Safety Assessment of Potatoes Resistant to Colorado Potato Beetle

Chapter 13

Safety Assessment of Potatoes Resistant to Colorado Potato Beetle 1

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P. B. Lavrik , D. E. Bartnicki , J. Feldman , B. G. Hammond , P. J. Keck , S. L. Love , M. W. Naylor , G. J. Rogan , S. R. Sims , and R. L. Fuchs 1

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Downloaded by UNIV OF ARIZONA on April 30, 2017 | http://pubs.acs.org Publication Date: October 5, 1995 | doi: 10.1021/bk-1995-0605.ch013

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The Agricultural Group, Monsanto Company, Chesterfield, MO 63198 Potato Products Division, Hybritech Seed International, Inc., Boise, ID 83706 College of Agriculture, University of Idaho, Aberdeen, ID 83210 2

3

Russet Burbank potato plants which resist damage by Colorado potato beetle (CPB) have been developed through Agrobacterium-mediated transformation. The CPB resistant potato plants produce two additional proteins, the Bacillus thuringiensis subsp. tenebrionis protein which confers resistance to C P B and the neomycin phosphotransferase II protein which serves as a selectable marker during the plant transformation process. Extensive studies have been performed to assess the environmental and food safety of the CPB resistant potatoes to gain regulatory approval for commercialization of potato varieties with this new trait. Data derived from the studies have confirmed the safety of the newly introduced proteins and demonstrated that the presence of these proteins and the process used to produce these plants did not alter the quality, composition or safety of the tubers from CPB resistant plants as compared to commercial Russet Burbank potatoes. Colorado potato beetle (CPB; Leptinotarsa deccmlineata) is the most damaging insect pest of the $2.3 billion U.S. potato crop (1.2). If untreated or poorly managed, the CPB can devastate potato production in some areas and lower yield by as much as 85 percent (3-5). Current CPB control primarily involves the use of a diminishing number of broad spectrum insecticides. These insecticides are variably effective due to environmental factors and insect susceptibility, with seasonal control costs which can exceed $200 per acre (6). Application of new technologies to control this pest is essential for continuance of potato production in some areas. 4

Current address: Calbiochem-Novachem, 10394 Pacific Center Court, San Diego, CA 92121 0097-6156/95/0605-0148512.00/0 © 1995 American Chemical Society

Engel et al.; Genetically Modified Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Through genetic modification, Russet Burbank potato plants have been produced which are virtually immune to damage by CPB (7). The CPB resistant plants were produced by inserting the gene coding for the CPB-active protein from Bacillus thuringiensis subsp. tenebrionis (B.t.t.) into the potato genome using Agrobacterium tumefaciens-mGdmted transformation (7). Field trials conducted during the past four years with CPB resistant potatoes have demonstrated effective, season long control of feeding damage by all stages of the C P B , including the recalcitrant adult, without insecticide application (7). Agronomic evaluation, consisting of plant growth characteristics, general insect and disease susceptibility, yield and tuber quality have shown the CPB resistant potatoes to be indistinguishable from control Russet Burbank potatoes (7). The superior CPB control offered by the plants will enable growers to significantly reduce the amount of chemical insecticide now applied to their crop and promote increases in beneficial insect populations which can help reduce other potato pests not directly controlled by the CPB-active protein.

Safety Assessment of CPB Resistant Potatoes As with other food crops improved through biotechnology, the United States Department of Agriculture (USDA), the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) exercise joint regulatory oversight for these genetically improved potatoes. The USDA, under the jurisdiction of the Plant Pest Quarantine Act, regulates the impact of the genetically modified plant on the environment and production agriculture according to guidelines published in 1993 (8). The EPA, under the jurisdiction of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), regulates the environmental and food safety, respectively, of both the pesticidally active (e.g. B.t.t. protein) and inert (e.g. NPTII protein) ingredient introduced into the plant according to proposed guidelines (9). The FDA, under the jurisdiction of the Federal Food, Drug and Cosmetic Act (FFDCA), oversees the safety and wholesomeness of the food and/or feed according to the new plant variety food policy published in 1992 (10). CPB resistant potatoes are the first genetically modified crop possessing a pesticidal trait to be reviewed for full approval by the regulatory agencies and can thereby serve as a case study for the overall safety assessment strategy (//). The safety of the CPB resistant potatoes was assessed according to the published guidelines and policies of the three regulatory agencies. The concept of "substantial equivalence" (also referred to as "no material difference" - terms which encompass a demonstration that the new potato variety is comparable to traditionally bred potatoes in terms of nutritional quality, level of important natural products, and agronomic and environmental performance) was used to assess the safety of these potatoes (10,12). The studies focused on two primary items: (1) the safety of the additional expressed proteins to the environment and in food/feed, and (2) the composition and wholesomeness of the potato tuber. Safety assessments were conducted on seven independently transformed CPB resistant lines.



