Nitrate and ammonia in soil are forms of inorganic nitrogen that can be assimilated in all plants, whereas atmospheric nitrogen can be assimilated only in some, basically leguminous, plants with the help of microbes. If, like peas and soybeans, all plants could utilize nitrogen gas as the source of inorganic nitrogen, then the volume of nitrogen fertilizers applied to fields could be reduced, without losing the maximum biomass of crops. However, in spite of the intensive studies, a method to enable crops to fix atmospheric nitrogen has not been developed. Because all plants can assimilate inorganic nitrogen from soil, the nitrogen assimilation or the nitrate reduction enzyme has been overexpressed in transgenic plants; however, such studies did not succeed in promoting nitrogen assimilation. In several recent reports, overproduction of glutamine synthetase in transgenic plants improved nitrogen utilization efficiency by promoting recapture of ammonia released during photorespiration. This can occur because the amount of ammonia released during photorespiration is much greater than that of primary nitrogen taken up by the plant. However, overexpression of the enzyme also appeared not to influence net nitrogen assimilation.

II. Application of transcription factors to molecular breeding
Genes encoding enzymes have been frequently utilized in molecular breeding to endow plants with new characteristics. When a new characteristic is determined by one additional biochemical reaction, the simple transfer of a new gene encoding the enzyme for the required reaction would be sufficient. In addition, if the aim is activation of a specific pathway that is regulated only at one rate-limiting step, then overexpression of the enzyme catalyzing the rate-limiting step might result in an increase of the enzyme activity, thereby leading to activation of the pathway. Overexpression of a desensitized form of the enzyme might be more effective, because the pathway would be constitutively activated, independently of negative regulations. However, if modification of multiple cellular responses or promotion of multiple enzymatic reactions is necessary for a new trait, then altered expression of genes encoding regulatory proteins, especially transcription factors, would be very useful. Because a single transcription factor often triggers multiple cellular responses to a specific internal or external stimulus by simultaneously inducing expression of many genes functionally unrelated to one another (Fig. 1A), overexpression of a transcription factor might produce stronger responses to a specific stimulus. In addition, because a single transcription factor often regulates the coordinated expression of enzymes involved in a metabolic pathway (Fig. 1B), enhanced expression of the key transcription factor would be capable of activating the pathway that is only slightly promoted by overexpression of one enzyme.

Figure 1. Transcription factors (TF) might activate transcription from the promoters of multiple genes, each of which is involved in different responses (A). TF might coordinately regulate expression of multiple genes involved in a metabolic pathway (B).

Studies of the CBF/DREB transcription factors, which specifically bind to the A/GCCGAC sequence in drought, high-salt, and cold inducible promoters, suggest the potential of transcription factors in molecular breeding. Transgenic Arabidopsis plants overexpressing the CBF1 (DREB1B) transcription factor acquired enhanced freezing tolerance², and overexpression of the DREB1A (CBF3) transcription factor also led to improved tolerance to drought, high-salt, and freezing stresses³.

Strategy
To enhance nitrogen assimilation in plants, we developed a novel strategy using a transcription factor⁴. Nitrogen assimilation in plants needs not only inorganic nitrogen present in soil but also carbon skeleton (2-oxoglutalate, 2-OG), which is produced from photosynthetic metabolic intermediates (Fig. 2).

Figure 2. The metabolic pathway for nitrogen assimilation in plants. Abbreviations for metabolites are used: PEP, phosphoenolpyruvate; OAA, oxaloacetate; 2-OG, 2-oxoglutarate. Several key enzymes are also indicated by abbreviations: PEPC, phosphoenolpyruvate carboxylase; PK, pyruvate kinase; CS, citrate synthase; ICDH, isocitrate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; NIA, nitrate reductase. Expressing Dof1 increased expression of PEPC, PK, CS and ICDH genes in the Dof1 transgenic Arabidopsis plants. [Image from Ref. 4 Copyright Proceedings of the National Academy of Sciences, USA.]

Thus, it had been speculated that the increased production of carbon skeleton might stimulate nitrogen assimilation in plants. The genetic modification of carbon skeleton production had been thought to be difficult, because many enzymes are involved in carbon skeleton production. Transfer of multiple genes encoding enzymes seemed not to be practical, but transfer of a gene encoding a key transcriptional activator seemed possible. We presumed that the pathway for carbon skeleton production might be activated using a maize transcription factor, Dof1, which appeared to be a strong candidate for the master regulator for the pathway.

