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2 Scientific Aspects THE POWER OF BIOTECHNOLOGY The tools of biotechnology offer both a challenge and tremen- dous opportunity. They do not change the purpose of agriculture- to produce needed food, fiber, timber, and chemical feed stocks efficiently. Instead, they offer new techniques for manipulating the genes of plants, animals, and microorganisms. Biotechnology tools complement, rather than replace, the traditional methods used to enhance agricultural productivity and build on a base of understanding derived from traditional studies in biology, genetics, physiology, and biochemistry. Biotechnology has opened an exciting frontier in agriculture. The new techniques provided by biotechnology are relatively fast, highly specific, and resource efficient. It is a great advantage that a common set of techniques gene identification and cloning, for example are broadly applicable. Not only can we improve on past, traditional methods with the more precise modern methods, but we can explore new areas as well. We can seek answers to questions that only a few years ago we never thought to ask. The power of biotechnology is no longer fantasy. In the last few years, we have begun to transform ideas into practical ap- plications. For instance, scientists have learned to genetically alter certain crops to increase their tolerance to certain herbicides. Biotechnology has been used to design and develop safer and 16

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SCIENTIFIC ASPECTS 17 more effective vaccines against viral and bacterial diseases such as pseudorabies, enteric colibacillosis (scours), and foot-and-mouth disease. Yet we have barely scratched the surface of the potential benefits. Much remains to be learned, and continued advances will take a serious commitment of talent and funds (see Chapter 3~. This chapter briefly reviews the major uses of biotechnology in agriculture. It looks specifically at the progress and poten- tials of genetic engineering and other new biotechnologies in plant and animal agriculture and bioprocessing. These sections review traditional approaches, discuss examples of progress using biotech- nology, and highlight opportunities on the horizon. USING GENE TRANSFER TO ENHANCE AGRICULTURE Throughout the history of agriculture, humans have taken ad- vantage of the natural process of genetic exchange through breed- ing that creates variation in biological traits. This fact underlies all attempts to improve agricultural species, whether through tra- ditional breeding or through techniques of molecular biology. In both cases, people manipulate a natural process to produce va- rieties of organisms that display desired characteristics or traits, such as disease-resistant crops or food animals with a higher pro- portion of muscle to fat. The major differences between traditional breeding and molec- ular biological methods of gene transfer lie neither in goals nor processes, but rather in speed, precision, reliability, and scope. When traditional breeders cross two sexually reproducing plants or animals, tens of thousands of genes are mixed. Each par- ent, through the fusion of sperm and egg, contributes half of its genome (an organism's entire repertoire of genes) to the offspring, but the composition of that half varies in each parental sex cell and hence in each cross. Many crosses are necessary before the "right" chance recombination of genes results in offspring with the desired combination of traits. Molecular biological methods alleviate some of these problems by allowing the process to be manipulated one gene at a time. In- stead of depending on the recombination of large numbers of genes,

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18 A GRICULTURAL BIO TECHNOLOG Y scientists can insert individual genes for specific traits directly into an established genome. They can also control the way these genes express themselves in the new variety of plant or animal. In short, by focusing specifically on a desired trait, molecular gene transfer can shorten the time required to develop new varieties and give greater precision. It also can be used to exchange genes between organisms that cannot be crossed sexually. Gene transfer techniques are key to many applications of biotechnology. The essence of genetic engineering is the ability to identify a particular gene one that encodes a desired trait in an organism isolate the gene, study its function and regula- tion, modify the gene, and reintroduce it into its natural host or another organism. These techniques are tools, not ends in them- seIves. They can be used to understand the nature and function of genes, unlock secrets of disease resistance, regulate growth and de- velopment, or manipulate communication among cells and among organisms. Isolation of Import ant Genes . The first step in an effort to genetically engineer an organism is to locate the relevant genetic among the tens of thousands that make up the genome. Perhaps the researcher is searching for genes to improve tolerance to some environmental stress or to increase disease resistance. This can be a difficult task similar to trying to find a citation in a book without an index. This task is made easier with restriction enzymes that can cut complex, double-stranded macromolecules of DNA into manage- able pieces. A restriction enzyme recognizes a unique sequence in the DNA, where it snips the strands. By using a series of different restriction enzymes, an organism's genom~c DNA can be reduced to lengths equivalent to one or several genes. These smaller seg- ments can be sorted and then cloned to produce a quantity of genetic material for further analysis. The collection of DNA seg- ments from one genotype a gene library can be searched to locate a desired gene. Patterns can also be analyzed to link a particular sequence a marker to a particular trait or disease, even though the specific gene responsible is still unknown. Restriction enzymes are also used in cloning genes. To clone a gene, a small circle of DNA that exists separate from an organism's

