Ingard In Transgenic Plant

Control of insect pests represents one of the major input costs of world agricultural production and consumes billions of dollars annually, predominantly in chemical pesticides and lost production. Insect control with pesticides is, however, becoming more problematic in both developed and third world countries. Farmers and consumers are increasingly aware of the environmental and ecological impacts of excessive pesticide use. As the target insects are repeatedly exposed to the same pesticide chemistries, they often develop heritable resistance to those pesticides and require the use of higher and higher doses or more toxic insecticide blends to achieve economic levels of pest control. Plant breeders, seed companies, and farmers are now turning to biotechnological solutions to these important pest problems in agriculture because of the environmental and economic advantages of deploying inbuilt insect control strategies as opposed to external applications. Over the past 10 years, transgenic crops protected against insects have been generated in some of the important broadacre crops, including cotton and corn, as well as in horticultural crops such as potato, and have been released commercially (1).


The bacterium Bacillus thuringiensis has had a long history of use as a sprayable microbial biopesticide (2). It has been used predominantly in horticulture and to a smaller extent in broadacre crops, especially those grown organically. During sporulation, the bacterium produces a series of insecti-cidal proteins assembled into a parasporal crystal. A mixture of the dried spores and crystals is reconstituted with water and sprayed on plants. On ingestion by the target insects, the crystals are solubilized and activated by digestive proteases to form highly potent and specific toxins that bind to particular gut receptors. The toxins are thought to aggregate and form ion-permeable pores that lead to gut dysfunction, lysis of gut epithelial cells, and the eventual death of the insect (see review in Ref. 3). Individual crystals in the bacterium may be a complex mixture of toxins each with its own range of insect specificities. The individual proteins within the crystals are encoded by both plasmid and chromosomal Cry genes that were identified as early targets for incorporation into crop plants to protect them from insects. There are now hundreds of identified Cry genes, and there is a standard nomenclature to describe them based on sequence similarities (see http:// and indicated links).

Initial attempts to express Cry genes in transgenic plants were not particularly successful, and this was attributed to the bacterial origins of these genes and especially their high AT content, which resulted in low levels of insecticidal protein. Resynthesis of the Cry genes removing cryptic plant polyadenylation signals, putative messenger RNA (mRNA) destabilization signals, improving the codon usage bias, and increasing the GC content were strategies that increased expression to a useful level (0.2-0.3% of total soluble protein) in transgenic plants (4). Modified and unmodified, full-length and truncated Cry genes have so far been expressed in an array of plant species (from trees such as poplar, larch, and eucalypt; cereals like wheat, maize, and rice; legumes like chickpeas, soybeans, and peanuts; vegetables such as potato, tomato, cabbage, broccoli, and sweet potato; and fruits like apples and strawberries) (see references in Refs. 5-9), but intellectual property ownership has restricted commercialization to a few high-value agricultural species, such as cotton and corn and to a lesser extent potato.


A. Bt-Corn

The United States is the largest corn-producing country in the world. One of the significant pests of U.S. and Canadian corn is the European corn borer

(ECB) (Ostrinia nubilalis), a lepidopteran insect, whose larvae are difficult to control as they bore into the corn stalk, where they are protected from applied pesticides. Only a few percent of the total acreage of corn is sprayed for control of ECB, so economic losses from this insect can be high. Bt-corn varieties targeted at ECB control were first released in the United States in 1996 and were so rapidly adopted by farmers that over a quarter of all the corn sown by 1999 was Bt-corn. Five different types of Bt-corn are currently registered by the U.S. Environmental Protection Agency (EPA), each with either different Cry genes (CrylAb, Cry 1 Ac, or Cry9C) or different levels or patterns of expression. The Monsanto and Syngenta Yieldgard corns express truncated codon-modified CrylAb genes derived from B. thu-ringiensis subsp. kurstaki (10,11). The Syngenta corn line has been back-crossed into both field and sweet corn varieties and, as with the Monsanto line, the CrylAb gene is expressed throughout the plant, including the silks and kernels, but is particularly high in leaves. Bt-corns called KnockOut (Novartis) or NatureGard (Mycogen) also express CrylAb but use a novel two-gene strategy to target expression to ECB-susceptible tissues. One CrylAb is controlled by the corn phosphoenolpyruvate carboxylase (PEPC) gene (12) directing expression in green photosynthetically active tissues. A pollen-specific promoter from the maize calcium-dependent protein kinase (CDPK) gene (13) controls the second. The combination of PEPC and pollen promoters provides high CrylAb gene expression in leaves and pollen, where it is highly effective in controlling European corn borer. Changes in business structures have resulted in the phaseout of KnockOut and NatureGard corns, and their registration will not be renewed. All existing stocks of these events can now be used only until the 2003 corn-growing season.

