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5 Past Experience with the Introduction of Modified Plants: Molecular Genetic Techniques Contemporary methods of genetic modification offer unique ad- vantages for crop improvement. They complement existing plant- breeding efforts by increasing the diversity of genes and germplasm available for incorporation into crops. The directed transformation of commercial varieties and hybrids should significantly shorten the time to commercial release. The rapid progress that has been made in gene identification and isolation methods, plant tissue culture, and gene transfer techniques has now permitted the extension of specific genetic change (for exam- ple, by recombinant DNA methods) to more than 30 species of crop plants (Gasser and Fraley, 1989~. Today, nearly all major dicotyle- donous crop species, including row crops (cotton, soybeans), veg- etables (tomato, potato), forages (alfalfa, clover), and trees (poplar, pear), can be genetically mollified by molecular methods. New meth- ods have facilitated the development of transformation systems for use in corn and other monocotyledonous crops. Within the next few years, all major crops will probably be amenable to improvement through molecular approaches as a matter of course. 54

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as PROPERTIES OF MOLECULAR GENETIC MODIFICATIONS Methods of Gene Introduction A variety of techniques have been developed to introduce genes successfully into recipient plants. These techniques can be broaclly grouped into those involving biological carriers, vectors, and those involving physical, or nonvectored, methods. Physical methods for modification include introducing DNA fragments into cells by m~- croinjection (Crossway et al., 1986) or electroporation (Fromm et al., 1986) or ~ntroduc~g DNA bound to a metal m~croparticle that is accelerated into target cells (Klein et al., 1988; Johnston et al., 1988~. Delivering genes by physical methods can produce a "sim- ple" pattern of DNA insertion (a single DNA fragment inserted at one chromosomal location) or a ~complex" pattern (multiple DNA insertions at one or more genetic loci). One of the vectored methods commonly used for plant modifi- cation utilizes nature's own genetic engineer, Agrobacterium [umefa- ciens (E`raley et al., 1986; Bevan, 1984~. By deleting the genes that modify normal cells into tumorigenic cells and leaving hit act those genes that are responsible for transferring DNA from the bacterium to the plant cell nucleus, modified A. tumefaciens cells can vector desirable genes into appropriate plants cells. Between 70 and 80 percent of the transformed cells produced by this method have a single target gene inserted at a single locus (simple pattern). Other vectored methods for introducing genes include the use of plant DNA (Blossom et al., 1984) and RNA (French et al., 1986) viruses. Gene insertion, whether transferred by vectored or nonvectored methods, appears to be a random event. Methods have not yet been developed to target the insertion of a gene to a specific chromosc)ma location, although progress is being made (Paszkowski et al., 1988~. Because of the random nature of gene insertion, some transgenic cells or plants may exhibit more or less gene expression than others. Thus, it is usually necessary to evaluate several different transgenic plants to choose those with the desired levels of gene expression. This type of selection is reminiscent of the evaluation done by plant breeders as plant lines are developed. ~ most modification methods, transgenic cells are selected in some manner (generally by resistance to an antibiotic or by screening with an appropriate gene reporter system) and exposed to altered cultural regimens to induce regeneration of plants. Regenerating