Results
We generated the transgenic Arabidopsis expressing Dof1 and investigated the effects of Dof1 on gene expression associated with the carbon skeleton production pathway. The results of semi-quantitative RT-PCR revealed that the expression levels of phosphoenolpyruvate carboxylase (PEPC) genes and pyruvate kinase (PK) genes, whose promoters contained putative Dof1-binding sites, were elevated in the Dof1 transgenic plants. In addition, the preliminary result with DNA microarrays suggested that expression of other genes, including the citrate synthase (CS) and isocitrate dehydrogenase (ICDH) genes, was also increased in the transgenic plants (Fig. 2). Thus, expressing Dof1 appeared to synchronously activate the expression of multiple genes involved in carbon skeleton production. Significant increases in PEPC and PK activities were also observed in the transgenic plants, although increases of enzyme activities were smaller than increases of the corresponding transcripts.

We initially evaluated the effects of expressing Dof1 on nitrogen assimilation by comparing amino acid concentrations in Dof1 transgenic and control plants. The amount of total free amino acids was clearly higher in transgenic Arabidopsis plants (approximately double) and the increase in glutamine (a sharp nitrogen utilization marker) was most remarkable. Glutamine concentration was 4.5 µmol/g FW in the control plants, whereas it was 12.5 µmol/g FW in the Dof1 transgenic plants. Furthermore, direct evidence of enhanced net nitrogen assimilation was obtained by measuring nitrogen content with whole plants. Nitrogen content was higher in transgenic plants by approximately 30%. Interestingly, the N/C ratio was constant in Dof1 transgenic plants and control plants, because the carbon content was also elevated in Dof1 transgenic plants.

By measuring concentrations of organic acids, it was shown that malate concentration decreased in Dof1 transgenic plants. However, despite the larger amount of assimilated nitrogen, which absolutely needed production of a larger amount of carbon skeletons (2-OG), elevation of the 2-OG level was not large in Dof1 transgenic plants. Thus, 2-OG appeared to be rapidly consumed for amino acid biosynthesis in the Dof1 transgenic plants. In addition, a significant reduction was observed in glucose content but not in sucrose content. A marked increase in ammonia was also observed in Dof1 transgenic plants, whereas the amount of nitrate was similar in the Dof1 transgenic plants and control plants. Ammonia concentration was 2.5 µmol/g FW in control plants, whereas it was 8.0 µmol/g FW in Dof1 transgenic plants. These results suggest that expressing Dof1 altered not only amino acid concentrations but also concentrations of various metabolites.

Figure 3. Control plants and transgenic Dof1 plants grown on the plates containing 0.3 mM nitrogen (0.1 mM NH4NO3 and 0.1 mM KNO3). [Images from Ref. 4 Copyright Proceedings of the National Academy of Sciences, USA.]

Because the Dof1 transcription factor from maize can improve nitrogen assimilation in Arabidopsis, this strategy might similarly work in a wide range of plant species. To evaluate this possibility, we also generated the Dof1 transgenic potato plants. Amino acid content in transgenic potato plants was higher than that in the control plants, suggesting applicability of this strategy to various plant species.

Discussion
Our results suggest it is possible to modify nitrogen assimilation in plants using the Dof1 transcription factor, although it will be necessary to investigate whether expressing Dof1 is really effective in improving nitrogen assimilation of crops in the field. Most importantly, enhanced nitrogen assimilation appeared to sustain growth under low nitrogen conditions, suggesting that the Dof1 factor is of use in molecular breeding for developing environmentally-friendly agriculture.

In transgenic plants, promotion of amino acid production, coupled with reduction of glucose, was observed, indicating cooperative modification of carbon and nitrogen metabolism. Because the metabolic alteration caused by expressing Dof1 is enormous, further analyses will be necessary to completely reveal it. In spite of the large alteration in amounts of various metabolites, the N/C ratio was not changed, presumably because the N/C balance is very fundamental. The analyses of Dof1 transgenic plants might provide an opportunity to reveal the mechanism maintaining the N/C balance in plants as well.

