Novel Food Information: Simplot Innate Potato Event Gen2-Z6
On this page
- Background
- 1. Introduction
- 2. Development of the Modified Plant
- 3. Characterization of the Modified Plant
- 4. Product Information
- 5. Dietary Exposure
- 6. Nutrition
- 7. Chemistry/Toxicology
- Conclusion
Background
Health Canada has notified J.R. Simplot Company (Simplot) that it has no objection to the food use of Simplot Innate® potato event Gen2-Z6. The Department conducted a comprehensive assessment of this potato event according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.
The following provides a summary of the notification from Simplot and the evaluation by Heath Canada and contains no confidential business information.
1. Introduction
Simplot has developed a genetically modified potato (Solanum tuberosum) event Gen2-Z6 (developed from Snowden), which exhibits reduced levels of free asparagine in tubers, as well as lowered levels of reducing sugars (i.e., glucose and fructose) in tubers. The reduction in asparagine significantly decreases acrylamide formation in tubers when subjected to various forms of thermal processing (e.g., baking, frying, etc.). Lower levels of reducing sugars further decreases acrylamide formation and limits heat-induced browning. This potato event also exhibits reduced expression of polyphenol oxidase 5 enzyme (Ppo5), resulting in a decreased incidence of black spot bruising in tubers. Lastly, this potato event expresses a resistance protein (R-protein) found in wildSolanum species Solanum venturii and Solanum phureja (VNT1). The VNT1 protein enables the potato plant to detect an effector protein, Avr-Vnt1, which is produced by Phytophthora infestans; an oomycete that causes late blight in potatoes. Detection of the effector protein allows the potato plant to initiate its native immune response to resist infection by P. infestans.
With the exception of late blight resistance, the other novel traits (i.e., reduced asparagine levels, lower levels of reducing sugars, and reduced black spot bruising) are achieved through the transcription of inverted repeat sequences containing small fragments of DNA from five different endogenous genes (i.e., Asn1, Ppo5, PhL, R1, and VInv), which results in the reduced level of mRNA transcripts (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway. As mentioned above, the late blight resistance trait is achieved through the expression of the VNT1 protein.
Simplot Innate® potato event Gen2-Z6 is considered by its developer to be a ‘Generation 2.0’ plant. The first generation of Simplot Innate® potato events (i.e., Gen1-E12, Gen1-F10, Gen1-J3, Gen1-J55, and Gen1-V11) exhibit lower levels of reducing sugars, have lower potential to form acrylamide during heat processing, and exhibit resistance to black spot bruising. The second generation of Simplot Innate® potato events possess all the same traits as their Generation 1.0 predecessors, while also exhibiting resistance to late blight disease
The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada’s Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how this potato variety was developed; how the composition and nutritional quality of this variety compared to non-modified potato varieties; and the potential for this potato variety to be toxic or cause allergic reactions. Simplot has provided data that demonstrate that Simplot Innate® potato event Gen2-Z6 is as safe and of the same nutritional quality as traditional potato varieties used as food in Canada.
The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division B.28). Simplot Innate® potato event Gen2-Z6 is considered a novel food under the following part of the definition of novel foods:
“c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that:
- the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.”
2. Development of the Modified Plant
The petitioner has provided information describing the methods used to develop Simplot Innate® potato event Gen2-Z6 and the molecular biology data that characterize the genetic change, which results in reduced levels of free asparagine in tubers, lower levels of reducing sugars (i.e., glucose and fructose) in tubers, reduced expression of polyphenol oxidase 5 enzyme (Ppo5), and expression of the VNT1 protein. This phenotype was achieved by transformation of the conventional potato variety Snowden with two different T-DNA constructs containing inverted repeat sequences consisting of specific DNA fragments from five endogenous genes (i.e., Asn1,Ppo5, PhL, R1, and VInv) and the Rpi-vnt1 gene derived from S. venturii encoding the VNT1 protein. The five endogenous target genes were chosen based upon their biochemical roles in asparagine production, accumulation of reducing sugars, or black spot bruising. The T-DNA inserts in Simplot Innate® potato event Gen2-Z6 contain two native promoters (i.e., pAgp and p Gbss) that drive the transcription of three inverted repeat sequences, primarily in tuber tissue. Expression of these inverted repeat sequences results in the reduced level of mRNA (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway
The Asn1 gene encodes an asparagine synthetase 1 (Asn1) enzyme that catalyses the conversion of glutamine to asparagine by transferring the side-chain amine (NH2) from glutamine to aspartate to form asparagine. Asparagine is a substrate of the Maillard reaction which converts amino acids and reducing sugars to acrylamide during high-temperature processing. Reduction of Asn1 and asparagine levels in potato tubers results in a decreased potential for acrylamide formation in cooked potato food products.