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56 plants from single cells, chili, or explants may produce somaclon- ally variant plants that, because of the cell culture or regeneration process, have a different phenotype than the parent. For example, somaclonaDy variant plants may be altered in terms of ploidy, steril- ity, or patterns of plant development. It Is ~rnperative that variants be recognized as such and not be confused with the direct products of modification per se. Genetic Stability of the Alteration Genetic modification usually involves the introduction of DNA into nuclear chromosomes and expression of the gene as a dominant trait. Such introduced genes, studied in a number of different set- tings, have been found to be inherited with stability equal to that of other nuclear genes. For example, Nelson et al. (1988) performed a Iimuted field test with tomato plats that were stable as fourth- generation progeny after modification. Other workers have reported similar finings with genes introduced into other plants (WalIroth et al., 1986; Deroles and Gardner, 1988~. No evidence suggests that introduced genes are lost more or less frequently than other plant nuclear genes. In addition, no evidence suggests that gene insertion with Agrobacterium-based vectors imparts any plant pest character- istics to the recipient plant. The vast majority of plant modifications target nuclear chro- mosomes; however, attempts are being made to modify organelIar genomes, those of chIoroplasts and m~tochondria. Whereas modific~ tion of Chiamy~omonas chioroplasts has been demonstrated (Boyn- ton et. al., 1988), similarly reproducible results with higher plant cell chIoroplasts remain to be established. When these are achieved, the nature of inheritance of the introduced gene will be changed because chioroplasts are transrnttted maternally, but usually not through pollen. Modification of organelles wait probably be important for engineering such traits as herbicide resistance aIld male sterility. There currently are a few replicon-based (autonomously repli- cat~ng) vector systems for plants (Brisson et al., 1984; French et al., 1986; Grimsley et al., 1987~; however, these have not achieved the same utility as in bacteria and yeast. It is likely that replicons based on RNA-containing or DNA-conta~ning plant viruses will be developed to induce desired proteins or nucleic acids in plants. In some cases, part or all of the viral genome may be integrated into the nuclear genome and then regulated for expression in specific cell

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57 types. Tm other instances, a modified virus or part thereof may be introduced and expressed as a replicon per se. Because few plant viruses are efficiently seed-borne, such replicon-based systems will probably not be used widely for introducing agronomic genes into plants. ~ all gene delivery and gene expression systems discussed (other than viral-based replicons), it Is highly unlikely that the new gene will be transrn~tted to different plant types other than through sexual means. Thus, while A. tumefaciens modification involves the use of the modified bacterium to deliver the gene, the bacterium itself is re- moved after gene introduction is completed. This is accomplished by treating the transformed cells and regenerating plantlets with antibi- otics that kill the bacterium. Collecting seed from transgenic plants excludes A. tumefaciens, which further ensures that the bacterium does not contaminate the progeny. No evidence to date exists that stably integrated DNA Is likely to be transferred by mechanisms other than hybridization under natural conditions, by either insects or rrucroorga~isms. Thus, there is no logical basis for more concern with the unusual transfer of an introduced nuclear gene than with any other nuclear gene transfer. The types of genetic alterations that have been achieved to date include the transfer of large segments of DNA (a segment as long as 50 kilobases of DNA); an upper size limit for transfer has not been determined. Generally, much smaller segments of DNA, from less than 2 to 10 kilobases, are introduced. Gene transfers could theoretically include many genes, although practical considerations generally mitigate against transferring more than four or five genes at any one time. Multiple transformation of a single Individual could produce a plant with many introduced genes, as does sexual hybridization of individuals that carry genes at distinct alleles. Whereas genes are commonly introduced to add new traits, it has not been possible to inactivate or remove a specific gene by homologous recombination or msertional activation. However, an alternative approach that emphasizes antisense gene constructs has been successful In eliminating or reducing the expression of endoge- nous genes. Several applications of antisense (nucleic acid sequences that are complementary to sequences that code for a protein) technol- ogy in plant systems have been described, including the alteration of chalcone synthase genes (Van der Krol et al., 1988) and alterations to produce tomato fruit deficient in polygalacturonase that retain firmness for an extended period (Sheehy et al., 1988~.

