GM Crop Database

Database Product Description

BT6, BT10, BT12, BT16, BT17, BT18, BT23 (NMK-89812-3, NMK-89175-5, NMK-896Ø1-8, NMK-89167-6, NMK-89593-9, NMK-899Ø6-7, NMK-89675-1)
Host Organism
Solanum tuberosum (Potato)
Trade Name
Russet Burbank 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 View
Canada 1995 1996 1996
Japan 2001 View
Korea 2004 View
Mexico 1996 1996
New Zealand 2001
Philippines 2003 2003 View
United States 1995 1995 1995

Introduction Expand

Transgenic Russet Burbank potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 (trademark NewLeaf™ ), were bioengineered to be resistant to attack by Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), a major insect pest of potatoes. These novel potato lines contain a CPB resistance gene (cry3A) that encodes an insecticidal crystalline Cry3A delta-endotoxin protein, derived from the soil bacterium Bacillus thuringiensis subsp. tenebrionis (B.t.t). Insecticidal activity is caused by the selective binding of Cry3A to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause gut paralysis, ultimately leading to bacterial sepsis and death. Delta-endotoxins, such as the Cry3A protein expressed in CPB resistant potato lines, exhibit highly selective insecticidal activity against a narrow range of coleopteran insects such as CPB, elm leaf beetle and yellow mealworm. Their specificity of action is directly attributable to the presence of specific receptors in the target insects. There are no receptors 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 1 for BT6, BT12, BT17, and BT23; 2 for BT10, BT16 Native
cry3A cry3A delta-endotoxin IR double enhanced CaMV 35S 3' poly(A) signal from pea ribulose-1,5-bisphosphate carboxylase, small subunit (rbcS) gene 1 for BT6, BT12, BT17, and BT23; 2 for BT10, BT16 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

CPB-resistant potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 were produced by Agrobacterium-mediated transformation of potato cultivar 'Russet Burbank'. The transfer-DNA (T-DNA) region of a "disarmed" form of the bacterial tumour inducing (Ti) plasmid was modified to contain genes encoding Cry3A delta-endotoxin and NPTII. As a matter of course, Ti plasmids used for plant transformation are "disarmed" such that they do not contain the virulence genes (virA) that are normally associated with the pathogenicity and disease-causing properties of A. tumefaciens.

A 5'-truncated copy of the cry3A gene, obtained from B. thuringiensis, ssp. tenebrionis (Btt), was modified by site-directed mutagenesis to enhance expression by generating plant-preferred codons. These modifications in nucleotide sequence did not alter the expected amino acid sequence of the Cry3A protein. Expression of the cry3A gene was under the regulatory control of the 35S promoter from cauliflower mosaic virus (CaMV) with a duplicated enhancer region and the 3' nontranslated termination region of a pea ribulose-1,5-bisphosphate carboxylase, small subunit (rbcS) gene. Expression of NPTII, encoded by the neo gene, was regulated by the CaMV 35S promoter and termination sequences from the nopaline synthase (nos) gene from A. tumefaciens.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from transgenic potato lines BT6, BT12, BT17, BT18, and BT23 indicated that single copies of T-DNA were integrated at a single insertion site. Analysis of B10 indicated that two copies of the cry3A gene were inserted in tandem while BT16 contained two copies inserted at separate genetic loci. The data provided showed that there was no incorporation of any coding region from outside the T-DNA borders.

Genetic Stability of the Introduced Trait

The stable inheritance of the novel traits in potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 was demonstrated over four generations of vegetative propagation.

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 inframe 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.

Expression of the B.t.t. protein Cry3A protein was quantified for the seven lines and found to average from 14.4 to 19.1 µg/g (f.w.) of leaf tissue and 0.8 µg/g (f.w.) of tuber tissue, corresponding to 0.09 to 0.12% of total foliage protein and 0.004% of total tuber protein. The mean whole plant expression for all seven lines was 6.64 µg/g (f.w.) of tissue, corresponding to 0.04% of the total plant protein.

Expression of NPTII was estimated to average 0.339 µg/g (f.w.) of leaf tissue, and 0.197 µg/g (f.w.) of tuber tissue, corresponding to 0.002% of foliage protein and 0.001% of tuber protein.

Environmental Safety Considerations Expand

Field Testing

These transgenic potatoes were field tested in the United States (1991-1994) and in Canada (1992-1995), and were compared to non-transgenic Russet Burbank 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 cultivar 'Russet Burbank'. Gene exchange between CPB-resistant potatoes and other Solanum species is very unlikely and is limited to cross-pollination with other potatoes.

