PI-005: Chapter 10 – Biotechnology

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Table of Contents

10.0 Biotechnology

10.1 Objective

To date, most potato cultivars have been developed using traditional plant breeding. Advances in biotechnology have allowed for the development of potato cultivars with novel characteristics, including enhanced genetic resistance to insects, diseases, and viruses. These cultivars are the result of converting traditionally susceptible varieties into varieties which are not only resistant but still maintain all of the other desirable characteristics for which the variety was initially developed. In Canada, the first transgenic potato cultivar was commercialized in 1996.

Chapter 10 will give inspectors a brief history of potato breeding, descriptions of the principles in potato transformation and the regulation of plants with novel traits (PNTs) in Canada.

10.2 Definitions

Biotechnology:
The application of science and engineering in the direct or indirect use of living organisms or parts or products of living organisms in their natural or modified forms;
Breeders Selection:
A potato crop which originates directly from true seed or from selected tubers for the purpose of evaluation as a potential variety for commercial use;
Confined release:
Release under conditions intended to minimize the establishment, spread and interaction of seed or genetic material from plants derived from the seed with the environment;
Crop:
Any breeders selection or a variety and class of seed potatoes growing in an aseptic environment, a protected environment or in one or more fields of a farm unit;
Dicotyledon:
A plant with two seed leaves;
Farm unit:
A single tract or a number of separate tracts of land under the same or different tenure operating as a single unit which uses common equipment, facilities or storage for the production and marketing of seed potatoes under the control of a grower;
Field:
The identifiable area of land on which seed potatoes of a particular variety and class are planted or have been produced;
GMO:
Genetically modified organism; transgenically modified through gene insertion or transfer of DNA;
Grower:
A person, cooperative, corporation or partnership that grows seed potatoes;
Laboratory Tests:
Tests that are conducted in a laboratory that is accredited Under the CFIA Laboratory Accreditation Program or equivalent;
Lot:
The quantity of harvested seed potatoes of a variety and class, including breeder's selection, that is identifiable by one certificate number;
Official protocols:
Protocols that must be followed by CFIA accredited laboratories to perform specific diagnostic tests;
PBO:
Plant Biosafety Office, CFIA;
PHPD:
Plant Health and Production Division, CFIA;
PLRV:
Potato Leaf Roll Virus;
PNTs:
Plant with Novel Traits; plant varieties/genotypes that are not considered substantially equivalent, in terms of their specific use and safety both for the environment and for human health, to plants of the same species in Canada, having regard to weediness potential, gene flow, plant pest potential, impact on non-target organisms and impact on biodiversity. PNTs may be produced by conventional breeding, mutagenesis, or by recombinant DNA techniques;
PSTVd:
Potato Spindle Tuber Viroid;
PVX:
Potato Virus X;
PVY:
Potato Virus Y;
Release:
Any discharge or emission of seed into the environment or exposure of seed to the environment; includes the growing and field testing of plants;
Seed potato:
A tuber, or any part of a tuber, that is certified according to the Seeds Regulations Part II for seed reproduction purposes;
Sister lot:
A lot or crop of potatoes planted with the same seed source identified by a unique certification number;
Tuber unit:
The separate pieces of one tuber that are planted consecutively in two or more hills in a row;
Unconfined release:
Release on an unrestricted basis.

10.3 Potato Breeding and Biotechnology

The potato, Solanum tuberosum, as it is presently known, differs substantially from its wild, bitter-tasting ancestors. These wild Solanum spp. provided the initial genetic information contained within the common cultivars of today. The main steps in the development of the cultivated potato included:
  • The domestication of its ancestors, the wild tuber-bearing diploid (two sets of chromosomes) species, in South America some 7000 years ago,
  • The emergence of a cultivated tetraploid (four sets of chromosomes) form and its introduction in Europe in 1570, and finally
  • Its emergence into an important staple crop in northern Europe by the end of the 18th century, followed by its distribution to other parts of the world.

Prior to the 1900s, potato plants were often grown from true seed originating from open pollination of compatible parents. With the advent of transportation and the use of equipment in the production of potatoes, the use of vegetative propagation became widespread. This resulted in observations on the quality of the potato stocks grown. Potato stocks were found to decline rapidly after a few generations of clonal (asexual) reproduction. Producers had to diligently select healthy clones which, in turn, led to the selection of many new improved varieties. It also led to the trade in seed tubers from different areas which were less affected by the problem of a loss of quality after several generations.

When the decline in the seed stocks was eventually proven to be associated with the accumulation of aphid transmitted viruses, the production of seed tubers in areas less favourable to aphid development was the logical option. This promoted the establishment of seed certification programs and protected seed areas.

