GM Crop Database

Database Product Description

B, Da, F
Host Organism
Solanum lycopersicum (Tomato)
Trait
Delayed softening through suppression of polygalacturonase (PG) enzyme activity.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
Zeneca Seeds

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1996 View
Mexico 1996 1996
United States 1994 1994 1995

Introduction Expand

The tomato lines B, Da and F were developed using recombinant DNA techniques to display the trait of delayed ripening of tomato fruit. These transgenic tomatoes lines contain a partial polygalacturonase (PG) gene that encodes for the PG protein, a pectin degrading enzyme derived from tomato. The lines differ slightly in that Da and F contain the partial PG gene in the sense orientation while line B contains a partial antisense PG gene, essentially a reverse copy. The presence of the partial PG gene, in either sense or antisense orientation, suppresses the expression of endogenous PG enzyme at the onset of fruit ripening.

In the case of line B, the mechanism of action is likely linked to the hybridization of antisense and sense mRNA transcripts, resulting in a decreased amount of free positive sense mRNA available for protein translation. For lines Da and F, which contain the truncated PG gene in the sense orientation, reduced PG expression may be due to coordinate suppression of transcription of both the endogenous gene and the introduced truncated gene.

In any event, reduced PG expression decreases the breakdown of pectin and leads to fruit with slowed cell wall breakdown, better viscosity characteristics and delayed softening. Tomato lines B, Da and F have improved harvest and processing properties that allow the transgenic tomatoes to remain longer on the vine to develop their natural flavour, maintain their firmness for shipping and produce a thicker consistency in processing.

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 transgenic tomatoes. 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
PG polygalacturonase DR CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region Truncated, sense and/or antisense
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens octopine synthase 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.

Modification Method Expand

The transgenic tomato lines B, Da and F were produced via Agrobacterium-mediated transformation of the inbred tomato line TGT7 (T7) (Lycopersicon esculentum var. esculentum). This transformation system was based on the disarmed binary vector Bin19, which was engineered to contain DNA sequences encoding a partial copy of the PG gene, as well as the NPTII gene sequences. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome.

The PG encoding gene was isolated from the tomato cultivar ‘Ailsa Craig’ and was inserted into the Ti binary vector in either the sense or antisense orientation, in each case under the control of the cauliflower mosaic virus (CaMV) 35S promoter and termination sequences from the nopaline synthase gene (3’ nos) from A. tumefaciens. The NPTII encoding gene was isolated from the Tn5 transposon of E. coli and its expression was regulated using nos promoter and octopine synthase terminator sequences from A. tumefaciens.

Tomato line F was further described to include the following DNA sequence elements; the origin of replication and internal fragment of gene III from bacteriophage M13, two lacZ gene fragments from E. coli and an ocd gene (encoding ornithine cyclodeaminase) from A. tumefaciens.

Tomato line F was used to develop the tomato hybrids 1401F, H282F, 11013F and 7913F.

Characteristics of the Modification Expand

The Introduced DNA

Data from Southern blot analysis of genomic DNA from transgenic tomatoes supported the conclusion that a single copy of the T-DNA was integrated into a single site within the host genome.

Expressed Material

The transformation events B, Da and F lead to the decrease (downregulation) of endogenous PG enzyme in fruit, at the onset of ripening. The only new protein expressed was NPTII, used as a selectable marker, which was detected in raw tomatoes from two of the hybrid lines derived from transformation event F, 1401F and H282F, at levels of 2.63 ± 0.89 µg/mg and 3.46 ± 0.72 µg/mg fresh tissue, respectively, but was not detected in tomato sauce following processing of either of these hybrids.

Environmental Safety Considerations Expand

Field Testing

Tomato lines B, Da and F were field tested in the United States from 1991 to 1994. The agronomic characteristics of lines B, Da and F were evaluated extensively in laboratory, greenhouse, and field experiments. It was determined that tomato lines B, Da and F did not exhibit weedy characteristics, nor did they have any 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 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, 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. However, it would be very unlikely for traits in tomato lines B, Da and F to naturally introgress into 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 large scale cultivation of tomatoes.

