GM Crop Database

Database Product Description
 
RBMT15-101, SEMT15-02, SEMT15-15
Host Organism
Solanum tuberosum L. L. (Potato) NewLeaf Y
 
Trait
Resistance to Colorado potato beetle (Leptinotarsa decemlineata, Say); resistance to potato virus Y (PVY).
 
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
 
Proposed Use
Production of potatoes for human consumption and livestock feed including potato process residue. All of these lines were commercialized, but only SEMT15-15 continues beyond 2001.
 
Company Information
Monsanto Company
Chesterfield Village Research Center (MO)
700 Chesterfield Parkway North
St. Louis
MO  USA
 
 
Summary of Regulatory Approvals
 
Country Environment Food and/or Feed Food Feed Marketing
Australia 2001  
Canada 1999 1999 1999  
Japan 2003  
Korea 2004  
Mexico 2001  
Philippines 2003 2003  
United States 1999 1998  
Click on the country name for country-specific contact and regulatory information.
Notes
Philippines Renewed December 22, 2008.

Introduction
 
The Russet Burbank cultivar, RBMT15-101, and Shepody potato cultivars SEMT15-02 and SEMT15-15 (NewLeaf Y ) were bioengineered to be resistant to two important potato pests, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say.) and the ordinary (O) strain of potato potyvirus Y (PVY-O).

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. Insecticidal activity is caused by the selective binding of Cry3A protein 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.

PVY is the type member of the potyvirus group and is an aphid-transmissible, rod-shaped, RNA virus that commonly infects potato, causing serious disease and economic loss. Pathogen-derived resistance to PVY was conferred by introducing the coat protein (CP) gene from the ordinary, or O-strain, of this virus. The coat protein forms a protective coat around the RNA genome of the virus and comprises 95% by mass of the virus particle. Although the exact mechanism is not fully understood, these transgenic potato lines exhibit resistance to infection and subsequent disease caused by PVY through a process that is related to viral cross-protection. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology.

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).

General Description
 
The NewLeaf-Y potato (Solanum tuberosum) lines SEMT15-02, SEMT15-15 and RBMT15-101 were developed through a specific genetic modification of cultivars Shepody and Russet Burbank to be CPB (Leptinotarsa decemlineata Say.) resistant and to resist infection by PVY. The novel lines produce a version of the insecticidal protein, CryIIIA, derived from Bacillus thuringiensis, as well as the coat protein (CP) from the ordinary (O) strain of potato virus Y (PVY-O). Delta-endotoxins, such as the CryIIIA protein expressed in Shepody and Russet Burbank NewLeaf-Y 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 paralysis and death. CryIIIA and related endotoxins are insecticidal only to lepidopteran or coleopteran insects and 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. PVY is the type member of the potyvirus group and is an aphid-transmissible RNA virus that 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. The genetically modified potato cultivars exhibit the trait of resistance to infection and subsequent disease caused by PVY through a process that is related to viral cross-protection.

The transgenic Shepody and 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 CryIIIA protein from B. thuringiensis subsp. tenebrionis and the CP from PVY-O. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII). The expression of NPTII activity was used as a selectable trait for screening transformed plants for the presence of the cryIIIA and PVY-O CP genes. Additional DNA outside of the T-DNA border sequences was incorporated into the genome of Shepody lines SEMT15-02 and SEMT15-15. These lines also contain the aad gene that encodes the enzyme 3'(9)-O-aminoglycoside adenylyltransferase, which confers bacterial resistance to spectinomycin and streptomycin. The aad gene was not expressed in plant tissue, but was present on the Ti plasmid as a selectable trait for screening bacterial colonies for the
presence of plasmid vector. The NewLeaf-Y Shepody and Russet Burbank cultivars produce three novel proteins: CryIIIA, PVY-O CP and NPTII.

The constitutive expression of CryIIIA protein was demonstrated in each of the transgenic NewLeaf-Y Shepody and Russet Burbank cultivars. On average, the amounts of CryIIIA protein produced in the leaves and tubers of SEMT15-02, SEMT15-15 and RBMT15-101 were comparable to the concentrations previously reported for NewLeaf Atlantic and Russet Burbank cultivars. The expression of PVY-O CP in either leaves or tubers from any of these transgenic lines was undetectable at a threshold of 2 ng/mg fresh weight tissue. In contrast, the accumulation of CP in plants naturally infected with PVY-O is readily detectable using enzyme linked immunosorbent assay (ELISA) or Western immunoblot analysis. It is not uncommon for commercial potato plantings to be significantly infected by PVY-O and thus the human
consumption of viral CP from these sources is likely to be much higher than the exposure due to
consumption of NewLeaf-Y potatoes. The presence of NPTII protein has been judged to be
insignificant with respect to any human health risk due to exposure. Solanine and chaconine are the principal glycoalkaloids commonly found in potato tubers. 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. Other than resistance to CPB and infection by PVY, the disease, pest and other agronomic characteristics of the NewLeaf-Y Shepody and Russet Burbank lines were comparable to their non-transgenic parental cultivars.
Reference: Office of Food Biotechnology, Health Canada

