Many photosynthetic microorganisms are capable of concentrating CO₂ in close proximity to Rubisco and thereby largely overcome the limitation imposed by the low affinity of the enzyme for CO₂². Higher plants of the C₄ and the CAM physiological groups can also raise the concentration of CO₂ at the site of Rubisco. Most crop plants, however, belong to the C3 physiological group, which do not possess this ability.

While characterizing high-CO₂-requiring mutants of cyanobacteria we identified a gene, designated ictB, probably involved in the ability to accumulate HCO₃^- in Synechococcus PCC 7942^2,3.

We constructed transgenic Arabidopsis and tobacco plants expressing ictB. Measurements of the photosynthetic characteristics with respect to CO₂ concentration showed that in both tobacco and Arabidopsis the rate of photosynthesis at a saturating CO₂ level was similar in the transgenic and wild type plants⁴. On the other hand, under air level of CO₂ or lower (such as experienced under water stress when the stomata are closed) the transgenic plants exhibited a significantly higher photosynthetic rate than the wild type⁴. The slope of the curve relating photosynthesis to intercellular CO₂ concentrations was steeper in the transgenic plants, suggesting that the activity of Rubisco was higher. This was confirmed in experiments where the activity of Rubisco, in situ, was assessed⁴. A higher internal CO₂ concentration close to the Rubisco sites could explain the faster photosynthetic rate under limiting CO₂ concentration and the higher Rubisco activity in the transgenic plants. Measurements of the CO₂ compensation point showed lower values in the transgenic plants (Table 1), in agreement with a higher intracellular CO₂ level in the transgenic plants⁴.

In view of the large impact of the expression of the foreign gene, ictB, on the photosynthetic activity, we performed growth experiments in which we examined the effect of air humidity (30% and 70% relative humidity) on the growth of the transgenic and wild type Arabidopsis plants (Figure 1). Naturally, growth was faster in plants well supplied with water and maintained under the high (70%) relative humidity conditions, and there was no significant difference between the wild type and the transgenic plants. On the other hand, the transgenic Arabidopsis plants grew significantly faster than the wild type under conditions of restricted water supply and low (approximately 30%) humidity.

Figure 1: Growth of transgenic (B,A,C) and wild type (WT) Arabidopsis plants. The plants were grown in 12 pots, each containing wild type and three transgenic plants and exposed to relative humidities of 30% or 70% and 21°C.

Our data demonstrated the potential use of the cyanobacterial gene, ictB, to raise plant productivity particularly under dry conditions where stomatal closure may lead to lower intercellular CO₂ level and thus growth retardation.

1. Woodrow IE and Berry JA. 1988. Enzymatic regulation of photosynthetic CO2 fixation in C₃ plants. Ann Rev Plant Physiol 39: 533-594.

2. Kaplan A and Reinhold L.1999. The CO₂ concentrating mechanisms in photosynthetic microorganisms. Annu Rev Plant Physiol Plant Mol Biol 50: 539-570.

3. Bonfil DJ et al. 1998. A putative HCO₃^- transporter in the cyanobacterium Synechococcus sp. strain PCC 7942. FEBS Lett 430: 236-240.

4. Lieman-Hurwitz J, Rachmilevitch S, Mittler R, Marcus Y, and Kaplan A. 2003. Enhanced photosynthesis and growth of transgenic plants that express ictB, a gene involved in HCO₃^- accumulation in cyanobacteria. Plant Biotechnology J 1: 43-50.

Bt cotton provides a fairly high degree of resistance to the American bollworm (Helicoverpa armigera), the major insect pest in India. The technology was developed by Monsanto and was introduced into several Indian hybrids in collaboration with the Maharashtra Hybrid Seed Company. Field trials with these Bt hybrids have been carried out since 1997 and, for the 2002/03 growing season, the technology was commercially approved by the Indian authorities. Its performance during the first commercial season in India is hotly disputed among biotechnology advocates and opponents, but an independent scientific assessment has not been conducted thus far.

For our analysis, we used data from on-farm field trials that were carried out during the 2001/02 growing season as part of the regulatory procedure. On 157 farms in three different states, Bt cotton hybrids were planted next to an isogenic line without the Bt gene and a local hybrid commonly grown in the particular district. All three plots were managed by the farmers themselves, following customary practices. Apart from official data that were collected by local researchers for biosafety evaluation, we used our own questionnaire to obtain details on input-output relationships from participating farmers.

