Decision Document DD96-06:
Determination of Environmental Safety of
NatureMark Potatoes' Colorado Potato Beetle (CPB)
Resistant Potato (Solanum tuberosum L.)

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Issued: 1996-01

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 Traitsand its companion document T-1-09-96 The Biology of Solanum tuberosum L. (potato), and the guidelines Dir95-03 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division and the Feed Section of the Animal Health and Production Division, and advice from the Pest Management Regulatory Agency, Health Canada, has evaluated information submitted by NatureMark Potatoes, a business unit of Monsanto, regarding seven transformed potato lines referred to as NewLeaf potatoes in the present document. These plants were transformed with genes conferring resistance to the Colorado potato beetle (CPB), a major pest of potato in Canada, and resistance to kanamycin as a selectable marker. CFIA has determined that these plants with novel traits should not pose concern to environmental safety. NatureMark Potatoes has developed and will implement a CPB resistance management plan.

Unconfined release into the environment and use as livestock feed of the NewLeaf potato lines BT06, BT10, BT12, BT16, BT17, BT18, and BT23 are therefore authorized. Any other Solanum tuberosum lines and intraspecific hybrids resulting from the same transformation events 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 (PNT's)
  2. Background Information
  3. Description of the Novel Traits
    1. Resistance to the Colorado Potato Beetle (CPB)
    2. Kanamycin Resistance
    3. Development Method
    4. Stable Integration into the Plant's Genomes
  4. Assessment Criteria for Environmental Safety
    1. Potential of the PNT's to Become Weeds of Agriculture or Be 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
    6. Potential for Development of CPB Resistance to the PNT's
  5. Nutritional Assessment Criteria as Livestock Feed
    1. Anti-Nutritional Factors
    2. Nutritional Composition of the PNT's
  6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNT's)

Designation(s) of the PNT: Newleaf potato lines BT06, BT10, BT12, BT16, BT17, BT18, BT23

Applicant: NatureMark Potatoes, a business unit of Monsanto

Plant Species: Potato (Solanum tuberosum L.)

Novel Traits: Resistance to Colorado potato beetle (Leptinotarsa decemlineata, Say); kanamycin (antibiotic) resistance

Trait Introduction Method: Agrobacterium tumefaciens-mediated transformation

Proposed Use of PNT's: Production of potatoes for human consumption (table and processing) and livestock feed, including potato process residue. These materials will not be grown outside the usual production area for potatoes.

II. Background Information

Monsanto has developed seven potato lines resistant to Colorado potato beetle (CPB), the most important insect pest of potato in Canada. These potato lines, referred to as Newleaf potatoes (lines BT06, BT10, BT12, BT16, BT17, BT18, BT23) in the present document, will provide an alternative for season-long control of larval stages and adults of CPB, thus resulting in a potential reduction of chemical foliar insecticide sprays.

The development of the Newleaf potato lines was based on recombinant DNA technology, by the introduction of two bacterial genes into the potato variety Russet Burbank. A gene conferring resistance to CPB was inserted, coding for an insecticidal protein active against a narrow range of Coleoptera. Another gene, conferring resistance to kanamycin, was also inserted; this gene is of no agronomic interest but was used to select modified plants from those that remained unmodified at the development stage.

These lines have been tested in Canada under confined conditions since 1992 in Prince Edward Island (1992-95), New Brunswick (1992-95), Ontario (1993-95), Manitoba (1993-95), Saskatchewan (1995), Alberta (1994-95) and British Columbia (1994-95). They have received complete approval for commercialization in the US. Health Canada has determined that food derived from these potatoes is substantially equivalent to that derived from currently commercialized potatoes (September, 1995).

Monsanto has provided data on the identity of Newleaf potatoes, a detailed description of the modification method, data and information on the gene insertion sites, copy numbers and levels of expression in the plant, the role of the inserted genes and regulatory sequences in donor organisms, and full nucleotide sequences. The novel proteins were identified, characterized, and compared to the original bacterial proteins, including their potential toxicity to livestock and non-target organisms with particular attention given to beneficial arthropods. Relevant scientific publications were also supplied.

