Click for next page ( 41


The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 40
Applying the Tools of Biotechnology to Agricultural Problems Effort is now under way to apply these cellular and molecular tech- nologies to specific agricultural problems. For instance, researchers are attempting to clevelop herbicide-resistant plants and plants less suscep- tible to environmental stresses, such as drought, salty soils, or climatic extremes. They are working on increasing the nutritional quality of feed crops. They are also trying to engineer soil microorganisms that can be used to supply nitrogen to crop plants, or else mitigate or combat soil diseases. Herbicide Resistance Some 420 million pounds of herbicides are used by farmers each year in the United States. Herbicides are used to kill the weeds that compete with crop plants. Unfortunately, they can also kill some crop plants as well. This restricts each herbicide to use on a resistant crop. Neverthe- less, crop Tosses from herbicides can still occur. A case in point is the herbicide atrazine, which is commonly used in the culture of corn. In Illinois, for instance, 90 percent of corn acreage is treated with atrazine each year. Corn can tolerate atrazine because the plant naturally contains an enzyme that rapidly breaks down and detoxifies the herbicide. Yet in the Midwest, corn is often used in rotation with soybeans, which are susceptible to atrazine. Sometimes winter climatic conditions cause res- idues of atrazine to remain in the soil the following spring. Such residues can dramatically reduce the yield of soybeans planted the following year. To avoid such losses and to broaden the range of each herbicide, plant breeders are interested in developing herbicide-resistant crops. An atra- zine-resistant soybean, for example, would be ideal for the Corn Belt. But before the trait can be transferred, it must be unclerstood. At 40

OCR for page 40
BIOTECHNOLOGY AND AGRICULTURE 41 Michigan State University, Charles Arntzen and colleagues have been trying to elucidate the genetic mechanism of atrazine resistance in weeds. Atrazine-resistant weeds began to appear spontaneously in the early 1970s, primarily in areas of prolongecl, steady use of the herbicide. Some 30 species of weeds have now become atrazine resistant. To find out how, Arntzen, a plant biochemist, chew on the work of plant physiol- ogists, other biochemists, and agroecologists, as well as molecular bi- ologists. Like the majority of herbicides, atrazine acts in the chIoroplasts where it disrupts photosynthesis. Photosynthesis is the process by which plants convert sunlight to chemical energy. Through photosynthesis, the sun's energy is used to support all higher forms of life on earth. In photosynthesis, sunlight is absorbed through chlorophyll, the green pigment in the chIoroplast. The sun's energy is used to force electrons to an excited state. In a complex series of reactions, the energy held by the excited electrons is used to build carbohydrates. One of the keys to photosynthesis is the transfer of solar energy from the chlorophyll, where it is absorbed, to the place where it is used. The excited electrons hold that energy. In bucket-brigade fashion, electron carrier molecules transport those electrons from one molecule to another. One of these carrier molecules is quinone. It is now known that atrazine kills plants by disrupting this electron transport, thereby blocking photosynthesis. It does this by competing with quinone, one of the carrier molecules. When atrazine is taken into the chIoroplast, it can take the place of the quinones on one type of protein in the membrane that holds the electron transport chain together. Without quinone to transport the electrons, photosynthesis is halted. Atrazine resistance arises from a mutation that alters that membrane protein so that it will no longer bind atrazine. The quinone, however, still binds to the altered membrane protein; consequently, electron trans- port remains undisturbed in the presence of atrazine. Using traditional biochemical procedures, Arntzen's group spent three years trying to purify that protein in orcler to study the nature of the mutation. They did not succeed. The task of determining the sequence of amino acids in a protein is difficult and time-consuming. It is easier to sequence the nucleotide bases in the gene that codes for the protein. From other studies, Arntzen knew that the protein was encoded by a gene in the chioroplast. Arntzen's group began collaborating with Lee McIntosh of Michigan State University and Lawrence Bogorad of Har- vard to study the genes of the chIoroplast. One of the chIorop:tast genes had already been isolated and cloned. It was the gene Arntzen was seeking.

