GM Crop Database

Database Product Description

RBMT21-129, RBMT21-350, RBMT22-082
Host Organism
Solanum tuberosum (Potato)
Trade Name
Russet Burbank NewLeaf® Plus
Trait
Resistance to Colorado potato beetle (Leptinotarsa decemlineata, Say); resistance to potato leafroll luteovirus (PLRV).
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
Canada 1999 1999 1999
Japan 2001
Korea 2004
Mexico 2001 2001
Philippines 2004 2004
United States 1998 1998 1998

Introduction Expand

The NewLeaf® Plus potato (Solanum tuberosum) lines RBMT21-129, RBMT21-350 and RBMT22-082 were produced by bioengineering the potato cultivar Russet Burbank to be resistant to the Colorado potato beetle (CPB) (Leptinotarsa decemlineata Say.) and to resist infection by potato leafroll virus (PLRV). These transgenic lines produce a version of the insecticidal protein, Cry3A, derived from Bacillus thuringiensis subsp. Tenebrionis, and the DNA sequences corresponding to open reading frames 1 (ORF-1) and 2 (ORF-2) from PLRV. Delta-endotoxins, such as the Cry3A protein expressed in Russet Burbank NewLeaf® Plus potatoes, act by selectively binding to specific receptors 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. The Cry3A protein expressed in these potato lines exhibits highly selective insecticidal activity against a narrow range of coleopteran insects such as CPB, elm leaf beetle and yellow mealworm.

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. PLRV is a spherical RNA virus belonging to the luteovirus group and is transmitted primarily by the green peach aphid (Myzus persicae). This virus has a worldwide distribution and commonly infects potato, causing serious disease and economic loss. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. These transgenic potato cultivars exhibit the trait of resistance to infection and subsequent disease caused by PLRV through an incompletely understood process that has been termed "replicase-mediated resistance", which may involve silencing of viral gene translation. 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 lines RBMT21-129 and RBMT21-350. 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). Line RBMT22-082 contained a copy of the 5-enolypyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from A. tumefaciens strain CP4 as a selectable marker. This gene was used as an alternative to antibiotic resistance selection in order to identify transformed plants during tissue culture regeneration and multiplication.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
cry3A cry3A delta-endotoxin IR arabSSU1A: A. thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit promoter A. tumefaciens nopaline synthase (nos) 3'-untranslated region Modified to enhance expression (plant preferred codon usage)
Rep replicase (RNA dependent RNA polymerase) VR figwort mosaic virus (FMV) 35S 77-nucleotide leader sequence from soybean 17.9 kDa HSP P. sativum (pea) ribulose-1,5-bisphosphate carboxylase small subunit E9 gene 3' non-translated region
Hel helicase VR figwort mosaic virus (FMV) 35S 77-nucleotide leader sequence from soybean 17.9 kDa HSP P. sativum (pea) ribulose-1,5-bisphosphate carboxylase small subunit E9 gene 3' non-translated region
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens A. tumefaciens nopaline synthase (nos) 3'-untranslated region Native
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase SM

figwort mosaic virus (FMV) 35S

77-nucleotide leader sequence from soybean 17.9 HSP, chloroplast transit peptide (CTP2) from A. thaliana

P. sativum (pea) ribulose-1,5-bisphosphate carboxylase small subunit non-translated region

present in RBMT22-82 only

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

These transgenic Russet Burbank potato lines were created through two separate Agrobacterium-mediated transformation events in which the transfer DNA (T-DNA) contained the genes encoding the Cry3A protein from B. thuringiensis subsp. tenebrionis and the ORF-1 and ORF-2 regions from PLRV. These two ORFs encode the putative viral helicase and replicase domains that are required for viral RNA synthesis. Regulation of the cry3A gene was via the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit promoter from Arabidopsis thaliana, while the figwort mosaic virus (FMV) 35S promoter was used for PLRV derived sequences. In addition, the T-DNA contained sequences encoding the enzymes neomycin phosphotransferase II (NPTII) for events RBMT21-129 and RBMT21-350, or 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) for RBMT22-082. The respective expression of either NPTII or EPSPS activity in RBMT21 and RBMT22 transformation events was used as a selectable trait for screening transformed plants for the presence of the cry3A and PLRV replicase genes.

