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3 Biotechnology in Agriculture WITH THE EXCEPTION OF THE pharmaceuticals industry, agri- culture is the commercial sector that has drawn the most attention from biotechnology. Many of the applications of genetic engineering in agriculture will probably take longer to emerge than in other areas, given the complexity of some of the problems that must first be solved. But the potential returns are unparalleled. Furthermore, several developments of the past few years have indi- cated that progress may be more rapid than was previously thought possible. Many of the products described in Chapter 2 for use in human health care have agricultural analogs. For instance, hormones, steroids, and antibiotics, which are all potential products of biotechnology, have long been used in agriculture. Biotechnology will also yield drugs, feed additives, and growth enhancers that have never before been available in commercial quantities. A particularly intriguing example is growth hormone, which may result in faster growing, larger, and leaner animals and which has increased milk production in dairy cattle by up to 40 percent. Products created through genetic engineering will also be used to diagnose and treat disease, which each year reduces the productivity of This chapter includes material from the presentations by Ernest G. Jaworski, Rudolf Jaenisch, and Philip Leder at the symposium. 30
BIOTECHNOLOGY IN AGRICULTURE 31 livestock and poultry in the United States by 20 percent. A genetically engineered vaccine for colibacillosis or scours, a diarrheal disease that kills millions of newborn calves and piglets each year, is already available. A subunit vaccine has been genetically engineered for foot-and-mouth disease in cattle, which remains a serious problem throughout South America, Africa, and the Far East. Other vaccines are being developed for rabies, swine and canine parvovirus, fowl plague, bovine papilloma virus, and many other diseases. Certain products of biotechnology will be used to detect and monitor the progress of disease, so that treatment can begin before economic losses occur. Researchers are developing monoclonal antibodies to diagnose bluetongue (a viral disease in sheep transmitted by gnats), equine infectious anemia, bovine leukosis virus, and a number of viral diseases that strike dogs and cats. Monoclonal antibodies can also fend off disease by conferring passive immunity to an infectious agent. Furthermore, as is the case throughout genetic engineering and biotechnology, researchers use such tools as monoclonal antibodies to learn more about the origins and mechanisms of diseases, which can in turn point toward more effective therapies. All the products mentioned above are made through fermentation processes or cell culture techniques. But biotechnology has another, fundamentally different, capability in agriculture. It can be used to genetically alter agriculturally important animals, plants, and mi- crobes, producing crops and livestock with characteristics that cannot be achieved through traditional breeding programs. For instance, microorganisms might be genetically engineered that provide nitrogen to important crops, greatly reducing the need for fertilizer. Plants might be produced that grow faster or in more places or that have larger and more nutritious yields. Animals might be able to secrete elevated levels of their own growth hormone so that they grow faster and larger. These are the truly revolutionary agricultural applications of biotechnology, and they are the subject of this chapter. Genetically Engineered Microorganisms in Agriculture Microorganisms in the environment affect the growth of plants and animals in a variety of ways, many of which are still poorly understood. As research progresses, it should be possible to genetically engineer these microorganisms to yield hardier and more productive crops and livestock. Given the unresolved difficulties involved in altering the genetic material of plants and animals, this may be the first direct application of genetic engineering in agriculture.