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SCIENTIFIC ASPECTS 19 main chromosomal complement-a plasrn~d is cut open using the same restriction enzyme that was used to isolate a desired gene. When the cut plasmid and the isolated gene are mixed together with an enzyme that rejoins the cut ends of DNA molecules, the isolated gene fragment is incorporated into the plasmid ring. As the repaired plasmid replicates, the cloned gene is also replicated. In this way, numerous reproductions of the cloned gene are pro- duced within the host cell, usually a bacterium. After replication, the same restriction enzyme is used to snip out the cloned gene, allowing numerous copies of that gene to be isolated. The ability to isolate and clone individual genes has played a critical role in the development of biotechnology. Cloned genes are necessary research tools for studies of the structure, function, and expression of genes. Further, specific gene traits could not be transferred into new organisms unless numerous gene copies were available. Cloned genes also are used as diagnostic test probes in medicine and agriculture to detect specific diseases. Gene Transfer Technology To transfer genes from one organism to another, molecular biologists use vectors. Vectors are the "carriers" used to pass genes to a new host, and they can mediate the entry, maintenance, and expression of foreign genes in cells. Vectors used to transfer genes include viruses, plasmids, and mobile segments of DNA called transposable elements. Genes can also be introduced by laboratory means, such as chemical treatments, electrical pulses, and physical treatments including injection with rn~croneedles. The basic principles behind these technologies are the same for animals, plants, and microbes, although specific modifications may be necessary. (The basic gene transfer methods are described in detail in the Appendix, "Gene Transfer Methods Applicable to Agricultural Organisms.") Vectors based on viruses, plasmids, and transposable elements have been adapted from naturally occurring systems and engi- neered to transfer desired genes into animals, plants, and mi- crobes. For plants, the classic example is the Ti plasmid from the soil bacterium Agro~acterium tumefaciens, which in nature transfers a segment of DNA into plant cells, causing the recipient cells to grow into a tumor. Scientists have adapted this plasmid

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20 A GRICULTUR24L BIO TECHNOLOGY by eliminating its tumor-causing properties to create a versatile vector that can transfer foreign genes into many types of plants. Similarly, the transposable P-element of the fruit fly Drosophi- [a melanogaster is an effective vector for gene transfer into Dro- sophila. This or similar transposable elements should prove to be adaptable to insects of agricultural importance. Animal viruses such as simian virus 40 (SV40), adeno, papilloma, herpes, vaccinia, and the retroviruses, all originally studied because of their role in disease, are now being engineered as vectors for gene transfer into animal cells and embryos. Plant viruses such as cauliflower mosaic virus, brome mosaic virus, and geminiviruses are similarly being exploited for their abilities to transfer genes. Cell Culture and Regeneration Techniques The ability to regenerate plants from single cells is important for progress with gene transfer into plants. Animals cannot be regenerated asexually, so the only way to introduce a foreign gene into all cells of an animal is to insert it into the sperm, egg, or zygote. Cell culture techniques are important for the regeneration of plants. They are also critical for fundamental studies on both plant and animal cells, and for the manipulation of microorgan- ~sms. The vegetative propagation of stem cuttings or other growing plant parts to produce genetic clones is common for some agricul- tural crops. Potatoes, sugarcane, bananas, and some horticultural species, for example, are cultivated by vegetative propagation. Techniques exist to propagate and regenerate whole plants from tissues, isolated plant cells, or even protoplasts (plant cells from which the cell wall has been enzymatically removed) in culture. This set of techniques is complete for some agricultural species, such as alfalfa, carrots, oilseed rape, soybeans, tobacco, tomatoes, and turnips. Progress on other crops, including major food species such as many cereals and legumes, has been slower. Cell culture techniques have taken on added importance as biotechnology has progressed. Genetic engineering requires an ability to manipulate individual ceils as recipients of isolated genes. Cell culture techniques allow scientists to maintain and grow cells outside the organism and thus expand their ability to perform gene

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SCIENTIFIC ASPECTS 21 transfer and study the results. In addition, cell culture allows sci- entists to regenerate numerous copies (clones) of the manipulated varieties, which is easier, more efficient, and more convenient, es- pecially for producing significant quantities of stock plants. A third use of cell culture is to regenerate "somaclonal variants," plants with altered genetic traits that can prove useful as new or improved crops. Thus, cell culture techniques are important to increasing the productivity and versatility of agriculture. However, there are some important limitations. Chromoso- mal abnormalities appear as cultures age. These changes are related to the phenomenon of somaclonal variation, which may prove useful to agriculture, but in many instances the changes are undesirable. Therefore, scientists must learn how to prevent chromosomal changes in cell cultures. Second, long-term cultures lose regenerative potential. As biotechnology expands, it will be critical to understand why different species have differing abilities to regenerate from cell cultures into plants and how factors such as the genetic or physiological origin of the cells and the culture conditions affect growth. Most plant cells appear to be totipo- tent, that is, they are in a reversible differentiated state that will permit them to regenerate into a whole plant under appropriate conditions. Understanding what these appropriate conditions are remains a fundamental question in the study of plant development and its genetic control. Monoclonal Antibody Technologies The development of monoclonaI antibody technology is based on advances in our ability to culture cells. Antibodies are the protein components of the immune system found in the blood of mammals. They have a unique ability to identify particular molecules and select them out. When a foreign substance (an antigen) enters the body, specialized cells called B lymphocytes produce a protein (an antibody) to combat it. To envision how antibodies work, think of a lock and key: The antibody key "fits" only the specific antigen lock. This marks the antigen for destruc- tion. Each of the specialized B lymphocyte cells produces only a single type of antibody and thus recognizes only one antigen. Apart from their natural role in protecting organisms via the immune response, antibodies are important scientific tools. They

