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DD2002-36: Determination of Environmental Safety of RBMT22-082 Colorado Potato Beetle and Potato Leaf Roll Virus Resistant Potato Line Developed by Monsanto Canada Inc.

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Issued: 1999-04

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08: Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document T-1-09-96: The Biology of Solanum tuberosum L. (potato), and the guidelines Dir95-03: Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office (PBO) of the Plant Health and Production Division, and the Feed Section of the Animal Health and Production Division, have evaluated information submitted by Monsanto Canada Inc. regarding RBMT22-082 (also known as NewLeaf™ Plus). These plants were modified to express resistance to Colorado potato beetle and potato leaf roll virus. The CFIA has determined that these plants with novel traits (PNTs) should pose no concerns with respect to environmental safety or the safety to livestock consuming feed derived from the PNTs, and are considered substantially equivalent to potato products currently approved for commercial production in Canada.

Unconfined release into the environment and use as livestock feed of the potato line RBMT22-082 are therefore authorized as of April 20, 1999. Any other Solanum tuberosum lines and intraspecific hybrids resulting from the same transformation event and all their descendants are also approved, provided that: i) no inter-specific crosses are performed; ii) the intended uses are similar; iii) it is known, based on characterization, that these plants do not display any additional novel traits and are substantially equivalent, in terms of their specific use and safety for the environment and for human and animal health, to potato currently being cultivated; iv) the novel genes are expressed at a level similar to that of the authorized line; and v) pest resistance management requirements described in the present document are applied.

Table of Contents

  1. Brief Identification of the Plants with Novel Traits (PNTs)
  2. Background Information
  3. Description of the Novel Traits
    1. Colorado Potato Beetle Resistance
    2. Potato Leaf Roll Virus Resistance
    3. Glyphosate Tolerance
    4. Development Method and Stability of the Trait
  4. Assessment Criteria for Environmental Safety
    1. Potential of the PNT to Become a Weed of Agriculture or Become Invasive of Natural Habitats
    2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential
    4. Potential Impact on Non-Target Organisms
    5. Potential Impact on Biodiversity
  5. Potential for Development of CPB Resistance to the PNTs
  6. Nutritional Assessment Criteria for Use as Livestock Feed
    1. Nutritional Composition and Proximate Analyses of the PNTs
    2. Anti-Nutritional Factors
  7. Regulatory Decision

I.  Brief Identification of the Plants with Novel Traits (PNTs)

Designation(s) of the PNT: RBMT22-082

Applicant: Monsanto Canada Inc.

Species: Potato (Solanum tuberosum L.)

Novel Traits: Colorado potato beetle and potato leaf roll virus resistance, and glyphosate tolerance

Trait introduction method: Agrobacterium tumefaciens-mediated transformation

Intended use of the PNT: Production of potatoes for human consumption (table and processing) and livestock feed, including potato processing residue. These PNTs will not be grown outside the usual production area for potatoes in Canada.

II.  Background Information

Monsanto Canada Inc. RBMT22-082 potato lines were developed by transforming the parental line Russet Burbank (RB) with three genes: 1. the cry3A gene from Bacillus thuringiensis (Bt) which confers resistance to Colorado potato beetle (CPB); 2. the open reading frames (ORF) 1 and 2 of the potato leaf roll virus (PLRV) replicase, PLRVrep, which confers resistance to the virus; and the CP4 EPSPS gene which encodes the enzyme 5-enolpyruvylshikimate-3-phosphate synthase and confers tolerance to the herbicide glyphosate. The CP4 EPSPS gene was used as a selectable marker to facilitate the selection of transformed cells in the laboratory. This line has been tested in Canada under confined conditions in British Columbia (1998-99), Alberta (1997-99), Saskatchewan (1998-99), Manitoba (1995-99), Ontario (1997-99), Quebec (1997-99), New Brunswick (1995-99) and Prince Edward island (1995-99).