The Ppo5 gene encodes a polyphenol oxidase 5 (Ppo5) enzyme that catalyzes the conversion of o-diphenols to o-quinones. Reactive o-quinone molecules auto-polymerize to form melanins, which are responsible for the colouration of oxidized plant tissues. A loss of Ppo5 activity limits the colouration of oxidized tissues (thus reducing black spot bruising).
The PhL gene encodes an a-glucan phosphorylase, starch phosphorylase L (PhL) that degrades starch by phosphorolytic release of glucose-1-phosphate from glucan chains. A loss of PhL activity limits reducing sugar accumulation (further contributing to the lower potential for acrylamide formation).
The R1 gene encodes a starch-related R1 protein (a-glucan, water dikinase) that catalyzes the transfer of the g- and b-phosphates of ATP (through a phosphor-histidine intermediate) to α-glucan and water, resulting in phosphorylated starch. R1 is mainly responsible for phosphorylation at the C6 position. Phosphorylation affects the degree of crystalline packing within the starch granule and makes it more accessible to degradation. Thus, loss of R1 activity impairs starch degradation, which reduces accumulation of reducing sugars (further contributing to the lower acrylamide potential phenotype).
The VInv gene encodes a vacuolar invertase (VInv) that hydrolyses sucrose into the reducing sugars glucose and fructose. Thus, loss of VInv activity reduces accumulation of reducing sugars (further contributing to the lower acrylamide potential phenotype).
Due to the nature of the inverted repeat sequences, their transcripts form double-stranded RNA (dsRNA) molecules through complementary binding. The dsRNA molecules act as a precursor for the plant’s own RNAi post-transcriptional regulatory pathway. A cellular RNase III enzyme (i.e. Dicer) recognizes and processes these precursor dsRNA molecules into small, 21-bp duplexes consisting of two individual strands (denoted as ‘guide’ and ‘passenger’, respectively) termed small interfering RNA (siRNA). The siRNA duplexes are subsequently bound by the RNA Induced Silencing Complex (RISC), which selectively degrades the ‘passenger’ strand of each duplex. The ‘guide’ strand (still bound to the Complex) serves to activate RISC and turn it into a silencing complex. In plants, the activated RISC binds to any messenger RNA (mRNA) that has complete complementary sequence to the bound ‘guide’ strand, and destroys the bound mRNA by enzymatic cleavage. The cleavage of the target mRNA can result in reduced expression of the associated protein (although this reduction in expression can vary for individual siRNA).
The first T-DNA construct (pSIM1278 T-DNA) contains the following genetic elements: the promoter for an ADP glucose pyrophosphorylase gene (p Agp) (1st copy), one of two convergent promoters that drive the expression of an inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, derived from Solanum tuberosum (potato) var. Ranger Russet; the inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, the promoter for the granule-bound starch synthase (pGbss) gene (in the reverse orientation) (1st copy), the second of two convergent promoters that drive the expression of the inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, derived from S. tuberosum var. Ranger Russet; the promoter for an ADP glucose pyrophosphorylase gene (pAgp) (2 nd copy), one of two convergent promoters that drive the expression of an inverted repeat sequence containing fragments of thePhL and R1 genes, derived from S. tuberosum var. Ranger Russet; the inverted repeat sequence containing fragments of the PhL and R1 genes, and the promoter for the granule-bound starch synthase (pGbss) gene (in the reverse orientation) (2nd copy), the second of two convergent promoters that drive the expression of the inverted repeat sequence containing fragments of the PhL and R1 genes, derived from S. tuberosum var. Ranger Russet. The pSIM1278 T-DNA construct was used in the development of Simplot Innate® potato events Gen1-E12, Gen1-F10, Gen1-J3, Gen1-J55, and Gen1-V11 and has been previously assessed by Health Canada (2016).