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22 AGRICULTURAL BIOTECHNOLOGY are used to detect the presence and level of drugs, bacterial and viral products, hormones, and even other antibodies in the blood. The conventional method of producing antibodies is to inject an antigen into a laboratory animal to evoke an immune response. Antiserum (blood serum containing antibodies) is then collected from the animal. However, antiserum collected in this way contains many types of antibodies, and the amount that can be collected is limited. Modern biotechnology has opened a door to a more efficient, more specific, and more productive way of producing antibod- ies. By fusing two types of cells, antibody-producing B lympho- cytes and quasi-immortal cancer cells from mice, scientists found that the resulting hybrid cells, called hybridomas, secreted large amounts of homogeneous antibodies. Each hybridoma has the ability to grow indefinitely in cell culture and thus can produce an almost unlimited supply of a specific "monoclonal" antibody. By immunizing mice with specific antigens, researchers can create and select hybridomas that produce a culture of specific, desired monoclonal antibodies. Thus, biotechnology has produced a way of creating pure lines of antibodies that can be used to identify complex proteins and macromolecules. Monoclonal antibodies are powerful tools in molecular analyses, and their uses in detecting low levels of disease agents such as bacteria and viruses are rapidly expanding. Beyond many diagnostic uses, hybridoma technology shows promise for unmunopurification of substances, imaging, and ther- apy. Immunopurification is a powerful technique to separate large, complex molecules from a mixture of either unrelated or closely related molecules. For imaging, easily visualized tags can be at- tached to monoclonal antibodies to provide images of organs and to locate tumors to which the antibody will specifically bind. Finally, new therapeutic methods- have been developed that use mono- clonal antibodies to inactivate certain kinds of immunological cells and tumor cells or to prevent infection by certain microorganisms. Although many applications of this technology are still in the experimental stages, the commercial agricultural use of mono- clonal antibodies has begun. For example, monoclonal antibod- ies are now on the market as therapeutics against calf and pig enteric colibacillosis, which causes neonatal diarrhea (scours) This approach is often more effective than conventional vaccines,

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SCIENTIFIC ASPECTS 23 and it supplements genetically engineered vaccines. Monoclonal antibody-based diagnostic kits can detect whether scouring ani- mals are infected with a particular strain of an Escherishia cold bacterium that causes scours, and thus help veterinarians deter- mine the appropriate therapeutic monoclonal antibody to use on an infected herd. Summary In its simplest form, genetic engineering involves inserting, changing, or deleting genetic information within a host organism to give it new characteristics. This technology will likely bring great benefits to agriculture, just as breeding has over several thousand years of human history. The development and use of new techniques is allowing researchers to manipulate the genetic character of organisms while overcorn~ng the complications and limitations of sexual gene exchange. Genetic engineering is re- ducing the amount of time needed to analyze genetic information and transfer genes. Both genetic engineering and monoclonal an- tibody technology, another major development in biotechnology, greatly increase the specificity and accuracy of analytical research methods. Further, these new technologies are permitting highly specific molecular analyses to be done and are opening new areas of inquiry. The tools of biotechnology, combined with traditional techniques in biology and chemistry, increase enormously both the power and the pace of discoveries in biological investigation. NEW APPROACHES TO CROP PRODUCTION In the past 50 years, agricultural production in the United States has more than doubled while the amount of land under cul- tivation has actually declined slightly. This impressive agricultural success is the result of many factors: an abundance of fertile land and water, a favorable climate, a history of innovative farmers, and a series of advances in the science and technology of agricul- ture that have made possible more intensive use of yield-enhancing inputs such as fertilizer and pesticides. Yet the productivity suc- cesses brought about by farm mechanization, improved plant vari- eties, and the development of agricultural chemicals may be harder to repeat in the future unless new approaches are pursued.

