**3. Agronomical improvements in soybean**

### **3.1. Herbicide tolerance**

that have been successfully optimized for stable soybean transformation include cotyledona‐ ry node–*Agrobacterium*-mediated transformation and somatic embryo–particle-bombard‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The *Agrobacterium*-mediated plant transformation method uses a soil dwelling bacteria spe‐ cies called *Agrobacterium tumefaciens* to transfer desirable genes into plants. Using this meth‐ od, a foreign gene can be placed within the T- DNA boarder regions of the bacterial plasmid which then integrates into a host plant's genome [1]. Wounded plant tissue gives off specific phenolic compounds which induce *Agrobacterium* to express a set of virulence (vir) genes. The expression of the vir genes results in the production of single-stranded DNA that is

There are several advantages of *Agrobacterium*-meditated plant transformation including straight forward methodology, minimal equipment cost, and reliable insertion of a single or a low copy transgene number. The first reported transformation of soybean with an *Agrobac‐ terium* strain used co-cultivation followed by organogenesis from cotyledonary nodes [2]. This work was followed by using *Agrobacterium* mediated transformation of immature coty‐ ledons [3], and embryogenic suspension cultures [4]. Since then, several groups have worked to improve these methods, in particular the transformation and regeneration from cotyledonary nodes. Cotyledonary node regions contain axillary meristems at the junction between cotyledon and hypocotyl. Generally, the cotyledonary nodes are pre-wounded and then co-cultivated with *Agrobacterium*. The axillary meristems proliferate and regenerate through the formation of multiple adventitious shoots on culture medium containing a cyto‐ kinin. In the United States, public facilities, including the Plant Transformation Facility at Io‐ wa State University and the Plant Transformation Core Facility at the University of Missouri, provide fee for service genetic transformation of soybean for public research,

The other widely used method of soybean transformation is somatic embryo particle bom‐ bardment-mediated transformation also called particle bombardment, or biolistic technolo‐ gy. This method directs small tungsten or gold particles coated with the desired genes toward the target plant cells with enough force to penetrate the cell wall and membrane [5]. Once inside the cell the DNA disassociates from the particle and becomes integrated into the

The particle bombardment transformation method was first used in soybean in 1988 by McCabe et al., who successfully transformed immature seed meristem [6] and was followed by the transformation of somatic embryonic tissue [7], and apical meristem [8]. Transforma‐ tion of somatic embryos has been the most successful method and is induced from imma‐ ture cotyledons cultured on medium containing moderately high concentrations of an auxin. These cotyledons are used to generate proliferative embryogenic cultures and to recover whole plants. A major advantage of the particle bombardment transformation method rela‐ tive to *Agrobacterium*-based methods is the removal of biological incompatibilities between tissues of many plant species and the *Agrobacterium* vector. However, it has been shown that certain genotypes are more susceptible to the formation of proliferative embryogenic tissue than others. Limitations of the bombardment process include the requirement of specialized

ment-mediated transformation.

Relationships

448

plant genome.

transferred and integrated into the plant genome.

mainly by cotyledonary node *Agrobacterium*-mediated transformation.

In 1970, glyphosate, a broad-spectrum foliar herbicide, was discovered [9]. Glyphosate in‐ hibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a crucial enzyme of the shiki‐ mate biosynthetic pathway that is responsible for the production of several essential aromatic amino acids. Glyphosate was commercially introduced in 1974 and by 1995 use had reached 4. 5 million kg in the United States. Due to popularity and broad use of this herbicide by farmers, glyphosate became a candidate for research in creating herbicide re‐ sistant soybeans and has led to one of the most well-known examples of herbicide tolerance: The Roundup Ready® soybean developed by Monsanto. Roundup Ready® soybeans were one of the first examples of a commercially viable transgenic plant. These transgenic soy‐ beans express functional EPSPS providing tolerance to the herbicide glyphosate (Round‐ upTM). The popularity of these soybeans grew with farmers since RoundupTM could be applied to a field of Roundup Ready® soybeans to significantly reduce weed populations while leaving the soybean crop unharmed. The development of Roundup Ready® soybeans offered farmers many advantages in a system that was relatively easy to use. The level and consistency of weed control allowed farmers to take advantage of a no-till system, and elim‐ inated the need for cultivation allowing growers to space rows more closely. Narrow row planting results in higher yields due to a more efficient use of space and may result in better weed control, as the canopy closes more quickly providing earlier competition against weeds. In addition, the window of application for RoundupTM is wider than for other post emergence herbicides currently used in soybeans, both in terms of the stage of soybean growth and the ability to achieve effective control of larger weeds. These factors contributed to the popularity of this weed control technology. At present, approximately 90% of the soy‐ beans farmed in the United States utilize this technology.