OCR for page 40
42 GENETIC ENGINEERING OF PLANTS Since then, the genes in both atrazine-susceptible and atrazine-re- sistant weeds have been sequenced. The only difference between the two is one nucleotide base: in the resistant weect, an adenine is replaced by a guanine. That one change spells one different amino acid, creating a slightly different protein but different enough to cause a "glitch" that prevents atrazine bincting. In short, one nucleoticle substitution in a single gene determines whether a plant is resistant or susceptible to this herbicide. Engineering an Atrazine-Resistant Crop Because resistance to atrazine is conferred by a single gene, this trait seems amenable to molecular gene transfer. But there are other, simpler approaches to develop herbicide-resistant crops. To date, the greatest success has come from classical plant breeding aided by knowledge garnered from molecular-level investigations. At the University of Guelph, Ontario, a research team including W. Bev- ersdorf and Vince Souza-Machado have bred atrazine-resistant strains of oiTseed rape (Brassica napus) and summer turnip rape (Brassica cam- pestris). This was accomplished through repeated backcrosses between rape and a closely related resistant weed, wild turnip (Brassica campestris). The key to this success was the knowledge that the trait is carried in the chIoroplast. In sexual crosses, the chIoroplast is transmitted by the maternal line alone; the mate contributes only nuclear DNA. That means the only way to generate a resistant crop is to use the resistant weed as the female parent and a crop plant as the source of pollen. The resulting resistant progeny are then fertilized with pollen from the crop plant, and the process is repeated for five to seven generations. "Finally what you get is a crop a new plant in which the cytoplasm, including the chIoroplast, is essentially donated by the weed, and the nucleus is donated by the crop plant," Arntzen described. Although backcrossing is laborious, it works. "Current estimates sug- gest that by 1985, with increased seed stocks, there will be close to one million acres of atrazine-resistant oilseed rape grown in Canada. It's a dramatic success story, and it didn't take one iota of genetic engineer- ing." Unfortunately, an identical approach is not feasible with many crops, as few are cross-fertile with weeds. There are, however, many herbicide- resistant weeds that are closely related tobut not cross-fertile with major crop plants. For example, the atrazine-resistant weed black night- shade (Solanum nigrum) is in the same genus as potato (SoZanum tuber- osum) and the same family as tobacco and tomato. That is where the

OCR for page 40
BIOTECHNOLOGY AND AGRICULTURE Atrazine-Resistant Weeds That Are in the Same Botanical Family as Crop Plants 43 Atrazine-Resistant Weed Crop Plant CHENOPODIACEAE Chenopodium album (common lambsquarters) Artiplex patula COMPOSITAE Senecio vulgaris (common groundsel) Ambrosia artemisifolia (ragweed) Brassica campestris (wild turnip) Solanum nigrum (black nightshade) Beta vulgaris (sugar beet) Beta vulgaris (red beet) Helianthus annuus (sunflower) Carthamus tinctorius (safflower) CRUCIFERAE SOLANACEAE Brassica campestris (turnip rape) Brassica napus (oilseed rape) Brassica oleraceae (cabbage) Solanum tuberosum (potato) Lycoperiscon esculentum (tomato) Nicotiana tabacum (tobacco) SOURCE: Charles T. Arntzen, Plant Research Laboratory, Michigan State University, East Lansing. new genetic technologies come in. Because the species are closely re- lated, their protoplasts can be fused in culture to create a hybric3. The first of these experiments was reported) in 1982 by Horst Binding in West Germany and lonathan Gresse} in Israe} and their colleagues, who fused protoplasts of black nightshade and potato. The goal was a resistant potato; unfortunately, the atrazine-resistant hybrid was more like the weed than the potato. A solution may be in sight, according to Arntzen. In a half dozen laboratories around the worId, researchers are now trying to inactivate the nuc:teus in the protoplast from the donor weed, so that just the weed's cytop:Lasm which contains the resistant chIoroplast will be introduced into the potato protoplast. "If somebody doesn't have a herbicide-resistant potato plant within the next year or two, 1'd be very surprised," Arntzen said. An alternative approach is to find a mutant in nature yet this par- ticular mutation rarely occurs. Arntzen and others are investigating methods to induce this mutation. The most powerful technique, if it can be mastered, will be to transfer the resistant gene from a weed into a crop plant using recombinant DNA technology. "There has been a Tot of progress along these lines," Arntzen said, "but we still have a great deal of work left to do." So far, the gene for herbicide resistance has been isolated and cloned inside a