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58 Types of Genetic Alterations During the past 5 years a variety of genes have been introduced into plants for research purposes, but relatively few have the potential for use In agriculture and food production. Those of likely importance to production agriculture (Boyce Thompson Institute, 1987) in the near future include . . plants that express a gene that induces accumulation of msec- ticidal proteins, including Bacillus thuringiensis endotoxins (Fischhoff et at., 1987; Vaeck et al., 1987) and a variety of protease inhibitors (Hilder et al., 1987~. Such proteins wiD limit the feeding of insects on the modified plants and reduce the need for chemical insecticides. plants that contain genes that encode the capsid protein of one or more plant viruses. The accumulation of viral capsid proteins protects these plants against the virus from which the gene was taken as weD as against closely related viruses (PoweD-Abe] et al., 1986; Tumer et al., 1987~. plants that are resistant to specific herbicides or classes of herbicides because they either detoxify the herbicide or resist its effects (Shah et al., 1986; StaLker et al., 1988; Haughn et al., 1988~. The resistance traits wait make it possible to use ~ agriculture normally nonselective but readily degraded herbicides that are safe to other life forms, thereby reducing weed control costs and long-lasting chemical damage to the environment. plants whose flower colors are altered (van der Kro} et al., 1988), fruits remain firm (Sheehy et al., 1988), and seed protein or of] compositions are altered (Beachy et al., 1985; Sengupta-Gopalan et al., 1985~. The rapidly expanding knowledge base in plant biology makes it likely that future targets for plant improvement via molecular genetic techniques wiD include resistance to environmental pressures that can affect plant productivity. This could mclude resistance to heat, drought, flooding and salt stresses, pathogenic bacteria, fungi, and parasitic nematodes. In addition, these tools should significantly increase our current understanding of plant development and gene expression (Goldberg, 1988~.

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59 CASE STUDIES OF PLANTS MODIFIED BY MOLECULAR GENETIC TECHNIQUES The field research an crops genetically modified has been less controversial than environmental introductions of other organisms. This may be attributed to the use of domesticated plants with which we have substantial experience regarding confinement during field research. During 1987-88, more than 20 trials were approved for field research with plants modified by molecular means including tomato (14 trials) and tobacco (7 trials) (Animal and Plant Health Inspection Service, unpublished, 1989~. Requests up to March 1, 1989, include an increasing number of agronomic crops: cotton (3), soybean (3), alfalfa (2), potato (2), and rice (1) as well as additional tomato (4) and tobacco (1) trials. Of the 36 approved thus far and of those requested trials as of March I, 1989, only one ~ from a noncommercial research group. These requests and approvals are mainly for additions of single genes for resistance to herbicides (18), msects (19), and viruses Gil, and a DNA sequence addition that enhances fruit quality (2~. Results of these introductions have not raised any additional safety concerns. The tests have taken place at diverse locations across the United States, including Illinois, Florida, California, Mississippi, Wisconsin, Delaware, and North Carolina. AD were reviewed in detail by the Department of Agriculture with review and inputs by other govern- mental agencies. The key consideration in approval of each test has been a scientific evaluation of its risk and environmental impact. The major issues that have emerged from these discussions are stability of the inserted genes, undesirable alteration in crop phenotype, environmental impact on nontarget species, potential for weediness of genetically modified crops, and ability to maintain the gene within the test site. Stability of Vertex Genes Crop plants modified by molecular techniques have been pro- duced either with A. tumefaciens Ti (for tumor inducing) plasrn~d vectors or by a variety of nonvector-mediated methods such as m~- croinjection, electroporation, particle guns, or calcium-phosphate precipitation. Tens of thousands of plants in over 30 different crop species have been studied in contained facilities with respect to gene