In response to Monsanto's hugely successful Roundup Ready® crops, Bayer Crop Science re‐ leased its own herbicide tolerant soybean known as Liberty Link® soybean [10]. Liberty Link® soybeans were developed to express a gene derived from the bacteria *Streptomyces vir‐ idochromogenes* called phosphinothricin-N-acetyltransferase (PAT). PAT is a glutamine syn‐ thetase inhibitor that binds to glutamate, making plants resistant to the broad-spectrum contact herbicide glufosinate ammonium. This herbicide causes cessation of photosynthesis and plant death by interfering with the biosynthetic pathway of the amino acid glutamine

and with ammonia detoxification. Glufosinate ammonium is the active ingredient in phos‐ phinothricin herbicides (Basta®, Ignite®, Rely®, Liberty®, Harvest®, and Finale®) used to con‐ trol a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Since glufosinate ammonium-based herbicides function by a different mode of action than glyphosate-based herbicides, the Liberty Link® system provides farmers with an alternative strategy for controlling weeds.

There are several other examples of transgenic soybeans expressing herbicide resistance traits that are in various stages of development. Pioneer has developed a transgenic soybean product that provides tolerance to two different classes of herbicides: glyphosate and aceto‐ lactate synthase (ALS)-inhibiting herbicides. These soybean plants express the glyphosate acetyltransferase (GAT4601) and modified version of a soybean acetolactate synthase (GM-HRA) proteins. [11]. The GAT4601 protein confers tolerance to glyphosate-containing herbi‐ cides by acetylating glyphosate and thereby rendering it non-phytotoxic. The GM-HRA protein confers tolerance to the ALS-inhibiting class of herbicides. The development of GM soybeans with characteristics controlled by multiple genes leading to the expression of two herbicides is a different approach than previous strategies involving single characteristics controlled by a single gene. These genetically modified soybeans express a combination of herbicides with different modes of action. Inherent crop tolerance will enable more effective management of weed populations.

BASF has used a similar method to introduce a soybean that combines herbicide-tolerant soybean varieties with the broad spectrum imidazolinone class of herbicides. These trans‐ genic soybeans contain the csr1-2 gene derived from *Arabidopsis thaliana* that encodes the imidazolinone-tolerant AHAS-Large subunit (also known as ALS). The AHAS-L subunit in‐ teracts with the endogenous soybean small regulatory subunit to form an enzyme complex that catalyzes the first step in the synthesis of branched-chain essential amino acids, valine, leucine, and isoleucine [12]. The AHAS enzymes occur ubiquitously in plants. Imidazoli‐ none herbicides inhibit the native enzymes, resulting in plant death.

To address the potential emergence of other herbicide resistant broadleaf weeds, Monsanto has developed a line of transgenic soybeans that are resistant to treatment with dicamba [13]. Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a low-cost, widely-used, broad leaf herbicide that is environmentally friendly. Soybeans transformed with a genetically engi‐ neered bacterial dicambamonooxygenase (DMO) gene were able to inactivate dicamba, making them resistant to this herbicide. Dicamba-resistant soybeans are in the advanced stages of research and development and are predicted to be commercialized soon.