OCR for page 40
44 GENETIC ENGINEERING OF PLANTS bacterium. Now the Michigan State and Harvard University groups are trying to achieve gene expression. The biggest hurdle will be finding a vector to carry the herbicide resistance into a crop, since the gene must be inserted into the chIoroplast. The only successful plant vector that has been developed to date the Ti plasmiddoes not work for chIo- roplasts. Bioengineered Microorganisms to Combat Plant Diseases One of the most promising, and relatively unexplored, applications of the new genetic technologies is in combatting soil-borne plant dis- eases, according to Milton N. Schroth, a plant pathologist at the Uni- versity of California at Berkeley. Some of the most destructive plant diseases, such as Fusarium wilts and Phytophthora root rots, are caused by microorganisms that inhabit the soil. Other, less virulent microor- ganisms also exact their toll: the presence in the soil of low-grade disease agents can lower yield significantly. For example, if strawberries are grown on fumigated soil, in which all the soil microorganisms have been destroyed, the yield is approximately 20 tons per acre, or four times higher than if they are grown on unfumigated soil. Because the effects of soil pathogens are sometimes insidious, it is difficult to estimate exactly the economic costs of soil-borne diseases, but they undoubtedly can be considerable. The increasing adoption of minimum or no-till farming practices pro- vides an extra incentive for developing effective controls. Both of these practices leave organic debris on the soil surface, which makes the soil both wetter and colder creating a more favorable environment for pathogenic microorganisms. There are several possible strategies. Fumigation of the soil effectively controls disease, but it is costly and impractical for large areas. Moreover, its benefits are transitory, as clisease-causing microorganisms can be easily reintroduced by wind or animals. Another approach is to breed resistant cultivars. Many resistant va- rieties have been developed, yet for unknown reasons the introduction of disease resistance often results in a loss in yield. Tobacco plants bred for resistance to Fusarium wilt fungus, for example, commonly show a nearly 6 percent reduction in yield as compared to susceptible plants. Resistant plants could also be developed through gene-splicing tech- niques. "it would be ideal if genes conferring resistance to pests could be introduced to the plant, expressed, remain stable, and not result in a cost to the plant," Schroth said. Unfortunately, demonstrated tech- niques for cloing this are not yet available. Moreover, it is not clear

