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THE IMPACT OF BIOTECHNOLOGY ON FOOD PRODUCTION Ernest G. Jaworski The application of recombinant DNA techniques to biological organisms, systems, and processes constitutes an exciting new biology that is being used to increase agricultural productivity and to improve the health of humans and animals. These advances coupled with those resulting from more traditional genetic and chemical approaches are having and will continue to have an enormous impact on the production of food throughout the world. These applications could each be described in some depth, but this would require more pages than are available in this volume. Therefore, this paper focuses mainly on the most recent advances in the transformation of plants, even though the creation of transgenic animals is actively being explored at the fundamental and applied levels. Advances in plant cell and tissue culture have made it possible in some cases to insert genetic information into the chromosome of an organism and then to regenerate whole plants from single cell cultures. Such techniques have been used to produce protoplasts, i.e., plant cells without a cell wall, which are useful in transformation. This significant development, coupled with the ability to clone pieces of functional DNA using bacterial systems and restriction endonuclease-generated DNA fragments, has 9

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provided the basic tools for the creation of transgenic plants. The key ingredient in the most recent advances in plant transformation resulted from the use of a naturally occurring bacterium (A~robacterium tumefaciens) that has the capacity to insert its DNA stably into the chromosome of plant cells (Fraley et al., 1986~. This system can be used in conjunction with plant cell culture to successfully produce whole plants containing foreign gene inserts. CELL TRANSFORMATION SYSTEM A~robacterium tumefaciens contains a large Ti, or tumor-inducing, plasmid that in its wild form is capable of creating crown gall tumors in plants. The Ti plasmid can transfer a small portion of its DNA (T-DNA) and stably insert it into the nuclear DNA of the transformed cell. Since the T-DNA contains genetic information responsible for the synthesis of plant hormones as well as novel metabolites called opines, its transfer and insertion creates the crown gall tumor when these genes are expressed. Although the mechanisms by which these transfer and insertion processes take place are not well understood, it has been possible to take advantage of A~robacterium's properties to genetically engineer the Ti plasmid into a useful transforming vector. Intermediate vectors containing selectable antibiotic resistance markers for the introduction of foreign genes into the Ti plasmid have been constructed and the tumor-inducing properties of A~robacterium deleted by removal of the plant hormone genes. The neomycin phosphotransferase (NPT) coding sequences from a bacterial transposon (Tn5) were joined to the 5' and 3' regulatory sequences of nopaline synthase--a gene derived from the Ti plasmid, which is known to be constitutively expressed in plants (Fraley et al., 1986~. This chimeric gene confers resistance to kanamycin, an aminoglycoside antibiotic that is lethal to plant cells. Thus this chimeric gene construct provided a selectable marker for the transformation vector. Direct cloning approaches using the Ti plasmids were not practical. It was therefore necessary to create intermediate or shuttle vectors either to integrate with 10

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a resident Ti plasmid by recombination or to replicate independently of the Ti plasmid as transvectors. The integrated vector was then used for the transfer of a number of foreign genes into A~robacterium cells. The characteristics of these plasmids include a segment of the pBR322 DNA for replication in Escherichia colt, a portion of a Ti plasmid (pTiT37) containing the functional nopaline synthase gene for ease in scoring transformed plant cells, a streptomycin/spectinomycin resistance determinant from Tn7 for selection in A~robacterium, a portion of DNA from another Ti plasmid (pTiA6) to provide homology for recombination with a resident octopine-type plasmid in A. tumefaciens, a synthetic multilinker containing unique sites for gene insertion, and the chimeric kanamycin resistance gene (NOS/NPT II/NOS). These plasmids and derivatives were introduced into A. tumefaciens by conjugation procedures and homologous recombination between the plasmid and the wild-type octopine Ti plasmid to produce cointegrates (Fraley et al., 1986~. Although this system was useful for the study of gene expression and inheritance of traits, it was not sufficiently efficient for routine production of transformed plants. A subsequent series of derivatives led to the formation of a variant and selectable T-DNA system, which was highly efficient in its transformation frequency (Fraley et al., 1986~. PLANT TRANSFORMATION SYSTEM Initially, an In vitro transformation was developed by incubating plant protoplasts directly in A. tumefaciens cell suspension (Fraley et al., 1986~. Protoplasts were prepared from a variety of leaf tissues by conventional enzyme digestion. The bacteria attached to the proto- plasts during cell wall regeneration and subsequently transferred the T-DNA into the plant cells by an unknown mechanism. Such cells were easily identified within 3 weeks by selection for kanamycin resistance. Since the protoplast system had a number of techni- cal drawbacks, an improved alternative procedure was developed to obviate problems in the isolation and regeneration of protoplasts. The modification involved cutting disks from leaves and infecting them with A~ro- bacterium. These disks were then placed on nutrient