While there was no significant difference in the number of sprays against sucking pests, Bt hybrids were sprayed three times less often against bollworms than the conventional hybrids. On average, insecticide amounts on Bt cotton plots were reduced by almost 70%, which is consistent with studies from other countries. The difference in India, however, is that use of Bt cotton also leads to a significant yield effect. During the field trials, average yields of Bt hybrids exceeded those of non-Bt counterparts and local checks by 80% and 87%, respectively.

Bt is a pest-control technology, so rather than an increase in the genetic yield potential, these effects have to be interpreted as avoided crop losses. Figure 1 confirms that, under Indian conditions, bollworms have a high destructive capacity which is not well controlled in conventional cotton. At average pesticide amounts of 1.6 kg/ha (active ingredients) on the conventional trial plots, crop damage in 2001/02 was about 60%. Bt does not completely eliminate pest-related yield losses. Yet, to achieve the same level of damage control without the technology would require a triplication of currently used pesticide quantities.

Figure 1: Insecticide use and crop losses with and without Bt technology

2001/02 was a season with high bollworm pressure in India, so that average yield effects will be somewhat lower in years with fewer pest problems. Moreover, although the trials were managed by farmers, experimental results cannot simply be extrapolated to commercial agriculture. But even when discounting for these aspects, yield advantages of Bt cotton will remain bigger in India than in the United States or China.

Analysis of factors influencing yield impacts of new, effective pest control technologies suggests that they depend on local pest pressure, availability of alternatives for pest control, and farmers' adoption of these alternatives. Generally, pest pressure in tropical and sub-tropical regions is higher than in temperate zones, while pesticide-use intensities are much lower, due to technical and economic constraints. In India, pesticides are available on local markets, but their effectiveness is limited because bollworms have developed resistance to many of the common products. Furthermore, small-scale cotton producers are often credit-constrained and do not have access to chemicals at the right point in time.

Given these linkages, we have a theoretical base to suggest that the case of Bt cotton in India might be more representative of GM crop impacts in developing countries than previous examples. Almost all GM crop technologies were initiated by commercial firms in the industrialized world, targeting the needs of farmers who are able to pay for them. Some varieties were transferred to the commercial sectors of Latin America and China, where agroecological conditions and pesticide application rates are similar. In all cases, yield effects have been low to medium, while there have been significant gains from pesticide substitution.

However, with careful adaptation and effective regulation, these same technologies can also be introduced to other developing-country regions, where yield effects will be more pronounced. Pest-resistant GM crops are easy to manage at the farm level, and they could substantially reduce current gaps between attainable and actual yields, especially in smallholder farming systems. Preliminary evidence from Indonesia and South Africa is in line with this hypothesis. Agricultural biotechnology offers many more applications for developing countries beyond pest control, but we show that the GM crops developed thus far can already have significant impacts. It is a major policy challenge to invest more in public research and address the existing institutional constraints, so that promising biotechnologies can reach the poor at affordable prices on a larger scale.

Qaim M and Zilberman D. 2003. Yield effects of genetically modified crops in developing countries. Science 299: 900-902.

Scope of research and development effort
The AquaBounty Atlantic salmon is the most widely publicized example of a large international effort aimed at developing transgenic aquatic and marine organisms (Table 1).

Table 1. Scope of effort for development of transgenic aquatic animals.

The most frequent application is for food production; GH genes have been inserted into over a dozen species⁴. Cecropin⁶, interferon, and other genes have been introduced to increase non-specific immunity to disease. Transgenic zebrafish, medaka, and other species are used as model systems for research on gene expression and embryological development. In a recent application, tilapia were engineered for use as bioreactors to express human coagulation factor VII in their blood⁷. Transgenic fish are under development for environmental biomonitoring, for example, for detecting environmental mutagens⁸. Several research groups are experimenting with transgenesis as a means of achieving reproductive confinement⁹. Development of transgenic mollusks and crustaceans is complicated by the inability of many P₀ founders to transmit the transgene⁵, although some progress has been achieved¹⁰.

Most transgenic lines are still in the development stage, although several are nearing readiness for possible commercialization. In addition to their possible benefits, the commercialization of transgenic aquatic organisms also poses a range of controversial issues, including food safety, environmental safety, and public policy.