Because many biochemical reactions are catalyzed by a number of enzymes, some difficulties in the genetic manipulation of metabolism arise from the frequent insufficiency of enhancing a single enzyme. Our results suggest that transcription factors may be powerful tools in genetic modification of metabolism in crops. By using transcription factors, we will be able to do more than in the past. Identification of transcription factors that are master regulators in respective pathways is a future challenge in molecular breeding.

References

1. Nosengo N. (2003) Fertilized to death. Nature 425, 894-895.

2. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O & Thomashow MF. (1998) Arabidopsis CBF1 overexpression inducees COR genes and enhances freezing tolerance. Science 280, 104-106.

3. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K & Shinozaki K. (1998) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol. 17, 287-291.

4. Yanagisawa S, Akiyama A, Kisaka H, Uchimiya H & Miwa T. (2004) Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low nitrogen conditions. Proc. Natl. Acad. Sci. USA 101, 7833-7838.

TRANSGENIC PLANTS WITH NO FOREIGN DNA
Tawanda Zidenga

A recent paper published in the Plant Physiology journal by researchers at the J.R. Simplot Company, Simplot Plant Sciences in Idaho, reports the first example of genetically engineered plants that contain only native DNA¹. The reported methods were used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. Polyphenol oxidase (PPO) is responsible for post-harvest enzymatic discoloration in many fruit and vegetables.

Too many hooks in the catch
Most of the early experiments on plant transformation were related to introduction of viral and bacterial genes. Plants resistant to antibiotics, herbicides, viruses, and bacteria, among other traits, were produced. Common examples include glyphosate-resistant soya beans and corn borer-resistant maize, both of which make use of bacterial genes. Foreign regulatory elements such as the constitutive 35S promoter from cauliflower mosaic virus were used to achieve high level expression of foreign genes. Transfer of DNA into plant cells has in most cases been achieved through use of a bacterium that has come to be known as nature's own genetic engineer, Agrobacterium tumefaciens. However, the Agrobacterium transfer (T-) DNA and adjacent backbone sequences are often inadvertently cotransferred with the T-DNA. This results in a transgenic plant with many foreign genetic elements, a scenario that has been described as leaving a hook in the catch and expecting customers to return². One public concern raised against the commercialization of genetically engineered organisms (GEOs) is the widespread presence of these foreign genetic elements. Last year, Kaare Nielsen³ of the Norwegian Institute for Gene Ecology proposed a system to categorize GEOs into different classes based on the genetic distance between target organism and the source of new variation. These categories are intragenic (within genomes), famigenic (species in the same family), linegenic (unrelated species), and xenogenic (laboratory designed genes).

Recent advances in plant molecular biology have greatly facilitated efforts to isolate plant genes associated with agronomic traits. Methods were also developed to remove selectable marker genes from the genomes of transgenic plants. While these efforts have been successful in reducing foreign DNA in commercial transgenic plants, they have not eliminated the "problem" and indeed have created new complications of their own.

From T-DNA to P-DNA
Rommens et al.¹ report replacement of Agrobacterium T-DNA by a plant DNA (P-DNA) fragment, thereby achieving a native DNA transformation approach. Putative T-DNAs were isolated from potato through PCR, and one region, later used as the P-DNA, was delineated by regions that shared homology with the left border of nopaline strains (21 of 25 bp) and the right border of octopine strains (22 of 25 bp). This P-DNA also lacked open reading frames and contained a high A/T content, which is believed to promote the DNA transfer process. When tested for its ability to support plant transformation against T-DNA, the P-DNA supported effective transfer of DNA from Agrobacterium binary plasmids to the genome of individual plant cells as illustrated below:

P-DNA mediated transformation. Left: Average number of calli that developed within 3 weeks of infection per tobacco stem explant. Right: Average number of shoots that arose from infected potato stem explants within 3 months of infection. Data are the mean ± SE of three experiments. pSIM108 is the P-DNA plasmid while pBI121 is the conventional T-DNA plasmid. [SOURCE. Rommens et al., 20041 Copyright Plant Physiology.]

