Ti to Tomato, Tomato to Market

JAN LEEMANS

Three presentations at the ferred into the plant genome. In the absence of the T• Miami Winter Symposium on DNA genes, such plant cells could be regenerated into 1 2 the "Molecular Genetics of normal and fertile plants. • The first practical system _I Plants and Animals" in Janu• for genetic engineering of plants was thus assembled. 0 ary, 1983 marked the launching These two research groups and a third one, di• CELEBRATING of plant genetic engineering. rected by Mary Dell Chilton of Washington Univer• A DECADE OF Researchers directed by Marc sity (St. Louis, MO) announced at the same time EXCELLENCE Van Montagu and Jeff Schell at another breakthrough: plant cells had been made the University of Gent (Belgium) and by Rob Fraley resistant to the antibiotic kanamycin by transferring a at the Monsanto Co. (St. Louis, MO), had indepen• bacterial neomycin phosphotransferase gene under dently "disarmed" the Ti plasmid of Agrobacterium the control of a promoter isolated from one of the 2 tumefaciens, a bacterium that can transfer a part of its Agrobacterium T-DNA genes. -4 Ti plasmid---{:alled T-DNA-into the plant genome. The experiment showed not only that foreign They had eliminated the crown gall disease-causing genes and proteins could be expressed in plants but genes from the T-DNA while leaving the DNA trans• also provided a widely used selectable marker gene fer mechanism intact. Substituting foreign genes for for cells and tissues into which genes have success• the tumor-causing-genes allowed them to be trans- fully been introduced. Today, Ti plasmid-derived vectors and marker genes are used routinely in labo• Jan Leemans is at Plant Genetic Systems N.V., Jozef ratories around the globe for transforming dicotyle• Plateaustraat 22, B-9000 Gent, Belgium. donous plant species. The research tools developed in 1983 not only marked the start of 0 applied plant biotechnology re• FIGURE1. Technical Commercial search; they also enabled tremen• Milestones in plant dous progress in the field of plant biotechnology. 1983 Ti plasmid disarmed'·' molecular biology. The use of Selectable marker for plants'·• transgenic plants became a power• 1985 U.S. allows plant patents ful tool in analyzing gene function and studying gene regulation5 and 1986 Coat protein-mediated virus resistance 23 First field trial approved protein targeting.6 in the U.S. and Europe Transformation of 24 25 1987 B.t.-based insect resistance • USDA/APHIS proposed 17 20 crop plants Herbicide resistance · guidelines for field testing Particle gun'' The pioneering gene transfer Cotton transformations experiments were performed in to• bacco and other Solanaceous spe• 7 14 1988 Soybean and rice transformation • cies. But within a decade, transfor• 33 34 Ripening control in tomato • mation protocols were established 40 Antisense in plants for all major crops. Agrobacterium

38 39 is now used to transform soybean 1990 RAPD-analysis • EC directive on deliberate Corn transformation '3 release & commercialization (the first of the "big four" crops-• Engineered male sterility27 soy, maize, wheat, and rice),7 cot• ton,8 sugarbeet,9 sunflower,10 oil• 1991 Revised UPOV convention seed rape, 11 and many vegetable accommodates biotech crops. Extensive efforts have been products undertaken to develop alternative 1992 Wheat transformation's Over 400 field tests methods to transform the world's performed worldwide most important cereal crops: com, 31 32 Modified carbohydrate composition • USDA/APHIS deregulates rice, and wheat, none of which are ripening controlled tomato natural hosts for Agrobacterium. Engineered fertility restoration's US~A!APHIS proposes The solution was direct gene s1mpler procedure for field testing six crop species transfer-no~, as was thought for 29 30 Modified fatty acid profile • FDA establishes framework many years, mto protoplasts-but for food safety evaluation into intact plant cells, which can be more efficiently regenerated into 1993 US patent on insect• whole plants. Researchers at resistant plants issued Cornell University (Ithaca, NY),

