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Biotecinolo~ and Agricaltarar! Research for Crop Improvemerlt CHARLES J. ARNTZEN INTRODUCTION Genetic improvement of crop plants has been underway for thousands of years. The first known organized plant cropping in the Iraqi Kurdistan (circa 6000 B.C.) involved seeding of wild wheat for cattle foraging. Since then our ancestors have harvested the largest and most desirable seeds or fruits of their favorite foods and planted some of these collections in organized agricultural efforts. During the last 50 years a better under- standing of basic genetics and crop physiology has paved the way to dramatic increases in productivity of agricultural crop commodities via improved genetic and cultural practices (hybrid seeds, improved fertil- izers, pesticides, and so forth). These approaches are being further refined at present in a wide range of academic and industrial settings, with continued, gradual increases in agricultural productivity. A series of economic changes is currently affecting agricultural prac- tices. Increased costs make it infeasible to expand irrigation, fertilizer, and other capital-intensive cultural inputs indefinitely. It is now rec- ognized that it would be desirable to change the genetic makeup of our crops to give them increased resistance to environmental and biological stresses (e.g., heat, drought, nutrient starvation, insects, diseases) so that there will be less reliance on amelioration of these stresses by Contribution number Il135 from the Michigan State University Agricultural Experi- ment Station. Research supported by a grant from CIBA-GEIGY and DOE Contract #DE-AC02-76ER01338 to Michigan State University. 52

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CROP IMPROVEMENT GT~ it. ~ .~ 1~ ~ 1 N-DNA 95+% >5O,OOO genes mt-DNA (1% -10-100 ct-DNA 3-5% -100 FIGURE 1 A diagram of the organization of a typical plant cell. The cell wall, surrounding the cell, is relatively rigid and defines the cell shape. Within the wall is the cytoplasm containing a vacuole (V), the nucleus (N), and several mitochondria (M) and chloroplasts (C). The structures diagramed within the nucleus are chromosones, the site of localization of 95 percent of the total DNA in the cell (corresponding to at least 50,000 individual genes). The rest of the DNA in the cell is in either the mitochondria (mt-DNA; about 10 to 100 genes) or the chloroplast (ct-DNA; also containing about 100 genes). 53 cultural means. Some of the desired traits were present to varying de- grees in the progenitors of existing crop plants but have been lost in generations of crop evolution. In other cases the desired combinations of traits have never existed. From the molecular biologist's standpoint, the current needs to rapidly add new physiological traits to our crops pose very interesting possibil- ities and problems. We must learn when we can use new biotechnologies to identify genes for desirable traits, and then put these traits into our present crops. Location of DNA Within the Plant Cell The genetic information of a plant resides in three different locations within the plant cell. Figure 1 shows in a stylized way what an individual leaf cell might look like in a crop plant. The cell has two major sub- divisions. One is the cell wall surrounding the cell. The wall is largely

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54 NEW FRONTIERS IN BIOTECHNOLOGY inert, containing cellulose, lignin, pectin, and some protein. It forms a box or enclosure that gives shape to the cell (and to the plant as a composite of many cells). Within the cell wall is the cytoplasm, the living portion of the cell. It has various inclusions. In plants, as opposed to animals, a large portion of the cell is filled by a vacuole (analogous to a storage bag containing water, enzymes, salts, and organic molecules). Another major compartment is the nucleus, which contains several chro- mosomes. These contain about 95 percent of the DNA (deoxyribonu- cleic acid) in the cell. This is enough genetic material to code for an estimated 50,000 different genes. It should be emphasized that it is not actually known how many genes are in the nucleus of crop plants or, in fact, what the vast majority of these genes do in controlling plant growth or development. It is known that the traits that are present have been selected through many gen- erations of crop evolution. It is also known that most crop-breeding improvements have involved manipulation of this nuclear DNA by care- ful, time-consuming genetic crossing. There are also two other sites where DNA is localized in plant cells. One is the mitochondria, the respiration centers of living cells (Figure 1~. While plant mitochondria contain less than 1 percent of the total cellular DNA and approximately 100 structural genes, these cellular inclusions are still genetically important. Mitochondrial genes are known to influence disease resistance and male sterility, for instance. The chloroplast is the third plant cellular compartment that contains DNA (Figure 14. These inclusion bodies also contain chlorophyll and are the site of photosynthesis (the conversion of solar energy to chemical form leading to the fixation of carbon dioxide [COW. The organic molecules formed during photosynthesis are the materials upon which all life in our biosphere is dependent. In other words, plants are the only avenue for the input of energy in our biosphere. About 3 to 5 percent of the total cellular DNA is in the chloroplast, organized to include approximately 100 structural genes. It is known that these genes control the synthesis of important proteins that are involved in photo- synthesis. CHOOSING A TARGET FOR BIOENGINEERING EFFORTS IN CROP IMPROVEMENT As was indicated earlier, plant cells contain tens of thousands of genes, each of which controls or influences a physiological process in the plant. At our present stage of research in molecular biology, we usually must deal only with one of these genes at a time. This limits the type of bioengineering efforts that can be contemplated in the near future.