A Dekalb Bt-corn called Bt-Xtra contains three genes, the CrylAc gene from B. thuringiensis subsp. kurstaki, the bar gene from Streptomyces hy-groscopicus, and the potato proteinase inhibitor pinll. While proteinase inhibitor genes expressed at high levels can inhibit insect digestive proteases and confer insecticidal properties to transgenic plants, the pinll gene in Bt-Xtra corn was truncated during integration into the corn genome and no protein expression is detectable. CrylAc is very similar to CrylAb but has a slightly different insecticidal activity spectrum. Since the purchase of Dekalb by Monsanto, the Bt-Xtra corn has been phased out in favor of Yieldgard. Finally, StarLink is a Bt-corn expressing a completely different type of Cry gene, the Cry9C from B. thuringiensis subsp. tolworthi. The Cry9C protein is active against ECB and some other lepidopteran pests of corn, including black cutworm (Agrotis ipsilon), but not the corn earworm (Helicoverpa zea). StarLink corn was registered only for animal feed and nonfood industrial use as there were some questions raised at registration concerning its potential allergenicity in humans. Subsequently, Cry9C was detected in corn chips and other corn food products, in violation of the original animal feed use registration. StarLink was voluntarily withdrawn from registration in 2000, and there is now an extensive program in place to track and remove remnants of StarLink corn from the human food chain. At the time of its withdrawal, it had been sown on less than 1% of the corn area in the United States. Thus, only two of the five original transgenic Bt-corn lines will be available commercially in the next few years.

Bt-corn varieties have been shown to be very effectively protected against ECB, but levels of infestation vary from year to year and region to region. It has been estimated that in 10 of the last 13 years growers would have received an economic benefit had they been growing Bt-corn when ECB pest pressures were high (14). Since their release, modest reductions (1.5%) in pesticide usage have been realized (15), but given that insecticides are rarely effective for ECB control and hence seldom used, this is still significant. Indirect benefits are also likely as the reduced ECB damage to Bt-corn is also likely to reduce sites for entry of pathogens, particularly those that produce mycotoxins that are health hazards to humans and farm animals eating the corn. The Cry]-expressing Bt-corn varieties have clearly allowed farmers to better control a very difficult agricultural pest (ECB), for which effective and affordable pest control options were unavailable. Yield losses from this insect have reached as much as 300 million bushels of corn in a single year. Such losses could be virtually eliminated using Bt-corn.

New additions to the current suite of Bt-corn varieties are likely in the next few years; the first, a CrylF-expressing corn (lepidopteran active), received conditional registration in 2001 (16). Of particular interest will be the first Bt-corn targeted at beetle pests, especially the corn rootworm (Dia-brotica sp.). Corn rootworm is really a complex of three or four different species of Diabrotica that are serious pests in the United States, with estimated costs of over U.S. $1 billion in chemical control and lost production. The larvae of these beetles attack the roots, and so control is primarily with soil insecticides, crop rotations, and foliar insecticide applications to kill the adult beetles and prevent further egg laying. Monsanto have developed a new transgenic corn (MaxGard) expressing the beetle-active Cry3Bb gene that appears to be very effective in controlling the larval stages of corn rootworm (17). The gene construct is believed to be a codon-modified synthetic Cry3Bb with a leader region from the wheat chlorophyll a/h binding protein gene, an intron from the rice actin gene, and a terminator from the wheat hsp]7 gene (18). Monsanto applied for full registration of a number of events of its MaxGard corn in late 1999 (19). The petition for nonregu-lated status was withdrawn in 2001, but Monsanto continues to do large-scale trials all over the U.S. corn belt. The transgenic corn will have to compete with other nontransgenic corn rootworm-resistant varieties (20). These nontransgenic varieties may be more acceptable to consumers who are concerned about GM corn products.