Colorado potato beetle (Leptinotarsa decemlineata Say.) is the most damaging insect pest of potato in Canada and, to date, no traditionally bred cultivars have been produced that are resistant to CPB. Both larval and adult stages of CPB feed on potato foliage and, when left unchecked, can completely defoliate potato plants resulting in yield reductions of as much as 85%. Current control of CPB is primarily achieved through the use of insecticides that are variably effective depending on insect sensitivity and environmental factors. Crop rotation, vacuum suction, propane flaming, polyethylene-lined trenches and trap plots are alternative but less effective management strategies for CPB.

Potato leaf roll virus, a luteovirus, is the causal agent of potato leaf roll disease. PLRV infects potatoes in most geographical locations where this crop is grown, resulting in yield losses in some cultivars of up to 75%. PLRV produces net necrosis, or death of the vascular tissues in the tuber, causing the tubers to discolour and consequently be rejected by potato processors. PLRV is introduced in a field by planting infected seed or by its natural vector, the green peach aphid (Myzus persicae.) PLRV is not transmitted mechanically and so in-field spread is via the insect vector. PLRV is managed through the use of virus-free seed stocks and the use of insecticides to control aphids.

Monsanto Canada Inc. has characterized the host and donor organisms, the plasmid vectors, the inserted genes and gene products including the number of insertion sites and copy numbers for RBMT22-082. The novel proteins were identified, characterized, and compared to the original bacterial and viral proteins, including their potential toxicity to livestock and non-target organisms with particular attention given to beneficial arthropods. Agronomic performance was evaluated by measuring yield and phenotypic changes to foliage or tuber. Stress adaptation was evaluated, including susceptibilities to various potato pests and pathogens including potato (poty) virusY (PVY), early and late blight, verticillium, rusts, leaf spot, aphids, leafhoppers, mites and cutworms.

The environmental consequences of the introduction of RBMT22-082 was evaluated in accordance with the following assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

The CFIA has consulted with the Pest Management Regulatory Agency of Health Canada on issues related to potential development of CPB populations resistant to the insecticidal protein produced by plants transformed with Bt genes. The CFIA requires that Monsanto Canada Inc. implement an insect resistance management plan to mitigate against the potential development of CPB resistance to the Cry3A insecticidal protein.

Health Canada has determined that food derived from these potatoes is substantially equivalent to that derived from currently commercialized potatoes (May, 1999).

The Feed Section of the Animal Health and Production Division, CFIA, has assessed the feed safety of RBMT22-082 in accordance with the following criteria for determining safety and efficacy of livestock feed, as described in Dir95-03:

Nutritional composition and proximate analyses of tubers were performed. Data to support the suitability of RBMT22-082 potato line as livestock feed was provided.

III.  Description of the Novel Traits

The transgenic Russet Burbank potato line RBMT22-082 was created through two separate Agrobacterium-mediated transformation events in which the transfer DNA (T-DNA) contained the genes cry3A and PLRVrep encoding the Cry3A delta endotoxin protein from B. thuringiensis subsp. tenebrionis and the ORF-1 and ORF-2 regions from PLRV, respectively. These two ORFs encode the putative viral helicase and replicase domains that are required for viral RNA synthesis. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII). The expression of NPTII activity in the RBMT22-082 transformation event was used as a selectable trait for screening transformed plants for the presence of the cry3A and PLRVrep genes. There was no incorporation of plant translatable plasmid DNA sequences outside of the T-DNA region and stable inheritance of the novel traits was demonstrated over multiple generations of vegetative propagation.

1.  Colorado Potato Beetle Resistance

Delta-endotoxins, such as the Cry3A protein, expressed in the RBMT22-082 transformation event 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. Cry3A is insecticidal only to Coleopteran insects and its 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.

2.  Potato Leaf Roll Virus Resistance

PLRV is a spherical RNA virus belonging to the luteovirus group and is transmitted primarily by the green peach aphid. 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. The genetically modified potato cultivars exhibit the trait of resistance to infection and subsequent disease caused by PLRV by interfering with the replication of the virus by a mechanism that has been termed replicase-mediated resistance.

