GM Crop Database

Database Product Description

5345
Host Organism
Solanum lycopersicum (Tomato)
Trait
Resistance to lepidopteran pests including, but not limited to, cotton bollworm, pink bollworm, tobacco budworm.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 2000
United States 1998 1998 1998

Introduction Expand

Insect-resistant tomato line 5345 was developed using recombinant DNA techniques to express the insecticidal protein, Cry1Ac, encoded by the cry1Ac gene from the soil bacterium Bacillus thuringiensis subsp. kurstaki (B.t.k) strain HD73. Insecticidal activity is caused by the selective binding of Cry1Ac protein to specific sites localized on the brush border midgut epithelium of susceptible lepidopteran species. Following binding, cation-specific pores are formed that disrupt midgut ion flow thereby causing gut paralysis and eventual death from bacterial sepsis. Delta-endotoxins, such as the Cry1Ac protein expressed in tomato line 5345, exhibit highly selective insecticidal activity against a narrow range of lepidopteran pests. The 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 this transgenic tomato. 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
cry1Ac Cry1Ac delta-endotoxin IR CaMV 35S 3' non-translated region of soybean alpha subunit of beta-conglycinin gene
aad 3"(9)-O-aminoglycoside adenylyltransferase SM bacterial promoter Not expressed in plant tissues
nptII neomycin phosphotransferase II SM CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region Native

Characteristics of Solanum lycopersicum (Tomato) Expand

Center of Origin Reproduction Toxins Allergenicity

The regions of Ecuador, Peru, and the Galapagos Islands.

Almost exclusively self-pollinating; hybridization with related Solanum species (e.g., S. lycopersicoides) requires human intervention.

Glycoalkaloids, primarily alpha-tomatine, but also solanine and chaconine. Also, lectins and oxalate.

Although not a major cause of allergic reactions, several glycoproteins from tomatoes are known to be allergenic.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Bacillus thuringiensis subsp. kurstaki cry1Ac

Although target insects are susceptible to oral doses of Bt proteins, there is no evidence of toxic effects in laboratory mammals or bird given up to 10 µg protein / g body wt. There are no significant mammalian toxins or allergens associated with the host organism.

Modification Method Expand

The transgenic tomato line 5345 was produced via Agrobacterium-mediated transformation of UC82B tomato using a protocol in which the transfer-DNA (T-DNA) of the bacterial tumour inducing (Ti) plasmid was modified to contain genes encoding the Cry1Ac delta-endotoxin from B. thuringiensis subsp kurstaki, NPTII, and another enzyme used as a bacterial selectable marker, 3"(9)-O-aminoglycoside adenylyltransferase (AAD). The AAD encoding gene, which was not expressed in the plants, was used during the development process to select for bacterial colonies that had been transformed with recombinant plasmid DNA.

The nucleotide sequence of the cry1Ac gene was modified via site-directed mutagenesis to contain plant-preferred codons in order to maximize protein expression in plant cells. These modifications did not alter the predicted amino acid sequence of the Cry1Ac protein. Expression of the introduced cry1Ac gene was regulated by including promoter sequences derived from the 35S transcript of cauliflower mosaic virus (CaMV) and a 3’ non-translated region of the soybean alpha subunit of the beta-conglycinin gene. The neo gene was regulated using the nos promoter and terminator sequences from A. tumefaciens.

Characteristics of the Modification Expand

Expressed Material

The transgenic tomato line 5345 expressed two novel proteins, a truncated version of the Cry1Ac delta-endotoxin and NPTII. While the AAD encoding gene was incorporated into the plant genome, it was not expressed because the promoter sequences were only active in bacteria.

Environmental Safety Considerations Expand

Field Testing

Tomato line 5345 was field tested in the United States from 1994 to 1997. Field trials were conducted to evaluate agronomic characteristics including plant height at crop maturity, yield, maturity dates and to assess inheritance and segregation of novel traits, protein toxin expression data, and its effects on both target and non-target organisms. Comparisons with non-transformed tomato lines determined that the agronomic traits for line 5345 were well within the range of unmodified tomato lines. The only significant consistent difference between the insect resistant tomato line 5345 and the unmodified parent variety was in the expression of the insect toxic protein Cry1Ac. Field data reports demonstrated that tomato line 5345 did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

Outcrossing

Cultivated tomatoes are self-fertile, and almost exclusively self-pollinating. Their unique flower and anther morphology makes tomato an essentially cleistogamous plant (self-pollination and fertilisation occur within an unopened flower). A low crossing rate between tomato varieties was demonstrated and attributed to the limited availability of pollen and poor foraging activity of insect pollinators.

Several related species are found as weeds in tomato fields, however, tomato (Lycopersicon esculentum var. esculentum) is generally sexually incompatible with all these weedy relatives. Two Solanum species, S. lycopersicoides and S. rickii, can be crossed with commercial tomato under specific, controlled conditions, but they do not naturally cross with L. esculentum and require the intervention of man. Neither Solanum species is a weed pest in the United States.