Summary of Introduced Genetic Elements
 
Code Name Type Promoter, other Terminator Copies Form
cry3A cry3A delta-endotoxin  (Bacillus thuringiensis subsp. Tenebrionis) IR arabSSU1A: A. thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit promoter
NULL
A. tumefaciens nopaline synthase (nos) 3'-untranslated region   Modified to enhance expression (plant preferred codon usage)
nptII neomycin phosphotransferase II  (Escherichia coli) SM nopaline synthase (nos) from A. tumefaciens
NULL
A. tumefaciens nopaline synthase (nos) 3'-untranslated region   Native
aad 3"(9)-O-aminoglycoside adenylyltransferase  (NULL) SM bacterial promoter
NULL
NULL   Not expressed in plant tissues
CP viral coat protein  (potato potyvirus Y (PVY) strain O (common strain)) VR figwort mosaic virus (FMV) 35S
;leader sequence from the Glycine max heat-shock protein, Hsp 17.9
3' poly(A) signal from pea ribulose-1,5-bisphosphate carboxylase, small subunit (rbcS) gene   Native

Characteristics of Solanum tuberosum L. (Potato)
 
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
 
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
 
The transgenic potato lines RBMT15-101, SEMT15-02 and SEMT15-15 were produced by Agrobacterium-mediated transformation of the potato cultivars 'Russet Burbank' and 'Shepody', respectively. 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, PVY-O coat protein, and NPTII. This plasmid was designated PV-STMT15. 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) strain BI 256-82, 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. Constitutive expression of the cry3A gene was controlled by the promoter region of the ribulose-1,5-bisphosphate carboxylase small subunit gene from Arabidopsis thaliana and the terminator sequence from the nontranslated 3' region of the nopaline synthase (nos) gene from A. tumefaciens.

During normal virus replication, the PVY-O CP is derived from a large poly-protein precursor by proteolytic cleavage. In order to express the PVY-O CP transgene in plant cells, the cloned complementary-DNA sequence corresponding to the CP gene was modified to contain a translation start codon (ATG). Expression of the PVY-O CP gene was under the control of the 35S promoter region of figwort mosaic virus, the leader sequence from the Glycine max (soybean) heat-shock protein, Hsp 17.9, and the terminator sequence from the nontranslated 3' region of the pea (Pisum sativum) ribulose-1,5-bisphosphate carboxylase small subunit gene E9 3'.

Expression of NPTII, encoded by the neo gene, was regulated by promoter and termination sequences from the nopaline synthase (nos) gene.

In addition to the plant expressible genes described above, plasmid PV-STMT15 also contained the following non-expressed elements: (1) oriV , which is the origin of replication from plasmid RK2 isolated from Agrobacterium strain ABI; (2) ori-322/rop , which is a segment of plasmid pBR322 which provides the origin of replication for maintenance of PV-STMT15 in Escherichia coli [rop] and the bom site, which enables conjugal transfer into Agrobacterium; (3) the aad gene from transposon Tn7 of E. coli, which encodes the enzyme 3"(9)-O-aminoglycoside adenylyltransferase and confers resistance to the antibiotics streptomycin and spectinomycin; and (4) the left (LB) and right (RB) sequences, which facilitate integration of the T-DNA into the plant host genome. Inclusion of the selectable marker aad gene, which was only expressed in bacterial cells, facilitated the isolation of bacterial colonies that had been transformed with recombinant plasmid DNA.

Characteristics of the Modification
 
The Introduced DNA
Line RBMT15-101 - insertion of the T-DNA occurred at three to four loci. At least one locus contained two copies of the T-DNA organized in inverted orientations. For two copies of the T-DNA, transfer was incomplete at the right border resulting in an incomplete copy of the figwort mosaic virus (FMV) 35S promoter associated with the PVY CP gene. One of the cry3Aa genes also lacked the Arabidopsis small subunit promoter and a portion of the 5' end of the gene. The NOS terminator region of this gene cassette was intact. One of the T-DNAs also had an incomplete NOS promoter region associated with an intact NPTII coding region. The coding regions of all the other genetic elements were intact. The analyses also showed that no plasmid sequences beyond the left and right borders were transferred.

