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SOYBEAN - APPLICATIONS AND TECHNOLOGY

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A seeder should place seed in an environment for reliable germination. The main objective of sowing is to put seeds at a desired depth and spacing within the row. Uniform seed distribution within the soil result in better germination and emergence and increase yield by minimizing competition between plants for available light, water, and nutrients. A number of factors affect seed distribution in soil. Seed metering system, seed delivery tube, furrow opener design, physical attributes of seed and soil conditions all play a part in determining seed distribution....

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  1. SOYBEAN MOLECULAR ASPECTS OF BREEDING Edited by Aleksandra Sudarić
  2. Soybean - Molecular Aspects of Breeding Edited by Aleksandra Sudarić Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access articles distributed under the Creative Commons Non Commercial Share Alike Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published articles. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Katarina Lovrecic Technical Editor Teodora Smiljanic Cover Designer Martina Sirotic Image Copyright George Burba, 2010. Used under license from Shutterstock.com First published March, 2011 Printed in India A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from orders@intechweb.org Soybean - Molecular Aspects of Breeding, Edited by Aleksandra Sudarić p. cm. ISBN 978-953-307-240-1
  3. free online editions of InTech Books and Journals can be found at www.intechopen.com
  4. Contents Preface IX Part 1 Molecular Biology and Biotechnology 1 Chapter 1 Protein Expression Systems: Why Soybean Seeds? 3 Kenneth Bost and Kenneth Piller Chapter 2 Optimizing Recombinant Protein Expression in Soybean 19 Laura C. Hudson, Kenneth L. Bost and Kenneth J. Piller Chapter 3 Virus-Induced Gene Silencing of Endogenous Genes and Promotion of Flowering in Soybean by Apple latent spherical virus-Based Vectors 43 Noriko Yamagishi and Nobuyuki Yoshikawa Chapter 4 Genetic Improvement: Molecular-Based Strategies 57 Aleksandra Sudarić, Marija Vratarić, Snežana Mladenović Drinić, and Zvonimir Zdunić Chapter 5 Integration of Major QTLs of Important Agronomic Traits in Soybean 81 Guohua Hu, Qingshan Chen, Chunyan Liu, Hongwei Jiang, Jialin Wang and Zhaoming Qi Chapter 6 A Versatile Soybean Recombinant Inbred Line Population Segregating for Low Linolenic Acid and Lipoxygenase Nulls - Molecular Characterization and Utility for Soymilk and Bioproduct Production 119 Yarmilla Reinprecht, Shun-Yan Luk-Labey and K. Peter Pauls Chapter 7 Breeding for Promiscuous Soybeans at IITA 147 Hailu Tefera Chapter 8 Characterization of Soybean-Nodulating Rhizobial Communities and Diversity 163 Yuichi Saeki
  5. VI Contents Part 2 Breeding for Abiotic Stress 185 Chapter 9 Proteomics Approach for Identifying Abiotic Stress Responsive Proteins in Soybean 187 Mohammad-Zaman Nouri, Mahmoud Toorchi and Setsuko Komatsu Chapter 10 Molecular Responses to Osmotic Stresses in Soybean 215 Tsui-Hung Phang, Man-Wah Li, Chun-Chiu Cheng, Fuk-Ling Wong, Ching Chan and Hon-Ming Lam Chapter 11 Genotypic Influence on the Absorption, Use and Toxicity of Manganese by Soybean 241 Andre Rodrigues dos Reis and Jose Lavres Junior Part 3 Breeding for Biotic Stress 259 Chapter 12 Resistance to Pythium Seedling Disease in Soybean 261 Rupe, J.C., Rothrock, C.S., Bates, G., Rosso, M. L., Avanzato, M. V. and Chen, P. Chapter 13 Phomopsis Seed Decay of Soybean 277 Shuxian Li Chapter 14 Soybean Rust: Five Years of Research 293 Arianne Tremblay Chapter 15 Detection, Understanding and Controlof Soybean Mosaic Virus 335 Xiaoyan Cui, Xin Chen and Aiming Wang Chapter 16 Evolution of Soybean Aphid Biotypes: Understanding and Managing Virulence to Host-Plant Resistance 355 Andrew P. Michel, Omprakash Mittapalli and M. A. Rouf Mian Chapter 17 Evaluation and Utilization of Soybean Germplasm for Resistance to Cyst Nematode in China 373 Ying-Hui Li, Xiao-Tian Qi, Ruzhen Chang and Li-Juan Qiu Chapter 18 Cell-Specific Studies of Soybean Resistance to Its Major Pathogen, the Soybean Cyst Nematode as Revealed by Laser Capture Microdissection, Gene Pathway Analyses and Functional Studies 397 Vincent P. Klink, Prachi D. Matsye and Gary W. Lawrence Chapter 19 Genetically Modified Soybean for Insect-Pests and Disease Control 429 Maria Fatima Grossi-de-Sa, Patrícia B. Pelegrini and Rodrigo R. Fragoso