32 BIOTECHNOLOGY The best known and most intensively studied relationship between microorganisms and plants involves the essential nutrient nitrogen. Plants cannot directly absorb and use the nitrogen gas that constitutes more than 75 percent of the atmosphere. It must first be fixed or converted into other nitrogen-containing compounds, either in indus- trial facilities that produce fertilizer or in certain bacteria and blue- green algae that live in the soil. The most agriculturally important nitrogen-fixing bacteria belong to the genus Rhizobium. These bacteria infect the roots of members of the legume family, including beans, peas, soybeans, peanuts, alfalfa, and clover, providing the plants with nitrogen and symbiotically receiving nourishment from the plants. This buildup of nitrogen-containing substances in turn increases the fertility of the soil for nonleguminous crops, an observation made as far back as Roman times. Researchers are trying to genetically engineer Rhizobium bacteria so that they will fix nitrogen more efficiently or infect other crops in addition to legumes. An important consideration in this work is the competitiveness of the genetically engineered bacteria. More produc- tive Rhizobium must be able to survive and to displace indigenous Rhizobium if they are to have an effect. Other microorganisms also fix nitrogen in the environment, and researchers are examining these to see if they could be adapted to supply nitrogen to crops. The challenges involved in this work are to engineer the organisms so that they will live in association with the desired crops and fix excess nitrogen beyond their own metabolic needs. Alternatively, researchers are investigating the possibility of trans- ferring the ability to fix nitrogen to microorganisms that already live in association with a given crop. The 17-gene complex that enables the bacterium Klebsiella pneumoniae to fix nitrogen was isolated, repro- duced, and introduced into Escherichia coli, which then became nitro- gen-fixing. But when the same genes were inserted into yeast cells, no nitrogen was fixed, indicating the difficulties likely to be encountered in trying to transfer this capacity to higher organisms. Other microorganisms affect plant growth in different ways. Some protect plants from bacterial or fungal infections or secrete compounds that regulate a plant's development. Others protect plants from such environmental conditions as acidity, salinity, and high concentrations of toxic metals. Some microorganisms are able to degrade toxic sub- stances used as pesticides, like 2,4-D. Others can kill weeds or other plants that compete with a crop for nutrients. As these and additional relationships between plants and microorganisms become better un- derstood, genetic engineering will turn to the production of altered microorganisms that enhance the vigor and growth of crops.
BIOTECHNOLOGY IN AGRICULTURE 33 Nitrogen-fixing Rhizobium bacteria form nodules on the roots of plants they infect, supplying the plant with nitrogen in return for nourishment from the plant. Genetic engineers are trying to alter Rhizobium bacteria so that they will infect plants other than legumes. Alternatively, researchers are seeking to transfer the ability to fix nitrogen into other microorganisms that live in association with crops. Genetic engineering will also be used to combat those microorga- nisms, such as certain bacteria and fungi, that harm crops. A particu- larly interesting example involves the bacterium Pseudomonas syringae. A protein on the surface of this widespread bacterium initiates the formation of ice when temperatures drop below freezing. If this protein were eliminated through recombinant DNA or conven- tional mutational techniques, temperatures could drop several more degrees before frost damage began to occur. Microorganisms amenable to genetic engineering also play critical roles in animal agriculture. For instance, some microbes are lethal to the insects that transport diseases into animals. An example is the
34 BIOTECHNOLOGY bacterium Bacillus thuringiensis, which produces a toxin that is deadly to mosquitoes and black flies. Other microorganisms perform their functions within animals. For example, ruminants can consume forage because it is fermented by microbes in their digestive tracts. It is even possible that the genetic engineering of these microorganisms could give animals the ability to digest foodstuffs that are now useless to them. The Genetic Engineering of Plants A more direct way to enhance the productivity of agriculturally important plants and animals is to alter the DNA that dictates their characteristics. At the most basic level, this is what plant and animal breeders have been doing since the dawn of agriculture. In recent decades, plant and animal breeders have developed sophisticated techniques to transfer traits among organisms that can interbreed. They have also developed a host of supporting technologies, such as cell and tissue