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60 expression and inheritance patterns. The cumulative results demon- strate that the introduced DNA sequences are incorporated into ran- dom sites in the genome, stably maintained through both vegetative and reproductive propagation, and neither excised nor transferred. All the evidence indicates that genes or traits introduced by molecular methods behave similarly to those introduced by classical techniques such as cell selection, mutagenesis, or sexuad hybridiza- tion that is, regular inheritance patterns ~ generations (Eraley et al., 1986; KuhIemeier et al., 1987~. Undesirable Alteration of Plant Phenotype Since gene insertion ~ random, inactivation of an important plant gene or genes could possibly result from the insertion pro- cess. the data accumulated to date, however, do not support this possibility. Efforts to introduce DNA to isolate genes by insertiona] inactivation reveal it to be an event of extremely low probability. The low frequency is understandable because less than 5 percent of the DNA in typical crop plant genomes constitutes actively expressed genes, and, in many cases, plant gene families may contain 5 to 10 functional members (Goldberg, 1988~. Inactivation of a single gene, therefore, is unlikely to produce an altered phenotype. There has been a recent report, however, of gene inactivation by transferred DNA (T-DNA) insertion In Arabidopsis thaliana (Feldman et al., 1989~. The Ara~oidFopsis haploid genome, however, consists of only 70,000 kilobases, which is about 1/80 the size of the wheat genome. The small genome size of ArabidFopsts greatly increases the likelihood that insertional mutagenesis wait lead to gene inactivation. Although the inserted gene or gene product might be able to im- pair some important plant process through pin unknown mechanism, such a risk is no greater than that associated with classical breed- ing. With the molecular modifications, the introduced sequences and their functions are known precisely, and their functioning in a new genetic background can be experimentally determined in greenhouse studies and in small-scale field tests. A variety of molecular probes are available to monitor the location, expression, and function of introduced genes. Recombination of DNA sequences is a normal consequence of sexual hybridization and an import ant contributor to the generation of new varieties arch hybrids as shown by restriction fragment length

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61 polymorphisms (RFLPs) (Tanksley et al., 1989). The existing prm cedures for plant breeding, field evaluation, and crop certification have evolved to deal with the consequences of genetic recombination. Off-types displaying undesirable phenotypes are removed (rogued) as standard procedure. In a history of 75 years of breeding and crop testing, crop breeders have been successful in protecting against the introduction of undesirable traits into crop varieties; the earlier de- scribed southern corn leaf blight, by contrast, was one example of an undesirable phenotype that went undetected. Envn~onmental Enpact on Nontarget Species Some people are concerned that crops modified by molecular techniques may have an adverse impact on the environment. These issues involve managed and natural ecosystems (which are addressed in this report) and the possible risk to anunal and human health (which is not considered! here). Risks to natural and managed ecosystems focus on the altered plants becoming weeds in succeeding crops or on the movement of genes to wild relatives that would increase the weediness of those relatives. These aspects were discussed in Chapter 4. Confinement ~ the key to minimizing the environmental unp act to nontarget species. Plant field tests to date have used removal of reproductive structures, the lack of non-cross-pollinating weedy rela- tives, and distance from related cross-poBinating varieties to prevent new genes or gene combinations from escaping beyond the control of the experiment. Established conditions for confinement of cIassicaBy modified plants in field tests are being used to limit movement of genes outside the test site, thereby minimizing effects on natural and managed ecosystem. AD modified plants that have been field-tested or are proposed for field research are highly domesticated with an established history In field testing. Potential for Weediness As discussed In Chapter 4, there are two major issues regarding weediness: (~) does the modified crop itself have weedy properties, and (2) does the modified crop have traits that if transferred to wild relatives would increase their weediness? The properties generally attributed to weediness include seed dormancy, long soil persistence, germination under diverse environ- mental conditions, rapid vegetative growth, high seed output, and

