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APPENDIX Gene Transfer Methods Applicable to Agricultural Organisms PhyIlis B. Moses INTRODUCTION The transfer of genes from one organism to another is a nat- ural process that creates variation in biological traits. This fact underlies all attempts to improve agriculturally important species, whether through traditional agricultural breeding or through the techniques of molecular biology. In both cases, human beings manipulate a naturally occurring process to produce varieties of organisms that display desired traits, for example, food animals with a higher proportion of muscle to fat, or disease-resistant corn. The major differences between traditional agricultural breed- ing and molecular biological methods of gene transfer lie neither in aims nor in processes, but rather in speed, precision, reliabil- ity, and scope. When traditional, or classical, breeders cross two sexually reproducing plants or animals, they mix tens of thou- sands of genes in the hope of obtaining progeny with the desired trait or traits. Through the fusion of sperm en c] egg, each parent contributes half of its genome (an organism's entire repertoire of genes) to its offspring, but the composition of that half varies in each parental sex cell and hence in each cross. In addition, because the traits desired usually come from only one parent and may be controlled by one or a few genes, many crosses are necessary before the "right" chance recombination of genes results in expression of the trait in the offspring. Even then, the progeny usually have to be crossed back to the parental variety to ensure stable adoption of 149

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150 AGRICULTURAL BIOTECHNOLOGY the new trait. Sometimes undesired traits derived from one parent of a new, improved variety persist whereas the desired traits are lost. Such are the difficulties and limitations of classical breeding. Molecular biological methods of gene transfer alleviate some of these problems by allowing the process to be manipulated at a more fundamental level. Instead of gambling on recombination of large numbers of genes, scientists can insert individual genes for specific traits directly into an established genome. They can also control the way in which these genes express themselves in the new variety of plant or animal. In short, by homing in on desired traits, molecular gene transfer can shorten the breeding time for new varieties and, in addition, lead to improvements not possible by traditional breeding. Laboratory methods to move individual genes between organ- isms capitalize on naturally occurring mechanisms of gene transfer other than sexual reproduction. These include uptake of DNA by cells and cell-to-cell transfer of packaged genetic material such as viruses. Scientists began by studying these mechanisms in sim- ple systems- bacteria and the viruses that infect them. Research has progressed at a remarkable rate. Now scientists can trans- fer genes into organisms as diverse as soybeans and sheep. Much work remains, however, to perfect gene transfer and its attendant technologies of embryo culture and plant regeneration. Scientists have relied heavily on favorite mode! organisms such as the bacterium Echerischia cold and the fruit fly Drosophila melanogaster, because of their ease of manipulation and the large body of scientific knowledge accumulated about them. Mode! systems are critical to the progress of research. Nevertheless, molecular biologists must extend their techniques to commercially important agricultural organisms. Movement in this direction will not replace all traditional agricultural breeding with molecular gene transfer. It will, however, expand the array of methods available to improve agriculturally important species. General Considerations Gene transfer occurs naturally among bacteria by a variety of mechanisms. Scientists learned in the 1950s and 1960s to exploit these mechanisms to study gene regulation in bacteria and in

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APPENDIX: GENE TRANSFER METHODS 151 the 1970s developed additional artificial gene transfer methods for bacteria. It turned out to be a relatively simple matter to get some types of bacteria to take up pieces of DNA from their surrounding medium. Genes contained on the new pieces of DNA could be stably inherited and expressed to give new characteristics to the host bacteria. Scientists then devised special conditions that improved DNA uptake, maintenance, and gene expression in the new hosts. Gene transfer is now a routine laboratory procedure for bacterial strains such as E. coli. The goals of gene transfer experiments with other organisms are the same as those of earlier work with bacteria to study gene regulation and to obtain stable inheritance and expression of new characteristics. The difference is that these other organisms are more complicated biological entities than are bacteria. Hence the experimental problems and procedures are more complicated. It has proved necessary to devise some very special conditions and tools to move DNA into the cells of other organisms. The explosion of knowledge in molecular biology is the direct result of certain basic biological discoveries that permit scientists to handle genes as macromolecules. Researchers can identify, iso- late, cut, and splice genes and transfer them from one species to another. Enzymes, obtained mainly from bacteria, enable scien- tists to perform the first four steps on genes from any organism, by procedures that are now standard in molecular biology. The fifth step, gene transfer, must be worked out individually for different organisms. Different species of animals, plants, and microbes vary widely in the ease with which gene transfer can currently be carried out. Plants have in general been more difficult to deal with than animals or microbes. The technology is improving rapidly, however, and it is likely that most organisms will in time be tractable targets for gene transfer. This report surveys the scientific status and short-term prog- nosis for gene transfer systems applicable to animals, plants, and microbes of agricultural importance. The development of these methods has hinged on scientists' understanding of underlying molecular mechanisms in organisms. These mechanisms are then exploited, as was originally done for bacteria, to develop methods