Transient selection system
Incorporation of antibiotic resistant genes in commercial transgenic plants has been a major area of controversy. Critics have argued that widespread use of antibiotic selection systems will make medical antibiotics less effective. However, progress in generating marker-free transgenic plants has been made⁴. Schaart et al.⁵ have recently reported a system for production of marker free transgenic plants that combines an inducible site-specific recombinase for precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. In the paper summarized in this report, Rommens et al.¹ describe the development of a transient selection method to generate marker-free transgenic plants. This method derived from the fact that co-transfer of two different DNA molecules from Agrobacterium to a single plant cell nucleus is not necessarily followed by the co-integration of both. The thinking, outlined in the figure below, was that plant cells could survive a temporary selection phase by virtue of the transient expression of a marker gene present in a conventional T-DNA. Upon release from selection, some cells integrate the P-DNA independently from the T-DNA molecule (indicated with D on the diagram below), thus offering marker-free P-DNA integration events.

Hypothetical model for use of markers to generate marker-free plants. The four different genotypes predicted to result from this coinfection are: P-DNA–/T-DNA– (A), P-DNA–/T-DNA+ (B), P-DNA+/T-DNA+ (C), and P-DNA+/T-DNA– (D). [SOURCE. Rommens et al., 20041 Copyright Plant Physiology.]

The paper also reports two-strain, marker-free transformation procedures using an omega mutated virD2 protein previously shown to impair the integration of transferred T-DNAs, while isopentenyl transferase cytokinin genes were used in negative selection against backbone integration. The all-native transformation methods are likely to bring a new twist to discussions and regulations regarding genetically modified foods if applied at a commercial level.

References

1. Rommens CM et al. (2004). Crop improvement through modification of the plant's own genome. Plant Physiology 135, 421-431

2. Elborough K & Hanley Z. Emerging plant biotechnologies: new ways to find needles in haystacks. ISB News Report, August 2001

3. Nielsen KM (2003). Transgenic organisms—time for conceptual diversification? (commentary) Nature Biotech. 21, 227-228.

4. Granger C. Pruning back transgenes. ISB News Report, May 2002.

5. Schaart JG et al. (2004). Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnology Journal 2(3), 233-240.

Tawanda Zidenga
Department of Plant Cellular and Molecular Biology
College of Biological Sciences
The Ohio State University

A NEW STRATEGY FOR GLYPHOSATE TOLERANT CROP PLANTS
Linda A. Castle and Michael W. Lassner

Introduction
Transgenic crops are integral to modern agriculture. In 2003, 81% of U.S. soybean, 73% of cotton, and 40% of maize acres were planted with genetically engineered varieties. Herbicide tolerance is the most widely planted transgenic crop trait, followed by insect tolerance. Nearly all transgenic soybeans are engineered for tolerance to the herbicide glyphosate. A large proportion of transgenic cotton, maize, and canola crops are also tolerant to glyphosate. Glyphosate acts by inhibiting enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), an enzyme in the pathway leading to biosynthesis of aromatic amino acids¹. Because this enzyme and pathway is unique to plants and microbes, glyphosate is not toxic to animals. Certain EPSPS enzymes are insensitive to glyphosate inhibition. When expressed in chloroplasts of transgenic plants, the insensitive enzymes confer tolerance to the herbicide. This mode of action accounts for all the commercial glyphosate-tolerant crops. Low cost, low toxicity, effective broad-spectrum weed control and availability of transgenic crop tolerance have resulted in glyphosate becoming the world's most valuable agrochemical.

An alternative to the insensitive target approach used to develop glyphosate tolerant crops is to engineer crops to produce an enzyme capable of detoxifying the herbicide. Phosphinothricin acetyltransferase (PAT or BAR) from Streptomyces detoxifies phosphinothricin- or bialaphos-based herbicides by adding an acetyl group². This trait is approved for use in the United States in canola, chicory, maize, rice, cotton, soybean, and sugarbeet. Because detoxification by acetylation is straightforward, requires no cofactors, and has proven to be a successful strategy for phosphinothricin detoxification, we sought an enzyme capable of carrying out the N-acetylation of glyphosate. Previous research demonstrated that N-acetylglyphosate is not herbicidal.

By utilizing the sensitivity and specificity of mass spectrometry (MS) to query natural abilities of common microbes, we were able to discover glyphosate acetyltransferase (GAT) enzymes capable of detoxifying glyphosate by N-acetylation. Because native enzymes exhibited poor kinetic properties, we used DNA shuffling^3-5 to create improved enzymes that exhibit higher turnover rates and increased specificity for glyphosate. The improved N-acetyl- transferase genes (gat), when expressed in plants, lead to a robust glyphosate tolerant phenotype as described in detail recently⁶ (Fig. 1).