The second T-DNA construct (pSIM1678 T-DNA) contains the following genetic elements: the native promoter for the R-protein, VNT1 (pRpi-vnt1) derived from S. venturii; the coding sequence of theRpi-vnt1 gene (Rpi-vnt1) derived from S. venturii encoding the VNT1 protein; the native terminator for the R-protein, VNT1 (tRpi-vnt1) derived from S. venturii; the promoter for an ADP glucose pyrophosphorylase gene (pAgp) derived from S. tuberosum var. Ranger Russet; the inverted repeat sequence containing fragments of the VInv gene derived from S. tuberosum var. Ranger Russet; and the promoter for the granule-bound starch synthase (pGbss) gene (in the reverse orientation) derived from S. tuberosum var. Ranger Russet. The pSIM1678 T-DNA construct was used to transform the Generation 1.0 Simplot Innate® potato events Gen1-E56 into the Generation 2.0 event Gen2-W8, and the previously assessed Generation 1.0 events Gen1-F10 and Gen1-J3 into Generation 2.0 events Gen2-X17 and Gen2-Y9, respectively.
Simplot Innate® potato event Gen2-Z6 was developed via Agrobacterium-mediated transformation based on Richael et al., 2008 Footnote 1 . C58-derived Agrobacterium strain AGL1 (Lazo et al., 1991 Footnote 2 ) carrying the pSIM1278 or pSIM1678 vector, was used to transform potato internode segments.
Simplot Innate® potato event Gen2-Z6 was developed by transforming the Gen1 event Gen1-V11 (previously assessed by Health Canada) with the pSIM1678 vector, creating the Gen2 event.
3. Characterization of the Modified Plant
Insert integrity and copy number of the pSIM1278 and pSIM1678 T-DNA inserts within event Gen2-Z6 was characterized using Southern blot analysis and complemented by several DNA sequencing techniques (i.e., Illumina and Sanger) where necessary to elucidate specific sequences within each insert. Several restriction enzyme digests were performed using sets of probes specific to the elements within each T-DNA insert (i.e., ASN, GBS, AGP, R1, INV, and VNT1) to detect the presence of these elements. The observed band products were compared by size, intensity, and number to the expected band products (based on the simple structure assumption). Where the observed bands suggested a simple insert structure, Illumina whole genome sequencing was used to confirm the insert sequence and verify sequence coverage across the simple insert. All of the observed bands suggested a simple insert structure for both T-DNA inserts.
Southern blot analysis and DNA sequencing techniques confirmed that within event Gen2-Z6, the pSIM1278 insert contains a nearly full-length T-DNA sequence and is identical to the pSIM1278 insert in its parental line (previously described for event Gen1-V11). In event Gen2-Z6, the pSIM1678 insert consists of a nearly full-length T-DNA sequence (with only minor deletions in the border sequences which is a common result of Agrobacterium-mediated transformation).
Furthermore, Southern blot analysis confirmed an absence of the pSIM1278 and p1678 plasmid backbone sequences in the event Gen2-Z6 genome.
The potato is a vegetatively propagated crop and reproduction does not involve meiosis. Thus, the T-DNA insertions within Simplot Innate® potato event Gen2-Z6 were expected to be genetically stable. Nevertheless, stability of the T-DNA inserts was confirmed over multiple propagations of this event by Southern blot analysis.
4. Product Information
Simplot Innate® potato event Gen2-Z6 differs from its traditional counterpart by the addition of three inverted repeat sequences containing sequence fragments of 5 endogenous genes (i.e., Asn1, Ppo5, PhL, R1, and VInv), and the converging promoters (i.e., pAgp and pGbss) that drive their expression. Expression of these inverted repeat sequences results in the reduced level of mRNA (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway of the host plant. Reduction in the protein expression of Asn1, Ppo5, PhL, R1, and VInv results in reduced levels of asparagine and reducing sugars (i.e., glucose and fructose) in tubers as well as lower levels of the Ppo5 enzyme. The reduction in asparagine alone significantly decreases acrylamide formation in tubers when subjected to various forms of thermal processing (e.g., baking, frying, etc.). Lower levels of reducing sugars further decreases acrylamide formation and limits heat-induced browning. Reducing levels of the Ppo5 enzyme results in a decreased occurrence of black spot bruising in tubers. These events also express the R-protein, VNT1, which confers resistance to the late blight-causing pathogen P. infestans.