Line SEMT15-02 - insertion of the T-DNA occurred at four to five loci. At least one locus contained two copies of the T-DNA organized in inverted orientations and one locus contained two T-DNAs linked by a complete copy of the plasmid backbone. For seven copies of the T-DNA, transfer of the T-DNA resulted in incomplete resolution of the right border, leaving incomplete copies of the FMV promoter associated with the PVY CP coding region. Plasmid sequences beyond the left and right borders, which include the aad gene and the oriV and ori322 plasmid elements, were inserted into this line. Integration of complete backbone elements occurred in two different ways: at one locus two T-DNAs were linked by a complete copy of the backbone; at two other loci, backbone integration was not associated with the left border flanking the NOS promoter of the NPTII encoding gene.

Line SEMT15-15 - insertion of the T-DNA occurred at four to five loci. At least one locus contained copies of the T-DNA organized in inverted orientations. For two copies of the T-DNA, transfer of the T-DNA resulted in incomplete resolution of the right border, leaving incomplete copies of the FMV promoter associated with the PVY CP coding region. One of the T-DNAs in this line had an incomplete NOS promoter region associated with an intact NPTII coding region. The coding regions of all the genetic elements were intact. Plasmid sequences beyond the left and right borders, which include the aad gene and the oriV and ori322 plasmid elements, were inserted into this line.

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 RBMT15-101, SEMT15-02 and SEMT15-15 potatoes.

The levels of expressed Cry3A protein were quantitated using enzyme linked immunosorbent assay (ELISA) of samples of leaf tissue from field grown plants and were found to range between 20-63 ?/g fresh weight tissue. Concentrations of Cry3A in tuber tissue (0.08 - 0.38 ?/g) were approximately 100 fold lower than that present in leaf tissue, equivalent to 0.0005 - 0.0019% of total tuber protein.

The transgenic expression of PVY CP was estimated to be less than the detectable limit of 2 ng/mg fresh leaf tissue. In contrast, the accumulation of PVY-O coat protein in naturally virus-infected potato plants was approximately 12 to 244-fold greater, in the range of 24-488 ng/mg fresh leaf tissue when measured by ELISA. NPTII was expressed in the tuber at levels, ranging from 0.003 - 0.01 ?/g fresh weight (equivalent to < 0.001% of the total tuber protein).

Environmental Safety Considerations
 
Field Testing
Potato lines RBMT15-101, SEMT15-02 and SEMT15-15 were field tested in the United States (1994-1998) and Canada. Field evaluations determined that vegetative vigour, overwintering capacity, insect and disease resistance (except for resistance to CPB and PVY), tuber yield and quality characteristics remained unchanged in comparison to commercial varieties.

Outcrossing
In general, the natural exchange of genetic material from potato is only possible with other varieties of potato (Solanum tuberosum). Since the reproductive characteristics of these transgenic potato lines were unchanged by the genetic modification, they should be no different than the respective parent cultivars 'Russet Burbank' and 'Shepody'.

The chances for successful hybridization between the transgenic potato lines and most wild relatives is extremely unlikely as potatoes 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 in the United States.

Field studies have shown that CPB and PVY resistance does not appear to be associated with increased weediness characteristics. Therefore, should CPB and PVY resistance genes be capable of introgression from these transgenic potatoes into wild species, the traits would be unlikely to provide a selective advantage sufficient to enable these hybrids to become serious weeds.

Weediness Potential
Resistance to CPB and PVY infection will not, in itself, render potatoes weedy or invasive of natural habitats, since none of the reproductive or growth characteristics have been modified. Field data reports indicated no obvious differences in traits likely to provide a selective advantage such as the number of volunteers, emergence from seed potatoes, and disease and insect susceptibility (other than to target pests, CPB and PVY). Traditional resistance genes for CPB and PVY, and resistance genes for other insect pests, have been identified in potatoes and other Solanum species with no reports of increased weediness potential.