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152 AGRICULTURAL BIOTECHNOLOGY for moving genes between organisms. This permits both funda- mental studies of gene function and the endowment of an organism with new, desired characteristics. Many factors must be considered in the design of gene transfer systems. The first requirement is an easily detected "tag" for the gene so that its progress into a new host can be traced. Sometimes the uniqueness of the foreign gene is sufficient: The gene can be identified by the new characteristic it confers or its physical pres- ence can be detected by a probe for its particular DNA sequence. Unfortunately, such direct identification methods are sometimes either impracticable or inconvenient and time consuming. In these cases the foreign gene can be tagged by attaching it to another gene whose presence in the host is easily and rapidly detectable. Genes for drug resistance are often used as tags. Host cells that incorporate these genes-along with the foreign gene can survive a drug treatment, whereas cells that have not taken up the genes will die. Another important consideration is the efficiency of gene . transfer. The probability of success must be high enough for transfer of the gene to be detected with a reasonable frequency. If drug resistance or other selection schemes are used, a lower fre- quency may be acceptable. In such cases many cells can be treated for gene transfer, but only those few that actually incorporate the foreign genes survive the selective treatment and are recovered. Special vectors can improve the efficiency of gene transfer. Foreign genes attached to the vector will be carried by it into the host cell. Vectors are often derived from circular DNA molecules called plasmids, or from viruses. Different transfer systems have particular features that can limit the size of foreign DNA segments that they are capable of transferring. Segment sizes are measured in base pairs, the funda- mental chemical units of DNA. A typical gene may be composed of anywhere from 1,000 to 50,000 base pairs. A few techniques for gene transfer can handle segments of DNA at the upper end of this range, but most current methods are limited to segments at the lower end. Because it may be desirable to transfer more than one gene at a time, scientists are working to develop more and better vectors that can handle multiple genes. When a DNA molecule is used as a vector for foreign genes, its own size sometimes limits how much extra DNA it can carry. Small

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APPENDIX: GENE TRANSFER METHODS 153 vectors, themselves less than 10,000 base pairs, are more often limited than are large vectors, which may be well over 100,000 base pairs. However, larger vectors require extra manipulations to equip them with foreign DNA. The final state of foreign genes inside the host cell is also im- portant. Genes can be maintained on vectors that are independent, self-replicating "minichromosomes," or they can be integrated into the larger chromosomes of the host cell. Depending on the exper- iment's purpose, independent maintenance or integration of new genes may be preferable. However, to ensure stable inheritance of transferred genes in intact animals or plants, the genes must usually be integrated. Related to the state of genes is the question of gene copy number, that is, how many identical copies of a foreign gene end up in the cell. Again, depending on the experiment's purpose, many copies or only one copy may be desired. For example, if gene transfer is used to engineer a cell line to manufacture large amounts of a commercially valuable protein, a high copy number, self-replicating minichromosome would be used. On the other hand, if the purpose is to equip an animal or plant with a new gene for disease resistance, only one or two copies of the gene might be needed in the organism's own chromosomes, where it would be properly expressed and inherited by succeeding generations. Transferred genes must be regulated so that their protein products are made In appropriate amounts at the correct time and in the right place. Genes are normally controlled by certain sequences in the surrounding DNA. These sequences in turn are affected by various factors within cells, for example, hormones. Transferred genes can be regulated by their normal control se- quences. Alternatively, scientists can equip them with new control sequences to either mimic the natural situation or achieve new effects. Before permanent genetic modification of an organism is at- tempted, it is important to study the gene of interest under various conditions to understand its normal function and regulation and to engineer any beneficial changes. These studies are conveniently done using a "transient expression" system, by which the activity of transferred genes can be rapidly measured inside cells, without waiting for stable, long-term genetic modification of the cells.