The cherry tomato, L. esculentum var. cerasiforme can be crossed with tomato, L. esculentum var. esculentum. However, it would be very unlikely for tomato line 5345 to naturally introgress into cherry tomatoes (var. cerasiforme) in the United States since the rate of outcrossing in tomatoes is low and cherry tomatoes are not common in areas devoted to large scale cultivation of tomatoes.

It was concluded that the chance of genetic exchange among tomato crops was small and outcrossing to other species, even more remote. In the event that an outcrossing event involving pollen from transgenic tomato line 5345 did occur, it was unlikely that the lepidopteran resistance trait would increase the plants weediness or change any physiology related to survival.

Weediness Potential

Tomatoes are not considered a weed pest. There was no evidence to indicate that the introduced lepidopteran resistance trait in line 5345 would convert these tomato plants into weeds. Tomato volunteers are not uncommon, but are easily controlled using herbicides or by mechanical means. Seed dispersal by birds or mammals away from the crop is insignificant. Furthermore, due to its tropical origin, tomato is very sensitive to temperatures below 10oC and winter cold will kill the majority of volunteer seedlings following harvest. Tomatoes are not persistent in undisturbed environments without human intervention. It was concluded that there was no likelihood that tomato line 5345 would have enhanced weediness traits compared to non-transformed tomatoes.

Secondary and Non-Target Adverse Effects

It was concluded that the genes inserted into transgenic tomato line 5345 would not result in any deleterious effects or significant impacts on nontarget organisms, including those that are recognized as beneficial to agriculture and those that are recognized as threatened or endangered in the United States. The Cry1Ac toxin protein is not known to have any toxic properties against non- target organisms.

Impact on Biodiversity

The transgenic tomato line 5345 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of tomato production. Since the risk of gene transfer to wild relatives in the United States is very remote, it was determined that the risk of transferring genetic traits of tomato line 5345 to species in unmanaged environments was not significant.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of the modified tomato line 5345 will be as both fresh and processed tomato products. Fresh market tomatoes are eaten whole and sliced or diced in a variety of foods. Processed tomatoes are consumed in the form of soups, preserves, ketchup, paste and prepared sauces. The genetic modification introduced into this transgenic line will not result in any change in the consumption pattern for fresh or processed tomato products. Line 5345 is expected to replace other tomato cultivars currently in use due to improved quality and handling characteristics. Hence, it will provide an alternate or additional choice to consumers and food manufacturers.

Nutritional Data

Compositional analyses were conducted to compare transgenic line 5345 tomato fruit with fruit obtained from control non-transgenic plants grown and processed under the same conditions. The measured parameters included total solids, protein, ash, carbohydrates, calories, vitamin A, vitamin C, and folic acid. There were no statistically significant differences for any of the parameters between insect-resistant 5345 tomatoes and control tomatoes. The presence of NPTII protein was judged to be insignificant with respect to any human health risk due to exposure. The consumption of this product will, therefore, have no significant impact on the nutritional quality of the food supply in Canada and the United States.

Tomatoes produce naturally occurring glycoalkaloids, alpha-tomatine being the most common. The level of alpha-tomatine decreases as the fruit matures so that the amounts in vine-ripened red tomatoes are negligible. The concentration of alpha-tomatine in immature tomatoes from transgenic line 5345 was 5.15 mg/100g fresh weight tissue, as compared with 3.79 mg/100g fresh weight tissue for the unmodified parental line. However, these levels evened out once the fruit ripened into mature red tomatoes and were within the range of alpha-tomatine levels reported in the literature for tomatoes (4.9-90 mg/100g fresh fruit).

Toxicity and Allergenicity

The likelihood of the Cry1Ac protein being allergenic was judged to be remote. No homologies were found when the deduced amino acid sequence of the introduced protein was 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, glycosylation, and molecular weight. The Cry1Ac protein does not possess these characteristics normally associated with food allergens.

Abstract Collapse

The tomato is a vine-like herb of the nightshade family (Solanaceae) that also includes potatoes, peppers and eggplants. Botanically this vegetable is a fruit (a berry), which although being a perennial plant in the tropics, is grown as an annual plant in northern climates. The tomato is a native of the Peru, Bolivia, and Ecuador area of the Andes Mountains. Its antiquity is uncertain in regard to cultivation but it was being cultivated when Europeans discovered America. However, it was not generally cultivated in the United States until 1835 because, until then, it was widely believed to be poisonous.

Tomatoes are propagated from seeds. In temperate regions seeds are generally started in greenhouses, hotbeds, or cold frames; the plants are set out in the fields when danger of frost is past. Fresh tomatoes are harvested by hand, while those destined for canning or for processing into soups, sauces and ketchups are harvested by machine. The numerous varieties differ greatly in plant form and fruit type, the latter ranging from a small currant size through cherry, plum, and pear forms to the large, nearly round fruits, 10 cm (4 in) or more in diameter, which are the most widely grown. All forms include red- and yellow-fruited varieties.