devised an instrument-the "particle gun," which is terial genes encoding enzymes that inactivate the • 17 18 The initial used to bombard plant cells with DNA-coated par• herbicide by acetylation, hydrolysis, or oxidation ticles12-a technique known as "biolistics." The (Monsanto, unpublished results), respectively. Toler• achievements microprojectiles penetrate the cell walls and deliver ance to glyphosate and to sulfonylurea, on the other DNA. Transgenic com (1990),13 rice (1988), 14 and hand, was engineered by introducing herbicide-insen• in plant wheat ( 1992)15 were produced this way. Also in 1992, sitive mutant forms of the target enzymes. 19-21Herbi• researchers at Plant Genetic Systems (Gent, Bel• cide-resistant transgenic crops have performed as gium), obtained transgenic corn lines by expected in field tests.22 biotechnology electroporating DNA into enzymatically wounded Gene transfer has been extensively used to en• immature embryos and into regenerable maize calli; 16 hance pest and disease resistance in crops. Research• were driven by the procedure is less genotype-dependent and requires ers directed by Roger Beachy, now at the Scripps a shorter period of tissue culture. Research Institute (La Jolla, CA) and then at Wash• the ington University (St. Louis, MO), demonstrated that Agronomic improvements viral coat protein genes expressed in transgenic plants Initial achievements in plant biotechnology were conferred resistance to virus infections.23 This ap• agrochemical driven by the agrochemical industries and focused on proach has been successfully applied to a variety of improving agronomic performance. The $6 billion virus/crop combinations. Virus resistance is impor• industries and global herbicide market attracted much attention. tant because it has positive effects on yield and may With the development cost for a new agrochemical reduce the need for chemical control of the insect focused on rising rapidly, researchers looked to plant engineering vectors that transmit the virus. as a way of gaining market share for a particular Reducing chemical insecticide use has been an• . . herbicide. Herbicide-tolerant crops were seen as a other important goal for plant biotechnology. Insect nnprovmg way of providing more effective, less costly, and more resistance was achieved in transgenic plants by ex• environmentally compatible weed control. The ap• pressing insecticidal proteins from Bacillus agronomic proach would allow reduced overall herbicide use by thuringiensis (B .t.).2 4·25 Insect-resistantcottonhas been shifting to broad-spectrum herbicides with high unit extensively field tested and will probably be the first performance. activity, low toxicity, and rapid biodegradation. crop with built-in insect resistance to be commercial• Two approaches to herbicide tolerance have been ized. Extensive screening of B .t. isolates has provided followed. Tolerance to phosphinothricin, bromoxynil, different B.t. genes encoding proteins that are highly and glyphosate has been achieved by expressing bac- active against various important pests such as the

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Colorado potato beetle. The• insecticidal proteins in• during seed production. teract with specific receptor molecules in the insect Biotechnology's midgut and insects can have receptors for different Industrial Improvements B.t. proteins.26 Alternating or combining the use of Biotechnology's promise to improve the agro• promise to several B.t. proteins that act through different recep• chemical performance of crops has lead several tors may be an important tool for managing the agrichemical companies to invest in the seed industry, emergence of resistance and safeguarding the long• sometimes by acquiring seed companies. Similarly, improve the term usefulness of B.t.-based insect control. opportunities to improve the quality of the harvested Major improvements in crop performance can product have lead large seed companies and food agrochemical generally be achieved through the production of hy• processors into a strategy of developing specialty brid varieties. Crosses between inbred plants often crops with improved quality traits. performance result in progeny with higher yield, increased disease Researchers at Calgene (Davis, CA) have made resistance, and enhanced performance in different significant progress in modifying plant oil composi• of crops has environments compared with parental lines. To pro• tion. By introducing new enzymatic activities or by duce hybrid seed, seed producers control pollination reducing the level of key enzymes in the biosynthesis to guarantee outcrossing and to prevent self pollina• pathway, fatty acid composition has been modified in lead several 29 30 tion. Several crops still lack an efficient pollination oilseed rape. • Similar approaches have been suc• control system. Researchers at PGS and UCLA (Los cessful in modifying carbohydrate composition. Ex• agrichemical Angeles, CA), used genetic engineering to create pression of an E. coli mutant gene encoding ADP• sterile male plants by expressing ribonuclease genes glucosepyrophosphorylase in potato increased the companies to under control of tightly regulated promoters.Z7 Subse• starch level/' while novel carbohydrates were pro• quently, they used a ribonuclease inhibitor to create duced when a bacterial cyclodextrin glucosyl• plants which, when crossed with sterile male plants, transferase gene was expressed. 32 invest in produce a hybrid crop in which fertility is fully re• The most widely publicized example of an engi• stored. 28 The method allows for the efficient produc• neered quality trait is the antisense inhibition of the seed 33 34 tion of hybrid oilseed rape, a major oil crop which polygalacturonase in tomato. · This and other ap• 35 37 today is grown as open-pollinated varieties. In com, proaches lead to fruits . with enhanced shelf-life, industry.... on the other hand, the method can replace the highly delayed spoilage, and better processing characteris• expensive practice of manual and mechanical re• tics that might ultimately improve flavor and texture. moval of the tassel, the male reproductive organ, Calgene plans to commercialize the first genetically