B. Bt-Cotton

Cotton is a major world crop grown primarily for its fiber but also for its oil and seed meal. Cotton is a demanding crop requiring significant inputs of both water and agricultural chemicals (insecticides, herbicides, fungicides, defoliants, and fertilizers). High inputs and high returns have made cotton one of the leading crops for the application of biotechnology, and by 1999-2000 about 12% of the world cotton area was sown with genetically modified varieties. In 2000 over 70% of the U.S. crop was sown with insect-and/or herbicide-tolerant GM cotton varieties: about 60% were herbicide-tolerant varieties and 40% were Bt varieties (about three quarters of the latter were stacked with a herbicide tolerance trait). The Bt varieties (Boll-gard) were first released in the United States in 1996 primarily for the control of Heliothis virescens (tobacco budworm, TBW) but also for other pests such as Helicoverpa zea (cotton bollworm, CBW, or corn earworm) and Pectinophora gossypiella (pink bollworm). Bollgard cotton varieties contain a single insertion of a codon-modified hybrid CrylAb/CrylAc gene from B. thuringiensis subsp. kurstaki (21). Three lines of Bt-cotton, 531, 757, and 1076, were initially deregulated, but only one, 531 (Bollgard), was released commercially in the United States. Initial sowings of Bollgard cotton in 1996 were about 1 million hectares (about 12% of the U.S. cotton area). This was a difficult year for a commercial launch because there were unusually high levels of CBW rather than the more usual cotton pest, TBW (CBW is less susceptible to CrylAb/c than TBW). Many thousands of hectares of Bt-cotton in the U.S. southeast became infested with CBW and had to be sprayed with pesticides. This poor performance was blamed on the increased corn area that year (which increased populations of CBW) and the hot dry weather that appeared to decrease expression of the CrylAb/c gene, particularly later in the season (22). Subsequent years proved less difficult for growers, and the performance of Bt varieties has improved as growers have adjusted their adoption and management of Bt-cotton to suit their particular pest problems and economics.

The benefits of Bt-cotton varieties in the United States have been difficult to quantify despite extensive surveys since their introduction. Attributing effects on yield, pesticide usage, and profits due to Bt is a statistical challenge as many other factors can influence yield and pesticide usage even on a single farm. However, a number of analyses (23) have indicated that there are at least three benefits to growers of Bt-cotton. First, there have been statistically significant reductions in yield losses due to the target pests in many areas (24). Pesticide usage has also declined in many areas, but again statistical analyses suggest that this decrease is small and mainly for the pesticides other than major organophosphates and synthetic pyrethrins (23). These increases in yields and decreases in pesticide usage translate to higher net returns to growers who adopt Bt-cotton technology.

Australia saw the first release of Bt-cotton in 1996, as INGARD varieties (Bollgard was already registered as a proprietary name for a different technology), on a relatively small area (30,000 ha or about 10% of total cotton area). In Australia, the main insect pests of cotton, as in the United States, are also the larvae of heliothine moths, in this case Helicoverpa armígera and H. punctigera. Infestations are normally extreme by comparison with that in the United States, and it is not unusual to spray 12-14 times per season to control these pests (compared with 2-3 in the United States). The total crop in 2000 occupied about 500,000 ha (about 180,000 of it INGARD) and insect control costs on conventional cotton often exceed AUS$100 million annually, for a crop worth about AUS$1.6 billion. Both H. armígera and H. punctigera are naturally less susceptible (by about 10-fold) to the CrylAb/c protein expressed in INGARD (or Bollgard) cotton and are much more abundant in cotton-producing areas than the similar pests in the United States. Early research trials indicated that although INGARD cotton controlled H. armígera well (Fig. 1), particularly during the early part of the season, efficacy declined late in the season when fruit was being set (25).

