GM Crop Database

Database Product Description

ATBT04-6, ATBT04-27, ATBT04-30, ATBT04-31, ATBT04-36, SPBT02-5, SPBT02-7
Host Organism
Solanum tuberosum (Potato)
Trade Name
Atlantic and Superior NewLeaf®
Trait
Resistance to Colorado potato beetle (Leptinotarsa decemlineata, Say).
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2001 2001 View
Canada 1996 1997 1997 View
Japan 1997 View
Korea 2004 View
Philippines 2003 2003 View
United States 1996 1996 1996

Introduction Expand

The transgenic cultivars of ‘Atlantic’ (ATBT04-6, ATBT04-27, ATBT04-30, ATBT04-31, ATBT04-36) and ‘Superior’ (SPBT02-5, SPBT02-7) NewLeaf® potatoes were genetically engineered to be resistant to attack by Colorado potato beetle (CPB). These lines were developed by introducing the cry3A gene, isolated from the common soil bacterium Bacillus thuringiensis subspecies tenebrionis (Btt), into the potato genome by Agrobacterium-mediated transformation.

The Cry3A protein expressed in these transgenic potato cultivars is identical to that found in nature and in commercial Bt spray formulations. Cry proteins, of which Cry3A is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow causing gut paralysis and eventual death due to bacterial sepsis. Cry3A is insecticidal only when eaten by the larvae of coleopteran insects such as CPB, elm leaf beetle and yellow mealworm, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of these plants. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
nptII neomycin phosphotransferase II SM CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region Native
cry3A cry3A delta-endotoxin IR double enhanced CaMV 35S or ats1A promoter of Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase 3' poly(A) signal from pea ribulose-1,5-bisphosphate carboxylase, small subunit (rbcS) gene 1-3 loci depending on line Modified to enhance expression (plant preferred codon usage)

Characteristics of Solanum tuberosum (Potato) Expand

Center of Origin Reproduction Toxins Allergenicity

South America, particularly the high plateau region of Bolivia and Peru

Only hybridizes with tuber forming Solanum species, which tend to be geographically separated from regions of potato cultivation

The glycoalkaloids, solanine and chaconine, are only known natural toxicants

No significant, reported allergens

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Bacillus thuringiensis subsp. Tenebrionis cry3A While beetles and other coleopterans are susceptible to oral doses of Cry3A protein, there is no evidence of toxic effects in laboratory mammals or birds. There are no significant mammalian toxins or allergens associated with the host organism.

Modification Method Expand

The transgenic Atlantic and Superior potato lines were created through two separate Agrobacterium-mediated transformation events in which the transfer DNA (T-DNA) contained the gene encoding the Cry3A protein from B. thuringiensis subsp. Tenebrionis. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII). The expression of NPTII was used as a selectable trait for screening transformed plants for the presence of the cry3A gene. Due to segregation effects, the NPTII encoding gene was not present in Superior cultivar SPBT02-5 submitted for commercial release. Additional DNA outside of the T-DNA border sequences was incorporated into the genome of Atlantic lines ATBT04-27 and ATBT04-36. These lines also contain the aad gene, which encodes the enzyme 3'’(9)-O-aminoglycoside adenylyltransferase that confers bacterial resistance to spectinomycin and streptomycin. The aad gene was not expressed in plant tissue, but was present on the Ti plasmid as a selectable trait for screening bacterial colonies for the presence of the plasmid vector. The data from at least 3 generations of vegetative propagation demonstrated the stability of the novel trait.


Characteristics of the Modification Expand

The Introduced DNA

The data provided showed various integration patterns depending on the line: the T- DNA was integrated at three sites for line ATBT04-6 and at one site for lines ATBT04-30 and ATBT04-31. No coding region from outside the T-DNA borders was incorporated into the genotype of these lines. One complete copy of the T-DNA was found at each of two insertion sites in line ATBT04-27, with one site also containing a bacterial gene coding for resistance to streptomycin and another partial copy of the cry3A gene. The streptomycin resistance gene is driven by a bacterial promoter and is not expressed in plants, as was demonstrated in these NewLeaf™ Atlantic potatoes. Finally, the transgenes were integrated at three insertion sites in line ATBT04-36.