  6. VII Contents Part 4 Recent Technology 453 Chapter 20 Spectral Characteristics of Soybean during the Vegetative Cycle Using Landsat 5/TM Images in The Western Paraná, Brazil 455 Erivelto Mercante, Rubens A. C. Lamparelli, Miguel A. Uribe-Opazo and Jansle Viera Rocha Chapter 21 Bio-Based Nanocomposites Composed of Photo-Cured Soybean-Based Resins and Supramolecular Hydroxystearic Acid Nanofibers 473 Mitsuhiro Shibata Chapter 22 Transgenic Residues in Soybean-based Foods 495 Mónica L. Chávez-González, Carolina Flores-Gallegos, Víctor M. García-Lazalde, Cristóbal Noé Aguilar and Raúl Rodríguez-Herrera
  7. Preface Soybean (Glycine max (L.) Merr.) is the leading oil and protein crop of the world, which is used as a source of high quality edible oil, protein and livestock feed. Various functional components derived from secondary metabolite have also received significant attention in terms of human health. Over the past three decades, the scientific and technological developments in most regions have increased soybean production on the global level. Nevertheless, soybean breeding has undoubtedly played a key role in production in- creases. Conventional breeding strategies have been very successful in improving soy- bean productivity and quality. In practice today, the field of soybean breeding is in tran- sition and changing rapidly. The incorporation of molecular aspects of genetic analysis and molecular marker-assisted selection is critical to understanding soybean breeding strategies and practices. Scientific discoveries in the area of structural and functional plant genomics lead to development of new soybean varieties with advanced nutritive properties and yield enhancement through greater resistance to various abiotic and bi- otic factors, better adapted to new market, production and environment demands. Based on the availability and combination of conventional and molecular technologies, a sub- stantial increase in the rate of genetic gain for economically important soybean traits can be predicted in the next decade. The book Soybean - Molecular Aspects of Breeding focuses on recent progress in our under- standing of the genetics and molecular biology of soybean and provides a broad review of the subject, from genome diversity to transformation and integration of desired genes using current technologies. This book is divided into four parts (Molecular Biology and Biotechnology, Breeding for Abiotic Stress, Breeding for Biotic Stress, Recent Technol- ogy) and contains 22 chapters. Part I, “Molecular Biology and Biotechnology”, (Chapters 1 to 8) focuses on advances in molecular biology and laboratory procedures that have been developed recently to ma- nipulate DNA and provide new genes of interest to soybean breeder. Chapter 1 considers the transgenic soybean seed as a unique platform for the expression and accumulation of desired proteins. Chapter 2 focuses on incorporating current knowledge for optimizing recombinant protein expression in soybeans. In Chapter 3, the authors describe the use of Apple Latent Spherical Virus (ALSV) vector for Virus-Induced Gene Silencing (VIGS) of endogenous genes at all growth stages of soybean plants and seeds. Chapter 4 reviews the technologies for molecular marker analysis and achievements in the area of genetic transformation (genetic modification) in soybean. Chapter 5 points out on the QTL meta- analysis for major agronomic traits in soybean (oil content, protein content, fatty acid, amino acid content, isoflavone content, fungal diseases resistance, insect resistance, cyst
  8. X Preface nematode resistance, 100-seed weight, lodging, plant height, growth stages). Chapter 6 describes development of a versatile soybean recombinant inbred line population segre- gating for low linolenic acid and lypoxygenase nulls, its molecular characterization and utility for soymilk and composite material production. Chapter 7 introduces the achieve- ments of soybean breeding work at the International Institute of Tropical Agriculture (Malawi) with emphasis on enhancement biological nitrogen fixation capacity of new breeding lines through the promiscuity approach as well as matching genotypes with effective inoculants strains. Characterization of soybean-nodulating rhizobial commu- nities and its diversity is the subject of Chapter 8. Part II, “Breeding for abiotic stress” (Chapters 9 to 11) covers proteomics approaches form as a powerful tool for investigating the molecular mechanisms of the plant responses to various types of abiotic stresses. It provides a path toward increasing the efficiency of indirect selection for inherited