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24 AGRICULTURAL BIOTECHNOLOGY Biotechnology offers vast potential for improving the efficiency of crop production, thereby lowering the cost and increasing the quality of food. The tools of biotechnology can provide scientists with new approaches to develop higher yielding and more nutri- tious crop varieties, to improve resistance to disease and adverse conditions, or to reduce the need for fertilizers and other expensive agricultural chemicals. The following paragraphs highlight some examples of how genetic engineering can be used to enhance crop production. The Genetic Engineering of Plants Perhaps the most direct way to use biotechnology to improve crop agriculture is to genetically engineer plants that is, alter their basic genetic structure so they have new characteristics that improve the efficiency of crop production. The traditional goal of crop production remains unchanged: to produce more and better crops at lower cost. However, the tools of biotechnology can speed up the process by helping researchers screen generations of plants for a specific trait or work more quickly and precisely to transfer a trait. These tools give breeders and genetic engineers access to a wider universe of traits from which to select. Although powerful, the process is not simple. Typically, re- searchers must be able to isolate the gene of interest, insert it into a plant cell, induce the transformed cell to grow into an entire plant, and then make sure the gene is appropriately expressed. If scientists were introducing a gene coding for a plant storage pro- tein containing a better balance of essential amino acids for human or animal nutrition, for example, it would need to be expressed in the seeds of corn or soybeans, in the tubers of potatoes, and in the leaves and stems of alfalfa. In other words, the expression of such a gene would need to be directed to different organs in different crops. P UTTING THE NEW TECHNOLOGIES TO WORK There are already successes that demonstrate how plants can be genetically engineered to benefit agriculture. Herbicide resis- tance traits are being transferred to increase options for control- ling weeds. Soon, the composition of storage proteins, oils, and starches in plants may be altered to increase their value.

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SCIENTIFIC ASPECTS . 25 One plant gene that has been isolated, cloned, and transferred Is for the sulfur-rich protein found in the Brazil nut, Berthallet~a excelsa. This protein contains large amounts of two nutritionally important sulfur-containing amino acids: methionine and cysteine. These are the very nutrients in which legumes, such as soybeans, are deficient. If the sulfur-rich protein gene were transferred into soybeans, it might enhance this legume's role as a protein source throughout the world. By purifying the Brazil nut protein and determining the order and kind of amino acids in the protein, scientists were able to synthesize an artificial segment of DNA coding for a section of this protein. This DNA "probe" was used to find and pull out the natural gene from the Brazil nut. Researchers then transferred the gene into tomato and tobacco plants, which were chosen because they are easier to manipulate than soybeans. Researchers have also transferred the gene into yeast cells. Early results show that the genetically engineered yeast do produce the sulfur-rich protein. Similar work is being done to improve oil crops. Oil crops produced in the United States in 1984 were worth $11.8 billion. Depending on their chemical composition, oils and waxes from plants have uses in feed, food, and industrial products such as paints and plastics. Chemical properties, and thereby the uses of plant oils, vary depending on the length of the fatty acid chains that compose the oil and their degree of saturation. Many of the enzymes controlling the biochemical pathways that regulate molecular chain length and degree of saturation have been well studied, and this reservoir of knowledge now makes it possible to genetically engineer the type of of! a crop produces. Although traditional breeding methods have succeeded in modifying the of! composition of some crops, genetic engineering opens a broader range of possibilities. Scientists have taken another important step in using genetic engineering to improve crop production: They have for the first time engineered plants to be resistant to powerful herbicides. One example is glyphosate (trade name: "Roundup"), a common, ef- fective, and environmentally safe herbicide. However, glyphosate indiscriminantly kills crops as well as weeds. Thus, it must usually be used before crop plants germinate. Yet by engineering crops to be resistant to glyphosate, scientists hope to expand the range of the herbicide's applications.

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26 AGRICULTURAL BIOTECHNOLOGY Scientists have isolated a glyphosate-resistance gene and suc- cessfully transferred it into cotton, poplar trees, soybeans, to- bacco, and tomatoes. The gene was derived from the bacterium Salmonella typhimurium. Similarly to other accomplishments in biotechnology, this success depended on extensive prior basic re- search on biochemical pathways in bacteria and plants, and so- phisticated gene cloning and transfer techniques. Field testing and commercialization of glyphosate-resistant crops should follow soon. Analysis of tomato growers' costs in California predicts that farmers could save up to $100 per acre in weed control costs if they used glyphosate in place of current herbicides, with concomitant reductions in labor, equipment, and environmental damage. This advance would also give farmers improved flexibility, yield, quality, and spectrum of weed control. LOOKING TO THE F UTURE With such promising examples already being realized, it is interesting to speculate about other possibilities. For instance, could scientists take naturally occurring chemicals that hinder plant growth-such an the compound crabgrass releases that pre- vents other grasses from invading its territory and engineer crop plants with their own ability to control weeds? Scientists have Tong known that some plants produce chern~cals that affect the growth of other plants; by studying these allelopaths, scientists may be able to engineer or breed plants that would give farmers new bio- Togical tools to fight weeds, in addition to mechanical cultivation and other cultural tools, and chemical herbicides. The potential value of research on biological methods of weed control is great, but the work is very complicated and significant advances are not expected quickly. One of the complicating factors that must be understood is how certain plants produce allelopathic molecules and at the same tune protect themselves against these chemicals. Observations of nature combined with abilities to engineer plants might also provide opportunities to manipulate plant growth and development. Through research, scientists have determined that flowering, dormancy, fruit-ripening, and a host of other growth and developmental processes come under the influence of a relatively few plant hormones or growth regulating substances.

