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NEW APPLICATIONS OF BIOTECHNOLOGY IN THE FOOD INDUSTRY Robert H. Lawrence In the past several years, biotechnology in the food industry has been the central theme of numerous scienti- fic reviews, national and international symposia, and several major reference works (Earle, 1984; Harlander and Labuza, 1986; Jarvis and Holmes, 1982; Kirsop, 1985; Knorr, 1987; Knorr and Sinskey, 1985; Moo-Young et al., 1985; Rehm anti Reed, 1983~. Reports of significant advances have come from the full spectrum of biotech- nology research and development resources: universities and institutes as well as genetic "biotiques" and large food corporations. Important business alliances continue to be formed on a worldwide scale, linking advanced biotechnology research skills with large producers and marketers of food products, principally in the United States, Japan, the United Kingdom, and Europe. These alliances include Amgen/Kodak, CalBio/American Home Proclucts, Genentech/Lilly, Genentech/Corning (Genencor), Interferon/Anheuser-Busch, Molecular Genetics/Upjohn, Synergen/Procter & Gamble, American Cyanamid/Pioneer Hi-Bred, Dupont/Advanced Genetic Sciences, W. R. Grace/ Cetus (Agricetus), Hoechst/Harvard, Monsanto/Genentech, Monsanto/Washington University/Rockefeller University, Roche/Agrigenetics, Beatrice/Ingene, Campbell/DNA Plant Technology (DNAP), Campbell/Calgene, CPC/Enzyme Bio- systems, Kraft/DNAP, General Foods/DNAP, Kellogg/ Agrigenetics, Heinz/ARCO, McCormick/Native Plants, Inc., Molson/Allelix, R]R-Nabisco/Escagen, and Seagram/ 19

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Biotechnica. Corporate boards and strategic planning groups of major food companies now understand the language of biotechnology and can perceive its utility and value; this has been the case with their corporate research departments for years. One thing is clear: The excite- ment and enthusiasm for biotechnology so characteristic of the pharmaceutical and medical areas in the early 1980s have now begun to hit the food industry with increasing force, and this momentum will likely establish this indus- try as the largest commercial arena for biotechnology. Companies involved include Archer Daniels Midland, American Home Products, Beatrice, Campbell, Cargill, Corn Products Company, Coors, Chr. Hansen's Laboratory, Firmenich, General Foods, Heinz, Hunt-Wesson, Kraft, LaBatt, McCormick, Nestle, Pillsbury, Purdue, Procter & Gamble, Ralston, RJR-Nabisco, Staley, Unilever, and Universal Foods. At least three important factors are responsible for this. First, in pharmaceuticals, the feasibility of the biotechnology promise has been established and the commer- cial reduction to practice (i.e., commercial application) is in place--in the marketplace! This was achieved by using many of the same technical concepts and strategies currently envisioned for food industry applications. Second, key advancements in technology continue to be made, principally in molecular genetics, cell technolo- gies, computer-aided protein engineering, bioreactor design, and biosensor/diagnostic technology. These advancements have substantially redefined the technical skills base and broadened the potential applications of biotechnology to foods. Third, within the food industry, reports of successful new applications of biotechnology (e.g., those reported here) add confidence to the predic- tion that biotechnology may well be the next key source of competitive leverage at the corporate and international levels, and may be the most important single technical consideration in consolidation strategies. The following paragraphs are a review of new appli- cations of biotechnology in each of the following food-related areas: enzymes, including the processing of cheese; fermentation, including brewing and wine making; agricultural raw materials (e.g., crop plants, meat, poultry, fish) with improved functionality; and plant cell bioreactors for food ingredient production. 20

