Appendix G
NRI Funded Contributions to Major Scientific Advances in Food, Fiber, and Natural Resources

Of the 3,070 proposals submitted to the NRI in 1995–1997, 745 were awarded with a total of $89,637,786 (R.Michael Roberts, NRI, Sept. 1, 1998, personal communication).

This appendix summarizes what NRI staff considered some leading examples of NRI-funded contributions to major scientific advances in food, fiber, and natural resources. The information presented here was supplied to the committee by the NRI staff.

NRI FUNDED RESEARCH THAT HAS LED TO MAJOR SCIENTIFIC ADVANCES

Biologically Safe Protection of Wheat from Take-all Disease by Using Soil Bacteria

Crop rotation is the best-known practice available to farmers for management of the soilborne plant pathogens responsible for potentially devastating root diseases, wilts, stem rots, and blights of crop plants and for such cosmetic diseases as common scab of potato. Growing unrelated crops in a rotation allows time (about 2 years or more) for the soilborne pathogens of a crop to die out or be eliminated by their natural enemies before the same crop is planted again in the same field. However, scientists have known that soil



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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Appendix G NRI Funded Contributions to Major Scientific Advances in Food, Fiber, and Natural Resources Of the 3,070 proposals submitted to the NRI in 1995–1997, 745 were awarded with a total of $89,637,786 (R.Michael Roberts, NRI, Sept. 1, 1998, personal communication). This appendix summarizes what NRI staff considered some leading examples of NRI-funded contributions to major scientific advances in food, fiber, and natural resources. The information presented here was supplied to the committee by the NRI staff. NRI FUNDED RESEARCH THAT HAS LED TO MAJOR SCIENTIFIC ADVANCES Biologically Safe Protection of Wheat from Take-all Disease by Using Soil Bacteria Crop rotation is the best-known practice available to farmers for management of the soilborne plant pathogens responsible for potentially devastating root diseases, wilts, stem rots, and blights of crop plants and for such cosmetic diseases as common scab of potato. Growing unrelated crops in a rotation allows time (about 2 years or more) for the soilborne pathogens of a crop to die out or be eliminated by their natural enemies before the same crop is planted again in the same field. However, scientists have known that soil

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research microorganisms can suppress soilborne pathogens after many years of a crop monoculture (that is, without crop rotation). Until recently, US agriculture has not taken advantage of microorganisms to protect crops against harmful pathogens. David Weller, R.James Cook, Jos Raaijmakers, and Linda S.Thomashow (CRGO/NRI funded scientists) have spent the last 2 decades studying the microbiologic basis of the decline of take-all disease after 12–15 years of wheat monoculture. Take-all is the most serious root disease of wheat worldwide because most of the world’s major wheat-growing areas, lacking economically suitable broad-leaf crops that could be grown in alternate years, use little or no crop rotation. Take-all is especially devastating where wheat is planted with reduced tilling to reduce soil erosion, and it has been known to wipe out entire fields. Soil laden with some strains of root-associated Pseudomonas, including P. fluorescens and P. chlororaphis, appears to suppress the fungal pathogen. These scientists have shown that those bacteria produce natural antibiotics (mainly 2,4-diacetylphloroglucinol and phenazine-1-carboxylate) that stop the growth of the fungus. Their research has provided the first proof that the ability of soil microorganisms to produce antibiotics is critical to their survival and activity in soil and that antibiotic-producing soil microorganisms constitute one of nature’s most effective methods for management of plant diseases. In addition to the knowledge of how to manage the beneficial bacteria through the cropping system, these strains of Pseudomonas can be grown in industrial quantities in fermentors and applied as a biologically safe seed coating that prevents take-all disease in wheat. Three patents have been issued, two more are pending, and two license agreements have been issued for use of specific strains in turf, as well as wheat. In 1997, Weller, Thomashow, Cook, and Raaijmakers received the Ruth Allen Award; this is the highest award for research given by the American Phytopathological Society and recognizes contributions to science that have changed the direction of research. CRGO and NRI Awards to Cook and colleagues: 1978; Biological Stress on Plants; $120,000; 3 years. 1981; Biological Stress on Plants; $60,000; 2 years. 1986; Biological Stress on Plants; $100,000; 2 years. 1989; Plant Pathology/Weed Science; $100,000; 2 years. 1991; Plant Pathology/Weed Science; $120,000; 2 years. 1991 (1993 renewal); Plant Pathology; $100,000; 2 years. 1994; Soils and Soil Biology; $212,000; 3 years. 1996; Plant Pathology; $ 116,041; 2 years. 1994 (1997 renewal); Soils and Soil Biology; $255,000; 3 years.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Patents: 4,456,684; June 26, 1984; D.Weller and R.J.Cook; Method for screening bacteria and application thereof for field control of diseases caused by Gaeumannomyces graminis. 5,955,298; September 21, 1999; L.S.Thomashow, M.Bangera, D. Weller, R.J.Cook; Sequences for production of 2,4-diacetylphloroglucinol and methods. 5,972,689; January 24, 1997; R.J.Cook, D.Weller, D.-S.Kim, L.S. Thomashow; Methods and compositions for the simultaneous control of the root diseases caused by Gaeumannomyces graminis, Rhizoctonia, and Pythium. Filed November 20, 1997; J. Raaijmakers, L.S.Thomashow, D.Weller, R.J.Cook; Biocontrol agents for take-all. Filed December 18, 1998; Z.Huang, L.S.Thomashow, D. v. Mavrodi, J. Raaijmakers, D.Weller, R.J.Cook; Transgenic strains for biocontrol of plant root diseases. Publications: Raaijmakers, J.M., D.M.Weller, and L.S.Thomashow. 1997. Frequency of antibiotic producing Pseudomonas spp. in natural environments. Applied and Environmental Microbiology 63(3):881–887. Bonsall, R.F., D.M Weller, and, L.S.Thomashow. 1997. Quantification of 2,4-dacetylphloroglucinol produced by fluorescent Pseudomonas spp. in vitro and in the rhizosphere of wheat. Applied and Environmental Microbiology 63(3):951–955. Raaijmakers, J.M., D.M. Weller, R.F.Bonsall, and L.S.Thomashow. 