Potatoes do not exhibit weediness characteristics and have difficulty becoming established outside cultivated fields. Small tubers left in the ground after harvest 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
It was concluded that the genes inserted into the transgenic potato lines RBMT15-101, SEMT15-02 and SEMT15-15 would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms. The plant-pesticide Cry3A was previously approved in several CPB-resistant potato plants. Prior studies on the toxicity of Cry3A to mammals, allergenicity, and environmental fate data on the impact to avian species, nontarget and beneficial insects, honeybees and nontarget organisms, determined that Cry3A would not negatively impact non-target organisms or effect threatened and endangered species. Similarly, the PVY CP is found in all PVY-infected plants, and there are no reports of this protein (or PVY-infected plants) having any toxic effects. The NPTII protein is ubiquitous in the environment and was determined not to have potential for toxicity.

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, there will be no transfer of novel traits to related species in unmanaged environments. Similarly, as the potential for 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 RBMT15-101, SEMT15-02 and SEMT15-15 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 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
 
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 transgenic potato lines RBMT15-101, SEMT1502 and SEMT1515 will not result in any change in the consumption pattern for potatoes. Due to their protection from CPB damage resistance and to infection by PVY, these 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 analysis of macro- and micronutrients from transgenic potato 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. It was concluded that the consumption of products from CPB-resistant potatoes has no significant impact on the nutritional quality of the Canadian and American food supply.

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

The amino acid sequence of the Cry3A protein expressed in these potatoes 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 sequences of the inserted Cry3A and PVY CP did not show homologies with known mammalian protein toxins and they were not judged to have any potential for human toxicity. The history of known safe consumption of PVY CP from virus-infected plant products provides additional evidence of lack of toxicity.

Allergenicity
The Cry3A protein and PVY CP do not possess characteristics typical of known protein allergens. There were no regions of homology when the deduced amino acid 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. The Cry3A protein and PVY CP are extremely unlikely to be allergenic.

Links to Further Information
 
Australia New Zealand Food Authority[PDF Size: 446007 bytes]
Draft risk analysis report: A384 - Food derived from insect and potato virus Y protected potato lines RBMT15-101, SEMT15-02, and SEMT15-15
Canadian Food Inspection Agency, Plant Biotechnology Office[PDF Size: 32341 bytes]
Decision Document DD2002-37: Determination of Environmental Safety of SEMT15-02, SEMT15-15, and RBMT15-101 Colorado Potato Beetle and Potato Virus Y Resistant Potato Lines Developed by Monsanto Canada Inc.
Monsanto Company[PDF Size: 108916 bytes]
Product safety description
Office of Food Biotechnology, Health Canada[PDF Size: 64478 bytes]
NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGY COLORADO POTATO BEETLE AND POTATO VIRUS Y RESISTANT POTATO LINES SEMT15-02, SEMT15-15, RBMT15-101
U.S.Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 21266569 bytes]
Monsanto Co. Petition for Determinaiton of Regulatory Status for NewLeaf Y Potato Lines RBMT15-101, SEMT15-02, and SEMT15-15
US Code of Federal Regulations Notice[PDF Size: 21657 bytes]
Monsanto Co.; Availability of determination of nonregulated status for potato genetically engineered for insect and virus resistance.
US Food and Drug Administration[PDF Size: 346569 bytes]
Memorandum to file concerning the industry consultation on CPB and PVY resistant potatoes
USDA-APHIS Environmental Assessment[PDF Size: 82544 bytes]
Monsanto Petition 97-339-01p for a Determination of Nonregulated Status for Transgenic Potato Lines Resistant to Colorado Potato Beetle and Potato Virus Y

References
 
Betz, F.S., Hammond, B.G. & Fuchs, R.L. (2000). Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regulatory Toxicology 32, 156-173.
Lawson, E.C., Kaniewski, W., Haley, L., Rosman, R., Newell, C., Sanders, P., Turner, N. (1990). Engineering resistance to mixed virus infection in a commercial potato cultivar: Resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Bio/Technology 8: 127-134.
Rogan, G.J., Bookout, J.T., Duncan, D.R., Fuchs, R.L., Lavrik, P.B., Love, S.L., Mueth, M., Olson, T., Owens, E.D., Raymond, P.J. & Zalewski, J. (2000). Compositional analysis of tubers from insect and virus resistant potato plants. J. Agric. and Food Chem. 48, 5936-5945.
US Food and Drug Administration (1994). Secondary Food Additives Permitted in Food for Human Consumption; Food Additives Permitted in Feed and Drinking Water of Animals; Aminoglycoside 3'-Phosphotransferase II; Final Rule. Federal Register, 59:26700-26711.


THIS RECORD WAS LAST MODIFIED ON WEDNESDAY, SEPTEMBER 25, 2002
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