Annual surveys of the usage of pesticides and economic returns to growers using Bt-cotton have been carried out in Australia since 1996. Because INGARD cotton can be only a proportion of the cotton on any one farm, detailed paired comparisons between the performance of INGARD and corresponding conventional varieties were possible (26). Pest control costs represent a significant proportion of the annual variable costs of cotton production in Australia, so the impact of INGARD on pesticide use appears to be much more apparent than in the United States. In all 4 years there was a significant reduction in overall use of pesticides (across all pesticide groups) and particularly those used against the Helicoverpa caterpillars. In 1999-2000, for example, there was a 40% decrease in pesticides targeted at all pests (down from 10.3 sprays in conventional cotton to 6.2 in Bt-cotton) or 47% reduction in pesticides targeted specifically at Helicoverpa species (from 9.7 down to 5.1 sprays on Bt-cotton) (Fig. 2). There was a 75% reduction in endosulfan use (a pesticide under threat in Australia because of its high toxicity to fish and the very low tolerance set for endosulfan contamination in rivers) and a 43% reduction in pyrethrins. This should

Gilbert Keyser

Figure 1 INGARD cotton provides good early season control of Australia's two main caterpillar pests, Helicoverpa armigera and H. punctigera. Research plots at the Australian Cotton Research Institute of a conventional variety on the left and its INGARD equivalent on the right. The fields were not sprayed at all for insect pests, and the conventional variety failed to yield any har-vestahle cotton as most flower buds and bolls were devoured by caterpillars. (Photo courtesy of Cheryl Mares, CSIRO.)

Figure 1 INGARD cotton provides good early season control of Australia's two main caterpillar pests, Helicoverpa armigera and H. punctigera. Research plots at the Australian Cotton Research Institute of a conventional variety on the left and its INGARD equivalent on the right. The fields were not sprayed at all for insect pests, and the conventional variety failed to yield any har-vestahle cotton as most flower buds and bolls were devoured by caterpillars. (Photo courtesy of Cheryl Mares, CSIRO.)

reduce the pressure on synthetic pyrethroids that have selected a high incidence of resistance among cotton pests.

The main concern to growers has been the high variability of lepidop-teran control in Bt-cotton crops. The number of effective days of Helicoverpa control (time from planting to first foliar spray of insecticide) varies from as little as 20 days up to 140 days with considerable differences in the performances of different transgenic varieties (26). Reduced control has been correlated with a decrease in both CrylAb/c protein and mRNA (27,28) and possibly increases in secondary metabolites that decrease the efficacy of Bt-toxins (29). Most of the benefit of Bt-cotton is realized in the first half of the season, when little insecticide now has to be used to control both major lepidopteran pest species. Careful crop monitoring and spraying when pests reach their economic thresholds (new thresholds were established specifically for INGARD varieties) has stabilized the performance of INGARD cotton. There is a strong tendency to sow INGARD cotton in environmentally sensitive areas (fields close to waterways and towns as Australia has very strong pollution control laws in relation to pesticide contamination of

Figure 2 INGARD cotton under commercial production required significantly fewer pesticide sprays for caterpillar pests than similar conventional varieties in the 1999-2000 season. An area of 191,000 ha of cotton was surveyed, of which 28% was INGARD cotton. Pesticide applications to INGARD were 47% less than on conventional fields, and there were no major differences in pesticides applied for other pests. (Adapted from Ref. 26.)

