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2 Introduction 4 Recent advances in biology have proceeded at an astonishing rate, and biologists now have the means, by directly modifying genes, to alter living organisms more quickly and more precisely than has been done by nature and humans over millennia. There is general agreement that this ability can yield far-reaching improvements in our environment and in medical and agricultural practice. However, field testing of prorn~s~g products of the new technology has been slowed by the absence of a full scientific consensus on the relative safety and risks of introducing modified organisms into the environ- ment. Furthermore, the specific questions that are most important to consider in making decisions have not been agreed on. Hence, this NRC committee was formed to attempt to determine a reasoned con- sensus about what scientific questions must be asked and how such questions can aid In the development of a decision-making process based soundly on the facts of science. The history of efforts to reach a common ground about the relative safety or hazard of genetic modification of organisms can be traced directly to the early 1970s, when advances In biological knowledge had given scientists the tools to recombine DNA ~ the laboratory into new sequences (see Appendix). 7
8 THE GENETIC MODIFICATION OF ORGANISMS: MERGING CLASSICAL AND MOLECULAR TECHNIQUES This report describes the properties of plants, rn~croorganisms, and the environment that must be evaluated when the introduction of a genetically modified organism into the environment ~ being planned. IN this introductory section we explore the basic biolog- ical principles that underlie both classical and molecular means of altering the genetic makeup of organisms and explain how our inter- pretation of these principles leads to the conclusion that the products of classical and molecular methods are fundamentally sirn~lar. Both methods of modifying DNA produce an organism (product) that is genetically different from the starting organism regardless of the method (process) used. The molecular techniques are often more precise than classical techniques and can modify single nucleotides of bacterial gnomes. Molecular mollifications surpass classical tech- niques in their ability to introduce a great variety of traits from a wide range of donor organ~srns into the recipient organisms. As a coronary, the molecular techniques can generate a greater range of phenotypes than the classical methods. These principles as they am ply to plants and rriicroorganisms are discussed in greater detail in the sections of this report dedicated to the two kinds of organisms. Plants and microorganisms contain nucleotides ~ combinations and arrangements that endow the organisms with genetic determi- nants for many traits. Other regions of DNA may control the expres- sion of the traits. The DNA provides the raw material upon which genetic modifications depend. The evolution of new forms of crop plants and microorganisms results from selecting organisms with desirable traits from populations that possess heritable variation. When genetic variants are selected to produce the next generation, the population is changed with respect to the frequency of individuals having the selected characteristic. In the terms used in population genetics, selective breeding or propagation changes gene frequencies, and the population differs in some aspect from its predecessor even though the change may be small. Modification of microorganisr~Ls and plants can be performed by either classical or molecular methods. No hard line exists between the two categories, especially with microorganisms. For this report, we generally include as classical those means of genetically modifying organisms that were used before recombinant DNA techniques were developed. One major distinction of classical methods is that they are relatively undirected modifications of the genome. Molecular
9 methods provide more flexibility and control and thus are more specific ~ directing the modifications toward a planned end product. Methodological and biological distinctions exist In culturing mu- croorgan~sms and plants, but one feature of the new genetic technolo- gies is that they permit us to manipulate plants at the cellular level. This technology provides new comrnonalities to plant and microbial breeding. Classical methods are those in which the genetic recombinations occur essentially ~ a natural way; desirable offspring variants are then selected in the laboratory or the field. Examples include spon- taneously mutating m~croorganisrns and sexually cross-bred plants. The term classical also includes some methods called that only be- cause they predate the introduction of modern gene-splicing tech- niques. The