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ENZYMES Food Industrv Uses A recent report on the U.S. market for enzymes indi- cated total sales of $185 million in 1985, 58% of which was in the food industry (Charles Kline & Co., 1986~. Of the classes of enzymes used by the food industry, the proteases and carbobydrases account for most of this market. The predominant protease sold is rennin (chymosin), which is used in cheese-making processes to Coagulate milk to form curds. Of the carbohydrates, those used in cornstarch processing (the so-called starch enzymes a-amylase, glucoamylase, and glucose isomer- ase) account for 85% of sales. The other enzymes have diverse applications, including flavor development (e.g., lipases in cheese making) (Arbige et al., 1986), improve- ment of extractions (e.g., pectinases in juice processing) (Kilara, 1982), and modification of food functionality (e.g., a-amylases in retarding bread staling) (Boyce, 1986~. The technology for improvement of food enzyme production by genetic engineering is clearly in place (Lin, 1986~. Genes for many of the important food industry enzymes have been cloned (Meade et al., 1987), and gene transfer systems that permit introduction and expression in generally recognized as safe (GRAS) organisms have been developed (Lin, 1986~. Two recent, important applications of genetic engineering to enzyme production are a-amylase and chymosin. a-Amylase High Fructose Corn SYrun Industrv. The first petition to the Food and Drug Administration (FDA) to affirm the GRAS status of a food-processing enzyme produced by recombinant DNA techniques was for a-amylase. This landmark petition was filed by CPC International, Inc., on July 9, 1984. a-Amylase is the enzyme used in the first step in the production of high-fructose corn syrup (HFCS), a widely used nutritive sweetener derived from cornstarch. The HFCS process was first developed in the United States between 1968 and 1972 by the Clinton Corn Processing Co. (Lloyd and Horwath, 1985) and involves three sequential 21

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enzymatic steps. First, raw cornstarch is liquefied by a-amylase hydrolysis to yield partially degraded starch chains called dextrins. The dextrins are then hydrolyzed by glucoamylase, which cleaves both the a-1,6 and a-1,4 glucosidic linkages to give corn syrup (glucose). This glucose hydrolysate is refined and then isomerized by immobilized glucose isomerase to give a mixture of glucose and fructose (42%) known as HFCS. This last step was commercialized in 1972 and represents the first large-scale use of an immobilized enzyme permitting a continuous process with significant cost reduction (Casey, 1977~. The 42% HFCS can then be further purified to yield second-generation syrups of 55% and 90% fructose (Coker and Venkatasubramanian, 1985~. The market for HFCS has grown dramatically. U.S. consumption increased from 2.3 kg/per person in 1975 to 20 kg/per person in 1985 (Newsome, 1986~. Today, produc- tion exceeds 4.54 billion kg annually. HFCS is used in many processed food products and is the principal nutritive sweetener of the soft drink industry. Grant (1986) recently discussed CPC's genetically engineered a-amylase and its GRAS affirmation petition with the FDA. His objective was to use Bacillus subtilis as a host system for the commercial production of a thermostable form of a-amylase that CPC had developed from Bacillus stearothermonhilus (Ishii et al., 191--an organism given GRAS status by FDA (Figure 1~. This heat- and acid-stable form of a-amylase is important for low-cost production of HFCS. Its production in B. subtilis was desired because of the ease with which B. subtilis can be used in commercial fermentations. According to CPC's petition, the strain designated as B. subtilis ATCC 39,705 was genetically derived from an asporogenic variety of B. subtilis ATCC 39,701, which lacked cx-amylase, by introducing genetic material from B. stearothermonhilus ATCC 39,709 for ~-amylase pro- duction. The genetically engineered B. subtilis contains DNA from a plasmid vector designated as pCPC720. The plasmid consists of a 2.4-kb portion of DNA comprising the ~-amylase gene from B. stearothermonhilus and a portion of DNA from plasmid pUBllO required for replica- tion of the new plasmid. pCPC720 does not contain the kanamycin resistance marker of pUB110, and transformed host cells are not resistant to kanamycin. 22

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A Petition for the Affirmation of the GRAS Status of Alpha-Amylase Derived from Bacillus subtilis ATCC 39,705 Filed July 1984 by CPC International Proposed commercial product: alpha-amylase Gags as a cell- free broth containing a thermostable form of enzyme B. sublilis (AMY-) ATCC 39,701 Asporogenic B. subtilis (AMY+) ATCC 39,705 Asporogenic (Thermostable alpha-amylase) B. stearothermophilus (AMY+ ) ATCC 39,709 (Thermostable alpha-amylase) pCPC720 (AMY=) ~ FIGURE 1 Genetically engineered a-amYlase. The CPC petition presents data characterizing the enzyme and recombinant organism to show that the genetically engineered enzyme is equivalent in every respect to that produced by B. stearothermonhilus and to establish the safety of the recombinant product. The enzymes would be used in the HFCS process only and would not be present in the food product. Regarding the CPC petition, Grant urged, "We need to have scientifically based regulatory decisions, and we need to have responsible industry actions.... Because this is the first of potentially many such petitions being reviewed by FDA with respect to a recombinant microorga- nism, the FDA has to be very careful and precise. The FDA ruling on our petition will set th'e policy for future rulings" (Grant, 1986, p. 22~. That is no doubt the case: CPC in September was awarded a patent covering the genetic engineering of _. subtilis to produce a thermo- stable pullulanase (Coleman and McAlister, 1986~. There are many similar developments, as shown in the following two examples. ChYmosin (Rennin): DairY IndustrY. Another enzyme that has been the focus of considerable genetic engi 23