Food safety
Foremost to many prospective consumers is the issue of food safety^3,4. Although cooking and digestion would break down most transgene products, three types of food safety concerns must be considered. First, bioactivity of the transgene product may pose concern, especially for pharmaceutical proteins. Second, allergenicity may prove hard to assess if the transgene comes from a non-food organism. Allergenicity assessment will be somewhat easier if the transgene comes from an organism representing known allergenic food groups, including fish and shellfish. In that case, the transgene product can be tested for reactivity against antisera from individuals with known food allergies. Third, toxicity potential is relatively easy to assess, and toxin genes would not be candidates for gene transfer.

The National Research Council (NRC) found that the level of food safety concern posed by products of animal biotechnology varies with the application³. For fish expressing a GH transgene, neither GH, GH fragments, nor hormones secreted in response to GH pose a risk to the human consumer. Hence, GH salmon likely pose little or no food safety risk. A comparative analysis of composition of products from GH and non-transgenic salmon is ongoing². The NRC has a broader study ongoing on unintended health effects of genetically engineered foods.

Environmental safety
A second set of issues concerns the environmental safety of aquatic GMOs. Escape from production facilities, such as floating net pens used for production of salmon, is likely. Interbreeding with wild populations poses genetic and evolutionary risks. Ecological risks are posed with a variety of species in the receiving ecosystem. The NRC attached a high level of concern to possible environmental impacts of transgenic fish³.

If transgenic individuals were to escape from confinement and interbreed with wild fish, would the transgene be purged from the population or would it persist? Empirical observations of particular transgenic lines^3,5 show higher oxygen consumption rate, lower critical swimming speed, higher willingness to risk exposure to predators, and lower viability of young. These observations suggest that transgenic individuals are less fit than non-transgenic individuals, that selection would remove the transgene from the receiving population, and that genetic impacts would be minor. However, some researchers question whether selection would remove the transgene from the population rapidly enough that impacts would be minor, and question the impact of recurrent introduction of transgenes into the population. Further, some researchers argue that single-trait models are simplistic, and that we must consider how the transgene affects fitness through the entire life cycle. For example, would a gain in mating success due to large size of a GH transgenic fish come at the cost of juvenile viability¹¹? Two net fitness models^11,12 predict that when there are tradeoffs in fitness traits through the life cycle, the transgene could spread through the population and, under certain conditions, threaten the viability of the population. Other possible tradeoffs would include: increased male mating success and reduced adult viability; increased adult viability, and reduced male fertility; and increased male mating success and adult viability but reduced male fertility. Current knowledge of possible genetic impacts of transgenic aquatic species is such that we cannot predict the outcome should a transgene be introduced into a wild population.

Since possible genetic impacts are plausible, reproductive confinement is appropriate. The proponents of the transgenic Atlantic salmon suggest production of all-female triploid stocks². This raises questions of whether 100% triploidy can be reliably achieved at the scale of commercial production and the level of sampling needed to assure that production stocks are indeed all triploid. The NRC is conducting a study of bioconfinement that should be helpful in addressing these questions.

Even with effective reproductive confinement, transgenic aquatic species pose ecological impacts on receiving ecosystems. Two key concerns are competition with and predation upon natural populations. Many interactions among aquatic species are mediated by size, particularly predation. We must determine for each case whether transgenics will exhibit a larger size distribution than non-transgenics. Ecological impacts can cascade through trophic levels in feeding webs. That is, predation affects the interactions of many species and influences the species structure of many aquatic communities. Aquaculture escapees can outnumber wild fish. For example, should a medium-sized farm with 100,000 fish lose 3% of the stock, these 3,000 fish might outnumber the wild population of the species, suggesting that competition and predation could become important interactions between the sterile stock, the wild stock, and prey populations. Recognition of these ecological risk pathways has led to greater discussion of aquaculture in on-land, indoor recirculating systems.

Public policy
The discussions of food safety and environmental issues posed by transgenic aquatic organisms have raised questions about the adequacy of regulatory oversight^3,4,13. Under the Coordinated Framework for the Regulation of Biotechnology, a transgenic fish is regulated by the Food and Drug Administration (FDA) as a "new animal drug" under the Federal Food, Drug, and Cosmetics Act. This approach fosters rigorous regulatory review of a product. Approval for marketing a product can be contingent upon adhering to given methods of production (e.g., use of all-female triploids or recirculating aquaculture systems). Commercialization would be followed by food safety and environmental monitoring, and approval for marketing could be withdrawn if found appropriate. However, the regulatory process is not publicly transparent. The existence and contents of a "new animal drug" application are confidential unless disclosed by the applicant. At the conclusion of regulatory review, FDA would publish its decision and rationale, without having offered opportunity for public comment. The closed nature of the procedure tends to decrease public acceptance of the regulatory process and of the product of biotechnology. Regarding possible environmental impacts of transgenic organisms, the Coordinated Framework invokes the National Environmental Policy Act. However, the act is procedural, requiring only that environmental impacts be formally assessed. Furthermore, FDA has limited environmental expertise.