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62 i high seed dispersion (Baker, 1974). These properties are usually thought to represent complicated, multigenic traits. Although it can be argued that only a few genes in certain crops separate them from weeds, crops derived by molecular methods are no more likely to evolve into weeds than crops produced by classical methods. The introduction of herbicide resistance into crops ~ receiv- ng research attention. Several crops such as tobacco, tomato, and oilseed rape that have been modified to resist active ingredients of herbicides, such as glyphosate, bromozynil, sulfonylureas, and phos- phinothricm, have been tested In the field. The benefit of such re- search will be increased flexibility ~ weed control, including benefits such as unproved weed control efficacy, reduced costs to farmers, the opportunity to replace currently used chemicals with more environ- mentally friendly chemicals, and the reduction of overall herbicide usage (Boyce Thompson Institute, 19873. Herbicide-tolerant plants have been feared to be able to develop into volunteer weeds or to spread resistance genes to weedy species. The key to evaluating that risk is to focus on specific products on a crop-by-trait basis. This involves determining the possibility that herbicide-resistant volunteer plants will become weeds in a subse- quent year, the potential for introgression of herbicide resistance genes into weedy relatives, and assessing the potential impact of herbicide-tolerant plants on the cropping and weed-control practices of particular geographic regions. Corn, wheat, and sugarbeets are examples of crops that can become volunteer weeds but are con- trolled In subsequent crops by cultivation and by different herbicide products. A glyphosate-tolerant volunteer corn plant In a soybean field would be controlled by normally used preemergent or postemer- gent herbicides. Similarly, a sulfonylurea herbicide-res~stant wheat plant could be controlled ~ either rotational crops or on fallow land with today's normal cultural practices. In addition. Dast exDerienc:e Tom breeding herbicide tolerance into crops-such as metribuzin resistance in soybean, atrazine resistance in canola, and acetanilide resistance in corn have shown that the phenotypes are stable, and these modifications have not increased the weedy characteristics of a given crop. The prunary U.S. crop targets into which herbicide tolerance is being engineered are corn, soybean, and cotton; none of these species outcrosses with weedy relatives In the United States or displays significant potential to develop into weeds themselves. ~A! _~^! _-- ~ _

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63 Specific Examples We cite as examples, field tests of tomato plants containing (1) the B. thuTingiensis insect-control protein, which In the laboratory killed caterpillar pests such as tomato hornworm, fruitworm, army- worm, and Worm (Fischhoff et al., 1987), and (2) coat-protein genes from tobacco mosaic virus (TMV), that confer resistance to infection by TMV (Nelson et al., 1988~. Scientific evidence available from published reports, expert opinion, and direct experimentation led to the conclusion that the field introduction of tomato plants tolerant to certain insects and viruses would have negligible environ- mental impact. 1. The genetically modified tomato plants were well characterized. Greenhouse testing confirmed stability of gene expression and inheritance. The plats were free of Agrobacterium spp. used for gene transfer. No unusual phenotypes were assO.ciated with genetically mod- ified plants. 2. The introduced genes and gene products were well characterized. The vector DNA contained no uncharacter~zed sequences. The B. thuringiensis protein produced in the plant has no effect on beneficial insects or mammals. ~ The TMV capsid protein has no effect on nontarget species. 3. Biological confinement at the test site was readily available. ~ Bt ~d TMV proteins decompose in the soil. . Tomato normally self-pollinates under field conditions and has no cross-hybrid~zing weedy relative in North America. In addition to the confinement afforded by the lack of cross- pollination and the absence of sexually compatible weed species, it was possible to physically contain plants at the test site by fencing to discourage seed dispersal by predators. Also, tilIage Ad chemical means were used to destroy volunteer tomatoes. Small-scale tests conducted in Blinois and Florida over the past 2 years have indicated the absence of environmental impact and provided the following new data. The introduced insect tolerance and virus-resistance traits were stable and not transferred to tomato plants as close as five feet away through cross-pollination.

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64 There were no significant differences In the nontarget insect populations collected in and around modified and control plants. Field control of caterpillar pests confirmed laboratory and greenhouse results; there was no extension of control beyond caterpillar species. Field control of TMV confirmed laboratory and greenhouse results. The plants containing the B. thuringiens~ endotox~n and coat-protein grew normally; there were no indications of any adverse phenotypes such as plants with increased susceptibil- ity to other viral or fun gel diseases. Although much field research is needed to evaluate the perfor- mance of insect-tolerant and virus-res~st~t tomato varieties under different conditions, the preliminary data confirm the predictable behavior of plants modified by molecular methods and tested in lam oratory and greenhouse. They also demonstrate that field research methods developed for crops mollified by classical methods "e also suitable for field research of crops modified by molecular methods. SUMMARY POINTS 1. Crops modified by molecular methods in the foreseeable future pose no risks significantly different from those that have been accepted for decades in conventional breeding. 2. The evaluation of plants modified by molecular techniques does not represent a unique concern. Under appropriate conditions of confinement, field-test evaluations can proceed with negligible risk.