Genetic Stability of the Introduced Trait

The NewLeaf™ potato lines were evaluated over several generations of vegetative propagation, used to generate potato seed, to determine if they expressed consistent levels of the Cry3A protein in leaf tissue. Expression of the Cry3A protein was found to be highly stable across multiple generations in all the lines tested.

Expressed Material
In B. thuringiensis, expression of the cry3A gene results in the production of two proteins, a full length 73 kDa (644 amino acids) Cry3A protein and a smaller molecular weight species (68 kDa; 597 amino acids) resulting from in-frame translation initiation at an internal ATG sequence. This latter protein, referred to as Cry3A band 3 protein, lacks 48 amino acids from the N-terminus but retains insecticidal activity, and corresponds to the form of the protein expressed in these transgenic potatoes.

Western immunoblot analysis of protein extracts from transgenic potato tissue demonstrated the presence of a 68 kDa immunoreactive species as well as a smaller molecular weight species of 55 kDa. Studies were conducted to confirm that the 68 kDa protein product of the cry3A gene transferred into potato plants undergoes processing or degradation in the plant cell into a smaller protein of 55 kDa. The two protein products seen in the plant cell appear identical to that observed in commercial B.t.t preparations.

Protein expression levels were quantitated by enzyme linked immunosorbent assay (ELISA) and found to average from 15.7 to 59.3 µg/g (f.w.) of leaf tissue and from 0.09 to 0.53 µg/g (f.w.) of tuber tissue, corresponding to 0.10 to 0.37% of total foliage protein and 0.0004 to 0.003% of total tuber protein. Likewise, production of NPTII was quantified in both leaf and tuber tissue at levels of 4.4-36.6 and 0.5-2.9 µg/g fresh weight tissue, respectively. The presence of NPTII protein has been judged to be insignificant with respect to any human health risk due to exposure.

Environmental Safety Considerations Expand

Field Testing

Over several years of field testing, these transgenic potatoes have been compared to non-transgenic ‘Atlantic’ and ‘Superior’ cultivars for differences in physical characteristics, disease susceptibility, and insect susceptibility. The field data reports indicated no obvious differences in the number of volunteers, emergence from seed potatoes, percent stand (emergence), overwintering capacity, and tuber yield and quality. Susceptibilities to diseases, other than to CPB, including early blight, late blight, verticillium, potato leaf roll virus, and potato virus Y were unchanged.

Field observations confirmed that Colorado potato beetle was controlled at all stages of development throughout the growing season and also indicated that potato flea beetles (Coleoptera: Chrysomelidae, Epitrix cucumeris Harris) were affected to some extent. Overall the field data reports and data on agronomic traits showed that the CPB-resistant potato lines had no potential to pose a plant pest risk.

Outcrossing

In general, the natural exchange of genetic material is only possible with other varieties of potato Solanum tuberosum. Since the reproductive characteristics of the CPB-resistant potato lines were unchanged by the genetic modification, they should be no different than the parent cultivars. Gene exchange between and CPB-resistant potatoes and other Solanum species is very unlikely and is limited to cross-pollination with other potatoes.

Weediness Potential

No competitive advantage was conferred to CPB-resistant potato lines, other than resistance to CPB, which will not, in itself, render potatoes weedy or invasive of natural habitats, since none of the reproductive or growth characteristics have been modified.

Potatoes do not exhibit weediness characteristics and have difficulty becoming established outside cultivated fields. Small tubers left in the ground after harvest (groundkeepers) may give rise to volunteer plants in the next crop but are usually killed by frost or drought. Surviving volunteers can be controlled with herbicides and cultivation. Furthermore, seed dispersal is limited as dissemination is by tuber and seed. Outside of cultivated areas, seedlings grown from true seed do not compete successfully and are not reported as a weed pest.

Secondary and Non-Target Adverse Effects

The host ranges and habitats of the nine coleopteran insect species currently listed or proposed as threatened and endangered were examined to determine if CPB-resistant potatoes might have an adverse impact on these species. None of these species were found to inhabit potato fields or feed on potatoes, and they usually occurred in specialized habitats. For example, some of these insects (i.e., the Kretschmarr Cave mold beetle and the Coffin Cave mold beetle) live in caves, and some (i.e., northeastern beach tiger beetle and puritan tiger beetle) live on beaches.
Other invertebrates, such as earthworms, and all vertebrate organisms, including non-target birds, mammals and humans, were not expected to be affected by the Cry3A insect control protein, because they do not contain the receptor protein found in the midgut of target insects.