3.  Glyphosate Tolerance

A microbial derived gene (CP4 EPSPS) which imparts field level tolerance to glyphosate, the active ingredient in Roundup™ herbicide, was introduced into RBMT22-082. A plant-derived coding sequence expressing an optimized chloroplast transit peptide was fused to the microbial EPSPS. This peptide facilitates the import of the newly translated EPSPS enzyme into the chloroplast, the site of both the shikimate pathway and glyphosate mode-of-action. The inserted gene codes for 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimic acid metabolic pathway for the production of the aromatic amino acids. The herbicide disrupts this essential pathway, leading to growth suppression or death of the plant. The introduced enzyme is glyphosate tolerant and continues to catalyze the production of aromatic amino acids in the presence of glyphosate. The expression of the novel enzyme in the plant was quantified by enzyme-linked immunosorbent assay (ELISA). The CP4 EPSPS gene was used as a selectable marker to facilitate the selection of transformed cells in the laboratory.

Roundup™ herbicide is not authorized for use in the production of potatoes in Canada and thus the herbicide will not be used in the field.

4.  Development Method and Stability of the Trait

The constitutive expression of Cry3A protein was demonstrated in the transgenic Russet Burbank cultivars at levels comparable with previously approved NewLeaf™ potato cultivars. The production of viral replicase protein in RBMT22-082 was undetectable in either leaf or tuber tissue, and is, therefore, likely to be less than for plants naturally infected with PLRV. The level of expression of CP4-EPSPS in all plant tissues from RBMT22-082 was comparable to the amounts measured in previously approved novel plants expressing this protein.

IV.  Assessment Criteria for Environmental Safety

1.  Potential of the PNT to Become a Weed of Agriculture or Become Invasive of Natural Habitats

The CFIA evaluated data submitted by Monsanto Canada Inc.on the reproductive and survival biology of the potato line RBMT22-082 and determined that vegetative vigour, overwintering capacity, insect and disease susceptibility (other than to CPB and PLRV), and tuber yield and quality, were within the normal range of expression currently displayed by commercial varieties. RBMT22-082 has no specific added genes for cold tolerance or winter hibernation.

The biology of Solanum tuberosum, described in T-1-09-96, is such that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by Monsanto Canada Inc. RBMT22-082 was determined not to be different from its counterpart in this respect. No competitive advantage was conferred to RBMT22-082 other than resistance to CPB and PLRV, and glyphosate tolerance. These traits would not be expected to render these potatoes weedy or invasive of natural habitats as the typical vegetative reproductive characteristics of these potatoes were not modified. Although limited distribution is possible through dispersal of tubers, volunteers will not persist in cultivated habitats in the presence of normal agronomic practices or compete in uncultivated habitats.

Based on the above considerations, the CFIA has concluded that RBMT22-082 potatoes have no altered weed or invasiveness potential compared to currently commercialized potato varieties.

2.  Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

There are no wild relatives in Canada that can naturally hybridize with S. tuberosum (see T-1-09-96) and the novel traits have not altered the inability of RBMT22-082 to outcross. The CFIA therefore concludes that gene flow from RBMT22-082 to relatives of S. tuberosum is not possible in Canada.

3.  Altered Plant Pest Potential

The intended effects of the novel traits in RBMT22-082 are unrelated to plant pest potential. The agronomic characteristics of RBMT22-082 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, leading to the conclusion that plant pest potential was not inadvertently altered.

Recombination:  Recombination is defined as an exchange of nucleotide sequences between two nucleic acid molecules. Recombination between viral genomes results in heritable, permanent change. The persistence of a recombined viral genome will depend upon its fitness with respect to its ability within the original host cell, its ability to replicate in the presence of parental viruses, its ability to spread systemically within the host, or its successful transmission to other host plants. Based upon the following information provided by Monsanto Canada Inc., the CFIA concluded that viral recombination of PLRV replicase gene and other viral RNAs is extremely unlikely to occur and thus minimal plant pest risk; the transgenic mRNA lacks the 5 and 3 ends of the PLRV genome, and does not contain any primary sequence homology to any other potential plant viral replicase binding region; since the transgenic replicase mRNA will not serve as a template for any known viral replicase, the likelihood of recombination is extremely unlikely because replication is required for recombination.