Figure 1. This composite shows steps taken to bring glyphosate N-acetyltransferase trait from discovery to proof of concept in the field. A. A panel of microbial colonies from a diversity collection screening plate. B. A model of glyphosate acetylation reaction carried out by GAT. C. A scheme of the shuffling process. The colored bars represent different starting genes for shuffling. In each iteration, genes are fragmented and reassembled. Recovered genes contain recombined portions in the same linear order from each of the starting genes. After screening for desired activity, best molecules are identified. The entire process is repeated using these selected progeny as parents for the next shuffling iteration. D. Maize plants expressing gat are tolerant to glyphosate spray under standard field conditions.

Discovery and Improvement of gat Genes
To discover an enzyme capable of acetylating glyphosate, we searched our microbial collection for an organism that could carry out the reaction. Since bacilli produce a wide variety of enzymes involved in secondary metabolism, we focused on Bacillus and related genera isolated from non-extreme environments. We developed a sensitive, high throughput (HTP) MS-based assay capable of detecting N-acetyl glyphosate in crude cell extracts. When incubated with glyphosate and acetyl CoA, strains of Bacillus licheniformis, a common saprophytic bacterium, catalyzed reproducible accumulation of the acetylated herbicide. To isolate the gene encoding GAT, recombinant E. coli, expressing genomic DNA fragments from B. licheniformis, were assayed by the MS method. N-acetyltransferase genes isolated from these strains are 93% identical. GAT enzymes are ~17 kDa, are most active at pH 7, and require only an acetyl donor and glyphosate for activity. Protein structure analysis confirms that GAT is a member of GNAT or GCN5-like N-acetyltransferase superfamily of enzymes.

Two kinetic properties, k_cat, a measure of reaction rate, and K_M, a measure of affinity of enzyme for substrate, were used to evaluate GAT enzymes. The K_M of GAT enzymes for acetyl donor substrate AcCoA is 1-2 mM. The three native GAT enzymes exhibit similar kinetic properties—K_M for glyphosate ranges from 1.2 _ 1.8 mM and k_cat ranges from 1.0-1.7 min^-1at pH 6.8. The ratio of k_cat/K_M can be used as a measure of enzyme efficiency and specificity. The average k_cat/K_M ratio of the parental enzymes is 0.85 min^-1 mM^-1. These enzymes are inefficient. Expression of gat genes in E. coli did not enable cells to grow on media supplemented with inhibitory concentrations of glyphosate. Similarly, gat expression in transgenic tobacco and Arabidopsis did not confer herbicide tolerance.

To develop enzymes useful for engineering herbicide tolerant plants, we used DNA shuffling to improve kinetic efficiency of GAT. DNA shuffling^3-5 is a process that recombines genetic diversity from parental genes to create libraries of gene variants that are screened to identify those progeny with improved properties. This recombination and selection process can be repeated using improved progeny as parents for the next iteration of shuffling. It is an effective technique for producing proteins with altered properties such as improved kinetics, altered substrate specificity, changes in temperature or pH optima, ligand binding, and solubility.

Libraries of shuffled gene variants were created, expressed in E. coli, and screened. Shuffled variants that specified the accumulation of more product than the parental controls using the HTP MS assay were selected for further analysis. In each iteration of DNA shuffling, we screened about 5,000 gene variants in HTP and further analyzed 24—48 purified enzymes to determine their kinetic properties. Typically, three to twelve improved variants exhibiting a high k_cat, a low K_M, or a high k_cat/K_M ratio were chosen to be the parents for the next iteration. Several GAT enzymes from the third iteration of gene shuffling were improved about 100-fold over original enzymes to k_cat/K_M = 80 min^-1mM^-1. This level of improvement was sufficient to allow growth of recombinant E. coli expressing gat on minimal agar medium supplemented with 1 mM glyphosate. However, these gene variants were unable to confer glyphosate tolerance to transgenic plants.