OCR for page 40
BIOTECHNOLOGY AND AGRICULTURE Reductions in Yield and Quality in Disease-Resistant Tobacco Lines in Comparison to the Susceptible Linea 45 Resistance Percent Yield Percent Price Reductionlha Reduction Tobacco mosaic 5.9 1.5 Fusarium wilt 6.9 1.9 Mosaic + Fusarium wilt 9.9 1.9 Mosaic + bacterial wilt 7.1 2.7 Mosaic + root knot nematode 5.0 1.8 Fusarium wilt + bacterial wilt 10.3 4.0 Fusarium wilt + black shank 6.6 3.2 a Based on data given in Chaplin, 1970. Agron. J. 62:87-91. SOURCE: Milton N. Schroth, Department of Plant Pathology, University of California at Berkeley whether the introduction of resistance through gene-splicing would re- sult in the same Toss in yield as does conventional plant breeding. The alternative genetic engineering approach is to harness and im- prove upon the beneficial microorganisms that inhabit some soils and use them to combat plant disease. In nature some soils, known as disease-suppressive soils, contain beneficial microorganisms that help to protect plant roots from pathogens. The mechanism of disease suppression in these soils is poorly understood, and it does not come from the beneficial microorganisms alone. Instead, it seems to be con- trolled by complex interractions among both biotic and abiotic factors. it has long been known, for instance, that physical conditions such as the salinity, acidity, temperature, and moisture levels of soils can render plants less or more susceptible to a disease. Nonetheless, beneficial microorganisms seem to play a major role. If a disease-suppressive soil is fumigated to destroy all microorganisms, the soil loses its capacity to suppress plant pathogens. But when some of the unfumigated sup- pressive soil is reintroduced, the disease-suppressive quality of the soil is restored. These naturally suppressive soils provide a substantial boost to crop yield. In Provence in southeastern France, the suppressiveness of the soil varies greatly from region to region. This has been determined by infesting the soil with pathogens and then comparing the severity of the ensuing disease. For centuries, muskmelons have been grown in the Chateaurenard area with little trouble from Fusarium wilt, even though the fungus is present in the soil. Yet in the neighboring two regions of Cavaillon and Carpentras, the disease can be so severe that the musk- melon crop sometimes has to be abandoned.

OCR for page 40
46 GENETIC ENGINEERING OF PLANTS Little is known about the microorganisms that inhabit the rhizosphere, or soil-root zone. Most research to date has focused on nitrogen-fixing bacteria because of their potential to reduce the need for chemical fer- tilizer (see Nitrogen Fixation, p. 48~. In just a few years, molecular bi- ologists have made great strides in understanding the genetic control of this trait. The same tools can be used for studying, and ultimately improving, these other beneficial microorganisms. Some of the most promising candidates for biocontrol agents are the root-colonizing bacteria, generically known as rhizobacteria. Some of these have the beneficial effect of promoting plant growth; others have deleterious or neutral effects. For use as biocontrol agents, the bacteria must be able to colonize the roots aggressively and have the potential to dominate the ecological niche. Finding such bacteria will be difficult, Pseudomonas colonizing the surface of a sugar beet root. This scanning electron micro- graph shows chainlike colonies of bacteria against the ribbed background of a sugar beet root (x 3000~. These beneficial bacteria suppress the growth of plant pathogens that could otherwise attack sugar beets. Courtesy of Milton N. Schroth, Department of Plant Pathology, University of California at Berkeley.

OCR for page 40
BIOTECHNOLOGY AND AGRICULTURE 47 as less than 5 percent of bacteria isolated to date from plant roots are able to colonize the roots effectively and promote plant growth. Several strains of Pseudomonas can. Rhizobacteria seem to work through two generic mechanisms. One is antagonism the bacteria compete with and displace the deleterious organisms on the plant root. The second is inhibition some rhizobac- teria produce antibiotics that inhibit a variety of pathogens. It seems likely that disease protection in soils is conferred through a variety of microorganisms and favorable environmental conditions. The hope is to identify some of the key beneficial microorganisms and adapt them to use as biocontrol agents in conducive soils, either by manipu- lating the soil environment or modifying these microorganisms to im- prove their efficiency. That, of course, depends on an understanding of their normal mode of action. Research to date has been promising. Though relatively little is known about rhizobacteria, their application to seeds and roots at planting time can increase plant growth and yield. In Idaho, California, and Penn- sylvania, potato yields increased 5 to 33 percent following application of Pseudomonas. It has also been effective on sugar beets and radishes. It might be simpler to introduce the microorganisms directly into the soil, but that approach is "unreachable" at this time and may remain but that approach is ''unreachable'' impractical for commercial agriculture, Schroth said. The biocontro} agents would have to compete with the other organisms already present in the soil. Those long-term residents are "well entrenched, and not easily displacecI by intruders." In such a scheme, vast amounts of inocuTum would be required on a regular basis; the cost might be prohibitive. Because strains of the same species are best adapted to occupying the same ecological niche, the ultimate approach may be to engineer the pathogen to control itself. This would entail inactivating the disease- causing agent from the microorganisms and then using this disarmed bug to displace its pathogenic relative. Eventually, when more is known about complex interactions in the rhizosphere, it may even be possible to manipulate the soil ecosystem to favor beneficial microorganisms. 1 , . ~ ldentitying and improving rhizo bacteria will require the combined efforts of bacterial ecologists, plant pathologists, biochemists, and ge- netic engineers. Specifically, they need to determine the genetic factors that govern root colonization. They need to identify the key factors that enable a microorganism to compete successfully in an ecological niche. One means of increasing their competitive ability might be to bioengineer them to tolerate greater moisture stress. Schroth cautioned against underestimating the complexity of the ag- roecosystem. It may be possible to design a strain of bacteria that gives

