton.

While the win on Agrobacterium transformation is a boon, achieving growth targets will still be an arduous challenge, because existing Monsanto products are quickly reaching market saturation, particularly in the United States. Robert T. Farley, Ph.D., Monsanto’s Chief Technology Officer, sites the company’s belief that the average number of Monsanto traits per acre of crop is 1.5 for cotton and 1.2 for corn in the United States. Round-Up Ready Soybeans are also near full market penetration in the United States. Monsanto will fall short of its growth targets without successful new product launches. Not surprisingly, the company’s less than stellar results recently have raised the stakes and further heightened the pressure to develop and bring to market new agbiotech products.

VISTIVE™ Soybeans May Close the Gap
In the search for new products, Monsanto reports positive progress on various R&D pipeline projects, such as second-generation Round-Up Ready Flex™ cotton, vitamin-enriched corn, and drought-tolerant crop varieties⁵. Yet, these products are still in development; the headline news for the 2005 growing season is the launch of VISTIVE™ low-linolenic soybeans.

VISTIVE soybeans, developed through conventional breeding, were designed to reduce the need for partial-hydrogenation when processing soybean oil. VISTIVE soybeans contain less than half the amount of linolenic acid normally present—3% as opposed to 8%—yielding a more stable oil with less need for hydrogenation⁶. Hydrogenated vegetable fat is favored by food processors because it is solid at room temperature and has a longer shelf life. However, the trans fatty acids produced by the hydrogenation process have prompted increased scrutiny as health concerns mount—research has suggested that trans fatty acids raise LDL (bad) cholesterol levels, leading to heart disease.

VISTIVE is posed to be an important new product that, if successful, will be critical to closing the gap between Monsanto’s current poor performance and future growth expectations. The new soybeans spell success for several reasons. First, the global market for soybean oil is large and growing. Currently soybean oil, together with palm oil, accounts for over half of all oil consumed in the world—and this proportion is on the rise. Consumers are increasingly switching to soybean oil as more studies suggest that soy not only lowers cholesterol, but may have other health benefits (for instance, see http://www.soyfoodsillinois.uiuc.edu/). To accommodate this growing demand, U.S. production of major crude vegetable oils is predicted to reach 8.6M metric tons in 2008, with soybean oil accounting for nearly 87%.

Second, beginning January 1, 2006, the U.S. Food and Drug Administration will require that all foods and dietary supplements it regulates list trans fat content on the nutritional facts panel. Such labeling will promote consumer awareness of the health hazards associated with trans fatty acids, which will certainly fuel a growing demand for foods with lower trans fat content. (Europe has yet to impose such rules, but pressure is building from consumer organizations.) As food producers search for ways to satisfy an increasing preference for healthier, lower trans fat foods, it is likely that VISTIVE will be able to command premium pricing and a booming market. "VISTIVE not only supports growing consumer demand for healthier food, but also represents an important investment in the future success of the soybean industry," said Kerry Preete, Vice President of U.S. Crop Production for Monsanto⁶. The development of VISTIVE soybeans indicates an important shift for Monsanto—by becoming more focused on consumer benefits, its bottom line may reap the benefits.

To bring the product to market, Monsanto has partnered with Cargill, Inc., a provider of food, agricultural, and risk-management products and services to 59 countries worldwide⁶. Monsanto will be relying on Cargill to contract with growers, process the VISTIVE soybeans at its facilities, and then market the VISTIVE oil to the food industry. Cargill announced that, for the 2005 growing season, it plans to contract 50,000 acres of VISTIVE soybean production in Iowa, close to its processing facilities in Iowa Falls, Cedar Rapids, and Des Moines, Iowa. Cargill has said it will pay a premium to producers who choose to grow VISTIVE soybeans. The new product will be stacked with the Round-Up Ready trait and will maintain performance parity with leading soybean varieties. It will be marketed under Monsanto’s Asgrow^® brand in 2005.