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peering research is chymosin, the active component of rennet used in the dairy industry to coagulate milk to form curds in the cheese-making process. Chymosin is an endoprotease that is highly specific in the hydrolysis of peptide in bonds in the V-fold of kappa-casein of milk, resulting in the destabilization of casein micelles and subsequent curd formation. Commercial sources are calf rennet extracted from the fourth stomach of young suckling calves and microbial rennets principally from the fungi Mucor miehei, M. ousillus, or Endothia narasitica. These fungi produce chymosin with slight differences in milk-clotting properties, and in heat and pH stability, as well as a different coagulation/proteolysis ratio from that of chymosin from calf sources. Thus, a demand exists for chymosin similar to calf rennet for use in the pro- duction of quality cheeses. The opportunity for microbial production of calf rennet chymosin has led several com- panies to develop strategies to clone the gene for chymosin from cDNA libraries derived from calf stomach mRNA and to achieve expression of the heterologous gene in various host organisms (Figure 2~. In vivo, chymosin is Strategies Gene Clone chymosin cDNA derived from calf stomach mRNA Signal -16 sequence 43 . . 365 _ (Protein) in Viva Secreted Zymogen Active Form Host Organisms Cheese Trials t perprochymosin | prochymosin | chymosin E. coil, B. subtilis (Fusion protein) S. cerevisiae, W - erornyces /actis Aspergillus nidulans E. co/l- derived chymosin vs. calf rennet Comparable cheeses Limitation Accumulation in cytoplasm, insoluble and inactive aggregates. Requires solubilization and refolding. Low yield, expensive. Companies -Codon, Genex, Unilever -Collaborative Research, Gist-Brocades -Genencor FIGURE 2 Genetically engineered chymosin production. 24

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produced as preprochymosin, which is secreted as the zymogen, prochymosin. In low-pH solutions, prochymosin is autocatalytically cleaved to chymosin (McGuire, 1986~. Escherichia coli-produced chymosin has several limitations (Pitcher, 1986~. The enzyme accumulates in the cytoplasm, requiring an expensive and low-yield process to derive the active enzyme. Two alternative host systems have been used: the so-called supersecretor strains of yeast (used by Collaborative Research, Inc.) and the filamentous fungi (used by Genencor, Inc.~. The Genencor strategy for Asner~illus production of bovine chymosin (Heyneker et al., 1986; W.H. Pitcher, Genencor, personal communication, 1986) involved the use of hetero- logous gene constructions in A. nidulans transformants, which secrete the gene product. Plasmid constructions consisted of the control regions of the A. nicer glucoamylase gene coupled to either bovine prochymosin or preprochymosin cDNA with a glucoamylase terminator. A. nidulans transformants secreted chymosin, which was similar to authentic bovine chymosin in molecu- lar weight and specific activity. Cheese trials using these chymosin preparations are being evaluated (Pitcher, 1986~. Thus, commercial production of chymosin similar to calf rennet appears to be technically feasible. Other applications of genetic engineering to enzyme production for the food industry include: lactase, to break down milk lactose; lipase and esterase, to develop cheese flavor; pectinase, to improve yield, reduce viscosity, and enhance clarification in fruit juice processing and wine making; protease, to serve as a malt substitute when used with barley; and carbohydrases, to facilitate carbohydrate metabolism in low-calorie beer production. FERMENTATION Brewinz Yocum (1986) of BioTechnica International has reported the development of a genetic engineering procedure suitable for polyploid industrial yeast (SaccharomYces cerevisiae) strains used in brewing. A new set of plas- mids for industrial yeast transformation was developed; 25