Syngenta and Bayer CropScience are co-developing HPPD-inhibitor tolerant soybeans, a novel herbicide tolerance trait for soy. The event consists of a molecular stack of a gene con‐ ferring tolerance to hydroxyphenylpyruvatedioxygenase (HPPD)-inhibiting herbicides as well as a gene for glufosinate tolerance. Inhibition of HPPD stops the catabolic degradation of tyrosine to plastoquinones which is important for photosynthesis, carotenoid biosynthe‐ sis, and tocopherol production [14]. This multiple herbicide tolerance stack will enable the use of multiple herbicides and will be an important new tool for soybean growers faced with increasing pressure from resistant weeds. In the future, other innovative molecular strategies can be expected to generate genetically modified (GM) soybeans with novel fea‐ tures to combat weeds and enhance weed resistance. These new GM soybeans will reduce environmental contamination risks and reduce costs for consumers and producers.

### **3.2. Insect resistance**

and with ammonia detoxification. Glufosinate ammonium is the active ingredient in phos‐ phinothricin herbicides (Basta®, Ignite®, Rely®, Liberty®, Harvest®, and Finale®) used to con‐ trol a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Since glufosinate ammonium-based herbicides function by a different mode of action than glyphosate-based herbicides, the Liberty Link® system provides farmers

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

There are several other examples of transgenic soybeans expressing herbicide resistance traits that are in various stages of development. Pioneer has developed a transgenic soybean product that provides tolerance to two different classes of herbicides: glyphosate and aceto‐ lactate synthase (ALS)-inhibiting herbicides. These soybean plants express the glyphosate acetyltransferase (GAT4601) and modified version of a soybean acetolactate synthase (GM-HRA) proteins. [11]. The GAT4601 protein confers tolerance to glyphosate-containing herbi‐ cides by acetylating glyphosate and thereby rendering it non-phytotoxic. The GM-HRA protein confers tolerance to the ALS-inhibiting class of herbicides. The development of GM soybeans with characteristics controlled by multiple genes leading to the expression of two herbicides is a different approach than previous strategies involving single characteristics controlled by a single gene. These genetically modified soybeans express a combination of herbicides with different modes of action. Inherent crop tolerance will enable more effective

BASF has used a similar method to introduce a soybean that combines herbicide-tolerant soybean varieties with the broad spectrum imidazolinone class of herbicides. These trans‐ genic soybeans contain the csr1-2 gene derived from *Arabidopsis thaliana* that encodes the imidazolinone-tolerant AHAS-Large subunit (also known as ALS). The AHAS-L subunit in‐ teracts with the endogenous soybean small regulatory subunit to form an enzyme complex that catalyzes the first step in the synthesis of branched-chain essential amino acids, valine, leucine, and isoleucine [12]. The AHAS enzymes occur ubiquitously in plants. Imidazoli‐

To address the potential emergence of other herbicide resistant broadleaf weeds, Monsanto has developed a line of transgenic soybeans that are resistant to treatment with dicamba [13]. Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a low-cost, widely-used, broad leaf herbicide that is environmentally friendly. Soybeans transformed with a genetically engi‐ neered bacterial dicambamonooxygenase (DMO) gene were able to inactivate dicamba, making them resistant to this herbicide. Dicamba-resistant soybeans are in the advanced

Syngenta and Bayer CropScience are co-developing HPPD-inhibitor tolerant soybeans, a novel herbicide tolerance trait for soy. The event consists of a molecular stack of a gene con‐ ferring tolerance to hydroxyphenylpyruvatedioxygenase (HPPD)-inhibiting herbicides as well as a gene for glufosinate tolerance. Inhibition of HPPD stops the catabolic degradation of tyrosine to plastoquinones which is important for photosynthesis, carotenoid biosynthe‐ sis, and tocopherol production [14]. This multiple herbicide tolerance stack will enable the use of multiple herbicides and will be an important new tool for soybean growers faced with increasing pressure from resistant weeds. In the future, other innovative molecular

stages of research and development and are predicted to be commercialized soon.

none herbicides inhibit the native enzymes, resulting in plant death.

with an alternative strategy for controlling weeds.

Relationships

450

management of weed populations.