The host organism, Solanum tuberosum (potato), is produced in all provinces and territories of Canada and is an integral part of the world’s food supply. Many varieties of this species have a long history of safe use as food. Simplot Innate® potato event Gen2-Z6 contains genomic DNA sequences derived from conventional potato and a wild potato species (Solanum verrucosum and Solanum venturii). According to the Organization for Economic Cooperation and Development (OECD), S. verrucosum is an acceptable source of genetic material used to improve potato varieties and has a history of safe food use. S. venturii is a wild potato species from Argentina and is a model system for virus-induced gene silencing studies. The use of genetic material from all three sources in Simplot Innate® potato event Gen2-Z6 would not be expected to pose a safety concern
With the exception of the VNT1 protein, the T-DNA inserts present in Simplot Innate® potato event Gen2-Z6 do not result in the expression of novel proteins, but rather the expression of dsRNAs that are further processed by the endogenous enzyme Dicer into smaller siRNAs that will subsequently activate the RNAi pathway, resulting in reduced levels of the mRNA transcripts encoding for five endogenous proteins (i.e., Asn1, Ppo5, PhL, R1, and VInv).
Transcript levels rather than protein concentrations were evaluated to link new phenotypic traits in the Simplot Innate® potato event Gen2-Z6 to changes at the molecular level (i.e., mRNA level). RNA was isolated from the various tissues of this event and its respective untransformed counterpart and used to carry out northern blot analyses. Three biological replicates of event Gen2-Z6 and its respective unmodified control were evaluated in each gel and blot.
The promoters used to drive the transcription of the inverted repeat sequences in the T-DNA expression cassettes (i.e., pAgp and p Gbss) are well-characterized and known to be highly active in tubers and stolons, and less active in photosynthetically-active tissues and roots (Nakata et al., 1994 Footnote 3 ; Visser et al., 1991 Footnote 4 ). Therefore, reduction in the levels of mRNA transcripts for the five target proteins was predicted by the petitioner to be higher in tubers, and less pronounced in leaves, stems and roots.
For event Gen2-Z6, the results showed reduced expression for all five target genes in tubers. No differences were observed in other tissues.
Expression of the Rpi-vnt1 mRNA transcript in event Gen2-Z6 is driven by its native promoter and terminator. Quantitative reverse-transcription PCR (qRT-PCR) was used to verify expression of the Rpi-vnt1 mRNA transcript in event Gen2-Z6 in leaves and tubers. Total RNA was isolated from these tissues of event Gen2-Z6 and its respective unmodified counterpart and subjected to qRT-PCR using Rpi-vnt1-specific primers.
Expression of the Rpi-vnt1 mRNA transcript was normalized to endogenous reference genes (elongation factor 1a and adenine phosphoribosyltransferase [APRT]), within each sample. These reference genes were chosen based on consistent expression in potato under various environmental conditions. The specificity of the qRT-PCR reaction was demonstrated using gene-specific primers. To serve as a positive control, expression of the Rpi-vnt1 mRNA transcript in S. venturii leaf tissue sample was analysed. For each sample type (including the S. venturii leaf tissue sample), three biological replicates were analysed in triplicate. Transcript expression results show that Rpi-vnt1 mRNA is expressed in event Gen2-Z6 roots, stems, leaves, flowers, and tubers, however at very low levels
Overall, expression levels of Rpi-vnt1 mRNA were similar in the leaf tissue of the potato event Gen2-Z6 and the S. venturii leaf sample while expression in the leaves of event Gen2-Z6 were significantly higher than in the tuber samples. This observation is consistent with other work on the expression levels of R-genes Footnote 5
It was noted by the petitioner that R-proteins are expressed at low levels in plants and in some cases are estimated to be as low as 18 ppt Footnote 6 . The VNT1 protein was detected in the tissues of event Gen2-Z6 when measured with a western blot assay sensitive to less than 500 ppb.
5. Dietary Exposure
While the intended purpose of Simplot Innate® potatoes is to provide a potato with lower potential to form acrylamide upon thermal processing, reduced blackspot bruising, and resistance to late blight, it is not expected that the introduction of event Gen2-Z6 into the marketplace will result in a significant change in the food use of potatoes.