traits. Chapter 9 describes recent methodologies for the extraction of proteins from soybean and then protein identification techniques related to the abiotic stresses. Chapter 10 is centered on the common and specific components of various types of osmotic stresses, tolerant germplasm-specific components, the current obstacles in this research area and the forward looking research strategies to tackle these problems. Studying anatomical and ultrastructural changes in response to manganese (Mn) nutritional disorders and deleterious effects of Mn stress on soybean is the subject of Chapter 11. Part III, “Breeding for biotic stress” (Chapters 12 to 19) addresses issues related to ap- plication of molecular based strategies in order to increase soybean resistance to various biotic factors (pathogens, insects, nematode). Chapter 12 reports the resistance to a num- ber of Pythium spp. that should be useful in reducing the risk of stand loss due to this group of pathogens. Chapter 13 introduces Phomopsis Seed Decay (PSD) with emphasis on application of SSR marker in identification resistant germplasm and using of the re- sistant cultivars as the most effective method for controlling PSD. Chapter 14 focuses on identification approaches to broaden the resistance to soybean rust caused by pathogen Phakospora pachyrhizi. In Chapter 15, authors describe Soybean Mosaic Virus (SMV), in- teraction between SMV and plant, as well as its current and future control strategies. Chapter 16 reviews the current status of soybean aphid biotypes and strategies for un- derstanding and managing virulence to host-plant resistance. Chapter 17 considers the advances of identification for resistance to cyst nematode, discovery novel gene from the resistant accession and resistant cultivar development. Cell-specific studies of soybean resistance to the cyst nematode as revealed by laser capture microdissection, gene path- way analyses and functional studies are the subject of Chapter 18. In Chapter 19, authors describe biotechnological insights using different molecules in order to decrease biotic stresses in soybean field. Part IV, “Recent Technology” (Chapters 20 to 22) reviews newer technologies into the realm of soybean monitoring, processing and product use. Studying the changes in the spectral behavior of the soybean crop, during the vegetative cycle, by spectral-temporal profiles of the mapped crop areas, using two vegetation indexes (Normalized Differ- ence Vegetation Index-NDVI; Greenness Vegetation Index-GVI) of multispectral images from the satellite Landsat 5/TM is the subject of Chapter 20. Chapter 21 describes the preparation and properties of the bio-nanocomposites composed of the ESO (epoxidized soybean oil) and AESO (acrylated epoxidized soybean oil) crosslinked by the photo-
  9. XI Preface polymerization and self-assembled HAS (hydroxystearic acid) molecules. Chapter 22 comments on a number of procedures for detection of transgenic residues in soybean- based foods. It becomes a very actual theme because the increase of products derived from genetically modified organisms in the market shelves and the consumer demands for more strict regulations about labeling of this kind of products. Each chapter of this book presents an excellent overview of a broad range of topics. The references at the end of each chapter provide a starting point to acquire a deeper knowledge on the state-of-the-art. While the information accumulated in this book is of primary interest to plant breeders, valuable insights are also offered to agronomists, molecular biologists, physiologists, plant pathologists, food scientists and students with an interest in plant breeding. The book is a result of efforts by many experts from different countries. I would like to acknowledge each of the authors who devoted much time and effort in delivering their chapter to this volume. We hope that this book will contribute to bringing about more informed modern techniques used in soybean breeding. Aleksandra Sudarić Agricultural Institute Osijek Osijek, Croatia
  10. Part 1 Molecular Biology and Biotechnology