Figure 2 INGARD cotton under commercial production required significantly fewer pesticide sprays for caterpillar pests than similar conventional varieties in the 1999-2000 season. An area of 191,000 ha of cotton was surveyed, of which 28% was INGARD cotton. Pesticide applications to INGARD were 47% less than on conventional fields, and there were no major differences in pesticides applied for other pests. (Adapted from Ref. 26.)

rivers etc.) to capture the significant environmental benefits of reduced pesticide applications. The economic benefit to the grower has been variable, in line with the variability in successful insect control. Of the paired comparisons carried out, 54% recorded some economic benefit and the rest were about break-even.

The Bt technology clearly has benefits, but some limitations have emerged in Australia (and presumably in Asia, where H. armigera is a major pest of cotton). In response to the poorer efficacy of Bt-cotton in Australia (and to enhanced H. zea control and better resistance management in the United States), a double gene product is being developed that contains two different Bt-toxin genes, the CrylAb/c gene (in INGARD and Bollgard) and a CrylAb gene also from Bacillus thuringiensis. These two gene cottons (Bollgard II) are still in the breeding stage, but initial field studies have indicated much better H. armigera control.

Bt-cotton is now approved for food use in Argentina, Australia, Canada, China, Japan, Mexico, and South Africa, as well as the United States. Bt-cotton was grown on about 0.5 million hectares in mainland China in 2000—much of it Bollgard, but also some Bt-cotton developed in China, e.g., (30)—with outcomes very similar to that observed in Australia (31), where the lepidopteran pests are similar. A number of Asian, European, and Latin American countries have experimental crops of both Bt-cotton and Bt-corn for precommercial evaluation.

C. Bt-Potatoes

In 1996 about 1% of the total U.S. potato acreage was planted to Bt-potatoes, and this rose to about 3% in 2000. The target pest for Bt-potatoes was the Colorado potato beetle (CPB) (Leptinotarsa decemlineata), whose adults and larvae defoliate potatoes, reducing tuber yields to the extent that potato production has had to cease in some areas. Although nonchemical methods of control are available, none are as cost-effective or practical as chemical pesticides. CPB easily develops resistance to chemical insecticides, and many populations are now resistant to a broad array of chemical types. Microbial biopesticides containing Cry3A Bt-toxins are effective in killing the young larvae but, in practice, are not very effective against the most damaging life stages, the third and fourth instar larvae and adults. Expressing the Cry3A gene in the plant has considerable advantages in targeting the most vulnerable stage of the insect's life cycle, neonates, but also, if the expression levels are high enough, in controlling larger larvae and adults. Several different transgenic cultivars of potato (sold as NewLeaf varieties) express the Cry3A gene from B. thuringiensis subsp. tenebrionis (32). Because potato is a vegetatively propagated plant, with low or no fertility, each cultivar or variety must be separately transformed, whereas with Bt-corn or Bt-cotton a single event can be backcrossed into several different elite cultivars or hybrids.

NewLeaf potatoes expressing Cry3A proteins were the first to be released, and control of CPB was very effective together with over 40% reduction in insecticide applications to the NewLeaf variety (15). In most potato-growing areas, aphids are also a significant pest, not only for the damage they can do to the plant but also because they transmit harmful plant viruses such as potato leaf roll virus (PLRV) and potato virus Y (PVY). The introduction of NewLeafPlus and NewLeafY potato varieties that have a built-in transgene protection against PLRV and PVY, respectively, as well as the Bt gene, might have made an even bigger impact on the use of Bt-potatoes in areas where these diseases are prevalent. However, large buyers of potato products have refused to buy GM potatoes and processors have followed suit, reducing the market for NewLeaf varieties despite good evidence that they provide considerable economic benefit to the farmer. After the poor economic performance of its NewLeaf potatoes, Monsanto appears to be reluctantly withdrawing completely from the transgenic potato market to concentrate on its other more successful biotech crops (33).