culture, embryo transfer, and artificial insemination, that facilitate these basic genetic manipulations. In this sense, genetic engineering will be building on a base of experience and expertise that has accumulated over centuries. But at the same time it will offer capabilities that have never before been available. According to Monsanto's Ernest Jaworski, three things are needed for the genetic engineering of plants: a host cell or tissue, a vector to transfer DNA into the host, and the segments of DNA that are to be transferred. Protoplasts have been a popular choice for hosts in the genetic engineering of plants. Protoplasts are cells taken from the leaves, stems, or roots of a plant that have been exposed to enzymes that dissolve the cells' tough outer walls. The "nakedness" of these cells makes it much easier to introduce DNA into them. The use of protoplasts as hosts is critically dependent on their ability to give rise to whole plants, a characteristic known as totipotency. Through exposure to the proper nutrients and plant hormones, protoplasts can be induced to regenerate cell walls and undergo cell division to form an undifferentiated mass of callus tissue. In some cases, this callus tissue can then be induced to differentiate into shoots, roots, or entire plants. However, it is not yet possible to regenerate whole plants from callus tissue for most of the agriculturally important food crops, and the factors controlling this process are still poorly understood. Once a protoplast host has been prepared, foreign DNA can be
BIOTECHNOLOGY IN AGRICULTURE 35 inserted into the cell in several different ways. Two protoplasts can be made to fuse, producing a hybrid cell that in some cases can be regenerated into a plant with novel characteristics. For instance, potato and tomato protoplasts have been fused to produce a hybrid dubbed the "pomato." Discrete segments of DNA, in the form of chromosomes, whole nuclei, or cell organelles (some of which contain their own nonnucleic DNA), can also be inserted into a cell mechani- cally. But the most powerful and versatile way of introducing DNA into a plant cell hinges on the properties of an unusual plant pathogen. "Nature was very kind to the plant molecular biologists," explains Jaworski. "It supplied us with a natural, soilborne organism called Agrobacterium tumefaciens. This soilborne organism invades plant tissues through wound sites and introduces genetic information, by a mechanism unknown as yet, into the chromosome of the plant cell. . . . This is one of the greatest systems for transforming plants that has been invented to date." Plant researchers have discovered that the agent in A. tumefaciens enabling it to perform this transformation is a large plasmid that has the ability to insert part of its DNA at a random location into the DNA of the cell nucleus. Normally, the genes inserted by this plasmid code for plant hormones that cause tumors in plants known as crown galls. But through the use of genetic engineering, researchers have deleted those tumor-inducing genes and have inserted genes of their own choosing. The first gene to be inserted in this way and expressed in a whole plantâin this case a petuniaâwas a gene conveying resistance to an antibiotic. Even some of the offspring of these plants were resistant to the antibiotic, demonstrating that the new DNA was passed on as a stable genetic entity. The genetic engineering of plants is clearly still in its infancy, but the early success of genetic engineering in some plants points the way toward a time when it may be possible to introduce desirable traits into many agriculturally important crops. For instance, researchers at Monsanto and elsewhere have been working with the genes that code for the enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase, often referred to simply as Rubisco. Rubisco, which is probably the most abundant protein in the world, is the key catalyst in photosynthesis, the process that allows plants to convert carbon dioxide from the atmosphere into other carbon-containing compounds the plants can use. Rubisco consists of eight large subunits encoded by genes in chloroplasts and eight small subunits encoded by genes in the nucleus. Although no vector systems exist to alter genes in the chloroplasts, researchers are genetically engineering the genes that encode the