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GENETICALLY MODIFIED FOODS

Figure 1. Plasmid used in the Agrobacterium-mçd\a.lQd transformation of Russet Burbank potato plants. T-DNA, region inserted into the potato genome, from the right border to the left border, the region contains: the E35S cauliflower mosaic virus promoter with the duplicated enhancer region; the crylllA gene which confers resistance to CPB; the E9 3' terminator of the pea ribulose-l,5-bisphosphate carboxylase; NOS 3' terminator from the nopaline synthase gene; the nptll gene which codes for neomycin phosphotransferase type II enzyme; and the 35S cauliflower mosaic virus promoter. Regions of D N A present in the plasmid but not transfered into the potato genome: aad, the gene for the enzyme streptomycin adenylyl-transferase that allows for bacterial selection on spectinomycin or streptomycin; ori-322/rop, a segment of pBR322 which provides the origin of replication for maintenance of the plasmid in E. coli and the replication of primer for conjugational transfer into Agrobacterium cells; oriV, origin of replication for the Agrobacterium. EcoRl, ////idlll, Xhol and Not\, endonucleases restriction sites and their location in base pairs.


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ET AL.


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Safety of Newly Expressed Proteins. The CPB resistant potato plants produce two additional proteins, the B.t.t. protein which confers protection to CPB and the neomycin phosphotransferase (NPTII) protein which enabled the selection of transformed potato cells expressing the B.t.t. protein in tissue culture. In assessing the safety of these newly introduced proteins in food/feed and to the environment, the safety of the donor organism as well as the potato transformation process was also considered. Safety of Donor Organisms and the Transformation Process. Two genes were introduced into the Russet Burbank genome, the crylllA and the nptll genes. The crylllA gene (13) was obtained from Bacillus thuringiensis subsp. tenehrionis (B.t.t.) strain BI 256-82 isolated by Krieg et al. (14) and encodes the CPB-active B.t.t. protein. This bacterium belongs to a large group of common spore-forming soil bacteria that produce proteinaceous parasporal crystalline inclusions that are lethal to specific classes of insects ( 15). For over 30 years, a number of species from this group of bacteria have been safely used as commercial microbial insecticide formulations (16). Microbial formulations of B.t.t. have been in commerce since 1988 with an equal record of safe usage (17,18). Based on the available scientific data, EPA and other regulatory agencies worldwide have determined that the use of registered Bacillus thuringiensis products, including B.t.t., pose no significant risks to human health or non-target organisms (19). The nptll gene was obtained from the procaryotic transposon Tn5 (20) and it codes for the neomycin phosphotransferase II enzyme which serves as a selectable marker protein during the plant transformation process. Organisms containing this protein are ubiquitous in the environment and within mammalian digestive systems (21). Many new plant varieties, improved through genetic engineering, utilize this enzyme as a selectable marker (22). The safety of this protein has been well established and the USDA, FDA and EPA have approved its use in crops (23-25). Introduction of the crylllA and nptll genes into the genome of seven CPB resistant lines under development was mediated by Agrobacterium tumefaciens harboring the double border binary vector, PV-STBT02, schematically shown in Figure 1. Southern hybridization analysis (26) of the DNA from each of the seven lines confirmed that the crylllA and the nptll genes, delineated by the right- and leftborders (depicted as the T-DNA region in Figure 1), were the only genes inserted into the potato genome. The analysis also demonstrated that complete insert integrity (gene size, composition and linkage) was maintained during the transformation process. Protein Expression Levels. The B.t.t. and NPTII proteins are expressed at extremely low levels in potato leaf and tuber tissues of all seven lines. Based on data obtained by validated enzyme linked immunosorbent assays (ELISA's) from plant tissue collected at seven field sites throughout the commercial potato growing regions of the United States, the expression level of the B.t.t. protein in the seven CPB resistant lines was estimated to range from 5 to 28 μg per gram fresh weight of