Concurrently, the concept of Mendel's law of gene inheritance led to the establishment of formal breeding programs, where true seed was produced from controlled crosses. This rigorous process was used to develop new potato lines with specific characteristics. Some techniques developed in those years, including flower induction, pollen collection and storage and seed extraction, are still used today.

Thus, human selection of naturally occurring genetic variation, resulting from mutation and sexual recombination (through crosses), led to the evolution of the modern potato crop.

The development of transformed potato lines carrying specific genes from unrelated organisms (e.g. different plant families, bacteria, viruses, and synthetic genes) is made possible through biotechnology. Traditionally, many crosses may be required to ensure that specific genes are passed on to their progeny. Transformation techniques allow breeders to directly transfer an isolated gene without transferring the entire donor genome in excess. The result is an efficient mechanism which allows the introgression of new genes into a crop species for which traditional hybridization barriers prevented genetic exchange.

The recent advent of biotechnology and emerging alternate methods for improving potato cultivars have added another dimension to the potato industry. The difficulty to the inspector, however, is that the cultivars, although genetically altered, have no visual changes. For instance, a genetically altered (transgenic) Russet Burbank potato cannot be distinguished from one that is unaltered unless sophisticated molecular techniques are employed.

10.4 Transformation of Potato Cultivars

Several transgenic potato lines have been obtained by the direct introduction of new genes into existing cultivars to confer desirable novel characteristics.

The gene is the unit of inheritance passed on to its progeny. Each gene generally carries the information representing a particular protein, and when expressed, these proteins determine the characteristics of an organism. In some cases, a gene may govern a particular trait, such as an organism's resistance to disease, while in other cases, a specific characteristic may be determined by many genes. In plant pest terms, these types of resistance are called horizontal (many genes) or vertical (single gene) resistance.

PNTs are defined by the following:

Potato varieties/genotypes that have been intentionally selected, created or introduced into a population possessing characteristics that do not demonstrate familiarity nor substantial equivalence to those present in a distinct, stable population with respect to specific use and safety and to both environment and human health.

PNTs may be produced by conventional breeding, mutagenesis, or more commonly, recombinant DNA techniques. Safety assessments regarding weediness potential, gene flow, plant pest potential, impact on non-target organisms and impact on biodiversity are required for all PNTs intended for importation and for environmental release in Canada.

Some examples of potatoes with novel traits include:

  • Potato plants containing viral genes that confer cross-tolerance to related viral infections (PVY, PVX or PLRV);
  • Potato plants that contain a bacterial gene that confers tolerance to attack from the Colorado potato beetle;
  • Potato plants that contain new genes that confer tolerance to bacterial and fungal diseases; and,
  • Potato with modified nutritional value (e.g. higher starch content).
Figure 10-1 A Demonstration of Gene Insertion

The following image is a diagram which provides a demonstration of gene insertion through cloning, using a source of DNA and host DNA.

Image - Processes involved in the regulation of plants with novel traits in Canada. Description follows.
Description for Figure 10-1

This image demonstrates gene insertion through cloning using a source of DNA and host DNA. The first part of the image shows the DNA before it has been cut; the source DNA is green and the host DNA is purple. The host DNA is then cut into two, and a portion of the source of DNA is cut out. This portion is then inserted into the Host DNA.

Genetic engineering of a potato plant begins with the identification and characterization of a target gene. The gene is then isolated from the donor organism, copied and cloned, using specialized enzymes, into another organism's DNA (see Figure 10-1). Transformation will introduce the new gene, with any additional DNA components required for its stable expression (e.g. a promoter, a terminator, and a specific marker gene). The promoter and terminator together regulate expression, that is, turning the gene on and off. In addition the promoter also controls where the gene will be expressed within the plant (i.e. only in the leaves, only in the roots, or throughout the entire plant). The marker gene must be inserted at the same time with the target gene to allow for the identification and selection of the transformed cells during development in the laboratory. Frequently, marker genes code for antibiotic resistance, allowing the recipient cells to be grown in the presence of certain antibiotics, unlike the unmodified cells.

In nature, Agrobacterium tumefaciens causes the development of crown gall disease by invading plant wounds and transferring a segment of its own external plasmid DNA. When using Agrobacterium to genetically modify plants, the disease-causing parts of the Agrobacterium are removed using restriction enzymes. The new genes of agronomic importance (such as improved nutritional value) are then cloned, or inserted, into the plasmid. The Agrobacterium is allowed to infect plant cells (usually segments of leaf, stem or mini-tubers) resulting in the introduction of the new gene into the host plant DNA. The transformed cells are then grown into full plants (see Figure 10-2). The plants exhibiting both the newly incorporated trait conferred by the new gene and the characteristics of the unmodified cultivars are then selected for further testing.