36 BIOTECHNOLOGY THE GENETIC ENGINEERING OF PLANT CELLS Agrobacterium tumefaciens Plant Cell f t Cleaved Tumor-Inducing Plasmid DMA Segment Containing Gene of Interest % + Transformed A. tumefaciens + Plant Protoplast Transformed Protoplast i\T1 j To genetically engineer plant cells, molecular biologists use an unusual soil pathogen, Agrobacterium tumefaciens. This bacterium contains a large tumor- inducing plasmid that can insert part of its DNA into the DNA of whatever plant cell the bacterium infects. If researchers replace the tumor-inducing genes of the plasmid with foreign DNA, the bacterium can be used as a vector to introduce novel DNA into plant cells. (Most often these cells are protoplasts, normal plant cells that have been exposed to enzymes that dissolve their cell walls, making it easier to introduce DNA into them.) Once transformed by A. tumefaciens, the cells can sometimes be regenerated into whole plants through exposure to the proper combinations of plant hormones and nutrients. small subunits. By increasing the efficiency with which Rubisco fixes atmospheric carbon dioxide, researchers hope to produce plants that will grow faster. In collaboration with researchers at Rockefeller University, plant molecular biologists at Monsanto have introduced into petunias the genes coding for Rubisco's small subunits in the pea. "It actually gets produced, processed and transported properly, and assembled as part of the petunia Rubisco holoenzyme," says Jaworski. "That, I think, is very
BIOTECHNOLOGY IN AGRICULTURE 37 encouraging." Furthermore, the bioengineered petunia Rubisco dem- onstrates many of the properties of Rubisco in the pea. "The gene is light-dark regulated: it is turned on by the light, it is not on in the dark. [Also] the gene is only expressed in the appropriate tissue. We did not find this pea small-subunit gene being expressed, for example, in the roots and stems of the plant, but only in the leaves." Another group of proteins that have been a focus of work by plant molecular biologists are the storage proteins in plant seeds. The seeds of legumes and cereal grains provide humans with an estimated 70 percent of their dietary protein requirements, but some of the most important storage proteins in these seeds are deficient in certain essential amino acids that must be made up in other ways. Researchers have consequently examined the possibility of genetically engineering the genes that code for these proteins to alter their amino acid composition. Such efforts quickly run up against a number of difficulties, accord- ing to Jaworski. "The storage proteins in crops such as corn and soybeans are very complex, multigene families. There are a number of pieces of information we don't have about exactly what happens when you genetically engineer a protein. Let's say we modify it with a single amino acid change. We don't know how that might affect the secondary and tertiary structure of the protein, which has to do with how it is going to be folded and deposited when it is being formed as a storage protein." Jaworski believes that a technically more feasible goal is the modification of leaf proteins rather than storage proteins, which are also commercially valuable as feed for livestock. It might be possible to alter these proteins to be more nutritious, or their concentration in leaves might be amplified through genetic engineering. But this, too, encounters certain difficulties. "We don't know what happens when we elevate natural proteins beyond a certain level," says Jaworski. "We know that in bacteria this can be lethal." The number of genes that are involved in determining a given characteristic is crucial to whether that characteristic is amenable to genetic engineering. For instance, it would be desirable in many cases to give crops the ability to fix their own nitrogen or to photosynthesize more efficiently. Crops might also be genetically engineered to produce higher levels of plant growth hormones or to have a greater ratio of harvestable to nonharvestable matter. Resistance to such environmen- tal factors as disease, insects, competing plants, flooding, drought, salinity, toxic metals, pesticides, heat, and cold are all potential goals of biotechnology. Unfortunately, many of these attributes are probably
38 BIOTECHNOLOGY the result of the interaction of many genes, making them difficult to decipher and transfer from one plant to another. But at least some of these traits are thought to be controlled by one or a handful of genes. For instance, researchers at Monsanto are trying to isolate the gene that codes for the enzyme EPSP synthase, which enables plants to resist an herbicide known as glyphosate that is sold by Monsanto under the trade name Roundup. By growing plant cells in the presence of increasing levels of the herbicide, the researchers were able to isolate a strain with a greatly amplified expression of the gene. They have now reproduced this gene and are trying to introduce it into new plants. Similarly, other plant researchers are trying to isolate and transfer genes that enable plants to make their own insecticides, resist infection by pathogens, or stand up better to a variety of environmental stresses. If successful, says Jaworski, "we can certainly change the geography of some of the cropping practices that limit us today to only specific areas of the world." Before these advances become a reality, several technical problems must be overcome. First, regeneration of whole plants from single- celled protoplasts has so far been accomplished only in a limited number of dicotyledons (flowering plants with two