Insect pest management through the use of chemicals has brought about considerable pro‐ tection to crop yields over the past several decades. Unfortunately, extensive and indiscrimi‐ nate usage of chemical pesticides has resulted in environmental degradation, adverse effects on human health and other organisms, eradication of beneficial insects, and development of pest-resistant insects. As farmers move forward with the objective of achieving greater crop productivity it will be imperative to replace chemical inputs with safer alternatives to man‐ age insect pests in agricultural ecosystems. Within agricultural biotechnology, insect resist‐ ance is a prime research area that has potential to improve agricultural productivity and provide much needed alternatives to pesticides while being effective against pests, innocu‐ ous to non-target organisms, and cost effective. With the advent of biotechnology, the ability to genetically modify plants for insect resistance on a commercial scale is within reach. One of the most extensively studied traits for insect resistance in soybeans involves the Bt gene.

*Bacillus thuringiensis* (Bt) is a common bacteria found in the environment. It has been used as a biological control agent against lepidopteran insects for more than 50 years. Bt targets a class of compounds responsible for insecticidal activity known as crystalline proteins, or cry proteins (Cry1), that are highly toxic after ingestion. The mode of action for Cry1 toxins is the disruption of midgut cellular membranes leading to cell death. One of the primary ad‐ vantages of using Bt genes for insect control in transgenic plants is the specific insecticidal action toward insects from the Lepidoptera order leaving beneficial insects, birds, and mam‐ mals unharmed. Thus, the insertion of Bt toxins into plants, by genetic modification, is an attractive model for the creation of insect resistant transgenic crops.

To date, many different plant species have been genetically modified to exhibit insect resist‐ ance using Bt. While the Bt trait has been commercialized in corn and cotton, it is still in de‐ velopmental stages in soybean. Transformation of soybean with Bt to induce resistance to lepidopteron species has been performed for over a decade. By 1994 fertile transformed soy‐ beans containing a synthetic Bt (Cry1Ac) were generated [15, 16]. Stewart et al., used detach‐ ed leaf bioassays to show that transgenic soybean lines were resistant to multiple soybean pests with less than 3% leaf defoliation compared to 20% observed in traditionally bred lepi‐ dopteron resistance soybean lines [16].

Other groups have used a similar strategy by evaluating soybeans engineered with Cry1Ac for resistance to lepidopteron species under field conditions. One example compared Bt lines to controls in the field using field cages and artificial infestation with lepidopteron lar‐ vae over a three year period [17]. In this case, Bt lines showed up to 9 times less defoliation from pets when compared to control plants. Similarly, Mcpherson and MacRae reported the evaluation of Bt soybean lines for suppression of lepidopteron species in the field over 2 years [18]. In this case, soybean plants expressing Cry1Ac were essentially absent of lepi‐ dopteron populations when compared to peak population densities of 20-30 larvae per row

in control plots. Furthermore, Bt lines showed <1.5% defoliation when compared to 53% de‐ foliation in control plants.

The utility for Bt soybeans has become evident. This has lead to pyramiding strategies using Cry1Ac with native plant resistance genes to increase plant resistance against insect-pests. Several quantitative trait loci (QTLs) from soybean lines have been described as showing an‐ tixenosis and antibiosis resistance towards lepidopteron insects [19, 20]. This work lead to the development of transgenic soybean lines by combining QTLs with synthetic Cry1Ac [21]. In this case, field evaluations and detached leaf bioassays were used to test this multi‐ ple resistance gene pyramiding strategy for antibiosis resistance. Based on defoliation in the field, as well as larval weight gain on detached leaves, soybean lines carrying a combination of Cry1Ac and the QTL were significantly more resistant to lepidopteron pests.

While Bt soybean varieties have not been commercialized this body of research has lead Monsanto to the development of soybeans that incorporate the Bt trait stacked with the sec‐ ond generation Roundup Ready germplasm [22]. Bt Roundup Ready 2 Yield seeds are cur‐ rently in Phase IV trials and are targeted for commercialization in Brazil in 2013. This pyramiding strategy would be the first in-seed insect protection for soybeans and is expect‐ ed to offer an important technology for farmers who face significant yield loss due to insect damage. Although not universal in its application and total in its protection, Bt will play a central role in protecting the crop from major insect pests.