Characteristics of the Modification Expand

The Introduced DNA

Line RBMT21-129 - insertion of the T-DNA occurred at two sites. One insertion site contained the right border of the T-DNA as well as the PLRV replicase gene cassette, the cry3A gene and the NPTII coding region, terminating within the NOS promoter. This T-DNA insertion was not completely resolved at the left border, resulting in the partial deletion of the 5' end of the NOS promoter used to express the NPTII encoding gene. The second insert consisted of the PLRV replicase gene and a partially deleted cry3A gene cassette. The ArabSSU1A promoter of the cry3A gene, as well as a portion of the 5' coding region of the cry3A gene, were deleted upon integration into the plant genome. The partial cry3A gene was still associated with its nos 3' terminator region. Northern and western blot analysis were unable to detect any protein or mRNA from the truncated cry3A gene. This T-DNA insertion was not completely resolved at the right border, resulting in the deletion of the FMV 35S promoter as well as a portion of the 5' end of the PLRV replicase gene. Northern blot analysis confirmed that the partial PLRV replicase gene did not give rise to any detectable mRNA.

Line RBMT21-350 - insertion of the T-DNA occurred at two sites. At one site, intact copies of all three genes were inserted. At the second site, a less than full-length copy of the T-DNA was inserted resulting in a truncated copy of the PLRV replicase gene, lacking the FMV 35S promoter region. Northern blot analysis demonstrated that no detectable RNA transcript was produced from the truncated PLRV replicase gene. The remaining two genes were intact.

Line RBMT22-082 - insertion of the T-DNA occurred at three sites. All three copies of the T-DNA contained intact coding regions for the PLRV replicase gene and the cry3A gene. Two copies of the T-DNA contained an intact coding region of the CP4 EPSPS gene. At one site, however, a less than full-length copy of the CP4 EPSPS gene had been inserted. Northern analysis was not able to detect any mRNA being produced from the truncated CP4 EPSPS gene, suggesting that, as the promoter region for the truncated CP4 EPSPS gene is intact, mRNA did not accumulate to any detectable levels. At the third insertion site, DNA sequences beyond the T-DNA right border were also inserted. This DNA was adjoined to the right border of the T-DNA and contained the aad gene and the ori-322 region. This result conflicted with PCR analyses, which were unable to detect the aad gene. The failure to detect the aad gene by PCR suggested that the gene was probably not intact.

Expressed Material

Lines RBMT21-129 and RBMT21-350 expressed three novel proteins, Cry3A, the PLRV replicase, and NPTII. The Cry3A protein was expressed in tubers at levels ranging from 0.11 - 0.9 µg protein/g fresh weight (equivalent to 0.0006 to 0.005% of the total tuber protein). The production of viral replicase protein was undetectable in both leaf and tuber tissue, although other evidence suggested that it was expressed. NPTII was expressed in the tuber, but at levels below the limit of detection (0.3 ng protein/g fresh weight). The levels of expression of Cry3A protein and PLRV replicase in RBMT22-082 were equivalent to the levels determined for lines RBMT21-129 and RBMT21-350. CP4 EPSPS was expressed in RBMT22-082 tubers at levels ranging from 0.21 - 0.78 µg protein/g fresh weight (equivalent to 0.001 to 0.004% of the total tuber protein).