Figure 10-2 Transformation of Potato Cultivars

The following image demonstrates the transformation of potato cultivars involving the infection of tissues which causes the transfer of DNA, the regeneration, and the final result which is the transformed plant.

Image - Figure 10-2 Transformation of Potato Cultivars. Description follows.
Description for Figure 10-2

The image first shows a petri dish containing plant tissues that are in a selective medium; it shows the transfer of the new gene into the plant tissues. This image then transitions to another with a red arrow. The next image demonstrates the regeneration of a whole plant. This image then transitions to a final image using a red arrow. The final image is of a potted-plant that is full-grown which demonstrates the overall transformed plant which would occur over time after the successful insertion of the new gene into the host plant DNA.

10.5 Regulation of Plants with Novel Traits (including potatoes)

Since 1988, regulators have overseen the importation and field release of a new class of crop plants derived through the techniques of biotechnology. Potato PNTs may be derived by mutagenesis or recombinant DNA techniques to insert specific genes from a variety of donor organisms. Regulatory evaluation is necessary due to limited familiarity with the novel traits that have been introduced. PNTs are regulated under the Plant Protection Act, Seeds Act, and Seeds Regulations Part V.

10.5.1 Regulation under the Plant Protection Act

Under the Plant Protection Act, potato PNTs are regulated in the same way as their traditional counterparts, in preventing the importation, exportation and spread of pests injurious to plants. Although these plants may differ from traditionally derived crop species by just a few genes, the CFIA assesses them individually based on the novel traits encoded by these genes, and on any unintended effects the actual genetic modifications may have imparted on the plants.

The performance of risk assessments is authorized under Section 6 of the Plant Protection Act, which states: "except as permitted under this Act or the regulations, no person shall move, grow, raise, culture or produce anything that there are reasonable grounds to believe is a pest; that is or could be infested with a pest; that constitutes or could constitute a biological obstacle to the control of a pest".

10.5.2 Regulation under the Seeds Act and Seeds Regulations Part V

In Canada the product based approach is used, such that potato PNTs are regulated on the basis of the characteristics of the product, not the specific process by which the product was made. The primary trigger of the regulatory process is the novelty of the plant species, its characteristics (traits) and use, in the Canadian context. Therefore products of traditional breeding or mutagenesis as well as the products of recombinant DNA technology may be considered novel and are regulated under the Seeds Act. The CFIA's Plant Biosafety Office (PBO) works within the Canadian Federal Framework for Biotechnology Regulation and under the Seeds Regulations Part V.

Potato PNTs are regulated under the Seeds Regulations Part V at two steps:

  • Confined field trials overseen by the Plant Health and Production Division (PHPD) PBO, and
  • Unconfined release into the environment authorized by the PHPD, PBO.

10.6 Importation

Potato PNTs require an import permit from the Plant Protection Division prior to import into Canada.

Information outlining potential plant pest characteristics, including the geographic origin of the novel plant material, and how it will be used and stored is required for all imports. Applicants must also identify the species and provide a general description of the novel traits and genes, the quantity of material they wish to import, and the purpose of the importation. No further notification will be required if the designated use is confined to a contained facility that prevents dissemination of any genetic material from the facility into the environment. Inspectors should refer to Regulatory Directive 2000-7: Guidelines for the Environmental Release of Plant With Novel Traits Within Confined Field Trials in Canada, PBO.

Specific conditions are incorporated on the permit, such that the material or material derived from it, must be contained in a research facility, and cannot be distributed or deliberately released without written authorization, only after an environmental risk assessment has been conducted by the PBO. These conditions are designed to prevent the environmental release of PNTs before a full risk assessment is conducted. For example, the conditions discourage the uncontrolled disposal of material onto compost heaps, where vegetative plant parts may survive. Applicants should use the Application for Permit to Import (CFIA/ACIA 5256) when importing plants with novel traits into Canada. The permit must accompany the shipment.

PNTs that have been determined not to pose a plant pest risk by the PHPD are generally subject to the same importation requirements as their unmodified counterparts. These can include plants with novel traits that have been authorized for unconfined, general environmental release following an environmental assessment and a pest risk assessment by the PHPD.

10.7 Confined and Unconfined Releases

Toward the time of commercialization, authorization for unconfined release will only be issued after the confined field trial assessments and the environmental risk assessment have been conducted by the PBO, under the Seeds Act and the Seeds Regulations Part V. This authorization must be obtained prior to the release (deliberate planting) of potatoes with novel trait(s) into the Canadian environment.