Gene transfer from CPB-resistant potato lines and other potato cultivars is limited. Multiple barriers, such as male sterility in the CPB-resistant potato lines derived from the male sterile cultivar 'Russet Burbank', prevent pollen production. Cross-pollination between CPB-resistant potatoes and male-fertile potato cultivars and subsequent seed production would be very unlikely due to limited pollen dispersal in potatoes. Should such an event occur, the resultant progeny must be male fertile in order for further introgression to occur.

The chances for successful hybridization between transformed potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 and most wild relatives is extremely unlikely as potatoes (Solanum tuberosum) are unsuccessful in forming natural hybrids with the native or introduced weeds of Solanum species that do not bear tubers. 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 gene introgression is excluded due to constraints of geographical isolation and other biological barriers to natural hybridization. No natural hybrids have been observed between these species and cultivated 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 as 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 (groundskeepers) 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 effects were 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 as 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, constituting up to 37% of the total average vegetable intake. The genetic modification present in the BT06, BT10, BT12, BT16, BT17, BT18 and BT23 transgenic lines will not result in any change in the consumption pattern for potatoes. Due to their protection from CPB damage, the CPB-resistant potato lines may 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 Russet Burbank tubers. The analysis of nutrients from each of the seven transgenic potato lines and non-transgenic potato did not reveal any significant differences in the levels of crude protein, ash, and 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 Russet Burbank. There were significant differences in carbohydrate, fat and fibre although in each case the measured concentration was within the range normally reported for 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 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. Line BT17 was found to have significantly higher glycoalkaloid levels (pThe 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. Additionally, acute oral toxicity studies with purified Cry3A protein did not reveal any deleterious effects when mice were administered a dose of 5220 mg/kg body weight.

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 potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 were genetically engineered to resist CPB by producing their own insecticide. These lines were developed by introducing the cry3A gene, isolated from the common soil bacterium Bacillus thuringiensis subspecies tenebrionis (Btt), into the potato cultivar ‘Russet Burbank’ by Agrobacterium-mediated transformation. The cry3A gene produces the insect control protein Cry3A, a delta-endotoxin.
The Cry3A protein produced by BT6, BT10, BT12, BT16, BT17, BT18, and BT23 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.

Transgenic potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 were tested in field trials in the United States (1991-1994) and Canada (1992-1995). Data collected from these trials demonstrated these potato lines were not different from conventional Russet Burbank. They grew normally and exhibited the expected morphology, reproductive and physiological characteristics of potatoes. Susceptibility to diseases and insects, other than CPB, remained unchanged and the transgenic potato lines did not exhibit enhanced weediness potential.

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. BT6, BT10, BT12, BT16, BT17, BT18, and BT23 were not expected to impact on threatened or endangered species.

Generally, varieties of S. tuberosum are capable of cross-breeding 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 BT6, BT10, BT12, BT16, BT17, BT18, and BT23 to other potato cultivars is unlikely. Multiple barriers, such as male sterility, prevent pollen production in the CPB-resistant potato lines as the transgenic lines are derived from the male sterile cultivar 'Russet Burbank'. Cross-pollination between BT6, BT10, BT12, BT16, BT17, BT18, and BT23 and male-fertile potato cultivars and subsequent seed production is restricted due to limited pollen dispersal in 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.

The food and livestock feed safety of transformed potato lines BT6, BT10, BT12, BT16, BT17, BT18, and BT23 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of the seven CPB-resistant lines was found to be equivalent to conventional potatoes as shown by the analyses of key nutritional parameters including protein, fat, ash, total dietary fibre, carbohydrates, calories, and micronutrients and trace elements (e.g., thiamine, niacin, riboflavin, vitamin C, calcium, iron and zinc). There were significant differences in carbohydrate, fat and fibre although in each case the measured concentration was within the range normally reported for potatoes. It was concluded that the consumption of products from BT6, BT10, BT12, BT16, BT17, BT18, and BT23 potatoes has no significant impact on the nutritional quality of the Canadian and American 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 toxicity of the Cry3A protein was also examined: in acute oral toxicity studies with mice Cry3A was found to have no negative effects, and the amino acid sequence of Cry3A was not homologous with those of known mammalian protein toxins.

The potential for allergenicity 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. 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, combined with the lack of amino acid sequence homology between Cry3A protein and known allergens, were sufficient to provide with reasonable certainty that Cry3A has no allergenic potential.

Links to Further Information Expand


This record was last modified on Friday, February 24, 2017