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40 AGRIOULTURAL BIOTECHNOLOGY These and other properties make it an ideal candidate to be genet- ically engineered to combat other diseases, both of humans and of agriculturally important animals. Vaccinia is basically a delivery system:- Given appropriate protocols, any gene can be moved into vaccinia and be carried into the recipient of the vaccine. This ability means the virus can be adapted to combat essentially any selected disease. Extensive work is necessary, however, to identify, isolate, and transfer the appropriate genetic material. So far, many foreign genes have been inserted and found to be active in vaccinia virus. Vaccinia is a large, complex virus that can simultaneously accornrnodate at least a dozen foreign genes and still successfully infect cells and replicate. Thus, a single vaccinia vaccine could immunize the recipient against a dozen different diseases. Researchers might someday develop "cassettes," carrying genes for various antigens of the primary infectious diseases in a given geographic area-one for Africa, South America, and so on. A single inoculation would confer immunity to the collection of diseases whose antigenic genes were packaged into the vaccine. Recombinant vaccinia virus vaccines are more efficient than conventional subunit vaccines that consist of only antigenic pro- tein. The difference is that vaccinia places the genes coding for the pathogen's antigen into the recipient's cells. Antigenic protein is then produced within the cells themselves. This method stimulates the vaccinated recipient's immune defenses more effectively than subunit vaccines, and immunity is longer lasting. Researchers have constructed vaccinia vaccines against a number of human diseases, including hepatitis B. herpes simplex, influenza, and malaria, and against some lethal animal diseases, including rabies and vesicular stomatitis virus. Extensive testing is under way. Animal agricul- ture will further benefit as scientists develop vaccines against other specific animal diseases. ALTERING INTESTINAL ORGANISMS A more speculative area of interest for genetic engineers lies in- side agricultural animals. Given appropriate research, could a way be found to alter the intestinal bacteria of ruminant farm animals to make them more efficient in utilizing plant waste fibers for food?

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SCIENTIFIC ASPECTS 41 Scientists are looking for ways to improve the microorganisms in- side an animal to create a more effective, natural, bioprocessing system. Application of biotechnology to this area is just begin- ning, but it provides a glimpse of the far-reaching possibilities that lie ahead for agriculture. BIOPROCESSING OPPORTUNITIES Several familiar age-old procedures are forms of bioproces- sing fermenting grape juice or leavening bread dough, for ex- ample. Yet bioprocessing also includes a range of technologies in which living cells or their components, such as enzymes, are used to cause the desired physical and chemical changes. Bioprocessing to produce industrial chemicals began during World War ~ when researchers developed alternative ways to pro- duce acetone and butane! using microorganisms. However, the growth of the petrochemical industry during World War IT replaced the microbial production of industrial solvents, and industrial bio- processing for bulk chemicals practically disappeared. The climate changed again, however, when it was discovered how well biological processes could synthesize complex molecules such as antibiotics, vitamins, and enzymes. The industry was transformed from one that produced high-volume, low-value industrial chemicals to one that produced lower-volume, high-value products. Advances in biotechnology have renewed interest in industrial uses of agricultural and forestry commodities. Bioprocessing of- fers innovative opportunities to create new products and foods, treat and use wastes, and use renewable resources (biomass) for fuel. Once developed, such processes could prove more economical as well as less environmentally damaging than current industrial processes. Alternative Fuels Many people have hoped bioprocessing could have a signifi- cant impact on fuel production, but the present economic situa- tion favors the extraction of natural reserves of petroleum, gas, and coal. Biomass energy, such as alcohol produced from grains and sugar, or methane (biogas) produced from animal manures and other waste products, has received some research attention. In the United States, gasohol (consisting of 10 percent alcohol and