Other acts might be invoked to apply the authority and expertise of federal agencies to issues posed by transgenic aquatic organisms^4,13. These include the Endangered Species Act (lead agencies are the U.S. Fish and Wildlife Service [USFWS] and the National Marine Fisheries Service), the Lacey Act (regarding injurious wildlife species, USFWS), the Non-Indigenous Aquatic Nuisance Species Prevention and Control Act (USFWS), Section 10 of the Rivers and Harbors Act (U.S. Army Corps of Engineers), and the Toxic Substances Control Act (Environmental Protection Agency). However, focusing only on the federal policy framework does not recognize that the states generally have lead authority for management of aquatic and marine resources to the three-mile limit offshore, plus valuable expertise on the species and ecosystems at issue⁴.

Three recent reviews of public policy covering transgenic aquatic organisms^3,4,13 stopped short of recommending that public policies be changed to strengthen regulatory oversight and improve transparency to the public. The Pew Initiative of Food and Biotechnology has a stakeholder forum that may make consensus recommendations¹⁴.

Conclusion
AquaBounty, the company seeking regulatory approval for commercialization of GH salmon, also has transgenic lines of rainbow trout and tilapia². Transgenic lines of GH tilapia and carp are under regulatory review in Cuba and China, respectively⁴. Scientific and regulatory issues posed by transgenic aquatic species will be debated for years to come.

Referring to the European Union's anti-GM food crusade, U.S. Trade Representative Robert Zoellick accused the organization of immoral behavior and claimed that some Member States had linked their aid to Africa with a rejection of GM foods. The Bush administration was also concerned about a domino effect with Asia, Latin America, and the Middle East following Zambia's lead in rejecting GM exports from the United States.

Impatient with the EU's glacial progress in ending its GM food moratorium and fearful that EU policy is creating a chilling effect around the world, U.S. officials came up with a plan. The United States would file an international trade case against the European Union in the World Trade Organization.

An Unsavory Alternative to the GM Food Moratorium?
The European Union has not allowed any new GM food or GM crop to enter its market since October 1998. This de facto moratorium has hit U.S. corn exporters the hardest by blocking an estimated $250-300 million in annual sales. Other U.S. crops have been hurt by the EU's exclusion: about 70 percent of U.S. soybeans and 40 percent of cotton crops are modified to withstand pesticides or to resist pests.

In an effort to end the moratorium, the EU enacted a measure (Directive 2001/18/EC) in October to pave the way for authorization of new GM crops. The new directive creates a comprehensive approval process based on the assessment of risks to human health and the environment, which must be followed before any GM product, or product containing a GM ingredient, can be released into the environment or placed on the market. The directive includes monitoring requirements and rules on mandatory labeling and traceability. The traceability regulations impose new obligations on business operators to retain information about each stage of market placement. The objective is to create records that document the progress of GM ingredients through the production and distribution chain.

At first, Europe's council of agricultural ministers failed to agree about the new rules. For instance, the European Commission had proposed that all food containing more than 1% GM ingredients should be labeled, but other Member States called for tighter limits. A meeting of Europe's environment ministers also failed to achieve an agreement about the rules.

Around the end of the year, the EU's effort to replace the moratorium with regulations bore fruit. The EU's agricultural and environmental ministers agreed to rules that would become law if authorized by the European Parliament. According to their proposal, products containing more than 0.9 percent EU-approved GM material will have to be labeled. All foods derived from GM material would have to be labeled, regardless of whether the final product contains detectable GM protein or GM DNA. For example, glucose syrup made from GM corn would have to be labeled. In addition, products that contain more than 0.5 percent of unauthorized GM material will be banned.

"Unenforceable and impractical" is how a spokeswoman for the United Kingdom's Food Standards Agency characterized the proposed rules. A lobbyist for the American Farm Bureau asserted that the labeling process would be so expensive that it would shut down exports. The rules did not sit well with the Bush administration either.

