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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Suggested Citation:"Gene Transfer." National Research Council. 1984. Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns. Washington, DC: The National Academies Press. doi: 10.17226/10.
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Gene Transfer The Background Most of the current excitement in agricultural research focuses on gene-splicing or recombinant DNA technology. Lawrence Bogoract de- scribed the development of this technology, its promise, and limitations. This technique had its impetus in research in the 1940s and 1950s on the molecular structure and function of genes. In the 1940s, Oswald Avery, Colin MacLeod, and Maclyn McCarty presented evidence that genes were made of deoxyribonucleic acid, or DNA. DNA is a molecule consisting of sugar, phosphate, and four bases: adenine, guanine, thy- mine, and cytosine (A, G. T. and C). At that time, no one could fathom how such a simple molecule could contain and transmit hereditary in- formation. Watson and Crick provided the answer in 1953. They described DNA as a two-stranded molecule, coiled in the now-famous double helix. The backbone of the molecule is a string of sugar and phosphate. A nucleo- tide base either an A, G. T. or C—sticks out from each of the sugars. The two strands are held together by weak bonds between these bases; A binds with T. and G binds with C. Thus, each strand is complementary to the other. The elucidation of that structure revealed how DNA passes on in- structions from one generation to the next. Prior to cell division, the two strands unwind. Each strand then serves as a template for the faithful replication of another DNA molecule, which is then passed on to progeny. DNA contains hereditary information but the major question is how is that information processed and turned into a trait? The answer lies in the "central dogma" of molecular biology- that is, information con- 15

16 GENETIC ENGINEERING OF PLANTS Old ~A. {= T ~ / .A~ Old New ,/ ~T A~ New Old Replication of DNA. The double-stranded DNA helix unwinds and each strand serves as a template for the building of a complementary strand. The resulting daughter DNA molecules are exact copies of the parent, with each double helix having one of the parent strands. From U.S. Congress, Office of Technology Assessment, Impacts of Applied Genetics: Micro-organisms, Plants, and Animals, U.S. Government Printing Office, Wash- ington, D.C., 1981. tained in DNA is copied into molecules known as ribonucleic acid, or RNA, and RNA specifies the synthesis of all proteins. Thus, DNA carries the instructions for proteins both for the structural proteins, such as those in the framework of membranes, and for the enzymes, which catalyze all the metabolic reactions of an organism. Proteins are chainlike molecules composed of a sequence of amino

GENE TRANSFER 17 acids. That is where the genetic code comes in: a series of three nu- cleotide bases in DNA codes for each amino acid in a protein. The sequence TAC, for instance, cocles for the amino acid methionine; the sequence TAT codes for the amino acid isoleucine. A single change in a nucleotide means that one amino acid in the protein is replaced by another. "That's a mutation in the gene and an alteration in the gene product," Bogorad explained. "It came as a surprise 20 years ago when it was discovered that a single amino acid change in a protein could greatly affect the way it worked." TRANSLATION Messenger R N A Cytoplasm The "central dogma." DNA carries the instructions for the synthesis of proteins. A series of three nucleotide bases in the DNA molecule code for a specific amino acid, the building blocks of protein. Each gene, a relatively short segment of a long DNA molecule, codes for a single protein. The genetic information is expressed in a two-step process, described by the "central dogma" of molecular genetics: DNA is transcribed to RNA, then RNA is translated to protein. During transcription, a strand of DNA serves as a template for the formation of a complementary strand of messenger RNA. Next, the messenger RNA moves from the cell nucleus to the cytoplasm. There ribosomes attach to the messenger RNA and direct protein synthesis by reading the genetic code and building a chain of amino acids.