D. Resistance Management and Bt Crops

One of the key concerns with deploying single Cry genes into crop plants is that the increased exposure will select for resistance in the target insects. This could destroy the potential for long-term control using this technology and affect other users of microbial pesticides. Unfortunately, field resistance to microbial Bt formulations has already occurred in both the Indian meal worm (Plodia interpunctella) in stored grains and the diamondback moth (Plutella xylostella) in a number of geographically isolated insect populations attacking vegetable crops (34). Laboratory-selected resistance has also been observed in a number of species that attack Bt-crops, including H. virescens (35) and more recently H. armígera (36) and P. gossypiella (37). These relatively isolated occurrences of resistant insects have, however, provided models for how to manage the development of resistance to Bt-crops in an agricultural setting (38).

The provision of non-Bt plants or refugia nearby to generate sufficient numbers of susceptible adults is the most commonly used resistance management strategy. This ensures that any rare homozygous resistant insects selected on the Bt crop will find only fully susceptible mates, thereby continually diluting the resistance genes in the target insects. For Bt-cotton, the refugia can be unsprayed or sprayed cotton or other crops that are hosts for the pest species and generate adult insects at the same time as those that might emerge on Bt-cotton. The area and location of these refugia relative to the Bt crop are critical and in Australia, at least, have been determined empirically for Bt-cotton from the numbers of pupae of H. armígera generated by crops treated in different ways. The requirements for refugia and other resistance management procedures are legislated into the registration label for INGARD cotton. The IPM approach adopted in Australia has been summarized (39). At present, refugia requirements are easily achieved as the total Bt area for any one farm can only be 30% of the total cotton planted. This area limit is likely to be lifted only when cotton with two Bt genes becomes available around 2003 or 2004. Similar refuges for Bt-cotton and Bt-corn have been strongly recommended by U.S. regulators in response to scientific concerns that the technology was at risk from overuse. The effectiveness of refuge strategies is dependent on many factors including the biology and population dynamics of the pest species, the frequency and mechanism of resistance, the placement and management of the crop and neighboring crops, and the expression level of the transgenes.

E. Nontarget Effects of Bt Crops

A second concern with Bt crops, or any other insecticidal transgenic plant, is that when released on a large scale they may have unintended, or even unnoticed, effects on other organisms in agricultural or neighboring ecosystems. Because Bt-corn is thought to exude Bt-toxins into the soil (40), where they can persist for relatively long periods bound to soil particles (41), and Bt-containing biomass is regularly returned to the soil during agricultural production, impacts on soil insects and microbes must also be considered. Two instances of possible nontarget effects have been described. First, Bt-corn has been reported to have unexpected effects on a beneficial insect, the green lacewings that feed on the European corn borer (ECB) (42). Green lacewings fed ECB that had eaten Bt-corn had a higher death rate and delayed development compared with the controls, and this was attributed to the poor nutritional quality of ECB larvae exposed to the Bt toxin. This study has not been extended to an agricultural setting.

In a second case of possible nontarget impacts of insecticidal crops, Bt-corn was believed to accelerate the demise of the endangered monarch butterfly (Danaus plexippus) in the United States (43). Monarch caterpillars feed on milkweed plants that often grow near cornfields. In a laboratory simulation, milkweed leaves were dusted with Bt-corn pollen and these leaves were fed to monarch larvae. After 4 days, only 56% of the larvae survived relative to larvae fed on plants dusted with non-Bt pollen or no pollen at all. Surviving larvae were also smaller than the controls. This laboratory study raised questions about the validity of the regulatory assessment of the environmental impacts of transgenic plants and prompted new field studies (summarized in Ref. 44) that did not confirm the laboratory results. The field study showed that Bt-corn would not have any adverse effects on nontarget lepidoptera such as monarch butterflies and demonstrated that the potential exposure of larvae to Bt-corn pollen was, in fact, very low. The risks to monarch butterflies from Bt crops must be considered small compared with the risks posed by habitat destruction and must be weighed against the effects of chemical pesticides on all environments adjacent to cropping areas. This incident highlights the need for field-based confirmation of any laboratory study that makes a claim for benefits or risks associated with any new biotech product.

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  • espedito
    When was ingard produced?
    2 years ago

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