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agar. Subsequently, callus formation was observed around the circumference of the disks. Within 3 to 4 weeks, plant regeneration occurred under appropriate conditions. Stable maintenance and expression of foreign genes (kanamycin resistance) were demonstrated in cells and plants derived from either the protoplast cocultivation or leaf disk systems. Subsequent progeny seed derived from the transformed plants inherited the kanamycin resistance in a simple Mendelian manner. GENE EXPRESSION The development of a transformation system for the stable and heritable introduction and expression of a foreign gene provided a tool for the analysis of gene expression in general. The first study in this effort involved light-regulated, tissue-specific gene coding for the small subunit of ribulose-1,5-bisphosphatecarboxylase (RuBPss) from peas (Fraley et al., 1986~. The investi- gators demonstrated that the genomic clone for pea RuBPss could be introduced into petunia cells by cocultivation with _. tumefaciens. Molecular analyses of transformed cells revealed that the small subunit gene of the pea was indeed expressed in the petunia under the control of its own promoter and was regulated by light in a manner identical to that seen for the endogenous gene in peas. The pea RuBPss retained its tissue-specific pattern of expression in leaves derived from regenerated transformed petunia plants. Subsequent studies using In vivo radio- labeling, followed by immunoprecipitation of ribulose-l, 5-bisphosphatecarboxylase, demonstrated that the heter- ologous RuBPss protein of the peas could be separated from the endogenous petunia RuBPss, thereby indicating that the pea RuBPss protein was correctly processed In vivo by petunia chloroplast (Fraley et al., 1986~. In vivo pea RuBPss was also recovered from the holoenzyme, which was immunoselected with the petunia antilarge subunit antibody, indicating that the small subunit of the pea could form a hybrid holoenzyme assembly with large subunits of the petunia. Two mammalian genes were also demonstrated to be expressed in plant cells. A cDNA clone encoding a-human chorionogonadotropin (a-hCG) under the 12

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control of the cauliflower mosaic virus 35s promoter and a mouse cDNA clone encoding a methotrexate-insensitive dihydrofolate reductase (DHFR) gene also under the control of the cauliflower mosaic 35s promoter expressed their gene products in transformed petunia cells. The results with ~x-hCG and the mouse DHFR indicate the broad utility of the A~robacterium system for the study of gene expression and regulation. Finally, the transfer of a legume storage protein gene into the petunia resulted in the tissue-specific accumu- lation of the storage protein in the seeds of the trans- formed plants. A cDNA clone encoding the soybean 7s c''-conglycinin protein was engineered into an appropriate A~robacterium plasmid and transferred into petunia cells (Fraley et al., 1986~. Analysis of sub- sequent petunia seeds indicated that there was regulated expression of the soybean storage protein gene in the petunia seed. This exciting model system for storage protein expression should permit the determination of the structural sequences required for protein translocation, glycosylation, and processing as well as the study of the regulatory sequences essential for seed-specific expres- sion. TECHNOLOGY APPLICATIONS Plants In the past 3 years, the gene transfer systems described above have led to important new insights into gene regulation and protein transport in plants. The basic applications of this technology should provide a means for developing deeper understandings of the speci- fic promoter/enhancer DNA sequences involved in gene expression and fundamental information on cats and bans gene regulation. Of specific interest, however, has been the recent demonstration of agronomically significant transforma- tions involving the generation of herbicide-tolerant, insect-resistant, and viral disease-resistant plants. The herbicide N-(phosphonomethyl~glycine, or glyphosate, is the active ingredient in Roundups . It inhibits the aromatic biosynthetic pathway at its sixth step; namely, 13