Agronomic characteristics such as yield of tubers, plant vigour, growth, colour, leaflet shape, flowering, were compared to those of unmodified potato counterparts. Stress adaptation was evaluated, including susceptibilities to various potato pests and pathogens. Processing qualities of tubers such as dry matter, specific gravity and French fry colour were also compared to qualities of unmodified counterpart tubers.

Composition analyses of tubers were performed, including total solids, sugars, vitamin C and minor constituents. Data to support the suitability of these Newleaf potato lines as livestock feed were provided. Proximate analyses to include crude protein, crude fat, crude fibre, ash and gross energy were supplied.

The potential development of CPB resistance to the insecticidal protein was discussed, and an insect resistance management plan will be implemented by NatureMark Potatoes, a business unit of Monsanto.

The Plant Biosafety Office of the Plant Health and Production Division, has reviewed the above information, in light of the following assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

  • potential of the PNT's to become weeds of agriculture or to be invasive of natural habitats,
  • potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or more invasive,
  • potential for the PNT's to become plant pests,
  • potential impact of the PNT's or their gene products on non-target species, including humans, and
  • potential impact on biodiversity.

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 the PNT's.

The Feed Section of the Plant Health and Production Division, CFIA, has also reviewed the above information in light of the assessment criteria for determining safety and efficacy of livestock feed, as described in Dir95-03:

  • potential impact to livestock, and
  • potential impact on livestock nutrition.

III. Description of the Novel Traits

1. Resistance to the Colorado Potato Beetle (CPB)

  • Bacillus thuringiensis subsp. tenebrionis (B.t.t.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal crystal Cry3A ð-endotoxins that are non-toxic to humans, other vertebrates, and beneficial insects. These proteins are used as environmentally acceptable foliar insecticides against CPB.
  • The CPB resistance gene engineered into Newleaf potatoes codes for one of these insecticidal crystal proteins, active against specific Coleoptera such as CPB, elm leaf beetle and yellow mealworm. Insecticidal activity results from the Cry3A protein binding to specific insect gut epithelium receptors, pore formation, loss of cations, and disruption of digestive processes following an osmotic pressure imbalance. Current knowledge indicates that only sensitive insects possess such receptors.
  • The cry3A gene is linked to a strong constitutive promoter. Protein production (referred to as B.t.t. protein in the present document) was quantified for the seven lines and found to average from 14.4 to 19.1 µg/g (f.w.) of leaf tissue and 0.8 µg/g (f.w.) of tuber tissue, corresponding to 0.09 to 0.12% of total foliage protein and 0.004% of total tuber protein. The mean whole plant expression for all seven lines was 6.64 µg/g (f.w.) of tissue, corresponding to 0.04% of the total plant protein. B.t.t. was expressed in foliage with little variation throughout the season.
  • The resulting B.t.t. proteins include a 68 kDa protein and two major proteolytic fragments of 55 kDa each. These two fragments account for 20-30% of the tuber B.t.t. protein, while only trace amounts are found in the leaves. A similar 55 kDa protein is also released in some B.t.t. registered microbial insecticides, and following tryptic digestion of the bacterial B.t. Cry3A protein purified from Escherichia coli.
  • The expressed 68 kDa protein and the 55 kDa fragments were compared to the bacterial proteins, and shown to be of similar molecular weight and immunological reactivity, indicating that there were no insertional or post-transcriptional modifications. Protein-bound carbohydrate residues were not detected, showing lack of glycosylation. The 68 kDa protein was shown to be of similar biological activity against CPB as the 68 kDa B.t. derived protein; preliminary data suggested that the 55 kDa fragments are more active than the 68 kDa protein.
  • Studies showed that the proteins were rapidly inactivated when subjected to typical mammalian acidic stomach conditions, i.e. in simulated gastric fluids. Aerobic soil degradation of the B.t.t. proteins is complete after 9 hours.
  • The gene nucleotide sequence and the enzyme amino acid sequence were provided. An amino acid database of allergens was used to search for homology between the B.t.t. protein and known sequenced allergens. No homology was found.