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these plasmids integrated the G418 resistance marker and targeted it for insertion at the HO (homothallism) locus. Multiple insertions were accomplished by a process that leaves the gene of interest integrated into the HO target locus but jettisons the G418 resistance gene. Yeasts transformed in this manner contain the new genes stably integrated into their chromosomes at homologous loci but with no remaining E. cold DNA sequences. For details of the plasmid contractions, refer to Figures 1 and 2 of Yocum (1986~. The BioTechnica group has demonstrated the commercial feasibility of this genetic engineering procedure in SaccharomYces for the production of light beer. BioTech- nica cloned the gene coding for glucoamylase from A. nicer and inserted the gene into brewing yeast. The glucoamylase expressed by the yeast during fermentation breaks down the soluble starch to glucose; this is metabolized by the yeast, resulting in a lower calorie beer without requiring the use of added enzyme prepar- ations. Wine Making Snow (1985) has recently proposed a strategy for genetic engineering of industrial yeast strains used in wine making to introduce the capability for malolactic fermentation. The primary fermentation that occurs in wine making is achieved through the use of yeast to convert sugar to alcohol. A secondary fermentation may be allowed to occur, particularly during production of red wines, which is catalyzed by bacteria in the genera Leuconostoc, Lactobacillus, and Pediococcus. During this secondary fermentation, malic acid is converted to lactic acid, which causes a decrease in wine acidity, brings finished wine into better acid balance, and develops more desirable flavor complexity. Procedures used to encourage the malolactic fermentation may increase the risk of wine quality loss. They also increase the costs of wine production. In the strategy proposed by Snow (1985) and experi- mentally investigated (Williams et al., 1984), the malolactic gene of Lactobacillus delbrueckii was introduced into a laboratory yeast strain. When this yeast was used to make wine in a trial fermentation, the 26

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malolactic gene was expressed and limited malate conver- sion occurred. Thus, the feasibility of this approach appears to have been demonstrated. Obviously, the yeast gene transfer system developed by BioTechnica would be of value in this approach. AGRICULTURAL RAW MATERIALS New applications of biotechnology are leading to notable improvements in yield and productivity in crop plants and animals (Jaworski, see paper in this volume). Crops may be specifically improved for functional attributes, such as nutrition, flavor, texture, and processibility. These improvements result in added value to the food processor as well as to the consumer. Cron Plants Figure 3 gives a food industry perspective of plant biotechnology. This discussion focuses on three areas: the central role of modern breeding strategies in crop development, new genetic tools and how they influence breeding strategies, and the functional attributes of crops along with the concept of utilization-side genetics and added value. Functionality Genes E s m - CL Functional Attributes ~ Culture, \< ~ Cell I GeneUC| ~ PrOtoplasts) ~ ~ "Fusion ~ if_ Transter I Traditional Added Value New Added Value FIGURE 3 Plant biotechnology--food industry perspective. 27

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Genetic Imorovement Strategies. Most contemporary approaches to crop improvement are centered on modern breeding strategies that use a wide range of genetic tools and germplasm resources to generate genetic variability and diversity for traits of interest and to construct genotypes with new gene combinations from which new plant varieties are developed and then selected through a series of trials and evaluations. To be effective in their critical strategic role, contemporary plant breeders must be proficient in the application of an array of genetic techniques, including several new technologies that are only now being integrated into plant breeding programs. These technologies have brought about a dramatic reorien- tation of the plant breeders' approach to the introduction of new genes into existing varieties and have greatly expanded the potential sources from which new, useful genes can be accessed. Conventional germplasm resources, including valuable wild plant populations, will remain a primary gene source. Techniques that facilitate inter- generic gene transfer will increase the importance of these germplasm resources. However, accessibility to genes from outside the plant kingdom (e.g., from bacteria and animals) is now possible and will require that plant breeders adopt a broader, interdisciplinary perspective. New hybridization systems for production of hybrid seeds are being developed; these involve cellular level manipulation of organellar genomes for cytoplasmic male sterility (Cocking, 1985) and the introduction of genes controlling self-incompatibility (Nasrallah and Nasrallah, 1985~. New hybrid seed production schemes have also been developed; these involve cloned parent lines produced by tissue culture techniques (Lawrence and Hill, 1982, 1983~. The ability to clone plants in large scale through somatic embryogenesis (Lawrence, 1981) and encapsulation to form synthetic seeds (Lutz et al., 1985; Redenbaugh et al., 1986) allow crops to be produced from unique geno- types, which cannot be economically reproduced through seeds. Thus, new options for crop establishment must be considered in developing breeding strategies. Plant breeding programs make extensive use of trials and evaluations, typically in greenhouses and field plots, for the characterization of genotypes and for making selections that will be subject to breeding advancement 28