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42 A GRICULTURAL BIO TECHNOLOGY 90 percent gasoline) made a brief, well-publicized appearance, but price changes in the oil market have undermined its competitive- ness. In Brazil, alcohol fuel is widely used; it is obtained primarily from the fermentation of sugarcane juice. However, producing energy from food crops is not yet prof- itable in most countries. Most of the sugar- and starch-containing plants, such as potatoes, corn, and cassava, that are easily con- verted into alcohol are relatively expensive. In addition, wide- spread and large-scale use of food crops for energy production could create food shortages, especially in developing countries. However, as scientists engineer microorganisms to feed on cel- lulose and develop efficient ways to break down the lignin (the tough compound that makes wood resistant to degradation) in woody plants, a fuel-alcohol industry based on less valuable plant materials (including trees, weeds, scrub, and wastes from pa- per manufacturing) might be developed. Similarly, the potential for bioprocessing to create methane lies in using microbes and wastes domestic sewage, manure, crop residues, and other cheap and available raw materials. Some scientists foresee a time when bioprocessing might also be developed to produce hydrogen for fuel. Progress in developing bioprocessing for alternative fuels will occur slowly because vehicles and markets adapted for such fuels are not developed, and there are no economic incentives for these markets to change. In addition, bioprocessing for bulk chemicals or for energy (e.g., methane, methanol, ethanol, etc.) is difficult to engineer even with a uniform feed stock such as sugarcane or corn. When a diversity of biomass materials is used, problems are compounded by the design of fermentation apparatus and the selection of microorganisms adapted to grow on different feed- stocks. Continued research on bioprocessing for bulk chemicals and alternative fuels, however, is important. Opportunities to use inexpensive by-products or wastes, or changes in economics based on the price of oil and gas, may make it economical in the long-term. Alternative Feed and Food Sources Bioprocessing also holds promise as a way to create unique sources of protein for an increasingly hungry world. For instance,

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SCIENTIFIC ASPECTS 43 scientists have found some unusually hardy microbes living in the Dead Sea, and one of these, Dunalielia bardawil, manufactures glycerol to counteract the pressures of its highly saline environ- ment. In Israel, this alga is grown and harvested in specially built ponds. In addition to glycerol, manufacturers obtain a compound called beta-carotene that is sold as a food coloring and a residue that is an excellent, protein-rich animal feed. In Finland, suIphite liquor from paper production is fed to certain molds, which not only purify the waste liquids but also yield a rich residue that is sold as animal feed. Similar techniques could be developed for waste materials from forestry, cheese-making, and other industries. Microbes have Tong played a role in food production. Cheese, pickles, bread, beer, and wine, for instance, all rely on bioprocess- ing. Molecular genetic techniques are being used to monitor the properties of microbes used in these processes to ensure product uniformity. Yet microbes can do more than preserve foods or alter their taste; the future might include a direct microbe-based food source: single-cell protein. People have consumed microbes in the form of algae as far back as the Aztecs. Modern biotechnology looks to single-cell protein primarily as an animal feed, but some scientists consider human consumption a possibility, too. Other Products Bioprocessing already contributes to our ability to produce vitamins, amino acids, enzymes, and more recently hormones, and this role should increase in the future. For instance, much of the supply of vitamins B2 (riboflavin) and BY (cobalbumin) comes from microbes. Researchers have adapted wild strains of a mold, Ashbya gossypii, to produce 20,000 times its original output of vitamin B2. Research has also intensified microbial production of vitamin BY over 50,000 times. Most cereal grains are deficient in two essential amino acids, lysine and methionine. These are usually added to animal feed to ensure an adequate diet. Methionine is made by chemical processes, but 80 percent of all lysine is produced by fermentation using bacteria. The amino acid derivative monosodium glutamate, which is used as a flavor enhancer in cooking, is produced by two bacteria through a bioprocess.

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44 AGRICULTURAL BIOTECHNOLOGY In the past, the use of enzymes has been limited by the expense of isolating them from natural sources and by their instability. Recent advances have provided ways to immobilize enzymes and use whole microorganisms as catalytic systems, thus yielding more stable and reusable enzymes and increasing the opportunities for their use. The biotechnological production of sugar substitutes is one example of a growing industry that has been made possible largely because of our increased ability to manufacture enzymes through microbial processes. Another area with potential for bioprocessing is waste treat- ment. As mentioned in previous examples, some bioprocessing systems can transform plant debris and other wastes into useful products, in effect creating an inexpensive and abundant renew- able resource. Another current example is a new strain of yeast genetically engineered with an enzyme that converts the lactose in whey, a dairy industry waste product, into ethanol, which has fuel energy value. On another front, bioprocesses are being developed to more efficiently treat municipal, industrial, and agricultural wastes. However, some problems remain in improving the depend- ability and design of these systems. To develop new approaches toward bioprocessing and to bring them into widespread use will require a great deal of research. First, systems must be designed to accomplish each goal. Success- fuT systems will require (1) a solid understanding of the organism involved, (2) an effort to develop the most productive strain of the organism and isolate the appropriate enzymes, and (3) intensive, specific research on the dynamics of each individual bioprocess. Next, the bioprocessing systems must be improved and perfected to offer economically competitive products. Research is needed to develop new industrial-scare methods to isolate products at the degree of purity appropriate for commerical use. Concentration of the final products is also important because separating them out after microbial conversion is often a major cost. CONCI`USIONS Benefits offered by biotechnology will not be fulfilled with- out a continued commitment to basic research. In fact, most of the prominent new biotechnologies are "spin-offs" from basic