With the onset and success of Bt crops other avenues have been explored for their possible roles in the development of transgenic insect resistant plants. These approaches include the use of plant defense proteins, lectins, α-amylase inhibitors, insect chitinases, and defensins. The development and implementation of engineered insecticidal soybean varieties is cur‐ rently in its infancy. The incorporation of a multiple gene stacking strategy will also be im‐ portant in the future development of insect resistant soybeans. Bt, in combination with other biopesticides, has the potential to drastically reduce the consumption of chemical pesticides, however it will be important to continue research and have a development strategy for a fu‐ ture generation of technology, to ensure that insects do not rapidly develop resistance.

### **3.3. Disease resistance**

The United States, Brazil, and Argentina are the three major soybean-producing countries in the world where more than 50% of all soybeans are harvested. Such a geographic distribu‐ tion facilitates the spread of insect-pests and diseases. Hence, soybean can be attacked by many different pathogens, including bacteria, viruses, fungi, and nematodes. These patho‐ gens and pests can cause damage in seeds, roots, leaves, stems and pods, and usually are tissue-specific. Therefore, disease resistance is another area of great interest for both re‐ searchers and farmers.

Disease control management is currently concentrated on agronomic practices, like planting under tillage, use of lodging resistant varieties, wide row planting, and rotation with nonhost crops. Chemical control has poor efficiency because of low penetration and uneven dis‐ tribution due to an already formed canopy. In addition, chemical application can be extremely expensive for farmers and unhealthy for the environment. There has been little success with conventional plant breeding for disease resistance in soybean leaving room for other approaches such as the use of biotechnology to produce genetically modified soybeans that have disease resistance.

### *3.3.1. Bacterial*

in control plots. Furthermore, Bt lines showed <1.5% defoliation when compared to 53% de‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The utility for Bt soybeans has become evident. This has lead to pyramiding strategies using Cry1Ac with native plant resistance genes to increase plant resistance against insect-pests. Several quantitative trait loci (QTLs) from soybean lines have been described as showing an‐ tixenosis and antibiosis resistance towards lepidopteron insects [19, 20]. This work lead to the development of transgenic soybean lines by combining QTLs with synthetic Cry1Ac [21]. In this case, field evaluations and detached leaf bioassays were used to test this multi‐ ple resistance gene pyramiding strategy for antibiosis resistance. Based on defoliation in the field, as well as larval weight gain on detached leaves, soybean lines carrying a combination

While Bt soybean varieties have not been commercialized this body of research has lead Monsanto to the development of soybeans that incorporate the Bt trait stacked with the sec‐ ond generation Roundup Ready germplasm [22]. Bt Roundup Ready 2 Yield seeds are cur‐ rently in Phase IV trials and are targeted for commercialization in Brazil in 2013. This pyramiding strategy would be the first in-seed insect protection for soybeans and is expect‐ ed to offer an important technology for farmers who face significant yield loss due to insect damage. Although not universal in its application and total in its protection, Bt will play a

With the onset and success of Bt crops other avenues have been explored for their possible roles in the development of transgenic insect resistant plants. These approaches include the use of plant defense proteins, lectins, α-amylase inhibitors, insect chitinases, and defensins. The development and implementation of engineered insecticidal soybean varieties is cur‐ rently in its infancy. The incorporation of a multiple gene stacking strategy will also be im‐ portant in the future development of insect resistant soybeans. Bt, in combination with other biopesticides, has the potential to drastically reduce the consumption of chemical pesticides, however it will be important to continue research and have a development strategy for a fu‐ ture generation of technology, to ensure that insects do not rapidly develop resistance.

The United States, Brazil, and Argentina are the three major soybean-producing countries in the world where more than 50% of all soybeans are harvested. Such a geographic distribu‐ tion facilitates the spread of insect-pests and diseases. Hence, soybean can be attacked by many different pathogens, including bacteria, viruses, fungi, and nematodes. These patho‐ gens and pests can cause damage in seeds, roots, leaves, stems and pods, and usually are tissue-specific. Therefore, disease resistance is another area of great interest for both re‐

Disease control management is currently concentrated on agronomic practices, like planting under tillage, use of lodging resistant varieties, wide row planting, and rotation with nonhost crops. Chemical control has poor efficiency because of low penetration and uneven dis‐ tribution due to an already formed canopy. In addition, chemical application can be

of Cry1Ac and the QTL were significantly more resistant to lepidopteron pests.

central role in protecting the crop from major insect pests.

foliation in control plants.