Overcoming the Moratorium at its Roots
Early this year, Bush administration officials said that they must file a case at the World Trade Organization against the EU's moratorium. The suit would center on allegations that the ban on GM crop and GM food approval acts as a nontariff trade barrier and a discriminatory practice under WTO rules. If a trade restriction, like the GM moratorium, is purported to be based on health concerns, then it must be backed up by scientific evidence. But there is little evidence to suggest that GM foods pose a health threat.

Robert Zoellick indicated that the United States was likely to bring its international trade case by the end of January. During early February, however, a Cabinet meeting that would have considered the suit was canceled. In light of the growing conflict in Iraq, the administration decided against antagonizing its European allies and postponed filing a case against the moratorium.

Suppose that the U.S. does file a complaint with the WTO and wins. Then the EU would face two choices: pay trade sanctions to compensate companies for the lost business, or lift the moratorium. In 1999, the U.S. fought and won a similar action when EU officials banned exports of U.S. beef products on the grounds that the meat contained growth hormones. But instead of allowing the beef products to enter its market, the EU decided to pay a $100 million fine each year. It is possible, therefore, that the EU would choose to pay sanctions while maintaining the GM product moratorium.

On the other hand, if the European Union disbands the moratorium under pressure from the United States and the WTO, U.S. exporters will probably face a consumer backlash in Europe. Furthermore, the ban would be replaced with traceability and labeling requirements many deem prohibitively expensive, escalating the cost of a Pyrrhic victory.

During a March 5 hearing of the U.S. Committee on Finance, Robert Zoellick said that, if the U.S. does bring a case against the EU, it would not be just a legal matter. Rather, the U.S. must "win the debate about biotech in world public opinion." To convince the EU to authorize GM products with agreeable rules on tracing and labeling, the U.S. will need to convince European consumers that GM food is safe, if not beneficial.

Porcine Panic: This Little GM Piggy Went to Market
In February, the Food and Drug Administration announced its efforts to track down 386 piglets that were part of genetic engineering experiments and that were sold into the U.S. food supply. Researchers at the University of Illinois in Urbana-Champaign had been engineering pigs with a bovine gene that increases milk production and a synthetic gene for improving a piglet's ability to digest milk. Their goal was to raise healthier, bigger pigs faster and without drugs.

Between April 2001 and January 2003, UI researchers tested the offspring of genetically engineered pigs. Piglets with the new genes were kept in the study, whereas those lacking the new genes were sold to a livestock broker. UI researchers insist that the non-transgenic pigs were not "investigational" and did not fall under FDA scrutiny. Both the FDA and the university assure that the sold pigs do not pose a risk to consumers. Nevertheless, the university faces the problem that FDA rules require the offspring of genetically engineered animals to be destroyed. The FDA may impose sanctions and fines on the university.

Patent Polemics
Turning to other porcine-related matters, the Court of Appeals for the Federal Circuit recently issued their decision on whether Schering-Plough Corporation's PrimePac vaccine infringes Boehringer Ingelheim's patent for producing Porcine Reproductive and Respiratory Syndrome (PRRS) virus. Following the severe financial consequences of the PRRS epidemic in the 1980s, Boehringer researchers discovered a new virus in tissue samples from diseased pigs. The company patented its process for growing and isolating the PRRS virus (U.S. patent No. 5,476,778). Boehringer sued Schering, which produces a vaccine against PRRS by attenuating the virus in cell culture. A jury decided that Schering infringed the patent, and the judge issued an injunction prohibiting the company from selling its infringing vaccine. On appeal, the Federal Circuit affirmed the decision.

In another patent case, the alleged infringer won. Plant Genetic Systems, N.V. (which became Aventis CropScience, N.V., which was then purchased by Bayer AG) sued DeKalb Genetics Corporation (now a part of Monsanto Company) for infringing U.S. Patent No. 5,561,236. The `236 patent claims transgenic plant cells, plants, and seeds; DeKalb made and sold transgenic corn seeds. After PGS lost in a district court, the company appealed to the Federal Circuit. A key issue in the case was whether PGS's broad claims should cover any transgenic plant or transgenic plant cell. The Federal Circuit agreed with the district court that the patent claims did not cover transgenic monocots, like corn. The court concluded that, in 1987 when PGS had filed the patent, stable transformation of monocot cells would have required undue experimentation. The teachings of the `236 patent, the Federal Circuit explained, supported methods for transforming dicots, but not monocots.