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engineered• tomatoes this year. and food products. In contrast, and despite European As an adjunct These are the first reported successes in modifying Community directives, national regulations in Europe plant metabolism through genetic engineering. More are still variable and ill-defined. This lack of clarity to to will follow as research groups devise strategies to and the associated regulatory costs could delay or improve the protein content of com and soybean to limit the introduction of biotechnology products in the conventional enhance their nutritional quality as animal feeds, to European Community. modify the ratio of fatty acids in vegetable oils to Public perception of biotechnology might become improve stability, shelf-life, and cooking properties the last but not least hurdle for commercializing plant breeding and to modify starches to address the specific needs of biotechnology. Anti-technology groups are highly the food and chemical industry. vocal and well-funded. While they represent directly programs, only a minority of the public, their non-actual presen• Molecular breeding tation of issues leads to public skepticism and threat• molecular The detection and exploitation of naturally occur• ens science-based regulations. The public must re• ring DNA sequence polymorphisms is a significant ceive adequate information to put the tangible ben• new development in plant breeding. Restriction frag• efits and the perceived risks of biotechnology in breeding will ment length polymorphisms (RFLPs) have been used perspective. to create linkage maps and have lead to the develop• increase the ment of indirect selection strategies for crop improve• Filling the inCormation vacuum ment programs. 39 Researchers at DuPont (Wilmington, There is no doubt that the new tools such as efficiency of DE) used PCR (polymerase chain reaction) technol• transposon or T-DNA tagging, the availability of ogy to develop a new tool for marker-assisted selec• genetic and physical maps, Y AC libraries, protein tion in breeding programs: RAPDs (random amplified microsequencing, and especially concerted efforts to progeny polymorphic DNA).38 Compared to RFLPs, RAPD characterize the Arabidopsis genome will be effec• analysis allows to detect polymorphisms at a higher tively used to isolate genes that determine important selection and frequency, is cost effective, and can be automated. plant phenotypes. By the end of its second decade, as Molecular breeding benefits germ plasm improve• an integrated and important component of agricultural considerably ment programs by providing the ability to identify, in and agro-industrial research, plant biotechnology will the progeny of a genetic cross, recombinants that be helping produce economical and high-quality food shorten received a (trans)gene of interest while retaining the and feed and contributing to sustainable agriculture on maximum genetic background of the elite recurrent a global scale. parent. For hybrid crops, molecular breeding is used development to predict the extent of heterosis by assessing the ReCerences degree of divergence between candidate inbred lines. I. Zambryski, P. et al. 1983. EMBO J. 2:2143-2150. times. Germ plasm screening allows the breeder to correlate 2. Fraley, R. et al. 1983. Proc. Nat/. Acad. Sci. allele frequencies with biochemical or agronomical USA 80:4803-4807. 