  11. 1 Protein Expression Systems: Why Soybean Seeds? Kenneth Bost and Kenneth Piller University of North Carolina at Charlotte and SoyMeds, Inc. United States of America 1. Introduction The global protein therapeutics market is approaching $100 billion in annual sales. Furthermore, the in vitro diagnostic market, which relies heavily on the use of recombinant proteins as analyte specific reagents, is approaching $50 billion in annual sales. Increases in each market sector are estimated to be 8% to 20% per year for the next decade. Therefore, the costs of protein-based therapeutics and diagnostics will continue to be a significant percentage of health care expenses for patients and for agricultural animals and pets. A platform technology, which produces recombinant proteins at greatly reduced costs, provides inherent advantages, and allows low-tech sustainability of product lines, represents a competitive innovation which could create significant wealth in this industry. The use of soybean-derived proteins has the potential to provide such advantages. Since the use of recombinant proteins is widespread in human and animal therapeutics and diagnostics, there are thousands of potential applications for such a platform technology. While no one technology is optimal for the expression of every transgenic protein (Brondyk, 2009), the research that we have performed to date demonstrates the utility and feasibility of commercial applications for proteins made in transgenic soybean seeds. Soybean seeds expressing transgenic proteins represent a novel, sustainable platform technology which overcomes some of the current limitations for producing recombinant proteins for diagnostics, therapeutics, and industrial applications. Advantages include low cost of glycoslyated protein production, greenhouse containment, highest protein/biomass ratio, marketable formulations which require no purification from soy, safety, accurate dosing, low cost of protein purification, low-tech sustainability of product lines, reduced risk of contamination, ease of scalability, minimal waste produced, as well as being a green technology. To our knowledge, no other protein expression technology compares. Despite the fact that the present protein therapeutic and diagnostic markets are growing rapidly, some applications continue to be limited by the current technologies employed (Brondyk, 2009). For example the cost of production and purification of certain recombinant proteins makes their use in particular applications non-profitable. Some proteins cannot be expressed by any current technology and require expensive purification from human or animal tissues. A platform technology which could alleviate these concerns would create significant opportunities for novel product development, or expanded applications for existing products. In short, there is a need for alternative technologies for expressing and purifying recombinant proteins which can advance their applications. We propose that
  12. 4 Soybean - Molecular Aspects of Breeding soybean seeds expressing transgenic proteins represent a platform technology that can be a solution for many of these problems. Recent research provides a strong basis for this supposition. These results support the utility of this technology, have demonstrated its advantages, and suggest additional benefits that are currently being explored. 2. Utility of glycosylated proteins produced in transgenic soybean seeds Roundup Ready soybeans were one of the first examples of a commercially viable transgenic plant (Padgette et al., 1995). These transgenic soybeans express a functional enzyme, 5-enolpyruvylshikimate-3-phosphate synthase, making the plants tolerant to the herbicide, RoundupTM. Presently, approximately 90% of the soybeans farmed in the United States are Roundup Ready. In addition to this value-added trait, there have been several recent successes in modifying soy crop lines aimed at imparting some commercial advantage. Transgenic soybean lines expressing the cry1A gene provide protection against Lepidopteran species (McPherson & MacRae, 2009). Transgenic soybean lines expressing an active APase, demonstrated increased phosphorus content when grown in soils with limited phosphate content (Wang et al., 2009). Alterations in seed oil content have been achieved using transgenic soybean plants expressing genes having lysophosphatidic acid acyltransferase activity (Rao & Hildebrand, 2009). Over-expression of an aspartate kinase in transgenic seeds allowed increased threonine levels to occur (Qi et al., 2010). While these examples are not a comprehensive listing of value-added traits that require the expression of a function enzymatic protein, they serve to demonstrate the amenability of soybean seeds to such transformations. While value-added traits are being exploited, transgenic soybean seeds have also been used as bioreactors to express a variety of foreign proteins. For example, Zeitlin, et al. (Zeitlin et al., 1998) successfully expressed functional