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enolpyruvylshikimate-3-phosphate (EPSP) synthase (Fraley et al., 1986~. This pathway is involved in the biosyn- thesis of phenylalanine, tyrosine, and tryptophan, and when the pathway is inhibited at the EPSP synthase level, the formation of these essential amino acids ceases. Early studies indicated that overproduction of a bac- terial EPSP synthase in bacteria results in herbicide tolerance (Fraley et al., 1986~. Subsequent efforts led to the development of a chimeric gene construct consist- ing of a petunia EPSP synthase cDNA flanked by the cauliflower mosaic 35s 57 promoter and the nopaline syn- thase, 3' regulatory regions. Transformation of petunia cells with this construct resulted in herbicide resis- tance and the overproduction of EPSP synthase 30- to 60-fold. Plants regenerated from these cell lines were found to be tolerant to Roundup ~ when sprayed at concentrations of 0.9 kg/hectare. Control plants were killed when sprayed with the herbicide at 0.22 kg/hec- tare. Similar strategies are currently being used to create plants tolerant to a number of other herbicides such as atrazine, the imidazolidinone series, sulfonyl- ureas, and phosphonotricine. Within the past few years, numerous crop plants, including canola, tomato, potato, tobacco, lettuce, sugar beets, and poplar, have become amenable to the transfor- mation technologies involving A~robacterium. On the basis of these rapid advances, it can be expected that practical demonstrations of the system are forthcoming. Within the past year, field tests were initiated with genetically engineered tobacco that is resistant to atrazine. The creation of a transformation vector for the production of viral disease resistance in plants was recently reported by the R. Beachy Group at Washington University (Powell-Abel et al., 1986~. The transfor- mation system described above was used to insert and express the tobacco mosaic virus (TMV) coat protein gene in both tobacco and tomato plants. The coat protein gene was engineered into a plasmid similar to the one described for herbicide tolerance. Both tobacco and tomato plants transformed with the coat protein gene were found either to be resistant to TMV infection or to demonstrate significant delays (weeks) in symptomology 14

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following infection. Control plants always demonstrated severe symptoms within days following inoculation with the virus. This is one of the first demonstrations of the genetic engineering of disease resistance in plants. Studies by AgroCetus have recently been carried into the field assessment stage with tobacco genetically engi- neered to be tolerant of soil-borne A~robacterium infections. Finally, a Belgian company, Plant Genetic Sciences, has recently demonstrated that the Bacillus thurin~iensis (B.t.) toxin gene could be inserted into the Ti plasmid system anti expressed in tobacco at sufficient concen- trations to protect the tobacco plants from attack by the tobacco hornworm. This was the first demonstration of engineering to protect a plant against insect damage by using biotechnology. These results demonstrate that single gene traits can be successfully introduced into plants for expression and that they can function at potentially economic levels. On the basis of these findings, it can be anticipated that useful single gene traits will also be inserted into plants in combination with other functionally important single gene traits. Furthermore, the stage has now been set for the examination of other genetic traits. MICROORGANISMS Transformations of microorganisms that colonize plants are also expected to be useful in enhancing the produc- tivity of plants. For example, transformed Pseudomonas florescens, which is a natural colonizer of roots in such major crops as corn and soybeans, has been engineered to carry and express the Bacillus thurin~iensis toxin gene mentioned earlier. Greenhouse tests have indicated that such microorganisms are capable of protecting the root systems of corn plants from attack by certain soil-borne insects. Similar advances can be expected in the development of both root-colonizing and leaf-colonizing organisms, which will protect plants from diseases, pests, and environmental stresses. Laboratory and greenhouse studies certainly promise that such organisms will effectively aid in plant productivity. Much further work will be needed to develop performance character- istics for these microorganisms under normal field 15