18 GENETIC ENGINEERING OF PLANTS The conversion of a gene to RNA and then to a protein product is called expression. If a gene is present and its protein product appears, the gene is said to be "on." If the gene is present but no product appears, it is said to be "off." Gene expression is a two-step process: first, the DNA is transcribed to RNA; then the RNA is translated to protein. Transcription is similar to DNA replication. The DNA molecule un- winds, but in this case the strands serve as a template for the formation of an RNA molecule. RNA contains three of the same nucleotide bases as does DNA A, G. and C. But in place of thymine, RNA has uracil, U. Thus, during transcription, G binds with C, and now A bincTs with U. Translation occurs when the RNA molecule, known as messenger RNA, leaves the nucleus and travels to the ribosomes, the site of protein synthesis in the cytoplasm. Here the RNA specifies the sequence of amino acids in a protein according to the triplet codes mentioned earlier. The Technique In theory, gene-splicing is relatively straightforward. In practice, it is far from routine. Bogorad outlined the basic procedure. The first step is to locate the desired gene among the 5 million or so in the cell nucleus. Each gene has three regions, all essential for successful functioning. The beginning is the promoter region, the series of nucleotides that is rec- ognized by the enzyme that triggers the transcription of DNA to RNA. The middle sequence of nucleotides contains the cocle, the instructions for producing a specific protein. The end series of nucIeotides, or ter- minator, is a signal to stop the transcription process. Next, the gene must be isolated from the others on the chromosome. For this task, the genetic engineer uses a restriction enzyme. These enzymes recognize specific nucleotide sequences and cut the DNA at precisely those points. It is these enzymes that allow researchers to snip a gene out of the DNA sequence from one organism and splice it into the DNA of another. In fact, the advent of recombinant DNA technology can be traced to the discovery of these restriction enzymes in the early 1970s. Now, a decade later, biological suppliers offer hundreds of re- striction enzymes for sale, each one recognizing a different sequence of nucleotides. Once the gene is isolated, it must be cloned, or duplicated, and in- serted into the host cell. Both steps are accomplished by inserting the gene into a plasmid. A plasmid is a tiny, circular piece of bacterial DNA. Plasmicis reside as separate units of DNA inside the cytoplasm of a bacterial cell. With the same restriction enzyme that was used to excise the gene from the donor cell, the genetic engineer cuts open the plasmid.

GENE TRANSFER LEA ~T...A~// /~ A...T~7 C .,/ 19 Eco Rl Hpa 1 —C G— —T. A— —T. A— —A, T— —A, T— ~ A —G C— —G C— —T A— T— —C G— Restriction enzymes. Restriction endonucleoses are enzymes that cleave DNA at specific sites. The illustration shows examples of cleavages made by two such enzymes, Eco RI and Hpa 1. Eco RI recognizes the DNA sequence CLANG and cleaves each strand between the G and A yielding single strand ends. Such "sticky ends" can readily join on to other DNA fragments created by the same enzyme. Hpa 1 does not leave "sticky ends"; it recognizes the sequence CGATT^=c and cleaves each strand between the A and T. Restriction enzymes can be used to isolate single genes.

20 GENETIC ENGINEERING OF PLANTS This leaves the plasmid with two "sticky" ends, which will now accept the foreign gene. After the foreign gene is inserted, another enzyme, called a ligase, is used to sew the plasmid together. Plasmids are ideal vectors for carrying the new gene into a host cell, because, in nature, plasmids are routinely passed from one bacterium to another, where they are readily accepted. When the recombinant molecule part bac- terial plasmid, part plant gene—is taken up by the bacterial cell, that cell is said to be transformed. As the plasmid replicates inside the host cell, it copies the foreign gene along with its standard gene allotment. The goal is not just to clone the gene, but to have that gene ex- pressed—to have the DNA transcribed to RNA, and the RNA translated into the desired protein in the host cell. Most work to date has in- volved the insertion of a gene from a higher organism—usually an animal—into a bacterial host, where the animal gene produces proteins such as insulin, interferon, and human growth hormone. The genes of higher organisms (eukaryotes) have different control signals—signals that turn genes on and off than do the genes of primitive organisms (prokaryotes) like bacteria, which lack a nucleus. These signals must be read correctly for the gene to be expressed. Gene expression has been achieved by removing the specific control signals from genes of higher organisms, in essence tricking the bacterium into accepting the foreign gene as a bacterial gene. Up to this point, the techniques for plant genetic engineering are similar to those used to design bacteria to produce insulin or other pharmaceuticals. In short, a gene is isolated, spliced into a vector, in- serted into a host cell, and expressed. Pharmaceutical applications de- pend upon a method of culturing large batches of these recombinant bacteria. By contrast, plant genetic engineering depends upon a means of regenerating a whole plant from the cells in culture. There are three tissue-culture techniques for regenerating plants from culture (see Cell Culture, p. 34~. For gene-transfer experiments, the pre- ferred route is the culture of protoplasts single cells from which the cellulose wall has been removed. That is because it is easier to insert genes into protoplasts than into cells containing the tough outer wall, which animal cells lack. Thus, before a foreign gene is introduced into a plant cell, that cell is treated with enzymes that dissolve the outer wall. The protoplast, containing its new gene, is then placed in a broth of plant hormones and nutrients that induce it to regenerate. It first re-forms a cell wall. By changing the nutrient mixture, the cells can be induced to multiply and form embryolike structures. Known as somatic embryos, these give rise to tiny plants, which then can be transferred to the soil.