1995. Primers and probes to detect soil pseudomonads that produce 2,4-diacetylphloroglucinol and phenazine-1-carboxylic acid. Phytopathology 85:1191 (abstract). Cook, R.J., L.S.Thomashow, D.M.Weller, D.Fuyimoto, M.Mazzola, G.Bangera, and D.Kim. 1995. Molecular mechanisms of defense by rhizobacteria against root disease. Proceedings of the National Academy of Sciences 92:4197–4201. Decreasing Milk Fever In Dairy Cows: A Major Advance Milk fever is an important metabolic disorder of dairy cows related to the onset of lactation when cows are unable to maintain normal blood concentrations of calcium. Serum calcium often decreases to a point that does not support normal nerve and muscle function. Cows then suffer severe appetite loss, generalized weakness or collapse, and, if left untreated, death. This disorder affects about 6–8% of all US dairy cows each year, directly costing the dairy industry up to $20 million.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research FY 1993, USDA Agricultural Research Service scientists J.P.Goff and R. L.Horst, at the National Animal Disease Center in Ames, Iowa, received an NRI grant to determine whether the potassium, sodium, or calcium concentration in the ration fed to cows just before calving influences their susceptibility to milk fever. At the time of the award, dietary recommendations urged restricting dietary calcium. However, field reports suggested that such a regimen might be unnecessary and possibly even detrimental. The researchers demonstrated that dietary calcium is not a major risk factor for milk fever. Diets high in potassium or sodium actually induced milk fever by increasing blood alkalinity. Cows on high-potassium diets had lower plasma concentrations of 1,25-dihydroxy vitamin D (important for intestinal calcium absorption) and lower plasma hydroxyproline (an index of the activity of bone calcium resorption). The results suggest that when the blood pH is high, the tissues become resistant to stimulation by parathyroid hormone, a calcium-regulating hormone. The stimulation of bone calcium mobilization and intestinal calcium absorption is then diminished, and normal blood calcium concentration cannot be maintained. Diets high in sodium are not commonly fed to dry cows, but potassium is a cation commonly found in high amounts in the forages included in dry-cow rations. This work also indicated that the partial success of previously recommended low-calcium diets was due primarily to a reduction in dietary potassium, not calcium. The expected benefits to the dairy industry are enormous. These findings provide an easily managed feeding approach to the problem of milk fever and already are changing how US cows are fed before calving. The results of this research will decrease the large economic loss due to milk fever. Additional economic benefits will include reductions in the incidences of other important dairy-cattle diseases, such as ketosis and mastitis, for which cows with milk fever are at higher risk. NRI Award to Goff and Horst: 1993; Milk Fever Risk Factors: Dietary Cation-Anion Differences; $80,000; 3 years. Publications: Goff, J.P., and R.L.Horst. 1997. Effects of the addition of potassium or sodium, but not calcium, to prepartum rations on milk fever in dairy cows. Journal of Dairy Science 80:176–186. Popular press publications: Because of the enormous applicability of the findings, the results have been widely disseminated in numerous industry publications, including Hoard’s Dairyman; Dairy Today; and Dairy Herd Management.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Storage-Protein Modification in Potato: Tuber Quality and Vaccine Production With funding initiated in the first year of the USDA Competitive Research Grants Office, William Park (initially at Purdue University, then at Texas A&M University) isolated and characterized the genes for the major potato tuber protein, patatin. Patatin accounts for about 40% of the total soluble protein in potato tubers, but under normal conditions it is not present in large amounts in leaves, stems, or roots. The work led to the development of tuber-specific expression vectors that can be used as a “molecular pickup truck” to direct the expression of any RNA or protein specifically to the economically important part of the plant. It should be noted that the promoters were not patented and thus are freely available in the public domain. The initial studies set the stage for studies by Dr. Park and others that have advanced understanding of fundamental plant processes, such as the key role of sugars in regulation of plant gene expression and of the mechanism of organ-specific gene evolution. In addition, the resulting potato transformation-propagation system has found practical application in, for example, improving potato cultivars, manipulation of carbohydrate metabolism, and—beyond plant science—production of pharmaceutically important proteins, such as edible vaccines. In the first instance, William R.Belknap and colleagues, at the USDA Agricultural Research Service, Albany, California, are using the patatin-control element as one of several promoters as they seek to develop improved potato cultivars. Preliminary results of plants currently in field trials indicate that the patatin-control element could have use in developing potatoes resistant to bruising damage. For development of edible vaccines, potatoes expressing a gene encoding the Escherichia coli heat-labile enterotoxin B subunit were fed to mice and to humans. In both, ingestion of the transgenic potatoes triggered a mucosal immune response. A second clinical study is now under way to test transgenic potatoes that express another diarrheal antigen, the Norwalk virus capsid protein. The work, by Charles Arntzen and colleagues (Boyce Thompson Institute) with support from the National Institute of Allergy and Infectious Diseases, offers a new strategy in development of safe and inexpensive oral vaccines against human diseases, such as diarrhea and other diseases for which a protective antigen has been defined; tetanus, diphtheria, and hepatitis B are examples. The strategy is being extended to diseases of agriculturally important animals. Avian influenza virus is under investigation by Hugh Mason (also of Boyce Thompson Institute) as a model system for oral vaccines for poultry. Awards to Park: 1981; Regulation of Tuber Protein Synthesis in Potato; $90,000; 2 years; Purdue Research Foundation. 1983; Regulation of Tuber Protein Synthesis in Potato; $80,000; 1 year; Texas A& M University.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research 1984; Regulation of Tuber Protein Synthesis in Potato; $73,000; 1 year; Texas A & M University. 19856; Regulation of Tuber Protein Synthesis in Potato; $78,000; 1 year; Texas A & M University. Award to Mason: 1997; Vaccines for Poultry Using Antigens Produced in Transgenic Plants; $100,000; 2 years. Publications: Mignery G.A., C.S.Pikaard, D.J.Hannapel, and W.D.Park. 1984. Isolation and sequence analysis of cDNAs for the major potato tuber protein, patatin. Nucleic Acids Research 12:7987–8000. Pikaard C.S., J.S.Brusca, D.J.Hannapal, and W.D.Park. 1987. The two classes of genes for the major potato tuber protein, patatin, are differentially expressed in tubers and roots. Nucleic Acids Research 15:1979–94. Bourque J.E., J.C.Miller, and W.D.Park. 1987. Use of an in vitro tuberization system to study tuber protein gene expression. In Vitro Cellular and Developmental Biology 23:381–6. Wenzler, H., G.Mignery, L.Fisher, and W.D.Park. 1989. Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants. Plant Molecular Biology 13:347–54. Haq, T.A., H.S.Mason, J.D.Clements, and C.J.Arntzen. 1995. Oral immunization with a recombinant antigen produced in transgenic plants. Science 268:714–6. Mason, H.S., J.Ball, J.J.Shi, X.Jiang, M.K.Estes, and C.J.Arntzen. 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proceedings of the National Academy of Sciences 93:5335–5340. Tacket, C.O., H.S.Mason, G.Losonsky, J.D.Clements, M.M.Levine, and C.J.Arntzen. 1998. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 4:607–9. Mason, H.S., T.A.Haq, J.D.Clements, and C.J.Arntzen. 1998. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16:1336–43.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Establishing Nutritional Requirements for Vitamin B6 and Folate in Humans Vitamin B6, folate, and vitamin B12 function both as coenzymes and as substrates in the one-carbon pathway that is required for the synthesis of nucleic acids and the methylation of DNA. Each can be marginal in the diets of the US population, and direct linkages to public health have been established. Jesse Gregory and colleagues, at the University of Florida, developed technologies to measure bioavailabity, metabolism, and metabolic function of vitamin B6 and folate in rodents and humans. They have provided new information on the bioavailability of dietary folate and daily folate requirements and developed a more quantitative understanding of the metabolic roles of folate and vitamin B6. Major accomplishments have included the following: Developed methods based on the use of nonradioactive- (stable-) isotope labeling to assess folate absorption, metabolism, and rate of turnover in humans and animals. Developed methods based on stable-isotope labeling to measure the normal rates and effects of folate and vitamin B6 deficiency on key reactions in one-carbon metabolism in humans. Demonstrated that the requirement for folate in nonpregnant women is about twice that previously believed. This observation was a major factor in the development of the much higher 1998 recommended dietary allowance for folate. Demonstrated that folic acid added to cereal-grain foods (breads, pasta, and rice) is effectively absorbed. The data proved that the recently adopted fortification program is effective in delivering available folate to the US population. CRGO and NRI Awards to Gregory: 1979; Food Composition and Processing Effects on Vitamin B6 Bioavailability; $ 100,000. 1981; The Bioavailability of Folacin in Foods; $100,000. 1983; Determination of Vitamin B6 and Folacin Bioavailability Using Isotopic Enrichment Methods; $135,000. 1986; Stable-Isotopic and Enzymatic Investigation of Folacin Bioavailability; $120,000. 1987; Stable-Isotopic and Radioisotopic Investigation of Folate Bioavailability; $175,988. 1991; Stable-Isotope Investigation of Folate Bioavailability and Nutritional Status; $166,842. 1992; Folate Nutritional Status and In Vivo Kinetics; $222,521. 1994; The Bioavailability of Folate in Foods; $227,618. 1996; Folate and Vitamin B6 Dependence of One-Carbon Metabolism; $198,586.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Production of Cattle and Swine Embryos in Vitro, a Prelude to Cloning Research in the 1960s and 70s developed procedures for test-tube (in vitro) fertilization (IVF) of eggs from some mammals, including the mouse and human. The achievement was illustrated most dramatically by the birth of the first IVF baby, Louise Brown, in 1977. Neal First’s laboratory at the University of Wisconsin pursued development of the procedures in livestock to allow immature eggs, obtained from ovaries collected from the slaughterhouse, to provide a vast source of embryos. The general approach was and continues to be collection of ovaries from the slaughterhouse, transportation of ovaries to the laboratory, aspiration of large numbers of oocytes (eggs) from many immature ovarian follicles, and finally in vitro maturation of the oocytes in special culture medium that mimics the conditions involved in egg maturation in the intact animal. There were other technical difficulties to overcome. Tissue-culture conditions had to be developed that allowed sperm from frozen semen to fertilize the matured oocytes and that permitted a fertilized egg to develop into a multicelled embryo. First and colleagues developed the basic methods to produce hundreds of embryos from slaughterhouse-derived material. The spinoff from this work has been enormous, including the following: It permitted the cloning of cattle by nuclear transfer from early-cleavage-stage embryos and was a prelude to the eventual demonstration of cloning by somatic cell nuclear transfer. The First laboratory holds the first patent in this field. It led to parallel developments in embryo manipulation and cloning in swine. It led to the improvement of transgenic techniques for cattle. The availability of many embryos has allowed gene expression to be studied during early development of livestock. For his achievements, First was awarded the von Humboldt Award for Agriculture in 1987 and the Wolf Prize (recognized as the equivalent of a Nobel Prize in agriculture) in 1997. CRGO and NRI Awards to First: 1985; Ontogeny and Control of Development of Bovine Preimplantation Embryos; $161,500. 1987; Ontogeny and Control of Development of Bovine Preimplantation Embryos; $142,500. 1988; Cellular Regulation of Meiotic State in Bovine Oocytes; $144,000. 1988; Development of Porcine Embryos After Multiplication by Nuclear Transplantation; $200,000. 1990; Cellular Regulation of Meiotic State in Bovine Oocytes; $70,000.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research 1992; Ontogeny and Control of Development of Bovine Preimplantation Embryos; $213,753. 