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or released for agricultural use. Under development are several diagnostic tools that permit evaluations and selections to be made in the laboratory (Frey, 1984~. These tools include isozyme analysis and protein electro- phoresis; DNA probes, molecular markers, and restriction fragment-length polymorphisms (RFLPs); and immunodiag- nostics. Applications of these techniques in plant breeding are of value in the attainment of several objectives: (1) breeding for Quantitative traits; (2) varietal identification and purity checks of seed lots; (3) screening for qualitative traits through marker linkage; (4) variety and genotype characterization for patent and Plant Variety Protection Certificate appli- cations; (5) predictions of combining ability to improve breeding productivity; and (6) characterization of the expression of genes introduced by molecular techniques (Frey, 1 984~. ~, ~do, , New Genetic Technioues. Several of the new genetic techniques currently being applied to plant breeding significantly extend the potential to manipulate crops genetically with greater efficiency and precision. These technologies include somaclonal variation, somatic cell genetics, gamete culture, protoplast fusion, and molecular approaches to gene transfer (Figure 3~. ., . ~. , . Although a considerable research effort in fundamental cellular and molecular biology has been required to develop these techniques as practical genetic tools, their strategic value in breeding must be considered within the context of the specific breeding objectives for a particular crop. A successful crop improvement program will generally require a balance in the use of more ... .. . . . . _ conventional approaches with predictable outcome, combined with more advanced tools with higher risks and less predictable utility. Both require a clear definition of traits targeted for improvement and a careful assessment of their commercial value. All the following techniques rely heavily on the universal capability of plant cells and tissues to be grown and manipulated In vitro. Literature on this subject constitutes an extensive knowledge base. The value of plant cell and tissue culture lies in the ability not just to access molecular and cellular genetic strate- gies, but also to use them in a practical way. The key is 29

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However, in the food industry the recent increase in research in crop biotechnology is building momentum on the other side of the equation--utilization-side or value- added genetics. Here, research is oriented toward the end use of agricultural raw materials, with a focus on added value and higher return. There are great opportunities to extend the value-added food industry component back to the farm (Figure 3~. Genetic improvement of specific crops for maximal return according to end use creates differ- entiated or noncommodity raw materials. The key to progress in this area will be research to develop a knowledge of the functionality of raw materials that is sufficient to be translated to genetic manipulation strategies. Companies that are successful in these efforts may generate an important new factor in technical leverage. The same may also be true at the international level. Utilization-side or value-added genetics will bring about a certain degree of restructuring in agricultural practices (e.g., emphasizing contract crop production and raw material identity channels). Whether these changes will favorably affect the family farm is an important question to be addressed by agricultural economists. Regardless of which side of the crop improvement equation is pursued, the issue remains: Which specific genes or traits are to be selected as economically feasible targets? Plant Cell Bioreactors Thirty years ago, Routier and Nickell (1956) at Pfizer received a U.S. patent for the use of plant cell cultures for the industrial production of natural products. Since that time, considerable progress has been made in several areas: (1) in the number of plant species that can be grown in culture, (2) in the production of a wide array of secondary metabolites (Dougall, 1985), (3) in our understanding of the biochemical pathways involved and their regulation, and (4) in bioreactor design and culture protocols (Shuler and Hallsby, 1985~. Despite these advances, there are only two commercial applica- tions, and these are very high-value medicinals and cosmetic ingredients--shikonin (Tabata and Fujita, 1985) and ginsengoside (Ushlyama et al., 1986~. 36

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Long-range projections for inexpensive production of secondary metabolites, based on future technical develop- ments, have been reported (Sahai and Knuth, 1985~. However, the outlook for economical production of food ingredients by cell culture is not optimistic for the near term. This is due primarily to their relatively low value, low levels of production by cells, and the high cost of the plant cell bioreactor approach. The most recent estimate at the current state of the technology is roughly $3,000/kg (Drapeau et al., 1987~. This appears to be a prime area for applying rDNA techniques to achieve the dramatic improvements required for commercial success. ANIMAL BIOTECHNOLOGY Most of the current applications of animal biotech- nology relate to the production side (Evans and Hollaender, 1986~; these include bovine growth hormone work, vaccine production, disease prevention, and embryo manipulations (sex selection, twinning, embryo storage, and transfer). Transgenic farm animals are still in the future. However, one area that relates to functional attributes for food is worth mentioning: genetic engi- neering of bovine milk proteins--the caseins. These are perhaps one of the most important and well-characterized groups of food proteins besides the seed storage proteins. Molecular work has advanced to the stage where systematic structure/function studies can be conducted on this class of proteins; this can lead to better under- standing of food protein functionality. Tom Richardson's group at the University of California, Davis, recently proposed a strategy involving protein engineering to change cascin structure to improve function in food products (e.g., caseins with additional chymosin sites to accelerate the rate of texture development) (Kang et al., 1986~. Commercial application of this strategy, however, must await progress in gene transfer and ~ expression In animals. CONCLUSIONS Biotechnolo~v Research in the Food IndustrY The food industry is characteristically conservative in the amount it invests in research (typically, 1.5% or less 37