GENE TRANSFER Current Constraints 21 Though major strides have been made in the past few years, only the barest beginnings have been made in the transfer of genes among higher plants. As Bogorad explained, the major limitation is the lack of knowI- edge about basic plant biology necessary to exploit this new technology. Each step of the process presents its own difficulties. For instance, just finding the desired gene is a monumental task—"one of the most difficult and challenging operations in molecular biology," Bogorad said. The plant genome is large and exceedingly complex. Some genes are located on chromosomes in the cell nucleus. Others are contained in two organelles the chIoroplasts and mitrochondria. Similarly, there are difficulties in identifying all the important parts of the gene, including any DNA sequences necessary to regulate expression of the gene; in developing an appropriate vector to carry the foreign gene into the plant cell; and, finally, in regenerating plants from the transformed cells in culture. Vectors One of the major challenges is the development of vectors to ferry foreign DNA into the plant genome. Only a few bacterial plasmids will work in plants. One of these is the Ti plasmid from the soil-borne bacterium Agrobacterium tumefaciens. It is the most promising vector to date for plant genetic engineering. Agrobacterium causes crown gall dis- ease: it infects the plant stem tissues, inducing tumors. The disease- causin~ agent is the bacterium's Ti (for tomor-ind~rin~N nln~mi~ Thin . . . . . . . . . . ~ plasmas does its damage by inserting itself into the plant cell's genome, where it is replicated and expressed along with the plant's DNA. The expression of the bacterial Ti plasmid genes causes the abnormal cell growth characteristic of crown gall disease. Actually, only a small piece of the Ti plasmid is inserted into the plant genome this piece is called T DNA (for transferred DNA). The Ti plasmid is a natural vector that routinely inserts new DNA into plant cells. Moreover, it comes equipped with a trait molecular biologists were seeking: its genes can be expressed in the environment of the plant genome; the regulatory signals of the bacterial genes can be read by the plant cell. Several scientists reasoned that the Ti plasmid could be tricked into carrying additional genes into the plant genome as well. For the past several years, there has been an intensive research effort to develop the Ti plasmid as a genetic engineering vector. Much of it

22 GENETIC ENGINEERING OF PLANTS Bacterial ~' e T P ~ ~ If Agrobacter/um tumefac/ens T-D NA Jon Chromosomes ii7 Transformed Plant Cell The Ti plasmid vector. In plant genetic engineering, the Ti plasmid can be used to carry foreign genes into plant cells. The Ti plasmid is the disease-causing agent of the soil- borne bacteria Agrobacterium tumefaciens. When the bacteria infect a plant, a part of the Ti plasmid called the T DNA is transferred to a plant chromosome. When the T DNA is expressed as part of that chromosome, it causes the plant cell to divide and grow abnormally. Researchers have recently developed procedures for removing the tumor- causing genes from the T DNA and replacing them with desirable genes. The Ti plasmid containing the altered T TUNA region can then he llCt°~ to insert the H~cirPH ~PnPc into plant chromosomes. ~ _ _ _ _ _ ~ ~ ~ ~ ~ ~ _ ~ ~ ~ ~ ~ _ _ _ v ^^ _ ~ ~t~ _ ~ ~ ~ v ~ has been performed by two research groups: one led by Mary Dell Chilton at Washington University and the other a European group led by Jeff Schell of the Max Planck Plant Breeding Institute in Cologne, Germany, and Mark Van Montagu of State University in Ghent, Bel- gium. They have addressed a number of questions, such as how to insert new genes into the T DNA region without disrupting the se- quences that control its insertion into the plant genome and how to remove the disease-causing part of the plasmid so that the vector could be used in practical as well as experimental gene transfer. Their efforts have paid off. In January 1983 the European researchers and another group at Monsanto Co. announced that they had used a Ti plasmid to carry a functioning bacterial gene into a plant cell. This was the first demonstration that a foreign gene could be inserted into