At the fifth shuffling iteration, we implemented two new strategies: (1) a functional prescreen based on resistance of gat-expressing E. coli to glyphosate; and (2) a dramatic increase in the diversity available for recombination. The functional prescreen allowed screening of more than 10⁶ gene variants with elimination of those with low activity prior to picking approximately 5,000 functional variants for assay. To increase genetic variance in our shuffling population, we incorporated diversity from hypothetical B. subtilis and B. cereus YITI proteins. We constructed a synthetic library⁵ using the best GAT variant from the fourth iteration as a template and incorporated diversity from the database-predicted YITI protein sequences. At the sixth iteration, we moved from product accumulation MS-based assay to a spectrophotometric assay that allowed us to screen for relative k_cat and K_M values in HTP without protein purification. We incorporated additional amino acid diversity in the eighth iteration library from more distantly related putative proteins predicted from L. inocua and Z. mobilis genome sequences. We completed an additional three iterations of multi-gene shuffling to recombine new diversity.

After 11 iterations of directed molecular evolution, the most efficient GAT variant had a k_cat of 416 min^-1 and K_M of 0.05 mM glyphosate, resulting in a k_cat/K_M ratio of 8320 min^-1 mM^-1, nearly a 10,000-fold improvement over parental enzymes. This protein is 76—79% identical to the original parental GAT proteins. Evolved GAT enzymes have kinetic values comparable to characterized N-acetyltransferases. Over time, residues that had a positive effect on k_cat or K_M were selected while residues with negative effects were eliminated. Because of the number of varying sites in any given iteration of shuffling, it was not clear which residues accounted for improvements. In the end, our ability to improve enzyme activity by DNA shuffling and functional selection outpaced and outperformed our attempts at sequence-function predictions.

Glyphosate Tolerant Plants
Our purpose in discovering and evolving the gat genes was to develop glyphosate tolerant crop plants. To be effective, GAT must convert the glyphosate to N-acetylglyphosate before cell death due to inhibition of EPSPS occurs. Like glyphosate, N-acetylglyphosate is stable and not metabolized in plants, however it is not herbicidal. The gat genes were introduced into Arabidopsis, tobacco, and maize. The transgenes were introduced into the nuclear genome and the proteins were expressed in the cytosol.

Glyphosate tolerant Arabidopsis plants were achieved with a fourth iteration improved gat gene. Improved gat variants from the fifth iteration enabled regeneration of highly tolerant transgenic tobacco plants. After glyphosate application at rates more than 20-fold higher than those used by farmers for weed control, the tobacco plants were morphologically normal and fertile. Fifth iteration gat genes also allowed production of glyphosate tolerant maize plants. For maize, glyphosate tolerance improved with increases in the catalytic efficiency of GAT. Most transformed plants expressing the best tenth and eleventh round gat gene were tolerant to glyphosate application six-fold higher than typical field application rates and showed no adverse symptoms. Efficacy trials of lines containing genes from several shuffling iterations are underway in the field to evaluate the commercial potential of this glyphosate tolerance trait.

References

1. Franz JE, Mao MK, and Sikorski JA, Glyphosate: A Unique Global Herbicide (Am. Chem. Soc., Washington DC, 1997).

2. De Block M et al. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. The EMBO J. 6, 2513-2518.

3. Stemmer WPC. (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. U.S.A. 91, 10747-10751.

4. Crameri A, Raillard S-A, Bermudez E, and Stemmer WPC. (1998). DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288-291.

5. Ness JE et al. (2002) Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 17, 1251-1255

6. Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho H-J, Duck N, Wong J, Liu D, and Lassner MW. (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science 304, 1151-1154.

Genetic engineering (GE) of plants, insects, animals, and microorganisms differs from traditional genetic selection for desirable traits in a number of ways: (1) GE by necessity involves in vitro genomic manipulations of the target organism; (2) GE requires the molecular engineer to have certain information (DNA sequences, or position or restriction enzyme sites) for the relevant region of DNA that is to be manipulated; (3) GE requires physical isolation of small useful pieces of DNA from the donor region of interest for manipulation and insertion; (4) GE typically involves the characterization of genotypes prior to the analysis of phenotypes, whereas in traditional breeding programs phenotypes are usually analyzed for desirable traits; (5) viruses, plasmids, microinjection, or gene guns are used to introduce foreign DNA into the target organism rather than recombination using gametes or compounds that induce mutagenesis from which viable organisms are then screened. Often the genetic constructs inserted into the recipient genome contain material that originated from organisms in different phyla or even kingdoms and the resulting organisms express traits that could never have been achieved with traditional genetic manipulations. Consequently, this new technology may present sets of unique inherent risks that have not been seen before with traditional genetic modification strategies.