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154 A GRICULTURAL BIO TECHNOLOGY The flexibility and rapidity of initial studies are also enhanced by "shuttle vectors," which are designed to replicate in both ani- mals or plants and in bacteria. By using shuttle vectors, scientists can easily grow and isolate genes in quantity from bacteria, mod- ify them in vitro, and then quickly transfer them into animal or plant cells to test their function. The transferred genes can also be reisolated from the anneal or plant cells, put back into bacteria, and grown in quantity again for further use. DIRECT DNA UPTAKE The earliest and still most widely used method for introducing DNA into animal cells grown in culture in the laboratory is direct uptake- of DNA from the surrounding culture medium. The con- ditions are in principle the same as those used for bacterial cells: DNA must enter the cell and become stably maintained and inher- ited in the cell line in such a way that its new genetic information is expressed to confer a new trait on the cell. The mechanics differ because animal cells diner structurally from bacterial cells. On the one hand, animal ceils have only a membrane surrounding their contents, whereas bacterial cells (and plant, fungal, and yeast celIs) have both a membrane and a wall. The rigid cell wall of the latter organisms often must be removed to allow DNA to enter the cell. On the other hand, most of the genetic information ~ animal, plant, fungal, and yeast cells is sequestered in the nucleus, an organelle surrounded by its own membrane. (Organisms that have cell nuclei are known as eucaryotes.) New genetic material usually must pass through this second membrane ~ order to be permanently added to a eucaryotic cell. Bacteria (known as procaryotes) lack an organized nucleus and usually accept new DNA more easily. The major advantages of direct DNA uptake (facilitated by chern~cal or electrical treatments, as will be described) are its simplicity and applicability to many organisms and cell types. Hundreds of thousands of cells may be simultaneously treated, in contrast to m~croinjection of DNA into individual ceils (described later), which ~ laborious and time consuming. Because it is so simple and rapid, direct uptake is extremely useful for basic studies of gene expression in cell culture. These studies are important for characterizing a genes function, before researchers attempt

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APPENDIX: GENE TRANSFER METHODS 155 elaborate and time-consuming gene transfer experiments in whole animals or plants. Foreign genes introduced by direct uptake are expressed in their new host cells after a short period, usually 1 or 2 days. Direct DNA uptake thus quickly reveals the function of newly isolated or engineered genes during this period of "transient expression." For long-term studies the genes must integrate into the cellos own chromosomes, or be carried ~ by the uptake of new chromosomes, to ensure that they are stably inherited. Integration occurs at a high frequency after direct DNA uptake into animal cells because so many copies of the foreign genes have been introduced. (Main- tenance on new chromosomes is discussed in the sections on Cell Fusion and Vector-Mediated Gene Transfer.) In addition, gene transfer into cultured cells by direct DNA uptake is user! for the commercial production of genetically en- gineered proteins. Drugs, hormones, food additives, and other valuable substances can be manufactured by cells into which the appropriate genes have been transferred. Human insulm for treat- ment of diabetics is now manufactured in bacteria in this way. The limitations of direct uptake, particularly for animals, cen- ter on the fact that intact organisms usually are not suitable re- cipients. Thus, gene transfer into an an~rnal embryo usually must be accomplished by other means. For plants this is not a strict limitation, as many species can be regenerated Into whole plants from a single cultured cell. Chemical Treatment Chemical treatments can induce animal cells to take up DNA from their medium; most frequently these ceBs are in culture rather than in living animal. In the supplest and most popular method, cells are mixed with DNA that has been precipitated with calcium phosphate (Graham and van der Eb, 1973~. This treatment compacts the DNA, so ceils take up many copies of the foreign genes. Alternatively, the chemical DEA~dextran may be used to facilitate DNA uptake (McCutchan and Pagano, 1968~. Cells in culture are relatively unspecialized and often do not correctly regulate genes as would the specialized organs of an in- tact animal. Researchers have therefore developed a technique to introduce DNA directly into intact organs, such as the liver or