latter include such human-mediated techniques as ex- posure of organisrrts to chemuca] mutagens or physical agents such as x-rays and ultraviolet radiation. We also include as classical those mechanisms of DNA transfer that occur without chemical treatment of a cell's envelope, such as transformation, conjugation, and trance auction In microorganisms. Molecular methods of genetic modification include the newer methods for modifying DNA In which one nucleotide can be substi- tuted for another at a predetermined site In a DNA molecule (site- directed mutagenesis). Molecular gene transfer methods are used for transfer of genetic material between donor and recipient cells that have diverged widely through evolution and probably do not exchange DNA without laboratory manipulation. However, it ~ im- portant to recognize that certain gene transfers thought impossible in nature a few years ago because of the phylogenetic distance between donor and recipient have now been shown to occur in the laboratory, and they may occur in nature. For example, there is evidence that a gene or genes for erythromycin resistance was transferred between the gram-negative bacterium Campylobacter and unrelated gram- positive bacteria (Brisson-Noe} et al., 19883. Recent laboratory ex- per~rnents have accomplished gene transfer between Eschertchia cold and streptomyces (Mazodier et al., 1989) or yeast tHeinemann and Sprague, 1989~. Another example relates to the natural transfer of DNA from the bacterial species Agrobacterium to plant cells (Nester et al., 1984~. Plasmid genes from this bacterium probably were trans- ferred into a species of tobacco early in the evolution of the genus Nicotiana, and they became integrated into the plant chromosome. These genes, or their remnants, have beers detected in a variety of
10 different species of Nicotiana, which presumably evolved from the original infected plant (Furner et al., 1986~. PLANT MODIFICATION~CIASSICAL TECHNIQUES Spontaneous and mutagen-induced variation in plants has pro- duced a great variety of genetic traits that may be used in plant breeding. The crop plants of today had their origins ~ the fields of early farmers who selected plants with desirable traits and perpetu- ated plants to meet agricultural needs. Controlled matings (hybridization) of plants through the sexual process is the cornerstone of classical plant breeding. Hybridization and selection of plants with new combinations of traits have been used to increase genetic diversity. By repeated hybridization and selection, new traits could be introduced into varieties already proven successful In agriculture. Hybridization is often possible between species, usually within the same genus. However, many interspecific hybridizations require human-mediated intervention to facilitate the sexual process. For example, developing embryos me excised and cultured on nutrient media before being grown as plants In the field. The male or female fertility of such hybrids ~ often reduced so that they themselves must be hybridized with one of the parents or with a closely related species. Alternatively, fertility can be restored by doubling the chromosome number. With sexual hybridization, the resulting progeny contain fuB complements of genes from each parent. The challenge for plant breeders is to select for the genes which result in a plant's exhibiting the desired combination of traits. Because interspecific hybrids, and even many intraspecific hybrids, have a parent that may be poorly adapted to survive and grow in an agriculturally useful way, consid- erable effort Is required to examine large numbers of plants to find the desired combinations of traits. Two major limitations exist with classical plant breeding. The first is an extraordinarily large degree of variability from which a low frequency of desired plants must be identified. Second, the gene pool-the source of genes accessible to the breeder is Tented to those species which can be sexually hybridized. PLANT MODIFICATIONS MOLECULAR TECHNIQUES TO principle, any gene can now be introduced into any plant by one of several possible molecular modification techniques. At