Dietary toxicity studies were performed using the 68 kDa microbial protein on beneficial insects (honeybee, ladybird beetle, green lacewing and parasitic wasp), and with eight non-target insect species representing the orders of Coleoptera, Diptera, Homoptera, Lepidoptera and Orthoptera (southern corn rootworm, yellow fever mosquito, green peach aphid, European corn borer, tobacco hornworm, corn earworm, tobacco budworm and German cockroach). No negative effect was observed, except for slightly higher mortality and reduced honeydew production of green peach aphids. Green peach aphids are major vectors of potato viruses, specifically of the potato leaf roll virus, and are chemically controlled in standard potato production systems. The effect of CPB-potatoes on these aphids was therefore negligible in terms of environmental impact.

The density of insect populations was investigated. The following beneficial and predacious arthropods were significantly more abundant in the transgenic potato plots than in those treated with conventional chemical insecticides: big eyed bugs, damsel bugs, minute pirate bugs, some Hymenoptera spp. and spiders. As a result, aphid populations may be reduced through predation by natural enemies. Field observations also showed that populations of the detritivorous collembolans were as abundant in CPB-resistant potato fields than in fields of non-transgenic potato counterparts, and more abundant than in those treated with conventional chemical insecticides.

Impact on Biodiversity

CPB-resistant potato lines have no novel phenotypic characteristics that would extend their use beyond the current geographic range of potato production. Since there is no occurrence of wild relatives of potato in Canada, there will be no transfer of novel traits to unmanaged environments. Similarly, as the risk of gene transfer to tuber-producing wild relatives in the United States is very remote, it was determined that the risk of transferring genetic traits from CPB-resistant potato lines to species in unmanaged environments was insignificant.

Other Considerations

In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and United States have required developers to implement specific Insect Resistant Management (IRM) programs. These programs are mandatory for all transgenic Bt-expressing plants, including CBP-resistant potato lines, and require that growers plant a certain percentage of their acreage with non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Potatoes are considered to be a staple food in North America, constituting up to 37% of the total average vegetable intake. The genetic modification present in the ATBT04-6, ATBT04-27, ATBT04-30, ATBT04-31, ATBT04-36, SPBT02-5 and SPBT02-7 transgenic lines will not result in any change in the consumption pattern for potatoes. Due to their protection from CPB damage, the NewLeaf™ Atlantic and Superior cultivars are expected to replace some existing commercial potato cultivars in all potato product applications. Hence, they will provide an alternate or additional choice to consumers and food manufacturers.

Nutritional Data

The major components of CPB-resistant potato lines were analyzed for nutritional constituents, proximate composition (protein, fat, ash, total dietary fiber, carbohydrate, and calories), internal quality characteristics (hollow heart and brown center, internal brown spots, vascular discoloration, and blackspot bruise), and French fry quality characteristics and compared with those of non-transgenic ‘Atlantic’ and ‘Superior’ tubers. The analysis of nutrients from each of these transgenic potato lines and from non-transgenic potato did not reveal any significant differences in levels of crude protein, ash, or starch. Similarly, the levels of micronutrients and trace elements, including thiamine, niacin, riboflavin, vitamin C, calcium, iron and zinc, were comparable to those of unmodified counterpart potatoes. It was concluded that the consumption of products from CPB-resistant potatoes has no significant impact on the nutritional quality of the Canadian and American food supply.

Toxicity

The glycoalkaloids, solanine and chaconine, are naturally occurring toxicants found in potato tubers, particularly in green tubers that have been exposed to sunlight. Analyses of total glycoalkaloid (TGA) levels in each of the transgenic lines demonstrated that in each case the level was below the administrative guideline of 20 mg/100g fresh weight that has previously been established for TGA in potato.

The amino acid sequence of the Cry3A protein expressed in CPB-resistant potato lines is closely related to the sequence of the same proteins that are present in strains of B. thuringiensis that have been used for over 30 years as commercial organic microbial insecticides. An analysis of the amino acid sequence of the inserted Cry3A protein did not show homologies with known mammalian protein toxins and was not judged to have any potential for human toxicity.