seed leaves), includ- ing tomatoes, tobacco, potatoes, and petunias. Regeneration has gen- erally not been possible with monocotyledons, the group of flowering plants that includes the important cereal grains. Work is under way to develop regeneration systems for these crops, but the continued lack of such systems will severely limit the range of plants that can be genetically engineered. Similarly, A. tumefaciens will only infect dicots, although plasmids similar to the one it carries might be induced to transform monocots. Consequently, researchers are intensively searching for other kinds of vectors that can introduce foreign DNA into plants. Pieces of DNA that can move about within the genome, known as transposons, are one possibility. Investigators are also looking at geminiviruses, which are single-stranded DNA viruses that may be able to transform some plant cells. But the most fundamental problem in applying genetic engineering to agriculture, according to Jaworski, is a lack of basic biochemical knowledge about plants. "We need to spend a lot more timeâand this is where I think we will see a great deal of activity in the next five to ten yearsâon identifying agronomically important traits and the genes that regulate those traits," he says. "If we cannot do this, we are not going to be very successful in really making the agronomic improvements that we desire to make." Even with such well-studied
BIOTECHNOLOGY IN AGRICULTURE 39 functions as photosynthesis, much more work needs to be done to understand the biochemical pathways of regulation at the genetic level. "We need a great deal more information about the signals that regulate tissue specificity, developmental specificity, temporal speci- ficity, and so on," says Jaworski. "We just don't have enough knowledge yet to understand how to regulate at will, and in a controlled fashion, the expression of a gene." The research needed to acquire this knowledge requires both greater cooperation between plant molecular biologists and traditional plant breeders and a commitment by the federal government to fund this kind of interdisciplinary effort, according to Jaworski. "There is a lot of basic research that has to be done in parallel with the applied research if we are going to be successful in moving the technology from the laboratory into the field." The Genetic Engineering of Animals Unlike plants, an animal cannot be regenerated asexually from cells plucked at random from certain parts of its body. Only one kind of cellâthe zygote formed by the fusion of a sperm cell and an eggâhas the capacity to develop into a fully formed animal. Therefore, to introduce a foreign gene into all the cells of an animal, including the germline cells that will pass on an animal's genetic heritage to its offspring, the foreign DNA must be inserted into the sperm, the egg, or the zygote. If a multicell embryo is exposed to foreign DNA, the resulting animal will be a mosaicâsome of its cells will carry the introduced genes and some will not. If foreign DNA is inserted into cells of the organism even laterâsay, after birthâa correspondingly smaller number of cells will be altered. There are several ways of introducing specific genes into the chro- mosomes of an animal's cells, according to Rudolf Jaenisch of the Whitehead Institute for Biomedical Research and the Massachusetts Institute of Technology. One of the most widely used is to insert copies of DNA directly into cells using a micropipette. This seems to work best when done to zygotes after the egg has been fertilized but before the genetic material of the egg and sperm have joined. "The success of deriving transgenic mice in this manner is variable," says Jaenisch. "In a good laboratory between 10 and 30 percent of the animals born will carry the foreign sequences in the germ line." Another way to transform animal cells with foreign DNA is by using retroviruses as vectors. Retroviruses are infectious agents that cause a wide variety of diseases in humans and animals, including some forms
40 BIOTECHNOLOGY of leukemia in nonhuman species. They have the ability to insert a single strand of DNA, derived from their own genetic material, into the DNA of the cells they infect. By genetically engineering certain kinds of retroviruses, researchers can replace their disease-causing genes with genes coding for other proteins. As with A. tumefaciens in plants, retroviruses can then incorporate these genes into the DNA of their hosts. In 1981 a rabbit globin gene became the first bioengineered gene to be inserted into an animal embryoâin this case a mouseâand repro- duced in all the cells of the mature animal. Since then a number of other genes, including oncogenes and genes coding for metallothionein, elastase, and immunoglobulin, have been inserted, expressed, and passed on to offspring in laboratory animals. A landmark experiment was the introduction into mice of a gene for growth hormone fused to the regulating DNA from a metallothionein gene