There is intense intellectual and practical interest in creating insects that are refractory to disease, in particular arthropod borne pathogens that cause human maladies. Several research laboratories are attempting to develop transgenic mosquitoes that contain novel genetic constructs that interfere with the transmission of pathogens that cause malaria, and dengue and yellow fevers in humans. Limited critical laboratory examination of the fitness of these transgenic mosquitoes has begun, and it is envisioned that field trials are still several years away. As potential future field trials with GMIs (e.g., mosquitoes and pink boll worms) become more likely, the movement of these organisms from secure laboratory facilities for release and establishment in natural systems raise critical issues regarding regulatory oversight, safety evaluation, risk assessment, and potential non-target impacts. With respect to these preceding issues, classical biological control, the deliberate introduction and release of exotic organisms for the control of non-native invasive pests, may provide guidance in developing protocols for issues pertaining to GMI releases. Many issues that proposed GMI releases will eventually face are fundamentally similar to releases of non-GMI classical biological control agents from secure quarantine facilities and include: (1) assessment of the potential benefits and hazards arising from release; (2) procedural assessments to adequately determine safety and explore risk to receiving ecosystems; (3) identification of non-target species that would be at risk from GMIs (i.e., either direct attack, food web perturbations, or gene transfer); (4) development of mechanisms that can be adopted to mitigate potential risk; and (5) assessment of public opinion on the acceptability of GMIs as a necessary management strategy in support of or replacement of traditional control practices such as pesticide applications for mosquito control.

Regulatory Oversight of Proposed GMI Releases
Currently in the US, there is no agreed upon process for assessing safety of GMIs and their risk to the receiving ecosystem prior to release, nor has it been definitively determined which state and federal regulatory agencies will be involved with deciding what constitutes acceptable data concerning safety and risk assessment and how these data should be assessed scientifically¹. Given the diversity of GMIs that could potentially be created for pest, vector, and disease management in a variety of ecosystems including natural, agricultural, and urban settings, the Food and Drug Administration (FDA), Environmental Protection Agency (EPA), and the US Department of Agriculture (USDA), may have input into regulatory oversight. As scientists begin the application process for permission to conduct field trials, overlapping jurisdictional boundaries across government agencies are likely to cause confusion, duplication of effort, and anxiety as to whether the necessary paperwork has been completed to satisfy all regulatory requirements. Coordination of oversight and division of responsibility across several government agencies is seen as an impediment in need of resolution before field testing of GMIs can be made possible².

New Zealand and Australia have some of the most stringent legislative requirements for regulating genetically modified organisms (GMOs which include plants, animals, and microbes). New Zealand's Hazardous Substances and New Organisms Act 1996 (HSNO) places incumbent obligations on proponents of GMOs requiring them to provide adequate data on which assessments for release can be based, and this includes consideration of international concerns that incipient programs may raise. This legislation (i.e., HSNO) provides a solid framework within which risks and benefits of proposed GMO use can be weighed, and decisions made in accordance with presented data. The Environmental Risk Management Authority (ERMA) administers the review process for the release of GMOs which includes extensive public consultation and consideration of concerns raised by Maoris, New Zealand's indigenous peoples. ERMA and HSNO also provide guiding frameworks for proposed importation and releases of exotic biological control agents in New Zealand.

In Australia, the Gene Technology Bill (2000) is the cornerstone legislative act that provides a national regulatory structure governing GMOs. Within this framework, the Gene Technology Regulator prepares a risk assessment and management plan for every proposed GMO release into the environment. Risk assessment includes the potential of the GMO of concern to cause adverse environmental impacts, to persist for inordinate periods of time, and to spread geographically or via exchange of genetic material. As with ERMA, extensive consultation with stakeholders is mandatory³. The regulatory frameworks adopted by New Zealand and Australia may provide legislative inspiration to federal regulators in the US who are facing similar hurdles as releases of GMIs draw near.