6. Nutrition
To evaluate if there were any unanticipated consequences of the genetic modifications in the Simplot Innate® potato event Gen2-Z6, the nutritional and anti-nutritional components of the event Gen2-Z6 potato were analysed and compared to the non-genetically modified (non-GM) parental control, Snowden. This was done as part of field trials conducted in 2018 at 4 different sites in the United States (Canyon, Idaho; Bonneville, Idaho; Montcalm, Michigan; Waushara, Wisconsin). The field trial was a randomized complete block design with 4 replicates/blocks per site and each block containing the event Gen2-Z6 potato and the parental control, Snowden. While there were no Canadian sites, the petitioner notes that these sites were located at similar latitudes and conditions to the potato-growing regions of Canada.
The nutritional compositional analytes measured in the event Gen2-Z6 potato and non-GM Snowden control were: moisture, protein, total fat, ash, crude fibre, carbohydrates (including sucrose and the reducing sugars fructose and glucose), calories, vitamins (i.e., vitamin B3, B 6, and C), minerals (i.e., copper, magnesium, and potassium); amino acids (18), and glycoalkaloids. These included all analytes recommended by the 2002 Organization for Economic Co-Operation and Development (OECD) Consensus Document on Compositional Considerations for New Varieties of Potatoes.
Of the analytes measured, the following showed a statistically significant treatment effect in the event Gen2-Z6 potato compared to the non-GM Snowden control: carbohydrates (higher in event Gen2-Z6), fructose and glucose (lower in event Gen2-Z6), sucrose (higher in event Gen2-Z6), calories (higher in event Gen2-Z6), moisture (lower in event Gen2-Z6), vitamin B3 (higher in event Gen2-Z6), vitamin C (higher in event Z6), glycoalkaloids (lower in event Gen2-Z6), and aspartic acid and asparagine (lower in event Gen2-Z6). The following amino acids were all higher in the event Gen2-Z6 potato compared to the non-GM Snowden control: alanine, arginine, glutamic acid and glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. Some of the differences in the levels of analytes in the event Gen2-Z6 potato when compared to the control were expected due to the intended effects of the genetic modifications (e.g., fructose and glucose, sucrose, and asparagine) and/or the analyte levels fell within the ranges of the levels of those reported in the scientific literature including the OECD (2002) and/or the ILSI Crop Composition Database (2019). These differences do not pose a nutritional safety concern. The remaining analytes were not statistically significantly different in the event Gen2-Z6 potato compared to the control.
The intended changes in the amino acid analytes are accompanied by certain predictable changes in the expression of the Asn1 gene that codes for the asparagine synthetase enzyme that converts aspartate to asparagine. Concomitantly, due to the lower asparagine levels, there were small but significantly higher levels of some amino acids in the event Gen2-Z6 potato when considered as a percent of total amino acids, with glutamine being the highest. Since these nutrient levels in the event Gen2-Z6 potato remain within ranges found in conventional varieties, there is no nutritional safety concern.
The lower levels of reducing sugars, fructose and glucose, in the event Gen2-Z6 potato compared to the control can be attributed to the partial down regulation of R1 glucan water dikinase, and down regulation of phosphorylase L and vacuolar invertase. Sucrose levels were significantly higher in event Gen2-Z6 potato at harvest compared to Snowden. This difference can be attributed to the down regulation of vacuolar invertase, which slows the conversion of sucrose into fructose and glucose in the vacuole. The lower levels of free asparagine and reducing sugars could result in lower acrylamide potential in event Gen2-Z6 potato.
The petitioner has demonstrated that Simplot Innate® potato event Gen2-Z6 has a similar nutritional composition to its control and/or conventional varieties that have been reported in the scientific literature. The intended changes in the analytes do not pose any safety concerns regarding the food use of event Gen2-Z6 potato from a nutritional perspective.
7. Chemistry/Toxicology
The petitioner provided a scientific rationale for the lack of toxicity of the event Gen2-Z6 potato based on previous Health Canada authorisations of potatoes for food use with these same genetic modifications (i.e., Simplot Innate® potato events Gen2-W8, Gen2-X17, and Gen2-Y9). Equivalency of the genetic modifications in event Gen2-Z6 and the previously authorised potato varieties was confirmed.