antibodies against herpes simplex virus-2 glycoprotein B in transgenic soybeans. More recently, proinsulin has been expressed and the storage vacuoles can accumulate mature polypeptide in these seed lines (Cunha et al., 2010). In our laboratories, we have successfully expressed the subunit protein antigens, E. coli, FanC (Garg et al., 2007; Oakes, Bost, & Piller, 2009; Piller et al., 2005), non-toxic, mutant forms of several bacterial toxins, and potential immunomodulatory proteins in transgenic soybean seeds, and are evaluating their usefulness as therapeutics. We have also successfully expressed particular full-length human proteins, and ongoing studies are aimed at demonstrating their substantial equivalence for use as analyte specific reagents in diagnostic assays. While this is not an exhaustive listing of the plant and foreign proteins which have been expressed in transgenic soybean seeds, these examples demonstrate the utility of this platform technology. There are several reasons for the success of such endeavors, but perhaps the most important lies in the biology of the soybean seed itself. One of the most important functions of the seed is to express and package proteins. This entails post- translational modifications, including glycosylation, and packaging which not only allows proper folding, but also provides an environment for stable, long-term protein storage. This conclusion is supported by the fact that many of the transgenic proteins expressed have enzymatic activity, ability to bind antigen, or the ability to be recognized by monoclonal antibodies specific for their native counterparts.
  13. 5 Protein Expression Systems: Why Soybean Seeds? 3. Soybean seeds represent the highest protein to biomass ratio Soybean seeds, by weight, are 40% protein with approximately 20% oil, 35% carbohydrates, and 5% ash (Liu, 1999). Most of the normal soy seed proteins are heat-stable and desiccant- resistant, in keeping with the ability of soybeans to remain germinate-capable following years of storage in ambient conditions. Soybean plants can produce as much as twice the protein per acre of any other major crop (see: http://www.soyatech.com/soy_health.htm). Protein production by soy is also more efficient than animal-derived protein when factoring the acreage required for grazing or feeding. As we will discuss below, this fundamental characteristic of soybean seeds to produce and store large amounts of protein may be exploited as a platform technology for expression. 4. High yield translates into a potential for low cost protein production Present and future use of recombinant proteins will be limited in large part by their cost of production. Presently, the expense of expressing and purifying some recombinant proteins prohibits or limits their practical or realistic use. This is true for some therapeutic, as well as diagnostic, applications in westernized societies, and such barriers are even greater for developing countries. Unless this economic burden can be overcome, barriers for product development will remain. For some applications, too much recombinant protein is needed such that the cost is prohibitive. For some applications, elaborate purification schemes make particular proteins unaffordable. For some applications, there is no source of some recombinant proteins, requiring isolation from human or animal tissues. A platform technology which could alleviate these concerns would create significant opportunities for novel product development, or expanded applications for existing products, and therefore, create significant wealth. Decisions to develop commercial products which include such proteins will depend largely on the practicality of having a cost-sustainable platform technology. Theoretically, expression of transgenic proteins in soybean seeds represents one of the most cost-efficient platforms, and recent work has demonstrated potential economic advantages. Presently we (Garg, et al., 2007; Oakes, Bost, et al., 2009; Piller, et al., 2005), and others (Cunha, et al., 2010; Ding, Huang, Wang, Sun, & Xiang, 2006; Moravec, Schmidt, Herman, & Woodford-Thomas, 2007; Qi, et al., 2010; Rao & Hildebrand, 2009; Wang, et al., 2009; Zeitlin, et al., 1998), have developed stable soybean lines that express 1% to 4% of their total soluble protein as the transgenic protein. Since soybean seeds are 40% protein by weight, an average sized seed weighing approximately 150 milligrams represents approximately 2.4 milligrams of transgenic protein per seed at 4% expression (Table 1). As will be discussed