3. Herrera-Estrella, L. et al. 1983. Nature 303:209-213. phenotypes to identify and introgress the loci that 4. Bevan, M. et al. 1983. Nature 304:184-187. contribute to a particular trait. As an adjunct to con• 5. Herrera-Estrella, L. et al. 1984. Nature 310:115-120. ventional breeding programs, molecular breeding will 6. Van den Broeck, G. et al. 1985. Nature 313:358-363. 7. Hinchee, M. et al. 1988. Bio!Technology 6:915-921. increase the efficiency of progeny selection and con• 8. Umbeck, P. et al. 1987. Bio!Techno/ogy 5: 263-266. siderably shorten development times. 9. D'Halluin et al. 1992. Bio!Technology 10:309-314. 10. Everett, N. et al. 1987. Bio!Technology 5:1201-1204. 11. Radke et al. 1988. Theoret. and Applied Genetics 75: Issues Cor commercialization 685-694. In the first decade of plant biotechnology, discov• 12. Klein, T. et al. 1987. Nature 327:70-73. 13. Gordon-Kamm, W. et al. 1990. The Plant Ce/12: 603-618. eries both in basic and applied research have lead to 14. Toriyama, K. et al. 1988. Bio!Technology 6:1072-1074. several prototype recombinant products, the stability 15. Vasil, V. et al. 1992. Bio!Technology 10: 667-674. 16. D'Halluin, K. ct al. 1992. The Plant Ce/112: 1495-1505. and performance of which have been demonstrated in 17. De Block, M. et al. 1987. EMBO J. 6:2513-2518. field trials. In the next decade, today's prototypes will 18. Stalker, D. et al. 1988. Science 242: 419-423. be developed into commercial products. The speed 19. Comai,L.etal.l985.Nature317:741-744. 20. Shah, D. et al. 1986. Science 233: 478-481. and the extent of those commercial introductions will 21. Lee, K. et al. 1988. EMBO J. 7: 1241-1248. be determined by technical, commercial, and regula• 22. De Greef, W. et al. 1989. Bio!Technology 7: 61-64. 23. Powell Abel, P. et al. 1986. Science 232: 738-743. tory issues. 24. Vaeck, M. et al, 1987. Nature 328: 33-37. Technical issues include the transfer of engineered 25. Fischhoff, D. et al. 1987. Bio!Technology 5:807-813. traits into agronomically relevant germ plasm and the 26. Van Rie, J. et al. 1990. Science 247:72-76. 27. Mariani, C. et al. 1990. Nature 347:737-741. adaptation of agronomic practices. Commercial is• 28. Mariani, C. et al. 1992. Nature 357:384-387. sues include the size and structure of the seed market, 29. Voelker, T. et al. 1992. Science 257:72-74. 30. Knutzon, D. et al. 1992. Proc. Nat/. Acad. Sci. the added value created by new genes, and the level of USA 89:2624-2628. proprietary protection for new technology. In suc• 31. Stark, D. et al. 1992. Science 258:287-291. cessful commercialization it will be costly to preserve 32. Oakes, J. et al. 1991. Bio!Technology 9:982-986. 33. Sheehy, R. et al. 1988. Proc. Nat/. Acad. Sci. USA 85: the identity of the product from seed to its fmal end 8805-8809. use. The new trait must contribute sufficient added 34. Smith, C. et al. 1988. Nature 334:724-726. 35. K1ee, H. et al. 1991. The Plant Ce//3:1187-1193. value to offset these costs. 36. Hamilton, A. et al. 1990. Nature 346:284-287. But it will be the regulatory climate that will 37. Oeller, P. et al. 1991. Science 254:437-439. influence the commercialization process the most. In 38. Williams, J. et al. 1990. Nucl. Acids Res. 18:6531-6535. 39. Tanksley, S. et al. 1989. Bio!Technology 7:257-263. the U.S., the regulatory agencies have developed a 40. Vander Krol, A. et al. 1988. Nature 333:866-869. coordinated framework for regulating engineered crops

526 BIO/TECHNOLOGY VOL. 11 MARCH 1993