Relationships

452

**3.3. Disease resistance**

searchers and farmers.

Bacterial infections are widespread diseases that occur mainly in the mid-to-upper and young leaves of the soybean plant. There are several bacteria which cause disease in soy‐ bean resulting in large amounts of yield loss and poor seed quality. While there has been promising research in the development of bacterial disease resistance with the use of bio‐ technology in other crop plants such as rice, tomato, banana, and tobacco, there has been less research on the development of bacterial disease resistance for soybean. This research may lead to new strategies for the development of bacterial disease resistance in soybean.

### *3.3.2. Viral*

The development of transgenic soybean that confers viral resistance has been studied a bit more extensively. Viruses in soybean are global pests. Significant resistance to several virus‐ es in a number of plant species have been achieved through pathogen derived resistance by the use of viral coat proteins which, when expressed *in planta,* can interfere with viral assem‐ bly. This is the same approach that has been used by several groups to develop transgenic viral resistance in soybean. One of the first groups to investigate this approach was Di et al., who produced a soybean that was resistant to bean pod mottle virus (BPMV) [23]. This was done by introducing a BPMV coat protein into the soybean genome. Transgenic events showed complete resistance to BPMV infection. Another study created soybean lines that were resistant to BPMV by inserting a BPMV capsid polyprotien. Events generated in this case were subjected to infectivity assays and not only exhibited resistance to virus infection, but also exhibited systemic infection, showing little to no visible symptoms [24]. Transgenic lines such as these could lead to future commercial cultivars with resistance to BPMV.

The development of soybean mosaic virus (SMV) resistant soybeans is important since SMV is found in all regions where soybean is grown and infection can cause yield loss up to 90%. Despite progress in other important crop plants, efforts to produce transgenic soybeans re‐ sistant to SMV have advanced slowly. In order to produce soybean lines that could confer pathogen derived resistance, plants were produced containing a coat protein gene and the 3' UTR from SMV [25]. Coat protein gene transcripts were detected in transgenic lines and two of the soybean lines were highly resistant to infections with the SMV virus. These results represent the first example of stable genetically engineered SMV resistance in soybean.

The sense coat protein gene of soybean dwarf virus (SbDV) was used to acquire SbDV-re‐ sistant soybean plants [26]. These insertions were classified into two types: overexpression of SbDV-CP mRNA, or repression accumulation of SbDV-CP mRNA, and siRNA by RNA analysis prior to SbDV inoculation. In both cases, after infection with SbDV, most plants of these transgenic lines remained symptomless, contained little SbDV-specific RNA and ex‐

hibited SbDV-CP-specific siRNA. The possible mechanism of the achieved resistance was thought to be RNA silencing. This same group later used RNA silencing to create resistance for SbDV using inverted repeat-SbDV coat protein (CP) genes spaced by a *β*-glucuronidase sequence [27]. Upon infection with virus, transgenic plants showed no symptoms of the dis‐ ease. Transgenic soybeans were shown to contain SbDV-CP-specific siRNA and little to no SbDV-specific RNA, suggesting that resistance to SbDV was achieved by an RNA silencingmediated process.

### *3.3.3. Fungal*

Fungi are the most common soybean pathogens and therefore represent targets for the de‐ velopment of disease resistant transgenic varieties in soybean. One of the more important fungal diseases affecting soybeans grown in the United States and Brazil is *Sclerotinia* stem rot (SSR) caused by the fungus *Sclerotinia sclerotiorum* (white mold). This mold has been as‐ sociated with the presence of oxalic acid (OA). Treatment of plants with OA induced symp‐ toms whereas metabolism of OA is correlated with fungal tolerance. Cunha et al., generated transgenic soybean lines that overexpressed oxalate decarboxylase (OXDC) [28]. When transgenic soybean lines were infected with white mold the disease progression showed sig‐ nificant reduction of severity that correlated with the level of transgene expression. Trans‐ genic events expressing high levels of OXDC showed complete resistance demonstrating the feasibility of this approach.