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of sales) and in its adoption of rapidly emerging areas of technology. As it now exists, biotechnology research is supported primarily by such typical power bases as DuPont and Monsanto and also by an increasing number of small research companies, the biotiques. Most of these small companies are quite skilled in their areas of expertise and are highly motivated, responsive, and productive. They represent the best of what we have come to recognize as the entrepreneurial spirit. What we are seeing in food industry biotechnology is the effective use of research alliances between large food processors and the biotiques. This has allowed food companies to quickly attain critical mass in specialized areas of research and has served to accelerate develop- ments in this area. Several of the examples used in this paper were the result of such alliances. This trend will continue. However, we are beginning to enter a new phase. As food companies become more familiar with the technology and begin to experience its success in the marketplace, we will see internalization of research skills and the full integration of biotechnology into the well-established food research disciplines. Consolidation in the Food Industry Acquisitions and mergers are common phenomena in the [ood industry. The result is the development of large corporations that are horizontally integrated across a broad spectrum of food sectors. As this occurs, along with the internationalization of biotechnology research skills, we will experience a strong movement toward vertical integration in the direction of our raw material base, which will position the "value-added cascade" to begin further back in the system, at the genetic level. This may represent the emergence of an important new fulcrum of competitive leverage in the food industry and may very well bring genetic biotechnology into its most productive arena. REFERENCES Abdullah, R., E.C. Cocking, and J.A. Thompson. 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technol. 4:1087- 1 090. 38

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Arbige, M.V., P.R. Freund, S.C. Silver, and J.T. Zelko. 1986. Novel lipase for cheddar cheese flavor development. Food Technol. 40:91-96, 98. Baenziger, P.S., D.T. Kudirka, G.W. Schaeffer, and M.D. Lazar. 1984. The significance of doubled haploid variation. Pp. 385-414 in I.P. Gustafson, ed. Genetic Manipulation in Plant Improvement. Plenum, New York. Boyce, C.O.L. 1986. Novo's Handbook of Practical Biotechnology. Novo Industri A/S, Bagsvaerd, Denmark. 125 pp. Carlson, P.S. 1973. Methionine sulfoximine-resistant mutants of tobacco. Science 180:1366- 1368. Casey, J.P. 1977. High fructose corn syrup: A case history of innovation. Staerke 29:196-204. Charles Kline & Co. 1986. American market for enzymes expected to reach $260 million by 1990. Genet. Eng. News. 6:18. Cocking, E.C. 1985. Somatic hybridization: Implications for agriculture. Pp. 101-113 in ~ Zaitlin, P. Day, and A. Hollaender, eds. Biotechnology in Plant Science: Relevance to Agriculture in the Eighties. Academic Press, Orlando, Fla. Coker, L.E., and K. Venkatasubramanian. 1985. Starch conversion processes. Pp. 777-788 in M. Moo-Young, H.W. Blanch, S. Drew, and D.I.C. Wang, eds. Comprehensive Biotechnology: The Practice of Biotechnology: Current Commodity Products, Vol. 3. Pergamon, Elmsford, N.Y. Coleman, R.D., and M:P. McAlister. 1986. Plasmids containing a gene coding for a thermostable pullulanase and pullulanase-producing strains of Escherichia cold and Bacillus subtilis containing the plasmids. U.S. patent no. 4,612,287. Collins, G.B., D.F. Hildebrand, P.A. Lazzeri, J.R. Myers, G. Benzion, M: Dahmer, and T.R. Adams. 1985. Cell culture systems for soybeans and clover with efficient plant regeneration via somatic embryogenesis. P. 26 in G.A. Galau, ed. Abstracts, First International Congress of Plant Molecular Biology. Organ~zed by the International Society for Plant Molecular Biology, Oct. 27-Nov. 2, 1985, Savannah, Ga. Center for Continuing Education, University of Georgia, Athens Ga. Comai, L., L.C. Sen, and D.M. Stalker. 1983. An altered aroA gene product confers resistance to the herbicide glyphosate. Science 221 :370-371. 39

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