Allergenicity

The likelihood of the Cry3A and NPTII proteins being allergens was judged to be remote. No homologies were found when the deduced amino acid sequences of the introduced proteins were compared to the sequences of known allergens.

In addition, the potential for allergenicity was assessed based upon the physiochemical properties of known food allergens, such as stability to acid and/or proteolytic digestion, heat stability, and glycosylation. The Cry3A and NPTII proteins did not demonstrate any characteristics normally associated with food allergens. Unlike known protein allergens, which are normally resistant to digestion, the Cry3A and NPTII proteins were rapidly inactivated and degraded when subjected to typical mammalian acidic stomach conditions, i.e. in simulated gastric fluids.

Abstract Collapse

Potato (Solanum tuberosum L.) is grown commercially in over 150 countries with a combined harvest of over 315 million metric tonnes in 2006. The major producers of potatoes are China, Russia, India, the United States, Ukraine, Poland and Germany. Potatoes are the fourth most important food crop in the world, providing more edible food than the combined world output of fish and meat. They are grown for the fresh and processed food industries, especially the frozen food sector. In North America, potato tubers are used primarily for French fries, chips, and dehydrated flakes. Other food uses of the crop include consumption of fresh tubers, and in the production of flour, starch and alcohol.

Colorado potato beetle (CPB; Leptinotarsa decemlineata [Say]) is the most destructive insect pest of potatoes in North America. The adult and all larval stages feed primarily on foliage and occasionally on stems. When the population of beetles is high, plants can be completely defoliated. Extensive feeding at any time during the growing season can reduce yield, as a reduction in leaf surface area decreases the plant’s ability to produce and store nutrients, which affects tuber size and number.

Commercial production of potatoes is nearly impossible without using insecticides to control CPB. Thirty-four percent of total insecticide use on potatoes is for control of CPB, more than used on any other insect potato pest. There are several insecticide classes that are available for Colorado potato beetle control including organophosphates, carbamates, pyrethroids, chlorinated hydrocarbons, insect growth regulators, chloronicotinyl, spinosads and abamectins. Colorado potato beetle has shown a tremendous ability to develop resistance to insecticides, including the arsenicals, organochlorines, carbamates, organophosphates, and pyrethroids. Cross-resistance to organophosphates and carbamates, and multiple resistance to organophosphates, carbamates, and pyrethroids has also been reported.

The transgenic cultivars of ‘Atlantic’ (ATBT04-6, ATBT04-27, ATBT04-30, ATBT04-31, ATBT04-36) and ‘Superior’ (SPBT02-5, SPBT02-7) NewLeaf® potatoes were genetically engineered to be resistant to attack by CPB. These lines were developed by introducing the cry3A gene, isolated from the common soil bacterium Bacillus thuringiensis subspecies tenebrionis (Btt), into the potato genome by Agrobacterium-mediated transformation.

The Cry3A protein expressed in these transgenic potato cultivars is identical to that found in nature and in commercial Bt spray formulations. Cry proteins, of which Cry3A is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow causing gut paralysis and eventual death due to bacterial sepsis. Cry3A is insecticidal only when eaten by the larvae of coleopteran insects such as Colorado potato beetle and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.
Over several years of field-testing, these transgenic potatoes have been compared to non-transgenic ‘Atlantic’ and ‘Superior’ cultivars for differences in physical characteristics, disease susceptibility, and insect susceptibility. The field data reports indicated no obvious differences in the number of volunteers, emergence from seed potatoes, percent stand (emergence), overwintering capacity, and tuber yield and quality. Susceptibilities to diseases, other than to CPB, including early blight, late blight, verticillium, potato leaf roll virus, and potato virus Y were unchanged. Field observations confirmed that Colorado potato beetle was controlled at all stages of development throughout the growing season and also indicated that potato flea beetles (Coleoptera: Chrysomelidae, Epitrix cucumeris Harris) were affected to some extent. Overall the field data reports and data on agronomic traits showed that the CPB-resistant potato lines did not otherwise differ from their non-transgenic counterparts.