below, soybeans can easily be converted into soy powder, and, one liter of this powder totals approximately 800 grams of seed material. At 4% expression, this one liter of soy powder contains approximately 12.8 grams of the unpurified, transgenic protein (Table 1). It is useful to compare this production with that for a liter of broth from bacterial (Zerbs, Frank, & Collart, 2009), yeast (Cregg et al., 2009), insect (Jarvis, 2009), mammalian (Geisse & Fux, 2009), or plant (Hellwig, Drossard, Twyman, & Fischer, 2004; Lienard, Sourrouille, Gomord, & Faye, 2007) cell culture. Since retail costs of commercially available recombinant proteins can easily be hundreds to thousands of dollars per milligram, extrapolations presented in Table 2 are enlightening. One liter, or 800 grams, of soy powder could represent as much as 12.8 grams of transgenic protein
  14. 6 Soybean - Molecular Aspects of Breeding at an expression level of 4%. As shown in Table 2, potentially, as little as one liter of total soy protein could contain the equivalent of millions of dollars of unpurified transgenic protein. Milligrams of transgenic Grams of transgenic Percent expression of the protein per seed (150 protein per liter (800 transgenic protein milligrams) grams of soy powder) 1% 0.6 milligrams 3.2 grams 2% 1.2 milligrams 6.4 grams 4% 2.4 milligrams 12.8 grams 8% 4.8 milligrams 25.6 grams Table 1. Estimated amounts of transgenic protein per seed or per one liter of soy powder Linear extrapolation of commercial value for Retail commercial cost of a theoretical 12.8 grams of the theoretical recombinant recombinant protein per milligram protein $10 $128,000 $100 $1,280,000 $1000 $12,800,000 $10,000 $128,000,000 Table 2. Extrapolation of the potential value for one liter of soy protein powder expressing a particular transgenic protein at a level of 4% Further extrapolations can be made for bulk production of transgenic soybeans in secure greenhouses. Such greenhouses provide containment and controlled conditions which allow optimal growth for maximal yields. Using such conditions, it is not difficult to obtain 60 bushels of soybeans per greenhouse acre (see http://www.soystats.com). The industry standard for an average weight of a bushel of soybeans is 60 pounds, with an average quantity of 2500 seeds per pound. This computes to approximately 9 million transgenic soybean seeds per acre. At an average weight of 150 milligrams per seed, this represents approximately 1,350 kilograms of seeds or 540 kilograms of total soy protein. As shown in Table 3, at already achieved 4% expression levels, this calculates to 21.6 kilograms of transgenic protein per greenhouse acre. Percentage Average estimated quantity of Calculated quantity of expression of the soybean protein per greenhouse transgenic protein per transgenic protein acre greenhouse acre 5.4 kilograms of transgenic 1% 540 kilograms total soy protein protein 10.8 kilograms of transgenic 2% 540 kilograms total soy protein protein 21.6 kilograms of transgenic 4% 540 kilograms total soy protein protein 43.2 kilograms of transgenic 8% 540 kilograms total soy protein protein Table 3. Estimated quantities of transgenic protein per greenhouse acre based on percent expression
  15. 7 Protein Expression Systems: Why Soybean Seeds? It should be noted that we have included 8% expression levels of the transgenic protein in Tables 1 and 3. While we have yet to achieve such levels in our laboratory, the use of transgenic soybeans to express foreign proteins is evolving [e.g. (Schmidt & Herman, 2008)]. Recent DNA sequencing of the soybean genome (Hyten et al., 2010; Schmutz et al., 2010), the development of better promoters, engineering of high protein-expressing seeds, and other coming advances, promise to further increase the efficiency of expression of transgenic proteins using this platform technology. Therefore it seems reasonable to conclude that future advances will only facilitate our ability to increase the level of protein expression. While future advances promise even higher percentages of transgenic protein expression, current levels will permit contained greenhouse to produce bulk quantities of particular proteins. Using theoretical costs for a recombinant protein as before, Table 4 extrapolates the potential value for an acre of greenhouse grown soybeans at an expression level of 4%. Again, these numbers serve to underscore the potential for high capacity of transgenic protein production using a confined growth space. Calculated quantity of Linear extrapolation for the Retail commercial cost