Environmental Safety Considerations Expand

Field Testing

These transgenic potato lines were field tested in the United States and Canada, and their agronomic characteristics were shown to be within the range of values displayed by conventional Russet Burbank. Susceptibilities to diseases such as PVY, early blight, late blight and verticillium were unchanged. Colorado potato beetle, the target insect, was controlled in test plots throughout the growing season. Field trial reports demonstrated that these potatoes did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

Outcrossing

In general, the natural exchange of genetic material from transgenic potato is only possible with other varieties of potato (Solanum tuberosum). Since the reproductive characteristics of these transgenic potatoes were unchanged by the genetic modification, they should be no different than the parent cultivar 'Russet Burbank'. The chances for successful hybridization between bioengineered potato lines 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. It was concluded that CPB and PLRV resistance were unlikely to increase weediness characteristics and that should CPB and PVY resistance genes be capable of introgression into wild species, the traits would be unlikely to provide a selective advantage sufficient to enable these hybrids to become serious weeds.

Secondary and Non-Target Adverse Effects

Studies demonstrated that Cry3A protein was not toxic to non-target organisms represented by larval and adult honeybee, ladybird beetle, green lacewing, parasitic wasp, Collembola sp., earthworm, mice and bobwhite quail. It was concluded that the genes inserted into these transgenic potato lines would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms.

Impact on Biodiversity

These 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 or the United States, there will be no transfer of novel traits to these species in unmanaged environments.

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 the potato lines described here, 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 new trait introduced into these transgenic potatoes was not expected to result in any change in the consumption pattern for potatoes. The occurrence of PLRV, including the replicase protein component, is ubiquitous in the agricultural environment and commonly occurs in the tubers of conventional potato plants. The use of replicase protein of PLRV and the genetic material necessary for its production was therefore determined not to pose a dietary risk under normal conditions. Due to their protection from CPB damage and resistance to infection by PLRV, these potato lines 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 analysis of macro- and micronutrients from these bioengineered lines revealed only small differences with the respective values from non-transgenic controls and in each case the level was within the normal range of variation reported for commercial potatoes.

Toxicity

The glycoalkaloids, solanine and chaconine, are naturally occurring toxicants found in potato tubers, particularly in green tubers that have been exposed to sunlight. The concentration of total glycoalkaloids (TGA) in transgenic tubers ranged between 4.3-17.1 mg/100g fresh weight tissue, with a mean value of 6.7 mg/100g fresh weight tissue. This was comparable to the TGA range of 2.5-16.1 mg/100g fresh weight tissue, previously measured in tubers from commercial non-transgenic cultivars of Atlantic, Gemchip, Norchip and Russet Burbank. In each case, the TGA concentration in transgenic tubers was below the administrative guideline of 20 mg/100g fresh weight that has previously been established for TGA in potato. The toxicity of the Cry3A, and NPTII proteins was previously assessed, using toxicity testing in mice, with no adverse findings. Furthermore, the amino acid sequence of the Cry3A protein expressed in these potato lines is closely related to the sequence of the same protein that is present in strains of B. thuringiensis that have been used for over 30 years as commercial organic microbial insecticides. Toxicity testing was not considered necessary for the PLRV replicase because of the long history of exposure of human beings to this protein through the consumption of PLRV-infected potatoes. An analysis of the amino acid sequences of the inserted Cry3A protein and PLRV replicase did not show homologies with known mammalian protein toxins and they are not judged to have any potential for human toxicity. Based on the evidence presented, it was concluded that all four novel proteins were non-toxic to humans.

Allergenicity

The Cry3A protein and PLRV replicase do not possess characteristics typical of known protein allergens. There were no regions of homology when the sequences of these introduced proteins were compared to the amino acid sequences of known protein allergens. Unlike known protein allergens, the Cry3A protein was rapidly degraded by acid and/or enzymatic hydrolysis when exposed to simulated gastric or intestinal fluids. It was determined that these two proteins were highly unlikely to be allergenic.