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leaf tissue (approximately 0.12% of the total leaf protein) and 0.4 to 2.0 μg per gram fresh weight of tuber tissue (< 0.01% of the total tuber protein). The expression level of the NPTII protein in both the leaf or tuber tissues was estimated to be approximately 2- to 5-fold lower than the expression of the B.t.t. protein. The expression of the B.t.t. and NPTII proteins in foliage remains relatively constant throughout the season with the expected decline at the onset of plant senescence. Safety of the B.tt Protein. The amino acid sequence encoded by the crylllA gene inserted into the potato plants produces a protein identical to that produced by the B.t.t. microbe found in nature and also present in registered commercial microbial pesticide formulations ( 7,27-29). This protein is selectively active against a narrow spectrum of Coleoptera without affecting non-target insects, humans or animals (30). Upon ingestion by susceptible insect species, the protein binds to specific receptors in the mid-gut, feeding is inhibited with disruption of the gut epithelium and eventual death of the insect pest (31). Since other non-target organisms, including other classes of insects as well as birds, fish, and mammals do not possess such receptors, the B.t.t. protein does not affect them (32,33). Even though the B.t.t. protein produced in CPB resistant potatoes is the same protein contained in commercial microbial products, many of the previous studies were repeated to verify the safety and selectivity of this protein. However, in contrast to previous studies which assessed the safety of microbial formulations, the present studies utilized highly purified B.t.t. protein as the test material. Since limited expression of this protein prohibited the isolation of large quantities of this protein from the potato tubers or potato plant directly, gram quantities of the B.t.t. protein were produced in E. coli engineered with the same gene present in the potato plants. Minor amounts of this protein, purified from the potato tuber and from E. coli, were shown to be chemically and functionally equivalent by a series of commonly used analytical assays, which included molecular weight, N-terminal amino acid analysis, immuno-reactivity, lack of glycosylation and insecticidal potency.

An acute gavage study was conducted in mice to confirm the mammalian safety of the B.t.t. protein. Following EPA guidelines, a high dose of the test substance (5000 mg of B.t.t. protein per kg body weight) was administered to the test animals. The dose was equivalent to over a 2.5 million-fold safety factor based on the average human consumption of potatoes and the level of the B.t.t. protein present in the tuber. No adverse effects were observed in test animals and there were no effects on food consumption, weight gain, or gross pathology. Additionally, purified protein was subjected to an in vitro digestion experiment which demonstrated that the B.t.t. protein has an extremely short half-life (less than 30 seconds) under simulated human gastric conditions (34). These studies confirm the safety of the B.t.t. protein to humans and animals. The specificity of the B.t.t. protein to CPB was confirmed by insect host-range studies, also using purified B.t.t. protein isolated from E. coli. Five non-target beneficial insects (including adult and larval honey bees, Apis mellifera; lacewings,


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ladybird beetles, Hippodamia convergens; and a parasitic wasp, were shown to be unaffected by high doses of the purified B.t.t. protein (greater than 100 times the lethal concentration to CPB). These studies confirm the specificity and the safety of the B.t.t. protein to nontarget insects. As with most common proteins, the B.t.t. protein is not expected to persist in the environment. Studies were conducted on the loss of bioactivity of the B.t.t. protein in potato plant tissue incorporated into soil (at levels approximately 35-fold higher than that estimated under field conditions) and in plant tissue without soil. Under both conditions, complete decay of biological activity was noted within 9 days. Therefore, a rapid degradation of B.t.t. protein bioactivity in potato plant tissues remaining in the field after potato tuber harvest is predicted. Chrysopa

carnea;


Nasonia vitripennis)