Tomatoes are a valuable source of food minerals and vitamins, and are low in calories. One medium-sized tomato provides 57% of the recommended daily allotment (RDA) of vitamin C, 25% RDA vitamin A, and 8% RDA iron, yet it has only 35 calories. Tomatoes are also rich in an anti-oxidant called lycopene, a carotenoid that has been found to protect cells from oxidants that have been linked to cancer. In laboratory tests, lycopene was found to be twice as powerful as beta-carotene in neutralizing free radicals. Lycopene has been linked to risk reduction for a number of types of cancers, including prostate, lung and stomach, pancreatic, cervical, colorectal, oral and esophageal cancers.

Tomatoes are subject to damage from many insects, nematodes, and fungal, viral, and bacterial pathogens. Tobacco budworm (Heliothis virescens) and tomato fruitworm (Helicoverpa zea) are destructive pests of tomato. Insect-resistant tomato line 5345 was developed using recombinant DNA techniques to express the insecticidal protein, Cry1Ac, encoded by the cry1Ac gene from the soil bacterium Bacillus thuringiensis subsp. kurstaki strain HD73. Insecticidal activity is caused by the selective binding of Cry1Ac protein to specific sites localized on the brush border midgut epithelium of susceptible lepidopteran species. Following binding, cation-specific pores are formed that disrupt midgut ion flow thereby causing gut paralysis and eventual death from bacterial sepsis. Delta-endotoxins, such as the Cry1Ac protein expressed in tomato line 5345, exhibit highly selective insecticidal activity against a narrow range of lepidopteran pests. The 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.

Tomato line 5345 was field tested in the United States from 1994 to 1997. Field trials were conducted to evaluate agronomic characteristics including plant height at crop maturity, yield, and maturity dates and to assess inheritance and segregation of novel traits, as well as protein toxin expression data. Comparisons with non-transformed tomato lines determined that the agronomic traits for line 5345 were well within the range of unmodified tomato lines. The only significant consistent difference between the insect resistant tomato line 5345 and the unmodified parent variety was in the expression of the insecticidal protein Cry1Ac. Field data reports demonstrated that tomato line 5345 did not exhibit weedy characteristics, and had no effect on non-target organisms or the general environment. The transformed tomato line was not expected to impact on threatened or endangered species.

Cultivated tomatoes are self-fertile, and almost exclusively self-pollinating. Their unique flower and anther morphology makes tomato an essentially cleistogamous plant, in which self-pollination and fertilization occur within an unopened flower. A low crossing rate between tomato varieties was demonstrated and attributed to the limited availability of pollen and poor foraging activity of insect pollinators.

Several related species are found as weeds in tomato fields, however, commercial tomato is generally sexually incompatible with these weedy relatives. Two Solanum species, S. lycopersicoides and S. rickii, neither of which is a weed pest in the United States, can be crossed with commercial tomato only under specific, controlled conditions requiring human intervention. The cherry tomato, L. esculentum var. cerasiforme can be crossed with tomato, L. esculentum var. esculentum. However, it would be very unlikely for tomato line 5345 to hybridize with cherry tomatoes in the United States since the rate of outcrossing in tomatoes is low and cherry tomatoes are not common in areas devoted to the large-scale cultivation of tomatoes. It was concluded that the chance of genetic exchange among tomato crops was small and outcrossing to other species, even more remote. In the event that an outcrossing event involving pollen from the transgenic tomato line 5345 did occur, it was unlikely that the insect-resistance trait would increase the plant’s weediness.

Tomatoes are consumed in both fresh (whole and sliced or diced in a variety of foods) and processed (soups, ketchup, paste, prepared sauces) forms, and the genetic modification introduced into the transgenic tomato line was not expected to result in any changes in consumption patterns.

Compositional analyses were conducted to compare transgenic line 5345 tomato fruit with fruit obtained from control non-transgenic plants grown and processed under the same conditions. The measured parameters included total solids, protein, ash, carbohydrates, calories, vitamin A, vitamin C, and folic acid. There were no statistically significant differences for any of these parameters between insect-resistant 5345 tomatoes and control tomatoes.

Tomatoes produce naturally occurring glycoalkaloids, alpha-tomatine being the most common. The level of alpha-tomatine decreases as the fruit matures so that the amounts in vine-ripened red tomatoes are negligible. The level of alpha-tomatine present in mature tomatoes from the transgenic line 5345 was within the range reported for conventional tomato varieties.

The potential for toxicity and allergenicity of the Cry1Ac protein was assessed by an examination of its physiochemical characteristics and amino acid sequence homology to known protein toxins and allergens. Unlike known protein allergens, the Cry1Ac protein was readily digested under conditions simulating the gastric and intestinal environment, and it was denatured upon heat treatment. There were no amino acid sequence homologies with known allergens or protein toxins. The Cry1Ac protein has a history of safe use, as demonstrated by its use in microbial Bt spray formulations in agriculture for more than 30 years with no evidence of adverse effects.

Links to Further Information Expand


This record was last modified on Wednesday, February 25, 2015