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conditions and soil types, and a special need exists to generate more basic data on the ecology of these microbes. In this regard, the E. cold lacZY genes (coding for p;-galactosidase and lactose permease) have been engineered into Pseudomonas florescens to create a well-marked microorganism for microbial ecology research. The microorganism contains four marker char- acteristics that make it possible to isolate it from soil samples and detect it at levels of one bacterium per gram of soil. The characteristics of the engineered pseudo- monad include its fluorescent properties, natural rifampicin resistance, ability to grow on a simple lactose media, and detection by the X-gal chromogenic dye. Thus this microorganism provides a very sensitive, selectable tracking system that should be extremely useful in ecological studies under natural environmental conditions. FUTURE NEEDS AND EXPECTATIONS The applications of genetic engineering and modern molecular biology have provided us with the ability to insert novel genetic traits into plants and into microorganisms that interact with plants. It can be predicted that this technology will have an impact on the production of food and on the efficiency of crop production throughout the world, since weeds, insects, and diseases create enormous losses in food production. Advances in the more qualitative traits in our food supply can also be anticipated and are in fact receiving attention at present. For example, extensive efforts are being devoted to the development of higher levels of solids in tomatoes by enhancing the concentration of natural polymers normally present in tomatoes. Protein engineering may well see its first application in simple amino acid codon shifts in seed storage proteins to enhance their content of lysine (e.g., in corn) or methionine (e.g., in soybeans). Gene engineering techniques may also be useful in improving the yield ot protein levels in a variety of crops, especially forage crops. Site-directed mutagenesis may be applied to the creation of more stable proteins for storage purposes. The loss of food products and gains in storage may be reduced by subsequent genetic engineering of the crops to create disease- and insect-resistant products. We now 16

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have the technologies and tools to approach some of these important food supply issues, but we should consider some of the future needs that could help advance progress more rapidly. Technical hurdles that still remain include cell culture procedures to broaden the base of crop species that can be regenerated, especially from protoplasts. Interestingly, a recent announcement by Japanese and French scientists indicates that it is now possible to regenerate rice plants from protoplast cultures. New transformation systems are needed, especially for monocot transformation. Other methods for the transfer of DNA into plant cells are being investigated, and there are indications that methods such as electroporation, microinjection, laser treatment, and the use of gemini virus vectors will soon have an impact on this field. Gene structure and function will continue to receive attention, especially with reference to the regulatory sequences affecting gene expression at the developmental and tissue-specific level. Among the most practical needs for the modification of our food supply will be the identification of additional agronomically important genes that can be used to create stress- and pest- tolerant plants. At present, the lack of knowledge about the basic biochemistry of plant systems remains one of the major limiting factors in the advancement and exploitation of the technology described. This knowledge is vital for our understanding of the genetic components involved in achieving such traits as frost tolerance, heat tolerance, drought tolerance, metal tolerance, disease resistance, and insect tolerance. Incentives have been provided to improve this situation, but additional resources must be directed toward improving plant tissue culture and regeneration; novel transformation systems, understanding of gene structure organization and function; the selec- tion, isolation, and characterization of agronomically important genes; and the development of unique plant- breeding techniques. Only then will the sociological and economic impacts of these exciting technologies and tools be fully realized. 17

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REFERENCES Fraley, R.T., S.G. Rogers, and R.B. Horsch. 1986. Genetic transformation in higher plants. CRC Crit. Rev. Plant Sci. 4~1~: 1-46. Powell-Abel, P., R.S. Nelson, B. De, N. Hoffman, S.G. Rogers, R.T. Fraley, and R.N. Beachy. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743. 18