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 CPB 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. Potato leafroll virus (PLRV) is a spherical RNA virus belonging to the luteovirus group and is transmitted in a persistent manner by aphids, primarily the green peach aphid (Myzus persicae). When an aphid picks up the virus from an infected plant, the virus circulates through the aphid, entering the salivary glands. The virus is then transmitted when the aphid probes (feeds on) other potato plants. Although acquisition of the virus by an aphid is rapid (few minutes of feeding), it generally requires 24-48 hours before the aphid can transmit it to another plant. This delay provides a window of opportunity to limit virus spread through the timely application of insecticides for green peach aphid control. Aphids are the only vector of PLRV transmission and infectious aphids remain so throughout their lifespan. The symptoms of PLRV infection include rolling upward of the top leaves and the presence of net necrosis (small brown strands of discoloured tissue extending throughout the stem end of the tuber after a month in storage) in the tubers of some cultivars. The transgenic Russet Burbank NewLeaf® Plus potato lines, RBMT21-129, RBMT21-350 and RBMT22-082, were produced using recombinant DNA techniques and contain two novel genes, whose individual expression results in resistance to attack by CPB and resistance to infection by PLRV. Resistance to attack by CPB was accomplished by introducing the cry3A gene from Bacillus thuringiensis subsp. tenebrionis, which encodes an insecticidal crystalline Cry3A delta-endotoxin protein. The insecticidal activity of Cry3A protein is due to its selective binding 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. Introducing DNA sequences corresponding to the ORF-1 and ORF-2 regions from PLRV conferred resistance to PLRV infection. These two ORFs encode the putative viral helicase and replicase domains that are required for viral RNA synthesis. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. These transgenic potato cultivars exhibit the trait of resistance to infection and subsequent disease caused by PLRV through an incompletely understood process that has been termed "replicase-mediated resistance", which may involve silencing of viral gene translation. The transgenic NewLeaf® Plus potato lines were tested in field trials in the United States and Canada. Data collected from these trials demonstrated these potato lines grew normally and exhibited the expected morphology, reproductive, and physical characteristics of ‘Russet Burbank’ potatoes. Susceptibility to diseases and insects, other than CPB and PLRV, remained unchanged. Transgenic NewLeaf® Plus potato lines did not pose a plant pest risk, or 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) in addition to nontarget species such as earthworms, mice and bobwhite quail. The results demonstrated that Cry3A did not negatively affect beneficial or nontarget organisms under study. These NewLeaf® Plus potato lines were not expected to impact on threatened or endangered species. Generally, varieties of S. tuberosum are capable of crossbreeding with each other. However, transgenic NewLeaf® Plus potato lines were derived from the male sterile ‘Russet Burbank’ potato, which does not produce pollen. As a result, it is not possible for these transgenic potatoes to cross-pollinate other potato varieties or related species. As a rule, successful hybridization between potatoes and Solanum species is only possible with other tuber-bearing species. 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. 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 NewLeaf® Plus potatoes was established based on several standard criteria. As part of the safety assessment, the nutritional composition of these transgenic potatoes was found to be equivalent to conventional potatoes as shown by the analyses of key nutrients including proximates (e.g., total protein, moisture, fat, ash, crude fibre, carbohydrates and calorie content), total solids, sugars, vitamin C, soluble protein, and natural glycoalkaloid toxicants. 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 levels were within the standard levels previously established for potatoes. Neither of the Cry3A nor PLRV replicase proteins shares amino acid sequence homology with any known protein toxins. Previous studies on the acute oral toxicity of Cry3A protein have established that this protein does not result in any adverse effects when fed to laboratory mice at doses up to 5220 mg/kg body weight. Toxicity testing was not required for the PLRV replicase because of the long history of exposure of human beings to this protein through the consumption of PLRV-infected potatoes. The Cry3A and PLRV replicase proteins do not possess characteristics typical of known protein allergens. There were no regions of homology when the amino acid sequences of these introduced proteins were compared to the amino acid sequences of known protein allergens, and unlike known protein allergens, the Cry3A protein was rapidly degraded by simulated gastric or intestinal fluids. It was determined that these two proteins were highly unlikely to be allergenic.

Links to Further Information Expand


This record was last modified on Wednesday, July 31, 2013