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CROP IMPROVEMENT 55 1. Identify a (single gene) trait of agronomic value. 2. Determine the molecular basis of the trait (identify a critical enzyme, a rate-limiting step in a metabolic pathway, or the target site of a small molecule that regulates an important metabolic pathway ). 3. Locate and isolate the gene that encodes the critical enzyme, or target site, and, where applicable, modify it. 4. Transfer the gene to a crop plant of choice. FIGURE 2 The series of steps involved in bioengineering for crop improvement. Ultimately, crop yields are what everyone would like to be able to influence (increase) via biotechnology. Unfortunately, the yield of har- vestable plant parts is determined by many traits (plant vigor, number of flowers, metabolic rates, and so forth). However, while there are many factors involved in the yield itself, we can try to limit our efforts to one specific item at a time. The central question concerning biotechnology and agriculture now being asked in many academic and industrial laboratories is simple: How can we identify a feature of a crop (or crops) that we can change via genetic engineering to obtain a superior variety? Answering the question requires study of the progression of steps summarized in Figure 2. At our current stage of bioengineering expertise, we must first find a single gene trait of agronomic importance and then identify the mo- lecular basis for the trait. This could entail identifying a specific enzyme that produces a new or unique product. It could also mean identifying a rate-limiting step in some metabolic pathway which, if improved, would increase the rate of an important process. Another approach could entail finding the target site for a molecule that affects or regulates a specific metabolic pathway. Once we identify a key enzyme or target site, we can begin the process of using the tools of genetic engineering to change it. To accomplish this, of course, it is necessary to locate and isolate the gene that encodes the critical component. There are now a variety of strategies that have been applied to this problem, each too complicated to enumerate in any detail here, but still very feasible with today's technologies. It is also becoming a routine procedure to be able to make desired alterations in this gene, such as causing it to produce a protein with a different amino acid composition. Once a gene is isolated, it is necessary to find a way to transfer the

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56 NEW FRONTIERS IN BIOTECHNOLOGY gene into the crop plant that we choose. This is an area of intensive research in many laboratories at the present time. Techniques (although limited as yet in applicability) are now available to achieve this gene insertion in certain crop plants. Advances in this aspect of biotechnology are needed and are certainly expected in the near future. BIOENGINEERING FOR HERBICIDE RESISTANCE: ONE EXAMPLE OF GENETIC ENGINEERING STRATEGIES Figure 2 delineated the general steps necessary for planning crop improvement via genetic engineering. The following material describes how this approach has been followed in one example. The vast majority of major crops produced in this country are treated with pesticides. This provides a very cost-effective means of controlling weeds, insects, and other pests that would otherwise reduce productivity or quality of the commodity. In the production of corn, greater than 95 percent of U.S. acreage is treated with herbicides to control weeds. The most widely used herbicide in corn has been atrazine. The reason that atrazine can be applied to corn is that this crop has a very efficient metabolic pathway that detoxifies this and other triazine herbicides. In contrast, when most weed seeds germinate in the fields treated with atrazine, the herbicide is taken up and the weeds are killed. The use of herbicides is not without its difficulties. One is carryover of the chemical in the soil. This results in problems when a farmer rotates crops so that a sensitive plant species is grown in a field that had pre- viously been treated with a chemical that is toxic to the new plant. A common example is soybeans planted in rotation with corn. The triazine herbicides that are effective in corn can, if they carry over in the soil, severely stunt the soybeans grown on the same field in a subsequent year. Normally, over the course of the growing season microorganisms in the soil degrade the triazines or other herbicides that are applied to the field. Under some conditions (such as a cold, wet spring), however, there is slow microbial activity. Problems arise in this situation, since the level of the residual herbicide can inhibit the crop to be planted. The genetic engineer can see this circumstance as an opportunity for a contribution. If one could find a way to put triazine herbicide resistance in soybeans, the triazine carryover problem would no longer exist. The result would be of substantial economic benefit to the farmer. Naturally Occurring Herbicide-Resistant Weeds A second problem encountered in the use of herbicides is more recent. It centers around the appearance of new weed biotypes that are resistant