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156 AGRICULTURAL BIOTECHNOLOGY spleen, of living animals. Calcium phosphate-precipitated DNA is injected directly into the organs, in combination with Tow concen- trations of enzymes that allow the DNA to enter (Dubensky et al., 1984~. This technique enables researchers to quickly study an isolated gene's function in the differentiated, specialized cells of an intact organ, which more accurately reflect the gene's proper function in an animal. A variation of the organ transformation technique involves injecting the calcium phosphate-precipitated DNA intraperitoneally, where it is taken up and expressed by ani- mal tissues, such as those of the liver and spleen (Benvenisty and Reshef, 1986~. Plant cells have been difficult to transform by chemical meth- ods, but recently breakthroughs have been made. Polyethylene glyco} has been used to obtain direct uptake and stable mainte- nance of DNA by protoplasts from a species of wheat, Triticum monococcum (Lorz et al., 1985), another monocot grass, Lolium muitifoTum (Potrykus et al., 1985a), and the dicots oilseed rape, tobacco, and petunias (Potrykus et al., 1985b). The frequency of integration of DNA after direct uptake is sometimes lower than for vector-mediated gene transfer into plants (discussed later), but there are no species restrictions on the type of host cell. However, protoplasts are used as recipients, so they must be capable of re- generating into plants for direct uptake to yield genetically altered species for agriculture. Insect, fungal, yeast, and bacterial cells are all amenable to variations of calcium phosphate or other chemical treatments for direct DNA uptake. Often, direct uptake is used to introduce vector DNA molecules containing engineered genes. Direct uptake procedures simply place foreign genes inside the cell; vectors can help to integrate the genes into the cell's chromosomes or stably maintain the genes within the cell on the vector's minichromosome (see the section on Vector-Mediated Gene Transfer). Electroporation A newer method that is being widely adopted is electropora- tion (Neumann et al., 1982; Potter et al., 1984~. Cells are mixed with DNA in solution and subjected to a brief pulse of electri- cal current. It is thought that the current pulse creates transient pores in the cell's membrane that allow DNA to enter efficiently.

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APPENDIX: GENE TRANSFER METHODS 157 Electroporation may work for any type of cell, even those that have resisted DNA uptake by chemical treatments, for example, cells of the immune system. Electroporation can introduce DNA into protoplasts of both major categories of plants dicots (e.g., carrots and tobacco) and rrlonocots (e.g., corn; Fromm et al., 1985~. Electroporation pro- vides a transient gene expression system for plants. As discussed previously, transient expression systems are very useful for pre- liminary characterization of new genes. The lack of such a system for plants had previously held up progress in characterizing plant genes. Electroporation also permits stable integration of genes into plant chromosomes. It has been used successfully to stably transform corn and tobacco cells (Fromm et al., 1986; Schocher et al., 1986; Shillito et al., 1985~. DNA MICROINJECTION DNA can be injected directly into single living cells using very fine glass pipettes (hollow needles). Experimenters use an elabo- rate apparatus consisting of a microscope and delicate microma- nipulators to view the cell, hold it steady, and inject a solution containing DNA. As with chemical or electrical uptake methods, foreign genes can be in the form of isolated molecules or attached to vectors. A disadvantage compared to direct uptake is that rela- tively few individual cells can be injected; however, the frequency of successful incorporation of DNA per injected cell is higher. Animals Microinjection has been very successful for delivering foreign genes into mouse embryos at an early stage of development. Usu- ally DNA is injected directly into a particular structure, the male pronucleus, of a fertilized mouse egg. This is the most receptive structure to the incorporation of foreign DNA. The embryos are subsequently reimplanted into foster mothers for development to term. Foreign genes are incorporated into the developing cells' chromosomes and are often present in every cell of the mature animal. Animals given new genes by this procedure are called "transgenic." Their new genes are usually passed on normally to their progeny. These foreign genes can be expressed, that is, make their protein products, which can confer new characteristics on

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158 A GRICULTURAL BIO TECHNOLOGY the animal. The now classic example is transgenic mice contain- ing foreign genes for growth hormone. Expression of these genes caused the mice to grow to up to twice their normal size (Hammer et al., 1984; PaIm~ter et al., 1983~. Many other animal genes have now been transferred into fer- tilized mouse eggs by m~cro~njection and correctly expressed in the resulting mature mice. These include the chicken transferrin gene expressed in the liver (McKnight et al., 1983~; a mouse im- munogIobuTin gene expressed in the spleen (Brinster et al., 1983~; the rat elastase gene expressed in the pancreas (Swift et al., 1984~; the rat skeletal muscle myosin gene expressed in skeletal muscle (Shari, 1985~; a chimaeric mouse/human ,B-gIobin gene in blood, bone marrow, and spleen (Chada et al., 1985~; and a swine histo- compatibility gene (Frels et al., 1985~. Traits of potential economic value to the farmer that might be transferred by m~croinjection Include increased levels of certain circulating hormones, antibiotic resistance, and ~mmunogIobulins (antiboclies) for "genetic vaccination" against pathogens. As noted previously, the introduction and expression of such genes has been successful in mice. A necessary supporting technology for in vitro microinjection of mammalian embryos is embryo transfer into surrogate mother animals, for in viva development of the embryos to term. Embryos of each livestock species must be handled in a slightly different manner, which must be experimentally determined. Hammer and his collogues (1985) reported the successful pro- duction of transgenic farm animals (rabbits, sheep, and pigs) by microinjection. The same foreign gene for growth hormone used to produce transgenic mice was used for these other species. New techniques were needed to visualize pronuclei for microinjection, because of differences in the fertilized eggs of each species. The m~- croinjected gene was integrated into the chromosomes of all three species, and was expressed in some of the transgenic rabbits and pigs. Scientists have been very successful in m~croinjecting genes into embryos of the laboratory fruit fly Drosophila melanogaster for studies on the molecular biology of this insect. Rubin and Spradling (1982) pioneered this approach with their transposable P-element vector (discussed in the section on Vector-Mediated Gene Transfer). This vector or others similar to it might be