Synergism: Synergism is noted when two viruses simultaneously naturally infect a plant and the symptoms are more severe than when either of the viruses infects the plant singly. Synergy was first described and is best studied with natural infections of potato (potex) virus X (PVX) and potato (poty) virus Y (PVY). Monsanto did not observe synergistic symptoms during field testing and seed certification programs.

Transencapsidation:  When a single plant cell is simultaneously naturally infected by two different strains of a virus (or two viruses), it may be possible for the genome of one virus to become encapsidated by coat protein of the second virus. If the virus is encapsidated by only one of the coat proteins, it is termed genomic masking or transencapsidation. Any changes are not inherited if such transencapsidated virions move to another host, so any effects are transient and pose no plant pest risk. The transgenic PLRV resistant potatoes contain the replicase gene from a naturally occurring PLRV isolate and do not contain any part of the PLRV coat protein gene; therefore, transencapsidation is not a plant pest risk.

Given the above, and that potato has no history as a plant pest in Canada, the CFIA has concluded that RBMT22-082 and RBMT22-082 do not display any altered pest potential.

4.  Potential Impact on Non-Target Organisms

Colorado potato beetle, the target insect, was controlled in test plots of RBMT22-082 throughout the growing season. Monsanto Canada Inc. demonstrated that Cry3A protein is 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.

The novel proteins were rapidly inactivated in simulated mammalian stomach fluids by enzymatic degradation and pH-mediated proteolysis. The proteins expressed in RBMT22-082 potatoes were shown to be equivalent to the native proteins. As previously stated, 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.

Screening the sequence of the expressed proteins against a database of known allergens revealed no significant homology to any known allergenic protein.

Based on the above, the CFIA has concluded that the unconfined release of RBMT22-082 will have no significant impact on non-target organisms, including humans.

5.  Potential Impact on Biodiversity

RBMT22-082 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of potato production in Canada. Since potato does not outcross to wild relatives in Canada, there will be no transfer of novel traits to unmanaged environments.

The use of RBMT22-082 potato plants could reduce the need for the insecticidal treatment of CPB and aphids, resulting in a reduction of chemicals released into the environment, an increase of non-target insect populations, and an increase in potential for the biological control of harmful insect pests. The CFIA has therefore concluded that the potential impact of RBMT22-082 on biodiversity would be insignificant and may be positive.

V.  Potential for Development of CPB Resistance to the PNTs

The potential for CPB to develop resistance to conventional chemical insecticides is well documented, and many of the chemical foliar sprays currently registered for CPB control are no longer effective. Bt foliar insecticides containing the Cry3A protein are registered in Canada for use on both potatoes and tomatoes. To date, resistance of CPB to Bt sprays has not been observed under field conditions. Resistance could develop as a result of increased use of Bt foliar sprays or as a consequence of the widespread adoption of Bt potatoes such as RBMT22-082.

RBMT22-082 potatoes produce high levels of Cry3A protein in all plant tissues throughout the growing season, resulting in extremely high mortality in CPB feeding on these plants. Target insects are consequently exposed to significantly higher levels of Bt than with the current foliar spray treatments, leading to high selection pressure for resistant CPB. In order to mitigate the eventual development of Cry3A resistant CPB, resistance management plans have been developed and implemented. A critical component of accepted resistance management strategies is the mandatory planting of refugia B proximal fields of non-Bt potatoes where CPB populations susceptible to Cry3A are maintained. Any resistant CPB that survive in Bt potato fields will be very rare and it is presumed will mate with susceptible CPB to produce heterozygotes susceptible to Cry3A. The efficacy of the high dose-refugia strategy for managing the advent of Cry3A resistant CPB has not been conclusively determined. All Bt potato lines should therefore be responsibly managed and CPB populations monitored for development of resistant individuals in a regular and consistent manner.