of a transgenic protein per potential value of 21.6 theoretical recombinant greenhouse acre at kilograms of the theoretical protein per milligram 4% expression recombinant protein 21.6 kilograms of transgenic $10 $216,000,000 protein 21.6 kilograms of transgenic $100 $2,160,000,000 protein 21.6 kilograms of transgenic $1,000 $21,600,000,000 protein 21.6 kilograms of transgenic $10,000 $216,000,000,000 protein Table 4. Extrapolation of the potential value for an acre of greenhouse soybeans expressing a particular transgenic protein at a level of 4% 5. Greenhouse containment for growing transgenic soybeans As shown in Tables 1, 2, 3, and 4, it is clear that at expression levels already obtained (e.g. 1% - 4%) there will be little reason to grow such transgenic plants in open fields. At production levels of 3-10 kilograms per acre, and with the potential for 3 separate growing seasons per year, propagation in contained greenhouses would impart few limitations, even for bulk production. Despite the fact the soybean plant is self pollinating, secure greenhouse growth would provide additional containment by eliminating transgenic seed escape (Traynor, 2001). As will be discussed below, processing of soybean seeds to soy powder can easily be accomplished in the greenhouse prior to removal of this non-germinating material for formulation and/or protein purification. Such containment procedures, therefore, provide management of these genetically modified crops from release into the environment. Propagation of transgenic soybean plants in secure greenhouses also provides advantages in addition to containment. Therapeutic and diagnostic proteins must be produced with good manufacturing practices (GMP) as dictated by approval agencies. Greenhouse growth
  16. 8 Soybean - Molecular Aspects of Breeding allows for ease of standard operating procedures to be implemented with respect to growth conditions, disease and pathogen monitoring, harvesting, and quality control. Stated simply, there are numerous advantages for greenhouse growth, and very few reasons for proposing open field propagation of transgenic soybean plants, that are destined to produce proteins for therapeutic and diagnostics purposes. 6. Costs to grow an acre of transgenic soybeans in contained greenhouses The efficiency with which soybean plants can be grown in the field or in greenhouses is well documented (Traynor, 2001). This understanding of maximizing crop yields serves to reduce the cost of producing seeds from transgenic plants. Current costs per acre to plant and harvest soybeans from open fields range from $300 to $600 per acre depending upon planting conditions, treatments, geographic area, etc. It has been estimated that greenhouse containment for soybean growth at Biosafety Level 2 (BSL-2) would increase this cost approximately 20 fold (Traynor, 2001). Despite this increased cost and the benefits that go with greenhouse production, the total expense for planting and harvesting remains a reasonable $6,000 to $12,000 per acre. Based on these estimates, it is possible to project a cost per milligram for production and harvest of soybeans expressing a transgenic protein using BSL-2 conditions (Table 5). Percentage Costs per milligram of Calculated quantity of transgenic expression of the protein assuming $12,000 protein per greenhouse acre transgenic protein production costs 5.4 kilograms of transgenic 1% $ 0.002 per milligram protein 10.8 kilograms of transgenic 2% $ 0.001 per milligram protein 21.6 kilograms of transgenic 4% $ 0.0005 per milligram protein 43.2 kilograms of transgenic 8% $ 0.00025 per milligram protein Table 5. Cost production projections per milligram of transgenic protein using BSL-2 greenhouse conditions and assuming $12,000 per acre total production costs It is clear to see the potential for large scale production of transgenic proteins at a very low cost of production when one considers such calculations. 7. Formulations which require no purification of the transgenic protein from soy The cost projections in Table 5 pertain only to the growth and harvesting of soybean seeds expressing the transgenic protein. As with all protein expression systems, additional costs are required for purification of the protein of interest. However, before we discuss purification costs and the advantages of soybean-derived proteins, an intriguing possibility is the use of formulations made from transgenic soybean seeds which would require no purification of the protein prior to its use.
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