GENE TRANSFER r - - -- - -A 23 a plant cell and be expressed. The Monsanto group included Robert Horsch, Stephen G. Rogers, and Robert T. Fraley. Both teams, working indenendentlv, inserted a bacterial gene for antibiotic resistance into the T DNA portion of a Ti plasmid. The Ti plasmid was then used to transform petunia cells in culture. The foreign gene was expressed: the cells in culture were resistant to the antibiotic. When a plant was re- generated from these cells, it retained the antibiotic resistance. Commenting on the widely heralded gene transfer, Robert M. Good- man of Calgene, Inc., interjected a note of caution. "Notwithstanding the excitement generated by the recent demonstration that the expected is possible, we are only at the beginning of a long period of research and development" on vectors. At this stage, biologists do not under- stand how the Ti plasmid works specifically, they clo not understand the signals that control the insertion anct expression of T DNA. Even without that knowledge, the Ti plasmid can be effectively used as a vector, as shown by the recent petunia experiment. But Goodman cautioned against becoming too intent on applications, on getting "too carried away with the short-term excitement that we overlook the need to invest in work that leads to an understanding of the underlying principles . . . that will allow the design of sophisticated genetic engi- neering vectors." For example, he said, "we must understand how the T DNA inserts if we ever hope to control the location and perhaps the multiplicity of the insertion." According to Goodman and others, adclitional work is necessary to improve the efficiency of Ti plasmid as a vector. In most work to date, only a small percentage of plant cells inoculated with Agrobacterium carrying the Ti plasmid are transformed. if the Ti plasmid is to be used in a practical gene-transfer system, then much higher rates of transfor- mation must be achieved. Such research has already begun. Other vectors will also be necessary. A major limitation of the Ti plasmicl is that it works only in those plants that the Agrobacterium normally infects. It does not infect plants in the grass family, which include the important cereal crops like corn, rice, and wheat. Conse- quently, there is now no vector available for their genetic engineering. It may be possible to modify the Ti plasmid so that it can infect grasses, yet other vectors having different host ranges should be explored. Another limitation is that the Ti plasmid can be used only to ferry genes into the nuclear DNA. Several agronomically useful traits, how- ever, are controlled by genes locater! outside the nucleus in either the chloroplasts or mitochondria. For example, male sterility in several cases is affected by genes in the mitochondria. Male sterility is a desired trait in plant breeding because it allows the inexpensive production of hybrid

24 GENETIC ENGINEERING OF PLANTS seecT. Yet at present, no vector is available to manipulate genes in the organelles. Plant viruses are also being studied as possible vectors. Viruses are tiny bundles of either DNA or RNA encased in a protein coat. Viruses are of interest because they somehow command the plant cell's ma- chinery to replicate the virus and express the viral genes. As with a plasmid, the idea is to insert foreign DNA into the virus and use it to transform a plant cell. Again, the virus would have to be "disarmed" so that it would not cause disease before it could be used as a practical vector. One advantage viral vectors offer is that almost all plants are susceptible to one virus or another. By contrast, the host range of the Ti plasmid is quite limited. Most work has been performed on the cauliflower mosaic virus, a small, double-stranded DNA virus. Researchers have inserted short pieces of foreign DNA into the virus. This virus has then been used to transform plant cells. In these experiments, the foreign DNA has been replicated inside the plant cell as part of the viral genome. But much work needs to be done to develop the cauliflower mosaic virus as a vector. Its major drawback is the size of the foreign DNA it can accept. So far, only pieces of DNA smaller than a gene have been stably inserted into the virus genome. It seems that the protein coat on the virus could be the limiting factor. But there are other plant viruses. RNA viruses, for example, can function without their protein coats and thus conceivably could accept larger pieces containing foreign genes. Viroids are another possibility, though their use is even more spec- ulative, both because of their size and because so little is known about them. ViroicTs consist of RNA without the protein coat. They are the smallest pathogenic agents known, smaller than the average gene. The potato spindle viroicT, for example, contains only 359 nucleotide base pairs, as opposed to about 8,000 in the cauliflower mosaic virus. Gene Expression Until recently, one of the key uncertainties of genetic engineering was whether a foreign gene would be correctly expressed in a higher or- ganism. The recent successful transfer and expression of a foreign gene into a plant cell answered that question. But, as Bogorad pointed out, achieving expression is only the first step. The next step is to control that expression so that the gene is switched on in the right place at the right time. That selective expression is what occurs in nature: although all cells contain the genes for photosynthesis, for instance, they are turned on only in the leaf, not the root. Molecular biologists have yet