It was concluded that the chance of genetic exchange among tomato crops was small and outside the species, remote. In the event that an outcrossing event involving pollen from transgenic tomato lines did occur, it was unlikely that the decrease in endogenous PG expression could affect seed persistence or weediness potential in progeny. Field testing determined that there were no changes to morphological or physiological characteristics which might affect pollination. It was determined that there was no likelihood that these modified tomato lines would gain a selective advantage to enable lines B, Da and F to become weeds or increase the weedy potential of another plant.

Weediness Potential

Tomatoes are not considered a weed pest. There was no evidence to indicate that the introduced trait, reduction in PG activity during fruit ripening, in lines Da, F, and B would convert them into weeds. Tomato volunteers are not uncommon, but are easily controlled using herbicides or by mechanical means. Seed dispersal by birds or mammals is insignificant. Furthermore, due to its tropical origin, tomato is very sensitive to temperatures below 10C 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 lines Da, F, and B would have enhanced weediness traits compared to non-transformed tomatoes.

Secondary and Non-Target Adverse Effects

The reduced levels of PG enzyme in genetically modified lines B, Da, and F should not have any toxic properties. Tomato flowers are unattractive to insect pollinators due to the limited availability of pollen. Bumblebee activity (Bombus terrestris) was found to be no different on modified and non-modified plants. Field testing determined that there were no significant differences in the susceptibility of modified and non-modified crops to pests and diseases. It was concluded that the genes inserted into transgenic tomato lines B, Da and F 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.

Impact on Biodiversity

The transgenic tomato lines B, Da and F have no novel phenotypic characteristics that would extend their 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 lines B, Da and F to species in unmanaged environments was insignificant.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of the 1401F, H282F, 11013F and 7913F hybrids will be mainly as processed tomato product in the form of soups, preserves, ketchup, paste and prepared sauces. The genetic modification of these novel hybrids will not result in any change in the consumption pattern for processed tomato products. The novel hybrids are expected to replace other tomato cultivars currently in use due to improved quality and handling characteristics. Hence, they will provide an alternate or additional choice to consumers and food manufacturers.

Nutritional Data

The analysis of nutrients from the novel 1401F hybrid line and the non-transgenic 1401 hybrid did not reveal any significant differences in the levels of macro- and micronutrients in either fresh tomatoes or processed tomato paste. 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.

An extensive analysis of fresh fruit and tomato paste samples derived from tomato line F was made and results compared to the range of values produced by non-modified, commercial tomato varieties. Results for the bulk components (soluble sugars, structural carbohydrate, moisture, ash, fat, carbohydrates including soluble sugars and dietary fibre, protein and oil) and for mineral (Na, K, Ca, Mg, P, Fe) and vitamin (A, E, B1, B2, niacin, B6, folate, C) content fell within the range expected of commercial tomato fruit and fruit purees.

Analysis of known anti-nutritional factors and potential toxins (lectins and alpha-tomatine, solanine, chaconine, tyramine, nicotine and serotonin) demonstrated that a similar range of concentrations of these compounds are present in both modified and unmodified fruit and tomato paste.

Alpha-tomatine is the principal naturally occurring glycoalkaloid in tomato, and the level of alpha-tomatine decreases as the fruit matures, so that the amounts in vine-ripened red tomatoes are negligible. Solanine and chaconine, which are the main glycoalkaloids occurring in potato, have been found in tomato in lesser amounts. The level of a-tomatine in tomato paste prepared from transgenic line F was 58 ppm as compared with 74 ppm for the unmodified TGT7 parental line. The additional glycoalkaloids, solanine and chaconine, were undetectable in either the transgenic or unmodified lines. An analysis of the levels of biogenic amines (tyramine, tryptamine and serotonin), which are considered as potential toxins in tomato, did not reveal any significant differences in the respective concentrations of these compounds in raw tomatoes from either transgenic line F or the unmodified parental line. Tomatoes from the novel hybrid lines 1401F and 282F contained undetectable levels of histamine and nicotine, as did their unmodified counterparts. Other than reduced polygalacturonase activity, the disease, pest and other agronomic characteristics of the 1401F, H282F, 11013F and 7913F hybrids were comparable to the unmodified hybrids.