1995; Effect of Bovine Embryonic Stem Cell Origin and Culture on Pluripotency and Totipotency; $120,000. Patent: 4,994,384; February 19, 1991; R.S.Prather, F.Barnes, J.Robl, N.L.First and V.F.Simmon; Multiplying bovine embryos. Corn from Cells, Not from Seeds When the Competitive Research Grants Office was formed in 1978, a grant was awarded to Ronald L.Phillips, of the first Genetic Mechanisms Program Panel, for tissue culture of corn. Phillips had already demonstrated that whole corn plants could be regenerated from cells in tissue culture, and this technology had been quickly adapted to other cereals. Most important, Phillips’s CRGO-supported research eventually allowed new genes to be introduced into corn. Before this breakthrough was achieved, however, a major problem had to be solved. Phillips recognized that the plants derived from tissue culture were not exact copies of each other (clones), but that variation was being induced by the tissue-culture procedure. On the one hand, this provided a way of producing new corn varieties; on the other hand, it was a drawback to the production of transgenic plants because the gene could not be introduced into a stable genetic background. The variability was due in part to the activation of mobile genetic elements during tissue culture and to alterations in the methylation of DNA. To circumvent the problem, Phillips used short culture times and then crossed the resulting plants to elite lines. Genetically engineered corn, particularly varieties that are resistant to insect predation, accounted for more than one-third of the crop in 1998, and its use is increasing. The most common genotype used by industry for producing transgenic crops traces back to Phillips’s research supported by CRGO. Tissue-culture regeneration technology of cereal crops has been a major tool in developing genetically engineered plants. CRGO and NRI grant awards to Phillips: 1978; Tissue Culture Genetic Systems in Corn; $110,000; 3 years. 1988; Tissue Culture Genetic Systems. 1992; Mapping and Isolation of Genomic Regions: Controlling Maturity in Maize; 2 years.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Cloning of Chitinase and Its Practical Application as a Biopesticide Karl J.Kramer, biochemist with the USDA Agriculture Research Service Grain Marketing and Production Research Center, Manhattan, Kansas, isolated the gene that codes for the chitinase enzyme from the tobacco hornworm Manduca sexta. Kramer and collaborators at Kansas State University—S. Muthukrishnan, Lowell Johnson, and Frank White—have cloned and incorporated the gene into tobacco and rice plants. They have demonstrated that coupling the chitinase gene with the bacterium Bacillus thuringiensis in genetically engineered plants can be a safe and effective biopesticide that substantially reduces insect growth and curtails foliar damage in plants. Chitinase works by breaking down chitin, a key component in membranes of insect skin and gut tissue. Without this chitin membrane, insects rapidly succumb to microbial infections. The chitinase must be ingested, so it poses a minimal threat to nontarget species because these organisms would have to chew on engineered plants. Because these species are not leaf feeders, they should not even contact the enzyme. Also, chitin is peculiar to insects and other invertebrates and not found in higher animals, so it is considered safe for humans and other mammals. Kramer and collaborators have received continuous USDA competitive grant funding since 1988 to support this research and have published at least 40 papers in scientific journals since the inception of the work. Most recently, they have patented the only known insect chitinase gene used in transgenic plants. Currently, NRI is supporting Kramer’s efforts to transform other plants, such as corn, wheat, and sorghum. Biotechnology companies—including Pioneer, Dekalb, and Prodigene—are working with these scientists to develop practical applications. CRGO and NRI Awards to Kramer: 1996; Development of Insect Chitinolytic Enzymes as Biopesticides; 2 years. 1998; Improvement of Insect Chitinase as a Biopesticide in Transgenic Plants; 3 years. Patent: 5,866,788; February 2, 1999; K.J.Kramer, S.Muthukrishnan, H.K.Choi, L.Corpuz, and B.Gopalakrishnan; Recombinant chitinase and use thereof as a biocide.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Publications: Kramer, K.J., A.M.Christensen, T.D.Morgan, J.Schaefer, T.H. Czapla, and T.L.Hopkins. 1991. Analysis of cockroach oothecae and exuviae by solid-state 13C-NMR spectroscopy. Insect Biochemistry 21:149–56. Corpuz, L., H.Choi, S.Muthukrishnan, and K.J.Kramer. 1991. Sequence of the cDNAs and expression of the genes encoding methionine-rich storage proteins of Manduca sexta. Insect Biochemistry 21:265–276. Christenson, A.M., J.Schaefer, K.J.Kramer, T.D.Morgan, and T.L. Hopkins. 1991. Detection of cross-links in insect cuticle by REFOR NMR Journal of the American Chemical Society 113:6799–6802. Hopkins, T.L., and K.J.Kramer. 1992. Insect cuticle sclerotization. Annual Review of Entomology 37:273–302. Kramer, K.J., L.Corpuz, H.Choi, and S.Muthukrishnan. 1993. Sequence of a cDNA and expression of the genes encoding epidermal and gut chitinases of Manduca sexta. Insect Biochemistry and Molecular Biology 23:691–701. Gopalakrishnan, B., S.Muthukrishnan, and K.J.Kramer. 1995. Baculovirus mediated expression of a Manduca sexta chitinase gene: Properties of the recombinant protein. Insect Biochemistry and Molecular Biology 25:255–265. Zen, K.C., H.K.Choi, K.Nandegama, S.Muthukrishnan, and K.J. Kramer. 1996. Cloning, expression and hormonal regulation of an insect N-acetylglucosaminidase gene. Insect Biochemistry and Molecular Biology 26:435–444. Wang, X., X. Ding, B.Gopalakrishnan, T.D.Morgan, L.Johnson, F. White, S.Muthukrishnan, and K.J.Kramer. 1996. Characterization of a 46-kDa insect chitinase from transgenic tobacco. Insect Biochemistry and Molecular Biology 26:1055–1064. Choi, H.K., K.Choi, K.J.Kramer, and S.Muthukrishnan. 1997. Isolation and characterization of a genomic clone for the gene of an insect molting enzyme, chitinase. Insect Biochemistry and Molecular Biology 27:37–47. Additional Information: A virtual article in Discovery Channel Online is available at http://www.discovery.com/news/archive/news990226/brief5.html?ct=36dafe7f. Dogs and Infectious Abortion in Cows: A Mystery Solved Neospora caninum is a protozoan parasite that is a major cause of infectious abortion in dairy cattle. The parasite is found throughout the United States and around the world. In California alone, Neospora caninum is diagnosed in 40% of aborted fetuses and costs dairy producers at least $35 million a year. This