Safety of the NPTII Protein. The NPTII protein was used as a selectable marker to enable the identification of potato cells containing the crylllA gene. The safety assessment of the NPTII protein has been discussed in detail in the FDA approval of the protein as a processing aid food additive (24,35) and in recent articles by Fuchs et al. (36,37). The latter articles describe acute gavage and digestive fate studies identical to those conducted for the B.t.t. protein, with similar results. No adverse affects were observed in a mouse acute gavage study in which mice were administered a dose (5000 mg/kg) of NPTII equivalent to 5 million-fold greater than the projected human consumption. Similar to the results observed with the B.t.t. protein, the NPTII protein was shown to rapidly degrade in the simulated digestive fate study, with a half-life of less than 20 seconds in gastric fluid. These studies confirm the mammalian safety of this protein. Composition and Wholesomeness of the Potato Tuber. Safety assessment of the tuber focused on whether the CPB resistant potatoes are "substantially equivalent" to the parental Russet Burbank variety. These varieties were evaluated in respect to composition, nutritional quality and in aspects that could impact the use, value, the environment, or food/feed safety of this product. In terms of nutrition, potato has long been valued as an excellent source of staple starch and contributes mainly to the carbohydrate calories of the diet. More recently, potato has also been valued for selected vitamins and minerals such as vitamin C, vitamin B , and potassium (National Potato Board). Tubers from CPB resistant potato plants were analyzed for these key components of interest as well as a number of other constituents. The compositional analyses were more extensive than the analyses carried out on new commercial potato varieties developed through classical breeding. The analyses included a total of 23 components: proximate composition (total protein, total carbohydrate, fat, dietary fiber and ash), vitamins (vitamin C, vitamin thiamine, niacin, folic acid and riboflavin), minerals (calcium, copper, iron, iodine, magnesium, phosphorous, potassium, sodium and zinc) and glycoalkaloids (solanines and chaconines), which are the important natural toxicants present in all potatoes. The levels of these constituents in each of the seven CPB resistant potato lines were comparable to the parental Russet Burbank control and 6


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Table I. Composition of C P B Resistant and Parental Russet Burbank Potatoes Russet Burbank Component

Mean


0

CPB Resistant" Range

Control Mean

Range

Published Range

Solids, % tuber fresh weight

19.6

18.0 -21.0

20.0

19.6 -20.5

16.8-24.5b

Carbohydrate, g/100g tuber

16.0

15.4 - 16.5

16.0

15.7 - 16.4

13.0- 17.0c

Protein, g/100g tuber

2.1

2.1 - 2.2

2.1

2.0- 2.1

1.4-2.9c

Vitamin C, mg/100g tuber

11.4

8.7- 13.6

11.6

11.0 - 12.3

10.3 -22.0b

Vitamin μg/100g tuber

97.2

75.4 - 119.0

97.2

89.2 - 105.0

140.0-280.0c

Folic acid, μg/100g tuber

6.7

5.7- 7.7

7.0

5.2- 8.7

4.0 - 20.0c

Potassium, mg/100g tuber

420

388 ••453

416

393 -•438

340 - 600c

3.1 - 16.1b Glycoalkaloids, 3.8 2.7- 5.8 2.7- 3.5 3.1 mg/100g tuber Values are the mean of tubers obtained from seven CPB resistant or parental Russet Burbank control plant lines grown at two field locations. At each field location, plots for each CPB resistant line were replicated six times. Taken from Russet Burbank tubers grown in Aberdeen, ID (Pavek, J. et al. Western Regional Variety Trial Report. 1980-1992, WRCC-27, University of Idaho, ID). cRange of values for white potatoes, Scherz, H. and Senser, F. In Food Composition and Nutrition Tables 1989/90, Deutsche Forshungsanstalt fur Lebensmittelchemie, Garching b. Muchen, Eds Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 1989, pp 542-544. a

b


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155

within the range of values reported in the literature. A comparison of the key components is summarized in Table I.