GENE TRANSFER 25 to master those controls. Until they do, a transferred gene could simply be on all the time in all the cells. In numerous laboratories, biologists are searching for the control ele- ments that regulate gene expression. If these controls can be identified and transferred to a plant along with the desired gene, they will permit gene expression to be "targeted" to specific organs and developmental stages. The regulation of gene expression is also of enormous theoretical interest, for the switching on and off of genes determines how a single cell differentiates into a plant. Another question is whether the introduction of a foreign gene will affect the expression of the other genes. The introduction of new genes through conventional plant breeding can have deleterious effects, sug- gesting that gene interaction is quite complex. For instance, in 1964 a strain of high-lysine corn was identified. Lysine is an essential amino acid in the diet of nonruminant animals. Though the strain has improved nutritional value for swine and poultry, it is not grown commercially because the yield is reduced 10 percent over other strains. Another drawback is that the kernels of the high-lysine strain do not have good storage quality. It is too soon to say whether molecular genetic engi- neering will involve similar trade-offs. Single and Mulligene Traits Based on the recent advances in identifying and isolating genes as well as advances with vectors, many molecular biologists are confident that they will be able to engineer traits controlled by a single gene or a small cluster of genes. Yet many commercially valuable traits, such as yield and stress resistance of various kinds, are controlled by numerous genes somehow acting in concert. These are called multigene traits. Just finding the genes will be difficult, as evidence suggests that they are scattered throughout the chromosome. Determining how the expression of these genes is regulated, and how their gene products interact, is an even more formidable task. Consequently, it is not clear that techniques can be developed to engineer multigene traits. Plant Regeneration Successful gene transfer ultimately depends on the ability to regen- erate plants from cells in culture. Yet protoplast culture is far from a proven technology. Although it works well in some species, such as carrots, tomatoes, tobacco, and petunia, some of the major crop species are notoriously difficult to regenerate from protoplasts. Potatoes and

26 GENETIC ENGINEERING OF PLANTS alfalfa have been successfully regenerated from protoplasts, but the technique does not work reliably for corn, wheat, or soybeans. Com- pounding the difficulty, no one knows exactly why. The secret lies in the signals that turn genes on and off during de- velopment. Cell culture is an attempt to mimic that process in the lab- oratory. As the ability of plants to regenerate reveals, cells are totipo- tent that is, each cell, such as a leaf cell, contains the instructions for the whole plant. In the differentiatec! state in the leaf, for example most of those genes are shut off. The trick is to induce the cell in culture to regress to an undifferentiated state in which the genes can be switched on ancT off again in proper sequence. The development of culture methods has been handicapped by the lack of knowledge about the regulation of gene expression during de- velopment. Research to understand the genetic mechanisms involved in regeneration is proceeding in tandem with efforts to develop practical culture techniques. Although an increasing number of plants are yielding to protoplast and the other culture techniques, these advances have stemmed as much from guesswork as from science. Whether or not a plant will respond in culture is influenced by several factors, including the composition of the nutrient broth, the specific genotype of the donor plant, and the site from which the explant is taken. In working with unresponsive species, biologists are often confined to juggling these factors perhaps screening hundreds of genotypes in search of one that will work. To a lesser degree, similar uncertainties surround the other two in vitro regeneration techniques: callus and suspension culture (see Cell Culture, p. 34~.

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