Dietary toxicity studies were performed using the Cry3A protein on four beneficial insects (honeybee, ladybird beetle, green lacewing and parasitic wasp), and eight non-target insect species (southern corn rootworm, yellow fever mosquito, green peach aphid, European corn borer, tobacco hornworm, corn earworm, tobacco budworm and German cockroach). No negative effects were observed, except for slightly higher mortality and reduced honeydew production of green peach aphids, which as vectors of damaging potato viruses, are normally controlled in potato fields by chemical means. The transgenic Atlantic and Superior potato lines were not expected to impact on threatened or endangered species.

Generally, varieties of S. tuberosum are capable of cross-hybridization with each other, but genetic exchange with other Solanum species is usually unsuccessful. In Canada there are no tuber producing wild relatives of Solanum. In the United States, tuber-bearing Solanum species include S. jamesii, S. fendleri, and S. pinnatisectum, however, the possibility of cultivated potato crossing with these species is remote because of geographical isolation and other biological barriers to natural hybridization. No natural hybrids have been observed between these species and cultivated S. tuberosum.

Gene transfer from the transgenic Atlantic and Superior potato lines to other potato cultivars is unlikely. Since the reproductive characteristics of the CPB-resistant potato lines were unchanged by the genetic modification, they should be no different than the parent cultivars. Gene exchange between and CPB-resistant potatoes and other Solanum species is very unlikely and is limited to cross-pollination with other potatoes.

Regulatory authorities in Canada and the United States have mandatory requirements for developers of Bt potatoes to implement specific Insect Resistant Management (IRM) Programs. The potential exists for Bt-resistant CPB populations to develop as acreages planted with transgenic CPB-resistant potatoes expand. Hence, these IRM programs are designed to reduce this potential and prolong the effectiveness of plant-expressed Bt toxins, and the microbial Bt spray formulations that contain these same toxins.

Potatoes are considered to be a staple food in North America, constituting up to 37% of the total average vegetable intake. The genetic modification present in the ATBT04-6, ATBT04-27, ATBT04-30, ATBT04-31, ATBT04-36, SPBT02-5 and SPBT02-7 transgenic lines will not result in any change in the consumption pattern for potatoes. Due to their protection from CPB damage, the NewLeaf® Atlantic and Superior cultivars are expected to replace some existing commercial potato cultivars in all potato product applications. Hence, they will provide an alternate or additional choice to consumers and food manufacturers.

The major components of CPB-resistant potato lines were analyzed for nutritional constituents, proximate composition (protein, fat, ash, total dietary fibre, carbohydrate, and calories), internal quality characteristics (hollow heart and brown center, internal brown spots, vascular discoloration, and blackspot bruise), and French fry quality characteristics and compared with those of non-transgenic ‘Atlantic’ and ‘Superior’ tubers. The analysis of nutrients from each of these transgenic potato lines and from non-transgenic potatoes did not reveal any significant differences in levels of crude protein, ash, or starch. Similarly, the levels of micronutrients and trace elements, including thiamine, niacin, riboflavin, vitamin C, calcium, iron and zinc, were comparable to those of unmodified counterpart potatoes. It was concluded that the consumption of products from CPB-resistant potatoes would have no significant impact on the nutritional quality of the food supply.

The glycoalkaloids, solanine and chaconine, are naturally occurring toxicants found in potato tubers, particularly green tubers that have been exposed to sunlight. Analyses of total glycoalkaloid levels in each of the transgenic lines demonstrated that in each case the levels were within the standards previously established for potatoes.

The potential for allergenicity and toxicity of the Cry3A protein was assessed based upon the physiochemical properties of known food allergens, such as stability to acid and/or proteolytic digestion, heat stability, and glycosylation. Cry3A did not demonstrate any characteristics normally associated with food allergens. Unlike known protein allergens, which are normally resistant to digestion, Cry3A was rapidly inactivated and degraded when subjected to typical mammalian acidic stomach conditions. The amino acid sequence of the Cry3A protein was not found to be homologous with those of known protein toxins or allergens. Additionally, the Cry3A protein has a history of safe use as demonstrated by its application in microbial Bt spray formulations in agriculture and forestry for more than 30 years with no evidence of adverse effects. These facts were sufficient to provide with reasonable certainty that the Cry3A protein, and thus the transgenic Atlantic and Superior potato lines, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand


This record was last modified on Friday, March 26, 2010