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APPENDIX: GENE TRANSFER METHODS 159 adapted for both beneficial and harmful insects of agronomic im- portance. Researchers routinely m~croinject genes into frog eggs, which are very large and metabolically active cells, for basic studies on gene expression in animals. More recently, m~croinjection was used to transfer DNA into the chromosomes of developing fish eggs (Chourrout et al., 1986; Zhu et al., 1985~. Projects are aimed at basic studies of fish molecular biology and questions of how fish respond to their environment at the molecular level, as well as at aquacultural applications. Both bovine and fish growth hormone genes have been in- jected into fish eggs. It has already been shown that injection of the purified protein hormones augments fish growth (Gill et al., 1985; Sekine et al., 1985~. Transferred genes should be even more effective than purified hormones in promoting fish growth. Re- searchers have injected metaDothione~n genes from both mammals and fish into fish eggs, with the goal of engineering fish resistant to toxic metals. They have Injected "antifreeze" genes obtained from winter flounder (also found in all antarctic fishes) to increase the cold tolerance of commercially valuable fish. Plants Microinjection can be used to deliver genetic material into plant cells. Segments of DNA, whole chromosomes, and even cellular organelles such as chioroplasts, which contain their own DNA molecules, can be micro~jected by methods used for animal cells, although certain physical properties of plant cells complicate the technique. Key elements for protoplast m~croinjection include m~cro- scopic resolution of the cell nucleus, which ~ enhanced by staining with dyes; immobilization of the cell by a holding pipette, embed- ding within agarose, or adhesion to glass surfaces; and efficient cell culture techniques. Researchers can successfully transform up to 14 percent of the cells they m~croinject with DNA (Crossway et al., 1986~. This high frequency might be increased further by using rn~cro~njection ~ conjunction with specially developed vec- tors, derived from the Ti plasmid or plant transposable elements (see sections on these vectors). Because of the high transformation frequency possible with microinjection, a direct selection scheme

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182 A GRI C UL TURA L BI O TECHNOL O G Y reported correct tissue-specific and developmental regulation of a gIobin gene (for the blood protein hemoglobins in transgenic mice (Chada et al., 1985; Magram et al., 1985~. The transferred gIobin gene was turned on for the first time in fetal blood-forming cells, and was expressed in adult mice only in the blood-forming tissues bone marrow and spleen. This pattern mimics normal regulation of the mouse gIobin gene. However, scientists have not yet devised a way to get genes to integrate at a specific site in animal or higher plant cell chro- mosomes, which would help in obtaining correct regulation and stability of introduced genes. Furthermore, with some current techniques genes are frequently inserted into chromosomes as mul- tiple copies, confounding these problems. DNA rearrangements, lethal insertions, male-sterile mutations, and mosaic organisms in which not all cells contain the new gene sometimes result. Moreover, changes In a gene's expression sometimes occur after transmission of the new gene to progeny. Researchers are working toward the ideal of targeted inser- tion of one stable gene copy that will be sexually transmitted and correctly expressed in all progeny. Fundamental studies of DNA recombination in mammalian ceils that may lead to targeted in- tegration are being carried out (Shau! et al., 1985; Thomas and Capecchi, 1986~. Another strategy to obtain correct gene regula- tion, for instance of gIob~n, is to insert a very large chromosomal segment that contains the gene surrounded by its usual neighbor- ing genes. Genes within such a cluster may be correctly regulated by complex sequences in the surrounding DNA. Currently, very large segments of DNA are difficult to handle and require special vectors to accomodate them. In most cases, researchers wish to keep inserted genes silent during early embryogenesis, and then activate them at the ap- propriate time in the organism's development. However, inserted genes are not always controlled correctly, even when their own regulatory sequences are still attached to them. For example, an inserted growth hormone gene controlled by its own promoter was not regulated correctly in transgenic mice, causing the female mice to be sterile. In contrast, when the same growth hormone gene was attached to the promoter from another gene, metallothionein, it could be turned on or off by raising or Towering the amount of trace metals in the transgenic animals' diets. Researchers could