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research parasite was first described about 10 years ago, but until last year veterinarians, dairy farmers, and researchers were frustrated by the disease because it was not known how it was transmitted. A major advance recently occurred in the battle to protect the dairy industry from this parasite. A team led by NRI-supported researcher Milton McAllister, at the University of Wyoming, in collaboration with researchers at Virginia Polytechnic Institute and State University and the Agricultural Research Service, demonstrated that the dog is a definitive host of Neospora. This discovery has paved the way for development of effective methods of disease prevention. Unraveling the mystery of Neospora transmission began with a 2-year seed grant in which McAllister and colleagues developed a protocol that would reliably induce formation of large numbers of encysted bradyzoites of Neospora in the brains of mice, gerbils, and sheep. The best results were obtained with immunosuppressed mice from which bradyzoites could be purified. Neospora has structural and genetic similarities to other protozoan species that are transmitted in the feces of carnivorous animals, and results from the 1994 NRI seed grant allowed the research team to obtain a small grant from the American Veterinary Medical Foundation to determine whether cats are definitive hosts; the results were negative. However, as a result of a later award from the NRI’s Animal Health and Well-Being Program, dogs were induced to pass the oocysts after being fed the tissues of mice infected with Neospora from both canine and bovine isolates. Scientific understanding of this parasite’s life cycle now points to practical ways to decrease its impact. Dairy cows are usually fed rations consisting of feed mixed in large batches. Feed is often piled on the ground in open areas during this process. If a dog shedding Neospora contaminates ration ingredients, an entire herd can be exposed to the parasite. The risk of Neospora abortion could be greatly reduced by simply maintaining animal feed in a fenced area or closed container. Fencing could also be used to prevent pregnant cows from eating feces-contaminated pasture. Thus, relatively simple methods can be used to solve a multimillion-dollar problem. The research team successfully completed its stated goal of discovering a definitive host for Neospora caninum. The information is being widely disseminated in the popular press (Multiple press releases, Colorado Dairy News, Hoard’s Dairyman, Parasitology Today, and so on). A valuable, new bovine isolate of Neospora that was also identified is now available to the research community for further investigations to diminish the impact of this infection. Further studies are needed to determine whether animals related to dogs, such as foxes and coyotes, also transmit the organism. Vaccine development would aid the dairy industry, particularly in management situations where eliminating working-dog and family-pet access to pasture and feed areas might not be possible. This research success highlights the importance of the NRI’s Strengthening Awards Program. Competition for these awards is open to faculty of small and

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research medium academic institutions and at institutions in the USDA Experimental Program for Stimulating Competitive Research. NRI Awards to McAllister: 1994; Induction of infectious cysts of Neospora caninum in rodents and fetal lambs; $45,000; 2 year Seed Grant. 1996; Determination of the Definitive Host of Neospora caninum; $111,727; 1 year Standard Research Grant. Publications: McAllister, M.M., S.F.Parmley, L.M.Weiss, V.J.Welch, and A.M. McGuire. 1996. An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen. Journal of Parasitology 82:354–355. McAllister, M.M., A.M.McGuire, W.R.Jolley, D.S.Lindsay, A.J. Trees, and R.H.Stobart. 1996. Experimental neosporosis in pregnant ewes and their offspring. Veterinary Pathology 33:647–655. McAllister, M.M., E.M.Huffman, S.K.Hietala, P.A.Conrad, M.L. Anderson, and M.Salman. 1996. Evidence suggesting a point source exposure in an outbreak of bovine abortion due to neosporosis. Journal of Veterinary Diagnostic Investigation 8:355–357. McGuire, A.M., M.M.McAllister, W.R.Jolley, and R.C.Anderson-Sprecher. 1997. A protocol for the production of Neospora caninum tissue cysts in mice. Journal of Parasitology 83:647–651. McGuire, A.M., M.M.McAllister, and W.R.Jolley. 1997. Separation and cryopreservation of Neospora caninum tissue cysts from murine brain. Journal of Parasitology 83:319–321. Dubey, J.P., M.C.Jenkins, D.S.Adams, M.M.McAllister, R. Anderson-Sprecher, T.V.Baszler, O.C.H.Kwok, N.C.Lally, C.Bjorkman, and A.Uggla. 1997. Antibody responses of cows during an outbreak of neosporosis evaluated by indirect fluorescent antibody test and different enzyme linked immunosorbent assays. Journal of Parasitology 83:1063–1069. McAllister, M.M., J.P.Dubey, D.S.Lindsay, W.R.Jolley, R.A.Wills, and A.M.McGuire. 1998. Dogs are definitive hosts of Neospora caninum. International Journal for Parasitology 28:1473–1478. McAllister, M.M., W.R.Jolley, R.A.Wills, D.S.Lindsay, A.M. McGuire, and J.D.Tranas. 1998. Oral inoculation of cats with tissue cysts of Neospora caninum. American Journal of Veterinary Research 59:441–444.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research NRI FUNDED RESEARCH WITH HIGH POTENTIAL FOR FUTURE SCIENTIFIC ADVANCES A Step Closer to Bioengineering Cold-Tolerant Plants Michael Thomashow and colleagues at Michigan State University used NRI funding from the Plant Responses to the Environment Program to create a cold-tolerant strain of Arabidopsis thaliana (a model plant organism). Researchers have known about cold-tolerant genes for close to 30 years, but they had not been able to bioengineer a cold-tolerant plant, because of the number of genes involved. At least 25 genes are associated with cold tolerance, and stable transfer of many genes is not yet possible. However, Thomashow was part of the research teams that discovered that the expression of these genes is regulated by proteins known as transcription factors. In theory, transcription factors can be used to enhance cold tolerance by turning on all the genes involved at one time. Thomashow tried to insert such a transcription factor into Arabidopsis and found that, indeed, cold tolerance was enhanced dramatically. Although it is true that the same cold-tolerant genes and a single transcription factor might not work in plants other than Arabidopsis, this is a first step in understanding how to control cold tolerance in plants. NRI Awards (directly related to this research): 1988; Plant Responses to the Environment Program; $140,000; 2 years. 1990; Plant Responses to the Environment Program; $110,000; 2 years. 1992; Plant Responses to the Environment Program; $160,000; 3 years. 1996; Plant Responses to the Environment Program; $243,393; 3 years. Publications (directly related to this research): Jaglo-Ottosen, K.R., S.J.Gilmour, D.G.Zarka, O.Schabenberger, and M.F.Thomashow. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280:104–106. Gilmour, S.J., D.G.Zarka, E.J.Stockinger, M.P.Salazar, J.M. Houghton, and M.F.Thomashow. 1998. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant Journal 16:433–442.