11 present, the most frequently used agent for DNA transfer is the common soil bacterium Agrobacterium (Nester et al., 1984~. This organism evolved a mechanism for transferring part of its plasmas into plant ceils, where it is integrated randomly into the chromosome (Peerbolte et al., 1986~. The introduced DNA is inserted within this plasmas DNA as a "hitchhiker. Once integrated into the plant's chromosome, the DNA ~ transmitted from parent to offspring and follows the pattern of Mendelian inheritance. Virtually all dicotyI~ donous plants are amenable to transformation by Agrobacterium, but most monocotyTedonous plants appear to be resistant. A technique frequently used to transform monocotyledonous plants, such as maize and rice, is electroporation; this technique requires removal of the plant cell wails before the DNA is added. These naked cells, or protoplasts, often do not synthesize new cell walls readily. Thus, regeneration of whole, fertile plants from pro- toplasts has limited use for molecular gene transfer, especially in cereal grasses. More recently, DNA-coated gold or tungsten parti- cles have been upshots into plant ceils, and stable, genetically trans- formed plants have been regenerated from the cells or organized tissue (Klein et al., 1987~. This technique may be suitable for introducing DNA into plant chIoroplasts (Boynton et al., 1988) and niitochondria (Johnston et al., 1988), as wed as into the nucleus. Current research is directed toward introducing DNA into specific plant tissues that have the greatest probability of regenerating genetically modified plants. COMPARISON OF CLASSICAL AND MOLECULAR TECHNIQUES IN PLANTS The major difference between classical and molecular techniques is the greater diversity of genes that can be introduced by molecular techniques and the greater precision of these introductions. From a single gene to more then 50 genes can be introduced with the Agrobacterium system, although the site in the plant chromosome at which the foreign DNA has been integrated appears to be random. The donor DNA can be derived from the same or different plant species, or even from m~croorganisrns or anneal cells. For example, the DNA from fireflies (Ow et al., 1986) and bacteria (Koncz et al., 1987) that cocles for luminescence has been inserted into plants. Thus, no species barrier exists, because the chern~cal nature of DNA is universal in its structure, irrespective of the organism of its origin.
12 After being integrated, the gene, to be useful, must be expressed in the host plant. Genes have regions at one end of their nucleotide chain that control when and under what conditions the gene wiD be expressed. These regions determine specific conditions for gene expression, for example, in the light, In specific tissues, or at cer- ta~n stages of development (Goldberg et al., 1989~. On the basis of this knowledge and recombinant DNA technology, one can attach the desired region of a gene to a bacterial gene and introduce the combination into a plant cell, where it will be expressed In a spe- cific tissue. Particular conditions, such as wounding, may be needed for expression of the added gene or genes, and knowledge of these conditions can be used to precisely control expression (Ryan, 1988~. GENOME MODIFICATION OF MICROORGANISMS CLASSICAL TECHNIQUES The classical methods of genome modification in microorganisms faD into two classes, selection of spontaneous and induced mutations and the exchange of DNA between (usually) closely related organ- isms. Spontaneous mutations result In a variety of heritable changes In the DNA, including the substitution of one nucleotide for another, the deletion or addition of one or more nucleotides, and other types of DNA rearrangements. Many spontaneous mut~ts appear to re- sult from the movement of transposable elements to new locations in the ceD's DNA. Transposable elements, first discovered in maize, also occur In other plants (McClintock, 1950), bacteria, and anunals. Another mechanism for generating variability in niicroorganisuts is through the introduction of new genetic information from either chromosomal or plasniid DNA. DNA from a donor organism's chro- mosome is integrated into the recipient genome. Plasrn~ds, being selfreplicating, do not have to integrate their DNA into the genome of the recipient. Consequently, plasmid DNA can be transferred to more widely divergent organisms than DNA from the chromosome of a donor organism. Plasmas movement can be monitored because the DNA often provides the genetic code for readily distinguishable traits, such as antibiotic resistance. In bacteria, gene transfer can occur by three different classical means: DNA-mediated transformation, in which the DNA Is trans- ferred as Naked DNA; transduction, In which the DNA is enclosed in a virus coat and the virus mediates the transfer; and conjugation, in which the DNA is transferred during cell-t~ceD contact between