that caused growth hormone to be expressed whenever the mice were exposed to certain heavy metals. The mice transformed by the growth hormone gene grew to more than twice the size of their normal siblings. The success of these experiments has generated great interest in the possibility of genetically engineering farm animals so that they would be more productive or more resistant to disease. To date, much of this interest has focused on the prospects for growth hormone. Experiments with injected growth hormone have suggested that animals producing elevated levels of their own growth hormone might grow faster, larger, leaner, and with less consumption of feed. Such advances would be particularly welcome in the production of swine, since they are gener- ally sold at an immature age and since consumers would likely favor leaner pork. Injections of growth hormone have also been shown to markedly increase the production of milk in dairy cows. Jaenisch warns, however, that several questions must be answered before the genetic engineering of farm animals becomes practical. For instance, researchers are still not certain whether elevated levels of growth hormone would have harmful side effects or whether such levels would even produce the increased growth expected. Swine are already bred for maximal growth, and it is not clear whether insertion of a growth hormone gene would further increase their size. Also, the mice transformed by the growth hormone gene showed signs of abnor- mally proportioned growth, and the female mice genetically engi- neered in this way were often sterile. "So there are a number of physiological consequences of inserting a gene that we really don't understand yet," Jaenisch says. "I think one has to be cautious about
BIOTECHNOLOGY IN AGRICULTURE 41 Dairy cows injected with growth hormone increased their production of milk up to 40 percent. Through genetic engineering, researchers hope to create cattle that will produce elevated levels of growth hormone endogenously, resulting in faster growth or increased milk production without the need for injections. drawing too many conclusions of what the value of this technique will be for general use." As in the genetic engineering of plants, an even more fundamental problem involves the regulation of the genes inserted into animal cells. The expression of an inserted gene can be influenced both by the regulatory sequences associated with the gene and by where the gene is inserted into the DNA of its host. At present, there is no way to control where a gene is inserted into the chromosome of either an animal or a plant cell. Yet this position of insertion can affect not only the expression of the inserted gene but also the regulation of the host
42 BIOTECHNOLOGY cell's DNA. For instance, inserted DNA can separate two sections of a functioning gene and block its action, causing genetic disease in interbred offspring if the gene is recessive (all of them have been to date). Inserted genes can also turn on even distant genes within the genome, causing a tumor if the activated gene is an oncogene. Such mutations are valuable in what they tell molecular biologists about the biochemical machinery of genetic regulation. But much more needs to be learned before it will be possible to insert genes in a predictable fashion and control the expression of those genes for desired ends. "The major scientific problem that confronts the geneti- cist is our inability in higher organisms to predictably, invariably, and inevitably replace and alter genes at will," says Philip Leder of the Harvard University Medical School. "It is not possible for us now to introduce genetic material into the mouse ... in a way in which the outcome of that experiment is absolutely predictable. It is not yet possible to correctly or predictably alter the amino acid composition of the major corn protein by introducing amino acids that are essential for human and animal nutrition, or to predict if that protein will be expressed in normal, or perhaps larger, amounts. To be able to do that predictably will open a new and important avenue for application and investigation and has to be viewed as one of our major scientific goals." Additional Readings Kenneth A. Barton and Winston J. Brill. 1984. "Prospects in Plant Genetic Engineering." Pp. 121-131 in Biotechnology and Biological Frontiers, Philip H. Abelson, ed. Washington, D.C.: American Association for the Advancement of Science. Winston J. Brill. 1981. "Agricultural Microbiology." Scientific American 245(Sep- tember):198-215. National Research Council, Board on Agriculture. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, D.C.: National Academy Press. National Research Council, Committee on Biosciences Research in Agriculture. 1985. New Directions for Biosciences Research in Agriculture. Washington, D.C.: National Academy Press. Thomas E. Wagner. 1984. The Implications of Genetic Engineering in Livestock Production. Knoxville: University of Tennessee.