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CROP IMPROVEMENT 57 to certain classes of herbicides. The most dramatic example is triazine herbicide resistance. This was first reported in 1970 but has now been documented for more than 30 species of weeds in various locations in the United States, Canada, Europe, and Israel. The problem is very analogous to insecticide resistance. It is well known that houseflies became resistant to DDT within a few years after this insecticide's introduction. In the case of herbicides, re- sistance to the chemicals has taken many more years to develop. This is largely because of the much longer life cycle of plants compared with that of insects. The new triazine-resistant weeds have frequently appeared in farms where corn has been grown in continual production with atrazine ap- plication year after year. It should be emphasized that the appearance of these weeds is currently more of an inconvenience to the farmer than it is a major problem. Weed control can still be achieved by switching to an alternative (although sometimes more costly) herbicide, since the resistance is specific for the triazine chemicals. The appearance of her- bicide-resistant weeds must be taken as a portent of the future, however. If weeds become resistant to more and more chemicals, our options for weed control via herbicides will become more limited. In the meantime, however, the molecular geneticist can see a silver lining in this cloud- if we can establish how weeds became resistant to triazines, perhaps we can do the same for soybeans or other crops. Research over the last five years has described in molecular detail the mechanism by which atrazine kills weeds. When the herbicide is taken up from the soil by germinating seedlings, it moves up from the roots to the leaves where it enters the chloroplasts. The chloroplast (Figure 3) has a very simple but important function for the biosphere. It harvests sunlight. Chlorophyll is bound to the membranes inside this cellular inclusion. These pigments catalyze con- version of light into chemical energy, which is used to drive the carbon fixation pathways. The leaf takes CO2 from the environment and con- verts it via photosynthesis into carbohydrates, amino acids, lipids, and so on. In this process the chloroplast also removes electrons from water, resulting in oxygen evolution, which is necessary for the respiratory activity of other living creatures in the biosphere. The conversion of radiant energy (sunlight) into chemical interme- diates involves many steps, each of which entails the movement of elec- trons along a chain of electron carriers (proteins with special functional cofactors) that are housed in the chloroplast membranes. When atrazine comes into the chloroplast, it binds to one of these electron carriers and blocks its function. This, of course, shuts off photosynthesis and thereby kills the plant by energy starvation.

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58 NEW FRONTIERS IN BIOTECHNOLOGY FIGURE 3 A chloroplast in the cell of a crop plant as observed in a thin-sectioned sample for electron microscopy. The oval-shaped chloroplast contains internal membranes (ar- rows) that are the sites of photosynthetic electron transport. One type of protein in these membranes binds atrazine and thereby blocks photosynthesis. As a result of mutation in the chloroplast DNA (the DNA molecules are too small to be visualized in this micro- graph), a change in the herbicide-binding protein of some weed species has occurred. This makes the plant herbicide resistant. A research goal is to transfer the altered gene from the weeds to crop plants so the latter will also be herbicide resistant. Molecular Basis of Triazine Herbicide Resistance When atrazine-resistant weeds were discovered, several laboratories began investigating why the weeds did not die when this herbicide was applied. It was found that atrazine entered the new weed biotypes and was metabolized at low rates (unlike in the corn plants). Exclusion or detoxification mechanisms were therefore ruled out as explanations for why resistance occurred. Only when the chloroplast membranes were examined did an explanation become obvious the atrazine could no longer bind to triazine-resistant chloroplast membranes. Obviously, it could not block photosynthesis and was therefore ineffective. To learn why the atrazine did not bind to resistant chloroplasts, sci- entists first identified the protein that contained the herbicide binding