13 donor and recipient cells. Presumably, all these mechanisms operate in nature (Freifelder, 1987~. GENOME MODIFICATION OF MICROORGANISMS MOLECULAR TECHNIQUES The range of techniques to mutate bacteria has expanded and become sophisticated in recent years. It now is routine practice to mutate specific genes (insertion mutagenesis) (Ruvken and Ausubel, 1981) as well as to alter specific nucleotides within a gene (site- directed mutagenesis) (Kunkel, 1985~. These techniques are possible not only for microbial genes, but, In principle, for genes from any organism. The range of microorganisms among which DNA can be trans- ferred has also been expanded through the use of new technologies. Thus, it is now possible to transform cells by physically altering their cell envelopes so that they become permeable to most DNA molecules. One such technique Is electroporation, in which recipient cells and the genetic material to be transferred are subjected to an electric current (Eromm et al., 1987~. The successful use of these techniques for genome modification requires that the entering DNA be able to replicate inside its new host. In principle, the techniques for performing these manipulations are straightforward. With such techniques, plasm~ds have been constructed that can replicate in both the bacterium E. cold and the yeast Saccharomyces cerev~siae (Ereifelder, 1987~. COMPARISON OF CLASSICAL AND MOLECULAR TECHNIQUES ~ MICROORGANISMS Recent molecular technological advances in mutagenesis and gene-transfer methods have opened new possibilities for expanding the range of rn~croorganisrns into which DNA from unrelated organ- isms can be introduced. The genus barrier and, indeed, the kingdom barrier are no longer complete obstacles. Recombinant DNA methodology makes it possible to introduce pieces of DNA, consisting of either single or multiple genes, that can be defined in function and even in nucleotide sequence. With -classical techniques of gene transfer, a v~iable number of genes can be transferred, the number depending on the mechanism of trans- fer; but predicting the precise number or the traits that have been
14 transferred ~ difficult, and we cannot always predict the phenotypic expression that will result. With organisms modified by molecular methods; we are in a better, if not perfect, position to predict the phenotypic expression. With classical methods of mutagenesis, chemical mutagens such as alkylating agents modify DNA In essentially random ways; it is not possible to direct a mutation to specific genes, much less to sped cific sites within a gene. ~deed, one common alkylat~g agent alters a number of different genes simultaneously. These mutations can go unnoticed unless they produce phenotypic changes that make them detectable in their environments. Many mutations go undetected un- ti! the organisms are grown under conditions that support expression of the mutation. SUGARY We have reviewed briefly the various means by which plants and microorganisms can be genetically modified by methods termed Classical or "molecular. Genetic variability in microorganisms and plants is enhanced by classical modifications such as spontaneous or mutagen-~duced variation, by hybridization, and by gene transfer. These methods are relatively ~rnprecise and undirected and less pow- erful than molecular techniques for modifying genes. However, no conceptual distinction exists between genetic modification of plants and microorganisms by classical methods or by molecular techniques that modify DNA and transfer genes. Figure 2-l graphically depicts this view. The difference in the modes of genetic modification are not deemed critical, and both methods are included in one box. This figure also illustrates that no distinction exists between so-called classical and molecular breeding methods at the steps of evaluation In laboratory, field, or large-scale environmental introduction. This understanding of the biological nrincinles has the f~ll~nn~ implications for the report: To r ~r^~~"~ ~ 1. The deliberations of the committees were guided by the conclusion (NAS, 1987) that the product of genetic modification and selection should be the primary focus for making decisions about the environmental introduction of a plant or microorganism and not the process by which the products were obtained. 2. Information about the process used to produce a genetically modified organism is unporta~t in understanding the characteristics
15 BIOLOGICAL SOURCE MATERIAL GENETIC MODIFICATION BY CLASSICAL OR CELLULAR/MOLECULAR METHODS g SELECTION OF DESIRED FORM - l EVALUATION IN LABORATORY AND/OR FIELD TESTS LARGE SCALE INTRODUCTION l FIGURE 2.1 Genetic modification of an organism and its introduction into the environment. Of the product. However, the nature of the process is not a useful criterion for determining whether the product requires less or more oversight. 3. The same physical and biological laws govern the response of organisms modified by modern molecular and cellular methods and those procluced by classical methods. Scientists have vast expe- rience with the products of classical modification, and the knowledge gained thereby is directly applicable to understanding, evaluation, and decision-mak~g about the relative safety or risk of field tests on products of molecular modification techniques.