2. Kanamycin Resistance

  • Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell.
  • The kanamycin-resistance gene, isolated from the bacterium E. coli, codes for an enzyme that phosphorylates kanamycin, thereby preventing it from binding to ribosomes and rendering the cells resistant.
  • The gene is linked to a constitutive promoter. Expression was estimated to average 0.339 µg/g (f.w.) of leaf tissue, and 0.197 µg/g (f.w.) of tuber tissue, corresponding to 0.002% of foliage protein and 0.001% of tuber protein.
  • This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids.
  • The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database.
  • The full nucleotide sequence and corresponding amino acid sequence were provided.

3. Development Method

Russet Burbank potatoes were transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector; the vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and replaced with genes coding for CPB resistance and kanamycin resistance. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was demonstrated in these Newleaf potatoes.

4. Stable Integration into the Plant's Genome

  • The data provided showed that there was no incorporation of any coding region from outside the T-DNA borders. Single copies of the T-DNA are integrated at a single insertion site in lines BT6, BT12, BT17, and BT23. Line BT10 contains two copies inserted in tandem and BT16 contains two copies inserted at separate genetic loci.
  • The transformed plants were propagated vegetatively, and CPB resistance was expressed for at least four vegetative generations.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT's to Become Weeds of Agriculture or Be Invasive of Natural Habitats

CFIA evaluated data submitted by NatureMark Potatoes on the reproductive and survival biology of Newleaf potato lines, and determined that vegetative vigour, overwintering capacity, male sterility, insect and disease susceptibility, other than to CPB, and tuber yield and quality, were within the normal range of expression currently displayed by commercial varieties. Newleaf lines have no specific added genes for cold tolerance or winter hibernation; no overwintered plants were recorded by NatureMark Potatoes in post-harvest years of field trials in Canada.

The biology of Solanum tuberosum, described in T-1-09-96, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by NatureMark Potatoes, Newleaf lines were determined not to be different from their counterparts in this respect.

No competitive advantage was conferred to Newleaf lines, other than that conferred by resistance to Colorado potato beetles. Resistance to CPB will not, in itself, render potatoes weedy or invasive of natural habitats, since the typical vegetative reproductive characteristics of potatoes were not modified. In addition, the background genotype of these NewLeaf lines is Russet Burbank, a male sterile variety that produces no seeds and that is not attractive to bees due to lack of nectar production. Although limited dispersal is possible through dispersal of tubers, volunteers would not persist under ordinary cultivation conditions or in unmanaged habitats.

The above considerations, together with the fact that the novel traits have no intended effects on weediness or invasiveness, led CFIA to conclude that Newleaf 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

The biology of potato, described in T-1-09-96, shows that there are no wild relatives in Canada that can naturally hybridize with S. tuberosum.

CFIA therefore concludes that gene flow from Newleaf lines to potato relatives is not possible in Canada.

3. Altered Plant Pest Potential

The intended effects of both novel traits are unrelated to plant pest potential, and potato is not a plant pest in Canada. In addition, agronomic characteristics of Newleaf lines, such as: plant vigour; growth; colour; leaflet shape; and flowering period, were shown to be within the range of values displayed by currently commercialized potato varieties. Susceptibilities to diseases such as: early blight; late blight; verticillium; potato leaf roll virus; and potato virus Y were unchanged, leading to the conclusion that plant pest potential was not inadvertently altered.

CFIA has therefore determined that Newleaf lines do not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

Colorado potato beetle, the target insect, is controlled in NewLeaf potato plots at all stages of development throughout the growing season. Field observations indicate that potato flea beetles (Coleoptera: Chrysomelidae, Epitrix cucumeris Harris) are also affected by these potatoes to some extent.

The following beneficial and predacious arthropods were significantly more abundant in NewLeaf potato plots than in those treated with conventional chemical insecticides: big eyed bugs, damsel bugs, minute pirate bugs, some Hymenoptera spp. and spiders. As a result, aphid populations may be reduced through predation by natural enemies. Field observations also showed that populations of the detritivorous collembolans were as abundant in NewLeaf potato fields than in fields of non-transgenic potato counterparts, and more abundant than in those treated with conventional chemical insecticides.