The previous evaluations noted that the small interfering RNA (siRNA) sequences involved in the RNAi do not share complementarity with human transcripts. This means that even in the unlikely event that they are absorbed into the human body, they would not have a target to act upon, so no toxicity would be expected. The VNT1 protein was found to lack homology with known toxins and no toxicologically relevant effects were observed in three 90-day toxicity studies in rats fed potato powders from the previously authorized potato varieties expressing this protein and possessing the same RNAi changes.
Updated homology searches (National Centre for Biotechnology Information [NCBI] database; March 2019) between the amino acid sequences corresponding to the introduced genetic material and known or suspected toxins did not identify any relevant homology. This suggests that the introduced genetic material does not produce known toxins.
The petitioner provided a scientific rationale for the lack of allergenicity of the event Gen2-Z6 potato based on previous Health Canada authorizations of potatoes for food use with the same genetic modifications (i.e., Simplot Innate® potato events Gen2-W8, Gen2-X17, and Gen2-Y9).
The previous evaluations noted that the VNT1 protein lacked homology with known allergens. The RNAi changes do not produce novel proteins and are therefore unlikely to elicit allergic reactions since almost all known food allergens are proteins.
Updated homology searches (AllergenOnline; 2129 sequences, ver. 19; Feb 2019) between the amino acid sequences corresponding to the introduced genetic material and known or suspected allergens did not identify any relevant homology.
Potato is not a priority food allergen; although some proteins found in potato (e.g., patatin, soybean trypsin inhibitors) are associated with food allergy.
Statistically significant decreases in the amino acid asparagine and increases in most of the other amino acids measured suggest possible slight changes in protein types or levels between event Gen2-Z6 potato and the parental Snowden variety. However, all amino acid values were within the combined literature range. This suggests that any changes in levels of allergenic proteins in the event Gen2-Z6 variety would be within the range of levels found across existing potato varieties.
Based on the information reviewed, there are no safety concerns regarding the food use of event Gen2-Z6 potato compared to conventional potato varieties from a toxicological or allergenic perspective.
Conclusion:
Health Canada’s review of the information presented in support of the food use of Simplot Innate® potato event Gen2-Z6 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this potato variety is as safe and nutritious as food derived from current commercial potato varieties.
Health Canada's opinion deals only with the food use of Simplot Innate® potato event Gen2-Z6. Issues related to its environmental release and use as animal feed have been addressed separately through existing regulatory processes in the CFIA.
This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.
For further information, please contact:
Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
hc.bmh-bdm.sc@canada.ca
Footnotes
- Footnote 1
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Richael, C.M., Kalyaeva, M., Chretien, R.C., Yan, H., Adimulam, S., Stivison, A., Weeks, J.T., and Rommens, C.M. (2008). Cytokinin Vectors Mediate Marker-Free and Backbone-Free Plant Transformation. 905–917.
- Footnote 2
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Lazo, G.R., Stein, P.A., and Ludwig, R.A. (1991). A DNA Transformation-Competent Arabidopsis Genomic Library in Agrobacterium. Biotechnology 9, 963–967.
- Footnote 3
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Nakata PA, Anderson JM, Okita TW (1994) Structure and expression of the potato ADP-glucose pyrophosphorylase small subunit. J Bio Chem 269: 30798-30807.
- Footnote 4
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Visser RGF, Stolte A, Jacobsen E (1991) Expression of a chimeric granule-bound starch synthase-GUS gene in transgenic potato plants. Pant Mol Bio, 17:691-699.
- Footnote 5
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Pel, M.A. (2010).Mapping, Isolation and Characterization of Genes Responsible for Late Blight Resistance in Potato. Wageningen University.
- Footnote 6
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Bushey, D.F., Bannon, G.A., Delaney, B.F., Graser, G., Helford, M., Jiang, X., Lee, T.C., Madduri, K.M., Pariza, M., Privalle, L.S., Ranjan, R., Saab-Rincon, G., Schafer, B.W., Thelen, J.J., Zhang, J.X., and Harper, M.S. (2014). Characteristics and Safety Assessment of Intractable Proteins in Genetically Modified Crops. Regul Toxicol Pharmacol. 69: 154-170.
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