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Reducing Processing Waste and Negative Environmental Impacts by Wood Modification Lignin, the complex polymer that cements cellulose fibers together and provides strength and protection for growing trees, is removed when wood is processed for commercial purposes. The removal process requires chemical treatments, and the chemicals and unseparated fibers become a waste product. Ronald Sederoff and other NRI-funded scientists at North Carolina State University have been working on modifying lignin composition in loblolly pine (a commercially important timber source) so that its mechanical strength is reduced and processing efficiency is increased. These researchers discovered that a mutant that produces a modified lignin exists naturally in loblolly pine populations. The mutant appears to change the composition and structure of lignin by blocking the enzyme, cinnamyl alcohol dehydrogenase. The enzyme converts cinnamyl aldehydes to cinnamyl alcohols, which are the precursors of lignin formation. These researchers also showed that laccase was another important enzyme involved in lignin formation, providing yet another enzyme that could be used to manipulate lignin formation genetically. NRI Grants (directly related to this research): 1988; Genetic Mechanisms/Plant Science Bio-Technology (joint); $66,000; 2 years. 1991; Plant Growth and Development Program; $110,000; 2 years. 1997; Wood Utilization Program; $111,000; 2 years. Patent: 5,824,842; October 20, 1998. J.MacKay, D.O’Malley, R.Whetten, R. Sederoff. Methods of providing and breeding trees having more easily extractable lignin due to the presence of a cinnamyl alcohol dehydrogenase (CAD) null gene. Publications (directly related to this research): O’Malley, D.M., S.Porter, and R.R.Sederoff. 1992. Purification and characterization of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine. Plant Physiology 98:1364–1371. Bao, W., D.M.O’Malley, R.Whetten, and R.R.Sederoff. 1993. A laccase associated with lignification. Science 260:672–674. O’Malley, D., R.Whetten, R.Bao, C.L., Chen, and R.R.Sederoff. 1993. The role of laccase in lignification. The Plant Journal 4:751–757. Whetten, R. and R.Sederoff. 1995. Lignin biosynthesis. Plant Cell 7:1001–1013.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Bioengineered Safe Resistance to Glyphosate Herbicide Weed control often requires the use of herbicides that can harm crop plants. Glyphosate (Roundup) is a common herbicide that kills grasses, sedges, and broad-leaf plants by blocking the biochemical pathway that produces essential amino acids (phenylalanine, tyrosine, and tryptophan). Only plants, fungi, and bacteria can make these essential amino acids. Animals (including humans) are insensitive to glyphosate, so its use is relatively safe. Plant resistance to glyphosate has already been genetically engineered with genetic material in the cell nucleus. However, there is now concern over the use of such plants because the resistant genes could be spread with the release of pollen. The release could lead to a decrease in the overall effectiveness of the herbicide against weeds and create “superweeds”. Henry Daniell and colleagues, at Auburn University, with the support of NRI funding have found a solution to the problem by using genetic material in the chloroplast to genetically engineer glyphosate-resistant tobacco. Chloroplast genetic material is maternally inherited and cannot be spread by pollen in most crops (with rare exceptions, such as pines). Chloroplast-derived resistance is also more resistant to glyphosate than nucleus-derived resistance. Application of glyphosate after crop emergence is now possible without the fear of uncontrolled spread of the resistance gene or herbicide damage to the crop. NRI Awards (directly related to this research): 1993; Plants Division; $10,648; 2 year Seed Grant. 1995; Value Added Products; $120,000; 2 years. 1997; Non-Food Characterization/Process/Product Program; $219,438; 3 years. 1998; Plant Genome; $160,000; 2 years. Publication (directly related to this research): Daniell, H., R.Datta, S.Varma, S.Gray, and S.B.Lee. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nature Biotechnology 16:345–348. Establishment of the Role of Steroid Hormones in Plant Growth and Development Steroid hormones are crucial for embryonic development and adult homeostasis in animals. Similarly, the insect steroid hormone ecdysone controls many developmental processes in insects. In plants, many steroids have been identified, but only one class of steroids, collectively called brassinosteroids

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research (BRs), has wide distribution throughout the plant kingdom and unique growth-promoting properties when applied exogenously. Useful agricultural applications of these compounds have been found, such as increasing yield and improving stress resistance of several major crop plants. Despite extensive research, mostly in the 1970s and 1980s, definitive proof that BRs are essential for normal plant growth had been lacking until recently. It is surprising that a clear role of BRs in plant growth and development came from genetic studies on photomorphogenesis in the model plant Arabidopsis, led by Joanne Chory, of the Salk Institute. A mutation in the DET2 gene resulted in plants that do not respond properly to light. The gene was cloned and shown to encode an enzyme (steroid 5α-reductase) involved in the biosynthesis of BRs. The mutant plant can be rescued by application of BRs. The identification of additional BR-deficient dwarf mutants in Arabidopsis and other plant species confirms the importance of these steroids in plant development. This work is important because it confirms the role of BRs as a major class of plant-growth regulators. Previously, only auxins, gibberellins, abscisic acid, ethylene, and cytokinins were considered “real” hormones, and BRs received little or no attention in botany textbooks or in general reviews of plant physiology and development. Now, because of the work of Chory and others, BRs are receiving a great deal of international attention. It is likely that greater understanding of the molecular mechanisms of BR action could have practical effects on the generation of transgenic crop plants of many species with altered growth properties (such as stature and yield). NRI Awards: 1993; Molecular and genetic analysis of arabidopsis Det 2 gene; 3 years. 1996; Molecular and genetic analysis of arabidopsis Det 2 gene; 3 years. Patents: Filed April 1996; Novel plant steroid 5α-reductase, DET2. Filed June 1997; Receptor kinase, Bin 1. Publications: Li, J., P.Nagapal, V.Vitart, T.McMorris, and J.Chory. 1996. A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272:398–401. Li, J., M.Biswas, A.Chao, D.Russell, and J.Chory. 1997. Conservation of function between mammalian and plant steroid 5α-reductases. PNAS 94:3554–3559.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Li, J. and J.Chory. 1997. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90:929–938. Fujioka, S., et al. 1997. Arabidopsis DET 2 mutant is blocked early in brassinosteroid biosynthesis. Plant Cell 9:1951–1962. Additional Information: Chory has been continuously funded by the NRI since 1991. She has also received funding from the National Institutes of Health, the National Science Foundation, the Department of Energy, and the Howard Hughes Medical Institute for studies on light-regulated gene expression and light signal transduction. Signal-Transduction Pathway of the Plant Hormone Ethylene The gaseous plant hormone ethylene has profound effects on plant growth and development. There are numerous responses to ethylene throughout the life cycle of the plant, including induction of ripening in climacteric fruits, promotion of seed germination, promotion or inhibition of flowering, abscission of various organs, and senescence. While she was a postdoctoral researcher in Eliot Meyerowitz’s laboratory at the California Institute of Technology, Caren Chang cloned the ETR1 gene from Arabidopsis, which was shown to be an ethylene receptor sharing the same “two-component” feature (a sensor and an associated response regulator) of bacterial regulators. It was the first plant hormone receptor to be cloned. Since moving to the University of Maryland in 1994, Chang has continued to work on the molecular and genetic mechanisms of ethylene signal transduction. Important discoveries include the previously undescribed association of a two-component receptor (ETR1) and the MAPK signaling cascade and the repression of responses by the receptors in the absence of ethylene (contrary to the typical signaling paradigms established in animals). This work is important not only because of the cloning of the first plant hormone receptor, but because it has made the ethylene response pathway one of the best understood signaling pathways in plants. It serves as a model for the study of other plant hormones. It also shows that plants do not necessarily transduce signals in the same way as other eukaryotes. Because numerous physiologic processes are mediated by ethylene, the manipulation of ethylene response is vital to the storage, transport, disease protection, appearance, and flavor of numerous plant products. The isolation of new ethylene signaling components will lead to new strategies for manipulating a variety of ethylene responses and thus help us to refine our ability to control plant growth processes.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research NRI Awards: 1995; Genetic Dissection of Ethylene Signal Transduction Based on the Arabidopsis ETR 1 Gene; 3 years. 1998; Arabidopsis Ethylene Signal Transduction: New Interacting Components; 3 years. Patents: 5,689,055; November, 18. 1997. E.M.Meyerwitz, C.Chang, A B. Bleecker. Plants having modified responses to ethylene. Publications: Chang, C. and R.C.Stewart. 1998. The two-component system: regulation of diverse signaling pathway in prokaryotes and eukaryotes. Plant Physiology 117:723–731. Clark, K.L., P.B.Larsen, X.Wang, and C.Chang. 1998. Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proceedings of the National Academy of Sciences 95:5401–5406. Chang, C. 1996. The ethylene signal transduction pathway in Arabidopsis: an emerging paradigm? Trends in Biochemical Science 21:129–133. Additional Information: Chang has been supported by the NRI since 1995. The NRI has been her sole source of federal support beyond her postdoctoral fellowship. Improvements in Plant Biotechnology by Manipulation of Gene Silencing The ability to use plants as “factories” to produce large quantities of valuable proteins is one of the most exciting and potentially useful developments to come out of modern biotechnology. Transgenic plants might be used to produce medically or industrially valuable proteins in a way that is less expensive, more environmentally friendly, and less reliant on the use of animals. In addition, growing such transgenic plants might provide improved economic opportunities for farmers. Until recently, the ability to use plants to produce large quantities of proteins has been hampered by the existence of a process in plants called gene silencing, which normally protects the plants from viral pathogens. As the name implies, gene silencing stops the production of proteins that would otherwise be produced at very high levels, such as viral proteins or proteins produced by “transgenes” in plants grown as protein factories.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research In 1995, the NRI Plant Pathology Program awarded $149,000 to Vicki Vance, at the University of South Carolina (award 95–37303–1815), for her work aimed at understanding the serious diseases caused by the interaction of two viruses in the same host plant (viral synergism). The award was renewed for $150,000 in 1997. In 1995, the NRI’s Plant Pathology Program also made an award to James Carrington, at Texas A & M, for $219,000 for his work on the intercellular movement of potyviruses (award 95–37303–1867). Using materials generated by Carrington’s group, Vance’s group showed that a protein encoded by one virus permitted a wide variety of unrelated viruses to accumulate to high levels and cause serious disease. They reasoned that the viral protein exerts this effect by paralyzing the host plant’s gene-silencing process (Pruss et al. 1997). Papers published independently by Vance’s group, and Carrington’s group, and a group in England (Anandalakshmi et al.1998; Kasschau and Carrington 1998; Brigneti et al. 1998) provided experimental data to show that this was indeed the case. Because of the practical implications of this work, Vance and her colleague at the University of South Carolina, Gail Pruss, made a successful US patent application through the University of South Carolina (application 08/827,575, “Method for enhancing expression of a foreign or endogenous gene product in plants”). The coinventors listed on the patent application were Laszlo Marton, Carrington, and William Dawson. Numerous companies have expressed interest in licensing the patent. Work aimed at understanding how the viral protein interferes with gene silencing continues to be supported through the NRI (award 98–35301–6078 made to Vance by the NRI’s Plant Genetic Mechanisms Program and award 98–35303–6485 made to Carrington by the NRI’s Plant Pathology Program). NRI Awards to Vicki Vance, University of South Carolina: 1995; Mechanisms of Plant Viral Synergism; 4 years. 1998; A Viral Suppression of Gene Silencing in Plants; 2 years. NRI Awards to James Carrington, Texas A&M University 1995; Intercellular Movement of Potyviruses; 2 years. 1998; Host Responses to Potyviruses; 3 years. Patent: 5,939,541; August 17, 1999; V.B.Vance, G.J.Pruss, W.O.Dawson, J. Carrington, M.Laszlo; Method for enhancing expression of a foreign or endogenous gene product in plants.