Toxicity and Allergenicity

The reduced synthesis of native PG and the presence of the novel protein NPTII were not judged to have any potential for human toxicity or allergenicity. Polygalacturonase is a natural component of all food plants. The partial sense PG gene present in transformation event F was identical to the corresponding endogenous tomato gene and conferred no additional risks. The antisense PG gene did not encode for any new protein products, and therefore should not have any toxic properties.

Heat processing ensures that the enzyme NPTII does not survive in a biologically active form. Regular human consumption of tomato products containing the heat-denatured protein has not caused recognized problems relating to toxicity or allergenicity. Neither effect would have been expected as judged by comparisons of amino acid sequences made with known antigens, the published lack of effects of the intact NPTII protein in chronic toxicity studies in rats and the recorded ease of degradation of this protein in the digestive tract.

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.

In the fresh market industry the tomato fruit is often picked at the mature green or breaker stages for long-distance shipping, and is then subsequently ripened by treatment with the gas, ethylene (12 to 18 h at 20ºC). For processing tomatoes the ethylene-producing compound, ethephon or Ethrel, is applied prior to harvest when only 10% of the fruit is ripe; this accelerates and concentrates fruit ripening and facilitates once-over machine harvest.

Pectin is a building block in plant cell walls and is what gives tomatoes their firmness. Fruit softening during ripening is due to the breakdown of cell wall pectin by an enzyme called polygalacturonase (PG). Tomato lines B, Da, and F were genetically engineered to express delayed softening by inserting a truncated version of the PG encoding gene in either the sense (lines Da and F) or the “antisense” (line B) orientation. The presence of the partial PG gene, in either sense or antisense orientation, suppresses the expression of endogenous PG enzyme at the onset of fruit ripening.

In the case of line B, the mechanism of action is likely linked to the hybridization of antisense and sense messenger-RNA (mRNA) transcripts, resulting in a decreased amount of free positive sense mRNA available for protein translation. For lines Da and F, which contain the truncated PG gene in the sense orientation, reduced PG expression may be due to coordinate suppression of transcription of both the endogenous gene and the introduced truncated gene.

In any event, reduced PG expression decreases the breakdown of pectin and leads to fruit with slowed cell wall breakdown, better viscosity characteristics and delayed softening. Tomato lines B, Da and F have improved harvest and processing properties that allow the transgenic tomatoes to remain longer on the vine to develop their natural flavour, maintain their firmness for shipping and produce a thicker consistency in processing.

Tomato lines B, Da and F were field tested in the United States from 1991 to 1994. The agronomic characteristics of lines B, Da and F were evaluated extensively in laboratory, greenhouse, and field experiments. It was determined that tomato lines B, Da and F did not exhibit weedy characteristics, nor did they have any effect on non-target organisms or the general environment. The transformed tomato lines were 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 these transgenic tomato lines 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 lines did occur, it was unlikely that the delayed-ripening trait would increase the plant’s weediness or probability of survival.

The transgenic line F was used to develop the hybrid lines 1401F, H282F, 11013F and 7913F, which, along with the parental transgenic line, were evaluated in comparison with the non-transgenic parental lines during the course of the food safety assessment.

The analysis of nutrients from the novel 1401F hybrid line and the non-transgenic 1401 line did not reveal any significant differences in the levels of macro- and micronutrients in either fresh tomatoes or processed tomato paste. An extensive analysis of fresh fruit and tomato paste samples derived from tomato line F was made and results compared to the range of values produced by non-modified, commercial tomato varieties. Results for the bulk components (soluble sugars, structural carbohydrate, moisture, ash, fat, dietary fibre, protein and oil) and for mineral (Na, K, Ca, Mg, P, Fe) and vitamin (A, E, B1, B2, niacin, B6, folate, C) content fell within the range expected of commercial tomato fruit and fruit purees. Analysis of known anti-nutritional factors and potential toxins, including lectins, glycoalkaloids (alpha-tomatine, solanine, chaconine) and biogenic amines (tyramine, nicotine and serotonin), showed that similar levels were present in both modified and unmodified fruit and tomato paste.

The reduced synthesis of native PG was not judged to have any potential for human toxicity or allergenicity. Polygalacturonase is a natural component of all food plants. The partial PG gene present in the transformed tomato lines was identical to the corresponding endogenous tomato gene and did not encode for any new protein products, and therefore was not expected to have any toxic properties.

Links to Further Information Expand


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