Dietary toxicity studies were performed using the 68 kDa microbial protein on beneficial insects (honeybee, ladybird beetle, green lacewing and parasitic wasp), and with eight non-target insect species representing the orders of Coleoptera, Diptera, Homoptera, Lepidoptera and Orthoptera (southern corn rootworm, yellow fever mosquito, green peach aphid, European corn borer, tobacco hornworm, corn earworm, tobacco budworm and German cockroach). No negative effect was observed, except for slightly higher mortality and reduced honeydew production of green peach aphids. Green peach aphids are major vectors of potato viruses in Canada, specifically of the potato leaf roll virus, and are chemically controlled in standard potato production systems. The effect of NewLeaf potatoes on these aphids is therefore negligible in terms of environmental impact.

Numerous oral acute and short term studies were undertaken with albino mice, Sprague-Dawley rats, northern bobwhite and bobwhite quails. The animals were fed NewLeaf potatoes or protein extracts. Treatment related effects were not observed. These observations were expected, as the novel proteins are rapidly inactivated in simulated mammalian stomach fluids by enzymatic degradation and pH-mediated proteolysis. The proteins expressed in NewLeaf potatoes were shown to be equivalent to the original microbial proteins produced by the common soil B.t.t. bacteria.

Based on the above, CFIA has determined that the unconfined release of these Newleaf lines, when compared with currently commercialized potato varieties, will not result in altered impacts on interacting organisms, including humans, with the exception of CPB and potato flea beetles.

5. Potential Impact on Biodiversity

Newleaf lines have no novel phenotypic characteristics which would extend their 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 NewLeaf potato plants could reduce the need for some foliar insecticidal sprays, resulting in a reduction of chemicals released into the environment, an increase of non-target insect populations, and an increase in potential for biological control of harmful insect pests.

CFIA has therefore concluded that the potential impact on biodiversity of these Newleaf potato lines, if any, would be positive.

6. Potential for Development of CPB Resistance to the PNT's

The potential for CPB to develop resistance to conventional chemical insecticides is well documented, and many of the currently registered chemical foliar sprays no longer control CPB. Foliar insecticides based on the B.t.t. proteins present in NewLeaf potatoes are registered in Canada for use on both potatoes and tomatoes. To date, resistance of CPB to B.t.t. has not been observed under field conditions. Resistance may develop as a result of increased use of these B.t.t. foliar sprays; resistance to the B.t.t. protein could also develop following continued exposure to the NewLeaf potatoes.

The development of such a resistance would result in the loss of these valuable B.t.t. tools for the control of CPB infestations in both potatoes and tomatoes.

NewLeaf potatoes steadily produce high levels of readily accessible B.t.t. proteins throughout the growing season, resulting in extremely high mortality in CPB feeding on NewLeaf. Target insects will thus be exposed to significantly higher levels of B.t.t. than through the current foliar spray treatments, leading to high selection pressures for resistant CPB individuals. A major component of the resistance management strategy linked to the use of these NewLeaf potatoes is the presence of non-selecting refugia (regular potatoes) in close proximity, where susceptible insect populations are maintained. Should resistant insects occur, they would then be able to mate with susceptible insects to produce heterozygotes, which are expected to be susceptible to the NewLeaf potatoes. Even though the majority of the scientific community agrees that this approach sounds effective in theory, it is very difficult at this point to predict the extent and rapidity of resistance development without field validation of the proposed strategy. These potato plants should therefore be responsibly managed and CPB populations monitored for development of resistant individuals in a regular and consistent manner.

Consideration must be given to the possibility that CPB populations developing resistance to NewLeaf potatoes could also be cross-resistant to other B.t. ð-endotoxins, resulting in the loss of other B.t. protein types that may be used for the control of CPB infestations. To date however, no B.t. proteins other than B.t.t. are registered for use against CPB in Canada.

The development of resistance to the B.t.t. proteins by non-target insect pests, that may then cause further problems in other crops, is another consideration. Potato flea beetles (Coleoptera: Chrysomelidae, Epitrix cucumeris Harris) can cause significant damage to potatoes, especially in the Maritimes (10-25% yield reduction), and can also visit cucumbers, eggplants, peppers and tomatoes. Tuber flea beetles (Coleoptera: Chrysomelidae, Epitrix tuberis Gentner) feed on potatoes and tomatoes. It is a very serious pest west of the Rockies and is also found in Alberta. Potato flea beetles (E. cucumeris) have been shown to be affected by the NewLeaf potatoes, but they can survive. This species and others in this genus may develop resistance to the B.t.t. insecticidal proteins. Presently, B.t.t. foliar sprays are not registered for control of these beetles in Canada, due to reduced efficacy, so even should resistance occur, control of these insects would not be compromised. CFIA has therefore concluded that development of resistance in non-target insect pests is unlikely to have an impact on the conventional control of these pests.