The CFIA believes that sound management practices can delay the development of resistant CPB populations. The resistance management plan for Bt potatoes to be implemented by Monsanto Canada Inc. must include, but is not limited to, the criteria outlined below:

  1. Monsanto Canada Inc. must ensure that each farm planted to NewLeaf™ Plus potatoes has no more than 80% of its farm acreage as NewLeaf™ potatoes (NewLeaf Plus potatoes and all other NewLeaf™ Plus potatoes transformed with Bt genes) and leaves at least 20% of potato acres as a refuge for CPB.
  2. Monsanto Canada Inc. must ensure that refuges for CPB are managed in a manner which supports a viable population of CPB sufficient to maintain the functionality of the refuge as a reservoir of Bt-susceptible beetles. The use of insecticides which effectively eliminate CPB from a refuge is not compatible with the management of a refuge.
  3. Education tools must be developed and provided to all growers and company field personnel. These will include information on product performance, resistance management, monitoring procedures and timetables, detection protocols for resistant CPB individuals, instructions to contact Monsanto Canada Inc. when support/information is required, and strategies to be followed if unexpected levels of CPB damage occur.
  4. Monsanto Canada Inc. will have procedures in place for responding to any reported instances of unexpected CPB damage. These procedures will include, where warranted, the collection of plant tissue and CPBs, and use of appropriate bioassays to evaluate suspected Cry 3A resistant individuals, and a protocol for immediate action to control resistant individuals.
  5. Detection of confirmed resistant CPB populations will immediately be reported to the Plant Biosafety Office, CFIA, and a procedure for control of resistant individuals must be ready for immediate implementation.
  6. Integrated Pest Management practices must be promoted, such as crop rotation and alternative control measures for CPB.
  7. Continued research in the area of resistance management of CPB using sound science will be conducted and the results will be made available to the CFIA.

The CFIA reserves the right to ask for modifications to any resistance management plan when warranted by the presentation of new information and in accordance with the Seeds Regulations, Part V, Section 112.

VI.  Nutritional Assessment Criteria for Use as Livestock Feed

1.  Nutritional Composition andProximate Analyses of the PNTs

Nutritional composition and proximate analyses of RBMT22-082 and the control variety Russet Burbank were completed. Total solids, soluble protein, dextrose, sucrose, glycoalkaloids, vitamin B6, niacin, vitamin C, potassium, copper, magnesium, and amino acids were analyzed to characterize the nutritional composition of the three transgenic lines. The proximate analyses included total protein, moisture, total fat, ash, crude fibre, carbohydrates, and calories. All components analyzed were statistically identical to the controls or within the levels reported in the literature for commercially available potato cultivars.

2.  Anti-Nutritional Factors

Solanine and chaconine are the principal glycoalkaloids commonly found in potato tubers. The concentration of total glycoalkaloids (TGA) in NewLeaf-Plus™ Russet Burbank 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 is comparable to the TGA range of 2.5-16.1 mg/100g fresh weight tissue that was 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.

VII.  Regulatory Decision

Based on the review of data and information submitted by Monsanto Canada Inc., and through comparisons of RBMT22-082 with unmodified Solanum tuberosum cv. Russet Burbank counterparts, the Plant Biosafety Office of the Plant Health and Production Division, CFIA, has concluded that the novel genes and their corresponding traits will not confer any intended or unintended ecological advantage to RBMT22-082 following unconfined release.

Based on the review of data and information submitted by Monsanto Canada Inc., the Feed Section of the Animal Health and Production Division, CFIA, has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of this line. Solanum tuberosum and several of its byproducts are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As tubers and plants of RBMT22-082 has been assessed and found to be nutritionally equivalent to traditional potato varieties RBMT22-082 and its byproducts are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment and use as livestock feed of the potato line RBMT22-082 are therefore authorized as of April 20, 1999. Any other Solanum tuberosum lines and intraspecific hybrids resulting from the same transformation event and all their descendants are also approved, provided that: i) no inter-specific crosses are performed; ii) the intended uses are similar; iii) it is known, based on characterization, that these plants do not display any additional novel traits and are substantially equivalent, in terms of their specific use and safety for the environment and for human and animal health, to potato currently being cultivated; iv) the novel genes are expressed at a level similar to that of the authorized line; and v) pest resistance management requirements described in the present document are applied.

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