To assess the overall wholesomeness of the CPB resistant potatoes, raw potato tubers from both the CPB resistant and Russet Burbank control plants were fed, along with the regular diet, to rodents in a 28-day study. In this study, rats consumed an average of 80 g of potatoes per kg of body weight per day, which is equivalent to a human consumption of 35 to 40 potato tubers per day. No differences in food consumption, growth rate, behavior, or gross pathology were observed during these studies. Assessment of the Allergenic Potential. Since potatoes are rarely known to cause allergic reactions, the allergenic potential assessment of CPB resistant potatoes focused primarily on the potential allergenic concerns of the two introduced proteins, the B.t.t. and NPTII proteins. These proteins have a long history of safe use and do not share the biochemical profile common to known allergenic proteins. Therefore, it is unlikely these proteins would pose any significant allergenic concerns. An assessment of the potential allergenic concerns for NPTII protein was considered by the FDA in their approval of the use of this protein as a processing aid food additive and found to pose no significant allergenic concerns (24). As discussed above, the B.t.t. protein expressed in CPB resistant potato plants is indistinguishable from the B.t.t. protein contained in commercial microbial formulations that have been used safely for the past six years on a variety of crops including fresh produce like eggplant and tomato, with no reported allergenic responses, thus supporting the lack of allergenic concern for the B.t.t. protein. Although there are no assays available to definitively predict the allergenic potential of proteins, the biochemical profile of the B.t.t. protein provides a qualitative basis for allergenic assessment when compared to known protein allergens. In general, protein allergens are present at high concentrations in foods that elicit an allergenic response; are stable to peptic and tryptic digestion and the acid conditions of the digestive system; and are often glycosylated (38-40). The B.t.t. protein does not possess any of these characteristics. The B.t.t. protein is present in CPB resistant potatoes at extremely low amounts; it was shown to be very labile in the mammalian gastric digestive system, minimizing any potential for this protein to be absorbed by the intestinal mucosa; and, the B.t.t. protein, as purified from the tuber of CPB resistant plants, is not glycosylated. Furthermore, a comparison of the B.t.t protein sequence by either a FASTa- (41) or Monte Carlotype (42) analysis showed no significant homology to any known protein allergens in protein data bases (Pir protein, Swissprot, and Genpept protein). Based on this qualitative assessment, we conclude that CPB resistant Russet Burbank potatoes pose no additional allergenic concerns than any other potato in commerce.


156



Conclusions CPB resistant Russet Burbank potato plant lines generated through genetic modification were assessed for environmental and food/feed safety based on the safety of the introduced proteins and the concept of "substantial equivalence." A direct safety assessment of the newly introduced B.t.t. and NPTII proteins, confirmed the safety of these components. The CPB resistant potato plant lines were shown to be substantially equivalent to traditionally bred potatoes in terms of nutritional quality, level of important natural products, and agronomic and environmental performance. Substantial equivalence was established for seven independently transformed CPB resistant plant lines with the parental Russet Burbank line which demonstrates a generic lack of effect by introduction of two genes in the potato genome. Literature Cited 1. 2. 3. 4.

Casagrande, R. A. Bull. Entomol. Soc. 1987, 33, 142-150. Potato Statistical Yearbook, National Potato Council, Englewood, CO, 1992. Hare, J. D. J. Econ. Entomol. 1980, 73, 369-373. Ferro, D. N.; Morzuch, B. J.; Margolies, D. J. Econ. Entomol. 1983, 76, 349356. 5. Shields, E. J.; Wyman, J. A. J. Econ. Entomol. 1984, 77, 1194-1199. 6. Ferro, D. N.; Boiteau, G. In Plant Health Management in Potato Production; Rowe,R.C.,Ed.; Am. Phytopath. Soc. Press: St. Paul, MN, 1992, pp 209-234. 7. Perlak, F. J.; Stone, T. B.; Muskopf, Y. M.; Petersen, L. J.; Parker, G. B.; McPherson, S. Α.; Wyman, J.; Love, S.; Reed, G.; Biever, D.; Fischhoff, D. A. Plant Molec. Biol. 1993, 22, 313-321. 8. U. S. Department of Agriculture (USDA). Federal Register 1993, 58, 1704417059. 9. U. S. Environmental Protection Agency (EPA). Federal Register 1992, 57, 55531-55532. 10. U. S. Food and Drug Administration (FDA). Federal Register 1992, 57, 22984-23005. 11. U. S. Environmental Protection Agency (EPA). Federal Register 1993, 58, 64582-64583. 12. Organization for Economic Cooperation and Development (OECD). Safety Evaluation

of Foods

Derived

by Modern

Biotechnology:

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Principles, Paris, 1992.

13. 14. 15.

Höfte, H.; Whiteley, H. R. Microbiol. Rev. 1989, 53, 242-255. Krieg, Α.; Huger, A. M.; Langenbruch, G. Α.; Schnetter, W. Z. Angew., Entomol. 1983, 96, 500-508. Aronson, A. I.; Beckman, W.; Dunn, P. Microbiol. Rev. 1986, 50, 1-24.


and

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23. 24. 25. 26. 27. 28.