OCR for page 40
48 GENETIC ENGINEERING OF PLANTS dramatic results in the laboratory. But in the field, competing with microorganisms that have evolved for hundreds of thousands of years, a successful laboratory strain might not perform well, or even survive. Nitrogen Fixation Nitrogen is an essential plant nutrient and a key determinant of crop productivity. Unfortunately, the nitrogen content of agricultural soils is quickly depleted. Farmers worldwide supplement the available nitrogen with some 60 million metric tons of nitrogen fertilizer annually. By the year 2000, an estimated 160 million metric tons of nitrogen fertilizer may be used each year. Producing that fertilizer is both expensive and energy . . Intensive. As farmers face the prospect of rising bills for nitrogen fertilizer, their crops are literally being bathed in nitrogen gas, as roughly 80 percent of the air is nitrogen. Yet plants are unable to use nitrogen from the air. Soybeans and other legumes are an exception; they have a symbiotic relationship with nitrogen-fixing bacteria, Rhizobium. In some soils, where Rhizobium are indigenous, the farmer need only plant the legume. In areas where the bacteria are not present, the farmer adds or inoculates the soil with Rhizobium. In either case, no nitrogen fertilizer is necessary. The Rhizobium infect the roots of the plants, causing nodules to form. Insicle the nodules, millions of bacteria convert the nitrogen that is in the air to ammonia, which the legume, like other plants, needs for protein synthesis. Agricultural yields could be sustained at tremendous savings if bio- Togical nitrogen fixation can be improved and extencled to major crops, such as corn and wheat, that now depenct on costly nitrogen fertilizer. The cluster of genes that control nitrogen fixation in microorganisms has been isolated and analyzed. In numerous academic and industrial laboratories, researchers are trying to understand how those genes are regulated and how they can be usecT in practical crop improvement schemes. Winston Brill of the University of Wisconsin and Cetus Mad- ison, Corp., described that work. The earliest payoff may come from attempts to improve the efficiency of nitrogen fixation in legumes. The approach is to engineer either the Plant or the Rhizobium or both to improve the symbiotic relation~hin . . . . ~ - .. . . . . ~ ~ . According to Frill, genetic manipulation of the bacterium is tar simpler than manipulation of the plant. He has used both standard mutagenesis and recombinant DNA techniques to develop improved strains of Rhi- zobium. When inoculated with the mutant strains, plants show increased vigor and growth.