CFIA believes that sound management practices can reduce and delay the development of resistant CPB populations. CFIA understands that NatureMark Potatoes has developed and will implement a pest resistance management plan that includes the following key components:

  • The early detection of resistant CPB populations is extremely important. Close monitoring for the presence of such populations, in NewLeaf potato fields and surrounding areas, is therefore warranted. Monitoring includes the development of appropriate detection tools such as visual field observations and laboratory bioassays, reporting schedules, education of growers, and enforcement procedures.
  • Education tools for growers and field managers will be developed, with descriptions such as: production practices; detection protocols for resistant CPB individuals; monitoring procedures and timetables; and strategies to be followed in case of detection of resistant insects.
  • Integrated Pest Management practices will be promoted, such as prediction of infestation problems from previous years, crop rotation, and use of trap trenches.
  • Detection of confirmed resistant CPB populations will immediately be reported to CFIA and a procedure for control of resistant individuals must be available for immediate action.
  • The strategy for resistance management of CPB when using plants that continually produce high concentrations of a B.t.t. ð-endotoxin and refugia has not been previously tested in the field on a large scale. Continued research in this area using sound science will be conducted.

The plans, information and data from the above are available to CFIA. CFIA has also strongly encouraged NatureMark Potatoes to develop novel CPB control systems with different modes of action that would offer additional or alternative management practices to growers.

If at any time, NatureMark Potatoes becomes aware of any new information regarding risk to the environment, including risk to agriculture such as development of CPB resistance, or risk to animal or human health, that could result from release of these materials in Canada, or elsewhere, NatureMark Potatoes will immediately provide such information to CFIA. On the basis of such new information, CFIA will re-evaluate the potential impact of the proposed release, and will re-evaluate its decision.

V. Nutritional Assessment Criteria as Livestock Feed

1. Anti-Nutritional Factors

Some sites reported high levels of glycoalkaloids in tubers in both control and modified lines. Composite means showed BT17 had significantly higher glycoalkaloid levels (p<0.05) than controls. This may be attributed to growing conditions during the confined trials.  Data for glycoalkaloids are reviewed by the National Potato Variety Registration Recommending Committee for all potato varieties recommended for registration.

2. Nutritional Composition of the PNT's

No differences in nutritional composition, i.e. crude protein, crude fat, crude fibre, were noted between the seven tuber lines and the control variety. With respect to crude protein, substantial equivalence of the B.t. lines to the parental line has been demonstrated. For carbohydrate, fat and fibre, substantial equivalence to the parent line has not been statistically proven. The mean values reported for these three characteristics, however, were within the published range for potatoes. Statistical analyses of the dextrose and sucrose concentration of replicated samples for each line from two sites were shown to be substantially equivalent to the control Russet Burbank lines. These results collectively demonstrate that the introduction of the novel genes into Solanum tuberosum resulting in Newleaf potatoes did not result in any secondary effects impacting on the composition or nutritional quality of the cultivar.

VI. Regulatory Decision

Based on the review of data and information submitted by NatureMark Potatoes, and through thorough comparisons of the Newleaf potato lines with unmodified potato counterparts, the Plant Biosafety Office of the Plant Health and Production Division, CFIA, has concluded that the novel genes and their corresponding traits do not confer to the Newleaf lines any characteristic that would result in intended or unintended environmental effects following unconfined release.

Based on the review of submitted data, the Feed Section of the Animal Health and Production Division has concluded that the novel genes and their corresponding traits do not in themselves raise any concerns regarding the safety or nutritional composition of the Newleaf lines. Potatoes, including potato process residue, are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As Newleaf potatoes have been assessed and found to be substantially equivalent to traditional potato varieties with respect to safety, Newleaf lines and their 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 NewLeaf potato lines BT06, BT10, BT12, BT16, BT17, BT18, and BT23 are therefore authorized. Any other Solanum tuberosum lines and intraspecific hybrids resulting from the same transformation events 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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