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APPENDIX: GENE TRANSFER METHODS 183 thus control both the amount of growth hormone made by trans- genic animals and the time at which it was made. Transfer of genes into cultured plant cells, from which trans- genic plants will be regenerated, presents an additional problem- the traits targeted for the mature plants may not be expressed in cultured cells and conversely, successful expression in cultured cells may not carry over to plants regenerated from these cells. For ex- ample, a salt-tolerant cell in culture may not yield a salt-tolerant plant when regenerated. The problem of selecting traits in cell culture extends to all plant gene transfer techniques performed on cultured cells, including direct DNA uptake, DNA microinjection, cell fusion, and vector-mediated methods. Scientists are therefore working on adapting existing systems to deliver genes into pollen grains, seeds, and seedlings, which can develop normally into ma- ture plants. This strategy has the additional acivant age of obvi- ating the need for plant cell culture and regeneration techniques for each individual species, which have been stumbling blocks for gene transfer into some agronomically important plants, notably the monocots corn and wheat, with recent progress being made for rice. Development of vectors for these species has also lagged behind, although adaptation of the Ti plasrn~d used for dicots, or transposons from monocots, may prove feasible. Alternatively, direct DNA uptake or rn~croinjection of pollen or embryos might be used. The problems of uptake and subsequent localization of DNA still impede research with some organisms, although these prob- lems are being overcome, for example, in the pathogenic fungi. These problems extend also to compartments of eucaryotic cells other than the nucleus that contain their own DNA mitochondria (the cell's energy powerhouses) and chIoroplasts of green plants (which harness the energy of sunlight through the process of pho- tosynthesis). For instance, the chIoroplast's DNA encodes proteins essential to photosynthesis, and often related to these, proteins involved In herbicide resistance. An important goal yet to be achieved is the directed transport of new DNA into the plant chIoroplast, although there is some experimental evidence to sug- gest that the Ti plasmas might be used (de Block et ale, 1985~. Other possible methods include microinjection of DNA directly into chIoroplasts and introduction of new genes on plasmas that would be stably maintained in chIoroplasts.

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184 AGRICULTURAL BIOTECHNOLOGY Additional basic arid applied research is needed to extend existing gene transfer systems to agriculturally important organ- isrns. Important practical detmIs cannot always be extrapolated from well-studied laboratory models. Furthermore, scientists still lack basic biochemical and genetic knowledge about many agri- culturally important species. This knowledge base is necessary to support more applied goals. Gene transfer systems require a supply of agriculturally useful genes, if such systems are to benefit the farming community and other segments of society. Scientists must devise ways to find and isolate genes of agricultural interest. This can often be facilitated by the very gene transfer methods that will later be used to move the genes so identified into new hosts. Scientists must also devise methods to measure the presence of genes that are not easily detected immediately after transfer. It should also be noted that current methods are applicable only to dominant or c>dominant genes, since transfer of a recessive gene cannot change a trait within an organism unless the normal, dominant gene can be inactivated. In summary, a variety of gene transfer methods is needed to accomplish diverse goals, which include fundamental studies of gene regulation, isolation of genes whose function and location are unknown, production of ~rc~t~in.~ in loran r,,,~nit.i"e arch ;._ tion of new traits. _ _ ¢ ~ ~^ ^_~ lo_ u ~ Ivy ~ at Ally V-~- REFERENCES Abdullah, R., E. C. Cocking, and J. A. Thompson. 1986. Efficient plant regeneration from rice protoplast~ through somatic embryogen- esis. Bio/Technology 4:1087-1090. Abel, P. P., R. S. Nelson, B. De, N. Hoffmann, 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. Ahlquist, P., R. French, M. Janda, and L. S. Loesch-Fries. 1984. Multi- component RNA plant virus infection derived from cloned viral cDNA. Proc. Natl. Acad. Sci. USA 81:7066-7070. Ahlquist, P., and M. Janda. 1984. cDNA cloning and in vitro transcription of the complete brome mosaic virus genome. Mol. Cell. Biol. 4:2876-2882. An, G., B. D. Watson, S. Stachel, M. P. Gordon, and E. W. Nester. 1985. New cloning vehicles for transformation of higher plants. EMBO J. 4:277-284. Anderson, W. F. 1984. Prospects for human gene therapy. Science 226:401- 409.

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