OCR for page 40

OCR for page 40
50 GENETIC ENGINEERING OF PLANTS The most efficient means of supplying nitrogen would be to transfer the nitrogen-fixing genes from the bacteria into the plant. It is also one of the toughest tasks imaginable. A number of laboratories have isolated the nitrogen-fixing genes from Klebsiella, bacteria similar to Rhizobium but easier to work with in the laboratory. Nitrogen fixation is a complex, multigene trait controlled by a cluster of 17 genes. These genes are broken clown into smaller units, each of which is regulated separately. To endow a plant with the ability to fix its own nitrogen would mean transferring all 17 genes, along with the complete collection of regulatory signals. PROBING THE MECHANISMS OF NITROGEN FIXATION Soybeans, alfalfa, and other legumes have a symbiotic relationship with the bacteria Rhizobium that enables these plants to obtain nitrogen from the soil. The increasing interest in extending the ability to fix nitrogen to other crops has spurred efforts to understand the unusual v , . . . . . relationship between Rhizobium and legumes. When Rhizobium intact plant roots they cause the cells to proliferate, giving rise to nodules on the roots. Rhizobium induce another, appar- ently unique, change in the plant cells: at the spot where the bacteria first come in contact with the root, the plant cells form a tubular~ike structure, known as an infection thread. These infection threads wind throughout the cells in the root nodule, providing a conduit through which bacteria migrate from one cell to another. Once inside the cells, the bacteria convert nitrogen to a chemical form the plant can use. The top photograph, a scanning electron micrograph (magnification x 2000), shows an infection thread traversing a ceil in an alfalfa root nodule. At the end of the thread (upper lefty bacteria are being released into the cell. Bacteria other than Rhizobium are not known to induce the formation . . .. ~ . . . . . ~ . . Of infection threads. Research is under way, using gene-splicing tech- niques, to transfer the genes that control nodule formation (the nod genes) and nitrogen fixation (the nif genes) from Rhizobium to other bacteria. If bacteria that infect nonieguminous plants can be endowed

OCR for page 40
BIOTECHNOLOGY AND AGRICULTURE with the properties of Rhizobium, it might offer a means of extending nitrogen fixation to other crops. The nod and nif genes have recently been transferred from Rhizobium to Agrobacterium tumefaciens. When the genetically engineered Agro- bacterium infected an alfalfa plant, it induced root nodules and infection threads to form (bottom photograph, magnification x 7601. The genes for nitrogen fixation, however, were not expressed. 51

OCR for page 40
52 GENETIC ENGINEERING OF PLANTS Brill predicted that this cluster of genes will soon be moved into dicots such as potatoes and tomatoes and later into monocots like corn and other cereals. "Is that the end of the story? Do we now have nitrogen- fixing corn?" he asked. The answer is no. The entire cluster of genes has already been transferred into yeast, but the genes were not ex- pressed. Not only must gene expression be achieved, but the genes must be inserted into the proper place in corn and turned on at the appropriate time. Nitrogen fixation requires an energy-rich, oxygen-depleted mi- croenvironment the corn's mitochondria or chioroplasts might be a suitable site for the genes. In addition, the chemical reactions involved in nitrogen fixation require far more iron and molybdenum than are normally found in most plant cells. It is too early to tell if the hurdles are simply formidable or if they are insurmountable. The Bottom Line The final test of these new agricultural products the improved va- rieties and bioeng~neered microorganisms, for instance will be their performance in the marketplace. The new products must offer an ad- vantage over existing ones if the farmer is to adopt them. As Schroth explained, the bioengineerecl products must improve the profit margin per hectare either by increasing yield or reducing production costs. That, in turn, depends on how well the new products can be integrated into existing agricultural techniques. Though disease-resistant plants can be bred, they often have reduced yield. It may be more economical for the farmer to use a susceptible variety and a fungicide to control the disease. Similarly, the benefit of a biocontroT agent will have to be weighed against a fungicide. Key factors influencing the commercial success of a biocontro! agent might be its shelf life and the ease with which it can be applied to the soil. So far, many researchers have been so caught up in what is scientif- ically possible that they have neglected the practical considerations, such as market analysis, Schroth added. He suggested that molecular biolo- gists work with plant breeders, agronomists, and pomologists in iden- tifying scientifically and economically attractive projects for genetic en- gineering. "It will not be a simple task to improve productivity per hectare," he said. "And it certainly will not be done by the unilateral efforts of one discipline." Bogorad concurred. "It is clear that we need molecular biologists plus plant pathologists and agronomistspeople who know about real plant problems. One of the difficulties we have today is that there are very, very few people who understand both sides of the problem."