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SCIENTIFIC ASPECTS 45 research efforts. As the examples in this chapter indicate, im- proved yields and reproduction, disease resistance, better quality products, reduced inputs, and similar advances are possible using biotechnology. However, society must be prepared to support the long-term efforts needed to transform these ideas into practical applications. Extensive laboratory and field research will be nec- essary to develop specific applications. This research will require considerable time and funding. Some of these new developments could dramatically transform agriculture and food production by increasing efficiency and productivity, thus lowering costs and im- proving competitiveness in the world marketplace. If we are to continue to make progress using genetic en- gineering to improve agriculture-whether by engineering the plants, animals, or the microorganisms and insects associated with agriculture research must focus on six important areas. 1. 2. 3. Gene identification locating and identifying agricultur- ally important genes and creating chromosome maps. Gene regulation understanding the mechanisms of reg- ulation and expression of these genes and refining the methods by which they may be genetically engineered. Structure and function of gene products-understanding the structure and function of gene products in metabolism and the development of agriculturally important traits. 4. Cellular techniques developing and refining techniques for cell culture, cell fusion, regeneration of plants, and other manipulations of plant and animal cells and em- bryos. 5. Development in organisms and communities under- standing the complex physiological and genetic interac- tions and associations that occur within an organism and between organisms. 6. Environmental considerations understanding the behav- ior and effect of genetically engineered organisms in the environment. GENE IDENTIFICATION Gene identification is crucial to the advancement of biotech- nology, because scientists need to understand what gene is respon- sible for the trait they want to alter. Basic research in biochemistry

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46 AGRICULTURAL BIOTECHNOLOGY and genetics is necessary to be able to identify specific genes and the traits associated with them. Only after the specific gene is identified can scientists alter it to benefit agriculture. Thus, it is important that our ability to identify genes be improved for future advancements in biotechnology. Chromosome Maps. Although they are merely general cata- Togs of a plant, animal, or microbial genome, chromosome maps are important guidelines for finding specific genes of importance to agriculture. Chromosome maps can show genetic engineers where to begin their search for specific genetic information. Chromosome maps identify "markers" that are often linked to important genes, such as the gene for a specific disease or physical trait, and they can be used to trace inheritance patterns. In humans, we have learned what markers, rather than specific genes, are linked to some inherited diseases such as cystic fibrosis. Researchers could provide a powerful too] to aid in the development of biotechnology if they would develop chromosome maps for the major crop species such as corn, wheat, and rice, and for important animal species such as cattle, swine, and poultry. GENE REGULATION Once a gene has been identified, the importance of under- standing gene regulation becomes clear. Part of manipulating a gene is getting it to be expressed appropriately. To accomplish that, scientists must understand how the gene is controlled-what turns it on and off, how it interacts with various hormones, and other factors. The science behind gene regulation is very intricate and requires a sophisticated understanding of molecular biology. Gene regulation becomes especially complex when several genes interact to control a trait. Such "multigenic" control is involved in some important agricultural traits, for instance in determining the storage proteins that contribute to the nutritional value of a crop or its hardiness in a particular environment. Advancing our understanding of gene regulation and expression will require basic research in biochemistry, physiology, and genetics, and will require intensive laboratory research, because each gene must be studied as an individual case.

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SCIENTIFIC ASPECTS STRUCTURE AND F UNCTION OF GENE PRODUCTS 47 The end products of the actions of genes are of prime interest in agriculture. The cellulose fibers of trees and cotton, the proteins in seeds or muscle fibers, and the carbohydrates and fats important in food and commerce are the end products of highly organized and regulated metabolic pathways. Genes code for the enzymes as well as for the structural and regulatory molecules that carry out the complex reactions that lead to these end products. The deficien- cies of our understanding in the biochemistry and physiology of metabolism and development are often the greatest constraints to applying biotechnology to agriculture. Understanding the linkage between metabolism and development and the genes that encode these processes will require progress on both fronts. The tools of biotechnology and techniques for isolating and manipulating genes can aid biochemical and physiological studies of metabolism. Con- versely, studies of metabolic pathways can help us identify genes and understand their regulation. CELLULAR TECHNIQUES The manipulation of plant and animal cells is part and parcel of strategies that involve genetic engineering, monoclonal anti- bodies, and bioprocessing. Although methods for cell culture, cell fusion, regeneration of plants from cells, and embryo manipulation exist for some species, these techniques must yet be successfully adapted to other species, which include important crops and live- stock animals. Moreover, specific microorganisms such as yeasts, fungi, viruses, and bacteria important to agriculture and biopro- cessing must be able to be cultured to allow both basic research and practical applications. DEVELOPMENT IN ORGANISMS AND COMMUNITIES Genetic engineering is more complex when it involves inter- actions among organisms. The symbiotic relationship between a microorganism and its host plant is intricate and raises many ques- tions for scientists. Gene identification remains important: What genes are involved in various stages of the relationship? Why does the microorganism colonize only one type of plant? Detailed study