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CROP IMPROVEMENT 59 site. It was then rapidly established that the gene for this protein resided on the chloroplast DNA. This led to the successful isolation of the gene encoding the herbicide-receptor protein. When the pair of genes from susceptible and resistant chloroplasts were analyzed to determine their nucleotide sequence, a single change was observed. This change causes the conversion of a serine in the protein of susceptible chloroplasts to a glycine in the protein of the resistant chloroplasts. We believe that this serine may play a central role in hydrogen bonding to atrazine and that it is thereby essential for the herbicide binding. Genetic Exchange Between Weeds and Crops Since the gene controlling the herbicide target site is altered in tria- zine-resistant weeds, the next question is: How can we make the same change in a crop plant? There is now one major success story along these lines. The work is the result of a novel breeding program conducted by university scientists at Guelph, Ontario. The Guelph scientists had identified a new triazine-resistant biotype of the weed Brassica campestris (commonly called bird's rape). Brassica campestris has a large flower that is very amenable to genetic manipu- lation. It is relatively simple to transfer pollen (the male germ line) from one plant to the female flower parts of another plant. It should be pointed out that the pollen contains only nuclear genes and does not contain chloroplasts or chloroplast DNA. The egg cell of the female flower contains both the nucleus and chloroplasts. The chloroplasts, therefore, follow strict maternal inheritance in almost all crop plants. The Guelph scientists used a novel approach at this point. They took pollen from the flowers of oilseed rape (an important oil crop in Ontario and a close relative of the weed) and fertilized the egg of the weed flower. This means that the progeny of this cross will have half of the genes in their nucleus coming from the crop plant and half from the weed. The novelty of these experiments lies in the fact that all the chloroplasts came from the weed, since it was the female parent. This is a special case where we can document the genetic contribution of a wild relative to a crop plant's physiological properties. The Guelph researchers continued this crossing, always taking pollen from the crop plant, for several generations. Since the genes of the weed were diluted out by 50 percent at each cross, they eventually ended up with a crop plant with cells containing the chloroplasts of the weed (which, of course, determine atrazine resistance). These scientists reg- istered the triazine-resistant seed of oilseed rape in 1980. They have accomplished the same thing with summer turnip rape, another impor- tant crop in Canada. Again the trick was simple. They took pollen from

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60 NEW FRONTIERS IN BIOTECHNOLOGY the crop plant and used it to backcross onto the weed and the subsequent progeny of this cross until there was a nuclear substitution in the weed cytoplasm. In both cases just about all of the genes in the nucleus come from the crop plant, but the chloroplasts from the weed are left, and the result is herbicide resistance. This novel crop-breeding program has opened up a new avenue for weed control in these two important crops. Unfortunately, this approach is not applicable to many other crop genetic systems, since there are no other cases where crops and triazine-resistant weeds are cross-fertile. This means that we must find other approaches, which is where new and novel bioengineering techniques are coming into play. Fusion of Cells from Weeds and Crops It is now routinely possible to isolate single cells from virtually all crop plants. When the cell walls are removed, the cells can be chemically induced to fuse. Current research in several laboratories is involved in fusing cells of Solanum nigrum (a common weed called black nightshade, which has developed herbicide resistance) and isolated cells of tobacco, potato, or tomato (crop plants that belong to the same family as that of Solanum nigrum). The object of these experiments is to donate chlo- roplasts from the weed to the cell line of the crop. In these experiments the nucleus of the weed cells can be inactivated chemically or by using X-irradiation resulting in new hybrid cells that contain the chloroplast of the weed plus the nucleus of the crop plant. There is one unconfirmed report that this strategy has been used to obtain a new tobacco plant that is triazine resistant. I anticipate the same success in the near future with potato and tomato and perhaps some other related crop plants. GENE TRANSFER VIA MOLECULAR TECHNIQUES The strategies for gene transfer outlined above (reciprocal crossing between cross-fertile weed and crop, and cell fusion to deliver weed chloroplast to crop plant cells) are straightforward. Unfortunately, these technologies are not now applicable to many major crops, including legumes (soybeans), cereals (wheat, rice), and other grasses. We must therefore project how these latter commodity crops can be manipulated. The most exciting approach will be to find a mechanism for transferring the gene of choice directly into a crop plant. In the past year great strides have been made in devising technologies for the incorporation of pieces of DNA (genes) into vectors (larger pieces of DNA, derived from a naturally occurring viruslike agent). These vectors can be induced to

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CROP IMPROVEMENT 61 move into plant cells where the DNA they deliver is incorporated into the plant nucleus. Unfortunately, the atrazine-resistance gene described above resides in the chloroplast, so we cannot rely on existing technology to accomplish the gene transfer needed for the triazine-resistance prob- lem. This only means a delay, however, since it seems certain that our understanding of the means of gene transfer into plants is only in its infancy. The triazine-resistance trait should, in fact, provide a tool with which biotechnologists can experiment to directly manipulate this and other genes. CONCLUSION It should be emphasized, in closing, that plant genetic engineering via biotechnology is a new and developing science that is going to sup- plement traditional agriculture. It will not replace standard methodol- ogies for crop improvements, but it will add new facets. Traditional crop breeding will continue to be the mainstay of our agricultural system in production and release of new varieties. What biotechnology will offer is ways to increase the speed at which crop improvement is made, as well as providing tools for introducing novel traits that cannot be achieved with traditional genetics. It will be exciting during the next 20 years to see how new aspects of biotechnology will affect American and inter- national agriculture.