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Environmental

Entwistle, P. F.; Cory, J. S.; Bailey, M. J.; Higgs, S., Eds.; John Wiley & Sons, New York, NY, 1993, pp 171-191. U. S. Environmental Protection Agency (EPA). National Technical Information Service (NTIS) PB 89-164198; U. S. Government Printing Office, Washington, DC, 1988. Beck, E.; Ludwig, G.; Auerswald, Ε. Α.; Reiss, B.; Schaller, H. Gene 1982, 19, 327-336. Nap, J. P.; Bijvoet, J., Stikema, W. J. Transgenic Research 1992, 1, 239-249. World Health Organization (WHO). Health Aspects of Marker Genes in Genetically Modified Plants. WHO Food Safety Unit Workshop Report, Copenhagen, Denmark, 1993, pp 1-32. U. S. Department of Agriculture (USDA). Federal Register 1992, 57, 4760847616. U. S. Food and Drug Administration (FDA). Federal Register 1994, 59, 26700-26711. U. S. Environmental Protection Agency (EPA). Federal Register 1994, 59, 49351-49353. Southern, E. M. J. Mol. Biol. 1975, 98, 503-517. McPherson, S. Α.; Perlak, F. J.; Fuchs, R. L.; Marrone, P. G.; Lavrik, P. B.; Fischhoff, D. A. Bio/Technology 1988, 6, 61-66. Herrnstadt, C.; Gilroy, T. E.; Sobieski, D. Α.; Bennett, B. D.; Gaertner, F. H. Biopesticide:

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Gene 1987, 57, 37-46.

29. 30.

31. 32. 33. 34. 35.

Donovan, W. P.; Gonzalez Jr, J. M.; Gilbert, M. P.; Dankocsik, C. Mol. Gen. Genet. 1988, 214, 365-372. MacIntosh, S. C.; Stone, T. B.; Sims, S. R.; Hunst, P. L.; Greenplate, J. T.; Marrone, P. G.; Perlak, F. J.; Fischhoff, D. Α.; Fuchs, R. L. J. Invert. Path. 1990, 56, 258-266. Slaney, A.C.;Robins, H. L.; English, L. Insect Biochem. Molec. Biol. 1992, 22, 9-18. Hofmann, C.; Vanderbruggen, Η. V.; Höfte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. Proc. Natl. Acad. Sci. USA 1988, 85, 7844-7848. Sacchi, V. F.; Parenti, P.; Hanozet, G. M.; Giordana, B.; Lüthy, P.; Wolfersberger, M. G. FEBS Lett. 1986, 204, 213-218. United States Pharmacopeia, United States Pharmacopeial Convention, Inc.: Washington, DC, Vol. XXII; pp 1788-1789. Calgene, Inc. FDA Docket Number: 90A-0416, 1990.


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37.


38. 39.

40. 41. 42.


Fuchs, R. L.; Heeren, R. Α.; Gustafson, M. E.; Rogan, G. J.; Bartnicki, D. E.; Leimgruber, R. M.; Finn, R. F.; Hershman, Α.; Berberich, S. A. Bio/Technology, 1993, 11, 1537-1542. Fuchs, R. L.; Ream, J. E.; Hammond. B. G.; Naylor, M. W.; Leimgruber, R. M.; Berberich, S. A. Bio/Technology, 1993, 11, 1543-1547. Taylor, S. L. Food Technol., 1992, 39, 146-152. Taylor, S. L.; Nordlec, J. Α.; Bush, R. K. In Food Safety Assessment; Finley, J. W.; Robinson, S. F.; Armstrong, D. J., Eds; ACS Symposium Series No. 484, American Chemical Society: Washington, D.C., 1992, pp 316-329. Metcalf, D. D. Clin. Rev. Allergy, 1985, 3, 331-349. Pearson, W. R. Meth. Enzymol., 1990, 183, 63-98. Needleman, S. B.; Wunsch,C.P.J.Mol.Biol., 1970, 48, 443-453.

R E C E I V E D June 7, 1995


Safety Assessment of Potatoes Resistant to Colorado Potato Beetle

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