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48 AGRICULTURAL BIOTECHNOLOGY is necessary to answer these sorts of questions about particular re- lationships. Researchers also need to understand the relationships under field conditions if they are to design organisms that can compete effectively once they are released. Another aspect of the asso- ciations between plants and microorganisms that needs research involves the mechanisms of infection. Knowing how a microorgan- ism attacks a plant is the first step in combating it. Without that basic understanding, genetic engineers will not be able to manipu- late the system to their advantage. Although genetic manipulation is becoming a reality, in far too many cases a lack of understanding of plant physiology and pathogen interactions limits its progress. ENVIRONMENTAL CONSIDERATIONS Many of the pending applications of biotechnology will require releasing genetically engineered plants, animals, and microbes into the environment. Clearly, the more that is known about the ecol- ogy and behavior of plants, animals, and microorganisms, the bet- ter are our chances of assessing the potential values and possible risks involved in introducing genetically altered versions into the field. Data on pathogenicity, mutagenicity, the ability to transfer genes, and other relevant factors can help predict the organism's effects on the ecosystem. Indeed, developing data and tools to support value and risk assessment is likely to become an increas- ingly important part of research efforts. The results of such work will help scientists understand the system, and will play a role in educating the public about both the risks and benefits offered by biotechnology. However, a detailed analysis of the regulatory as- pects of this important and controversial issue is beyond the scope of this report. RECOMMENDATIONS INCREASED EMPHASIS ON BASIC RESEARCH Basic research programs in physiology, biochemistry, genet- ics, and molecular biology within agricultural disciplines such as agronomy, entomology, and animal science need to be strength- ened and in many cases redirected to questions of identifying genes and understanding the regulation of their expression. Just

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SCIENTIFIC ASPECTS 49 understanding the regulation of their expression. Just as an enor- mous information base has provided a substructure for sweeping advances in biomedical science, a similar foundation of knowledge is now needed about the basic biochemistry, physiology, and ge- netics of such agricultural subjects as host-pathogen interactions, plant and animal developmental responses to environmental stim- uTi, enzymes and metabolic pathways, and molecular constituents and their patterns of organization in subcellular organelles. Ac- quiring such knowledge will affect the rate at which agriculturally valuable genes can be identified, isolated, and characterized, and is a prerequisite for applying the tools of biotechnology to agricul- tural problems. A similar call for augmented basic research within agricultural and related biological and biochemical fields was sounded in pre- vious reports (NRC, 1984, 1985a, 1985b; Winrock International, 1982~. Positive steps have been taken. Yet far more impetus is needed to ensure the continued success of American agriculture in an ever-changing world economy. IMPROVED TECHNIQUES AND APPLICATIONS The repertoire of molecular biology and cell culture techniques needed to implement advances in genetic engineering is incomplete. Methods for gene transfer in many plants, animals, and microbes; plant cell culture and regeneration; and animal embryo culture and manipulation are inadequate to support the goal of improving agricultural productivity. Increased efforts are needed to apply techniques developed for laboratory organisms to those plants, animals (including insects), and microbes relevant to agriculture. A national effort should be mounted by both public and pri- vate sectors to apply techniques of biotechnology to problems in the agricultural sciences. This effort should include research on: . Gene identification locating and identifying agriculturally important genes and creating chromosome maps. . Gene regulation understanding the regulation and expres sion of these genes and refining methods by which they may be genetically engineered. . Structure and function of gene products- studying the struc ture and function of gene products in metabolism and the development of agriculturally important traits.

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50 AGRICULTURAL BIOTECHNOLOGY . Cellular techniques developing and refining techniques for cell culture, cell fusion, regeneration of plants, and other ma- nipulations of plant and animal ceils and embryos. Development in organisms-using the new technology to study cell and organismic biology in intact organisms. . Development in communities understanding the complex as- sociations and interactions that occur among organisms. INCREASED ATTENTION TO THE ECOLOGICAL ASPECTS OF BIOTECHNOLOGY Both the public and private sectors should increase their ef- forts to develop an extensive body of knowledge of the ecological aspects of biotechnology in agriculture. In particular, studies must be done to further our understanding of the behavior and effects of genetically engineered organisms. In addition, the public must be educated about biotechnology. These efforts are essential to support future applications of biotechnology and to adequately inform regulators and the public about both the benefits and pos- sible risks involved.