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research Publications: Anandalakshmi, R., G.P.Pruss, X. Ge,., R.Marathe, A.Mallory, T.H. Smith, and, V.B.Vance. 1998. A viral suppressor of gene silencing in plants. Proceedings of the National Academy of Sciences 95:13079–13084. Brigneti, G., O.Voinnet, W.X.Li, L.H.Ji, S.W.Ding, and D.C. Baulcombe. 1998. Viral pathogenicity determinants are suppressor of transgene silencing in Nicotiana benthamiana. The European Molecular Biology Organization Journal 17:6739–6746. Kasschau, K.D., and J.C Carrington. 1998. A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95:461–470. Pruss, G., X.Ge, X.M.Shi, J.C.Carrington, and V.B.Vance. 1997. Plant viral synergism: the polyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9:859–868. A New BAC Library Vector for Transferring Large DNA Inserts to Plants Numerous potential improvements in the characteristics of agriculturally valuable plants are possible through identification of a desired trait in another organism and its expression in a crop plant. That is accomplished by locating the genes responsible for the trait and inserting them into the crop plant’s chromosomes. Some desirable genes, such as those for disease resistance in plants, can occur in clusters, creating the need for a reliable system for transforming large segments (>100 kb) of DNA into plants. The relatively large genomes of plants and the abundance of repetitive DNA sequences in them add to the need for an improved transformation system. To provide a solution to those long-standing problems in basic plant biology, Carol Hamilton, while at Cornell University, developed a binary-bacterial artificial chromosome technology, called BIBAC, to facilitate the development of new elite varieties of agronomic crops. BIBAC technology not only accelerates the identification of agriculturally important genes, but also makes it possible to introduce valuable traits of interest into plants without dragging along deleterious traits—a common problem for classical plant breeders. The success of the BIBAC system would not have been possible without enhancing the ability of Agrobacterium tumefaciens to effect DNA transfer to the plant chromosomes. That is, this work included basic research that has affected the plant-transformation community. Since the development of BIBAC technology, several groups have requested the virulence helper plasmids that made technology a success, not because they needed BIBAC technology itself, but because they were interested in improving the transformation efficiency for their plant system of interest. In general, this is a more common problem for agronomic crops than for model plants used for basic research. The critical elements of the new technology, the bacterial strains and plasmids, have been requested by hundreds of academic and industrial laboratories around the

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National Research Initiative: A Vital Competitive Grants Program in Food, Fiber, and Natural-Resources Research world. In addition, various parties have expressed interest in licensing BIBAC technology; these queries are directed to the Cornell Research Foundation. A US patent has been issued for the BIBAC vector, and foreign patents are pending. The Center for Advanced Technology/Biotechnology at Cornell University supported the construction and maintains a BIBAC Web site in support of BIBAC technology. NRI Award: 1995; Evalustion and Applicaton of a New BAC Library Vector Designed for Transfer of Large DNA Inserts; 3 years. Patent: March 31, 1998; C.M.Hamilton; Binary BAC vector. Publications: Hamilton, C.M., A.Frary, Y.Xu, S.D.Tanksley, and H.B.Zhang. 1999. Construction of tomato genomic DNA libraries in a binary-BAC (BIBAC) vector. The Plant Journal 18(2):223–229. Hamilton, C.M. 1998. BIBAC technology: progress and prospects. AgBiotech News and Information 10(1):23N-28N. Hamilton, C.M. 1997. A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene 200:107–116. Hamilton, C.M., A.Frary, C.Lewis and S.D.Tanksley. 1996. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proceedings of the National Academy of Sciences 93:9975–9979. Additional Information: Hamilton was funded in 1995 as an NRI new investigator at Cornell University with additional support from the National Science Foundation and the Department of Energy. She is now employed at Paradigm Genetics, Inc., in Research Triangle Park, North Carolina. The Center for Advanced Technology/Biotechnology maintains a BIBAC Web site at www.bio.cornell.edu/biotechn/BIBAC/BIBAC, that includes how to obtain BIBAC materials, general information, restriction maps, and several detailed protocols. Hamilton replies to all BIBAC correspondence and can be reached at chamilton@paradigmgenetics.com.