10.7.1 Confined Release

Confined release is the release of the PNTs into the environment under specified conditions which minimize any potential impact of the PNTs on the environment while providing the opportunity to fully evaluate the PNTs. Criteria are set to prevent potential gene flow from pollen, seed or vegetative parts, and to prevent the introduction of the PNTs into the feed and food systems. Typical terms and conditions for confined field trials relate to reproductive isolation, disposition and handling of plant material, monitoring, post harvest land use restrictions and maintenance of accurate records regarding the confined trials. Fields planted with PNTs potatoes under the confined field testing requirements must provide an isolation distance of one blank row and must be kept free of all potatoes for two years following the trial period.

10.7.2 Environmental Assessment: The Five Criteria

The environmental assessment conducted by the PBO takes into account the following five criteria:

  • Altered Weediness Potential
  • Gene Flow to Related Species (there are no sexually compatible wild relatives of potato in Canada)
  • Altered Plant Pest Potential
  • Potential Impact on Non-Target Organisms
  • Potential Impact on Biodiversity.

10.7.3 Unconfined Releases

Prior to unconfined releases, an assessment, similar to that of the confined field testing although in greater depth, must be done. This assessment provides a full description of the novel plant material concentrating on potential changes in the environment when compared to unmodified plants of the same species.

Characteristics of the potato PNTs that must be examined in the assessment include:

  • The un-modified host plant species (biology/ecology in Canada);
  • The novel trait;
  • The resulting novel plant;
  • Potential for different interactions; and,
  • Agricultural and silvicultural practices.

Other requirements may need to be met prior to the commercialization of potatoes with novel traits, even if it has been authorized for unconfined release. Additional requirements include a food safety assessment by Health Canada, a feed assessment by the Feed Section and like most agricultural crop kinds, the potato line must be registered as a variety by the Variety Registration Office, PHPD.

10.7.4 Biotechnology Fees

As of December 1, 1997 all applications for confined field trials and unconfined release are subject to fees and to submitting statistics on confined field releases. See below for details on the fee structure. Note: Once review of an application has been initiated, no refund will be issued.

Fees for Biotechnology Activities
  • New Submission for confined testing (each) - $400
  • Renewal Submission for confined testing (each) - $100
  • Each trial site (confined) - $100
  • Unconfined release (per submission) - $2000

10.8 Monitoring for Compliance

Field inspections have been an integral part of the overall regulation of PNTs confined field testing. The purpose of field inspections of these confined trials is to verify compliance with the imposed terms and conditions (e.g., confinement measures for reproductive isolation, presence of same and related species) designated to minimize gene flow to the same and related species. Other terms and conditions, such as handling and disposition of seed at seeding and harvest time, prohibition of food and feed use, and frequency of monitoring of the trials by the applicant and field managers, cannot always be verified by inspectors due to time restrictions on inspection staff.

There are two types of inspections for confined field trials:

  • Current harvest inspections, and
  • Post-harvest inspections.

10.8.1 Current Harvest Inspection

For an annual crop, such as potato, inspections occur the year that the crop is actually grown.

The following are noted at the time of inspection:

  • Growth stage of the PNTs and of the plants in border rows (if present),
  • The area occupied by the entire trial (approximate),
  • Verification that the confinement measures for the trial are actually being followed (e.g. integrity of border rows, isolation distance), and
  • Presence of related species (cultivated and weedy) on the trial site, including border rows (if applicable) and within the isolation distance.

If compliance problems are encountered, the inspectors are asked to suggest remedial actions to the trial manager/field contact. Trials may require re-inspection depending on the severity of the compliance problem and the complexity of the required remedial action. Whenever compliance problems are noted, inspectors are asked to immediately report this to the PBO. If the inspector was not able to reach the field trial manager/contact, PBO staff will contact the trial applicant to inform them of compliance problems and to request their cooperation in ensuring that remedial actions are performed immediately.

10.8.2 Post Harvest Inspection

Potato trial sites must be free of potatoes for two years from the trial date and all volunteers must be controlled. Post-harvest inspections occur the year (or years) following the harvest or the termination of a PNTs confined trial. The trial site (including the 10m isolation distance zone around the site) is inspected for the presence of volunteers and related species. The applicant is responsible for monitoring the PNTs site for volunteers and related species during the specified post-harvest period and to remove and destroy them prior to flowering or tuber set.

If compliance problems are encountered the inspectors are asked to suggest remedial actions to the trial manager/field contact. Whenever compliance problems are noted, inspectors are asked to immediately report this to the PBO. If the inspector was not able to reach the field trial manager/contact, PBO staff will contact the trial applicant to inform them of compliance problems and to request their cooperation in ensuring that remedial actions are performed immediately.

10.9 Plant Biosafety Office

Any requests for information regarding potato PNTs should be forwarded to:

Plant Biosafety Office
Plant Health and Production Division
Canadian Food Inspection Agency
59 Camelot Drive
Ottawa, ON K1A 0Y9

Phone: 613-773-2342
Fax: 613-228-6629
Canadian Food Inspection Agency - Contact Us

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