Safety Evaluations and Risk Assessment
Major concerns surrounding permanent establishment of GMIs in nature include the potential for creating new pests and for disrupting ecosystems, because they may transmit novel genetic material to wild relatives or exhibit an increased ability to inhabit areas that exclude non-transformed conspecifics. Realization of these negative impacts will depend on the fitness and competitiveness of GMIs, the dispersal ability of the GMI and its environmental tolerances, and the permissiveness of the receiving environment. Additionally, adverse effects may manifest themselves via non-target organisms that use the GMI as a resource or are displaced by competition. These issues are similar to those posed by the importation and release of exotic biological control agents. Evaluation of traits likely to enable GMIs to become pestiferous need to be identified and evaluated prior to release. Rigorous protocols similar to those used for weed biological control agents may provide important starting points for deliberation in the development of novel testing regimens for GMIs. A major new area of investigation will be concerning scenarios facilitating unwanted gene flow into unintended recipient populations and the outcomes should this occur. Consideration of factors promoting gene flow is a major departure from testing protocols for biological control agents. Interpretation of what constitutes acceptable levels of safety and risk will undoubtedly be interpreted differently depending on varying tolerance perspectives of the analyzing parties in the country of release and adjacent neighbors.

Mitigating Non-Target Impacts
Despite rigorous applied laboratory tests, small-scale field trials, and modeling of results, there is the potential for unintended consequences to manifest themselves, as answers to questions pertaining to safety and risk are influenced by temporal, spatial, and myriad biotic and abiotic factors that cannot be easily replicated experimentally. A logical step for mitigating non-target effects from GMIs would be built in mechanisms to prevent continued persistence and spread. One safeguard would be the inclusion of marker genes to readily identify GMIs from non-transformed conspecifics. This technology is already in place and would allow population monitoring and measurement of spread. An additional safeguard would be incorporation of safety devices that could be activated to disable GMIs thereby countering their adverse effects should they arise. Greater research into the development of safety options will most likely occur as GE technology advances and field trials become more likely.

Conclusions
Legislative guidelines and protocols for scientifically addressing issues of safety and risk of GMIs is in need of increasing attention, as organisms developed in the laboratory steadily approach the field testing phase. Similar issues concerning the outdoor release of GMIs have faced the biological control community. Growing disquiet generated by prominent ecologists and conservationists have increased awareness of non-target impacts and the difficulty of predicting unforeseen ecosystem perturbations caused by exotic natural enemies used for the biological control of exotic pests. Recognition of these issues has increased research effort by biological control practitioners to experimentally address factors pertaining to safety and unforeseen risk within prescribed experimental and regulatory arenas. Where consideration of analogous issues pertaining to the release and use of GMIs in the environment is necessary, lessons learned by the biological control community could form a sound starting basis for molecular biologists and vector ecologists developing good practice guidelines.

1. Minkel, J.R. 2004. Bugging for guidance. Scientific American 291 (1): 34.

September 20-21, 2004
Washington, DC

This two-day multidisciplinary workshop, sponsored by the Pew Initiative on Food and Biotechnology, will provide an exploration of the potential benefits and risks of genetically engineered insects and the public policy and ethical implications of releasing them. Representatives of government, academia, consumer and environmental groups, and policy leaders are encouraged to attend.

The Initiative believes that bringing people with diverse perspectives and expertise together at an early stage in the development of insect biotechnology will ensure that information is available in a timely fashion for decision makers and the public when specific transgenic and paratransgenic insect applications are evaluated for environmental release. Through activities such as this conference, our organization is providing a neutral forum for interested parties to convene and to explore issues of concern.

June 27 - 29, 2005
Nashville, Tennessee

A not-for-profit consortium of 37 leading agricultural research agencies and universities in North America has chosen the University of Tennessee and the University of Kentucky to co-host its annual conference in 2005. The 17th meeting of the National Agricultural Biotechnology Council will hold open discussions about the safe, ethical, and beneficial development of agricultural biotechnology.

The conference is expected to explore the theme "agricultural biotechnology: beyond food and energy to health and the environment." Agricultural producers and consumers as well as representatives from corporate, government and academic institutions and public-interest groups are expected to participate.

NABC works to define issues and public policy options related to biotechnology associated with food, agriculture, and the environment and to promote increased understanding of the scientific, economic, legislative, and social issues associated with agricultural biotechnology. They provide a network for member institutions to work together on the complex issues that arise regionally and nationally. Previous NABC meetings have addressed such issues as sustainable agriculture; food safety and nutritional quality; gene discovery, access, and ownership; world food security; and industrial consolidation.