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- SOYBEAN MOLECULAR
ASPECTS OF BREEDING
Edited by Aleksandra Sudarić
- Soybean - Molecular Aspects of Breeding
Edited by Aleksandra Sudarić
Published by InTech
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- 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
- 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
- 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
- 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
- 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-
- 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
- Part 1
Molecular Biology and Biotechnology
- 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
- 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.
- 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
- 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
- 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
- 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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