Much of the research in the development of fungal disease resistance has focused on over‐ expression of a single gene to confer protection, though such a method favors co-evolution and pathogenic resistance. An alternative strategy taken by Li et al., was to create multigene resistance by overexpressing multiple anti-fungal genes [29]. Two such genes previ‐ ously shown to be involved with fungal disease resistance are chitinase (CHI) and the barley ribosome-inactivating protein (RIP). While Li et al., successfully produced transgen‐ ic soybean overexpressing both traits, transgenic events were not challenged with fungal infection.

An alternative technology has shown promise with controlling fungal infection through the use of single-chain variable fragment (scFv) antibodies. While plants do not produce endog‐ enous antibodies, they can express and correctly assemble antibody fragments. In fact, anti‐ body production in soybean was first demonstrated in 1998 [30]. A similar antibody approach was recently taken by Brar and Bhattacharyya to control *Fusarium virguliforme* which is responsible for soybean sudden death syndrome (SDS) [31]. Using the pathogenic toxin Tox1 as a target, soybeans were transformed with an antibody gene encoding scFv an‐ ti-FvTox1 to create transgenic lines with enhanced foliar SDS resistance compared to control plants. Their results suggest that FvTox1 is a pathogenicity factor for the development of SDS and that expression of a soybean plant scFv antibody can reduce a toxin-induced plant disease. This biotechnology approach may be translational in fighting other plant diseases that are induced by pathogenic toxins.

To date there are no commercially available transgenic soybeans that confer resistance to disease, including fungal pathogens. In 2011 DuPont, Pioneer Hi-Bred, and Evogene an‐ nounced a collaboration to develop soybean varieties displaying in-plant resistance to soy‐ bean rust [32]. This is a major step in the direction of creating the first commercially available transgenic soybean variety that is resistant to a fungal pathogen.

### *3.3.4. Nematode*

hibited SbDV-CP-specific siRNA. The possible mechanism of the achieved resistance was thought to be RNA silencing. This same group later used RNA silencing to create resistance for SbDV using inverted repeat-SbDV coat protein (CP) genes spaced by a *β*-glucuronidase sequence [27]. Upon infection with virus, transgenic plants showed no symptoms of the dis‐ ease. Transgenic soybeans were shown to contain SbDV-CP-specific siRNA and little to no SbDV-specific RNA, suggesting that resistance to SbDV was achieved by an RNA silencing-

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Fungi are the most common soybean pathogens and therefore represent targets for the de‐ velopment of disease resistant transgenic varieties in soybean. One of the more important fungal diseases affecting soybeans grown in the United States and Brazil is *Sclerotinia* stem rot (SSR) caused by the fungus *Sclerotinia sclerotiorum* (white mold). This mold has been as‐ sociated with the presence of oxalic acid (OA). Treatment of plants with OA induced symp‐ toms whereas metabolism of OA is correlated with fungal tolerance. Cunha et al., generated transgenic soybean lines that overexpressed oxalate decarboxylase (OXDC) [28]. When transgenic soybean lines were infected with white mold the disease progression showed sig‐ nificant reduction of severity that correlated with the level of transgene expression. Trans‐ genic events expressing high levels of OXDC showed complete resistance demonstrating the

Much of the research in the development of fungal disease resistance has focused on over‐ expression of a single gene to confer protection, though such a method favors co-evolution and pathogenic resistance. An alternative strategy taken by Li et al., was to create multigene resistance by overexpressing multiple anti-fungal genes [29]. Two such genes previ‐ ously shown to be involved with fungal disease resistance are chitinase (CHI) and the barley ribosome-inactivating protein (RIP). While Li et al., successfully produced transgen‐ ic soybean overexpressing both traits, transgenic events were not challenged with fungal

An alternative technology has shown promise with controlling fungal infection through the use of single-chain variable fragment (scFv) antibodies. While plants do not produce endog‐ enous antibodies, they can express and correctly assemble antibody fragments. In fact, anti‐ body production in soybean was first demonstrated in 1998 [30]. A similar antibody approach was recently taken by Brar and Bhattacharyya to control *Fusarium virguliforme* which is responsible for soybean sudden death syndrome (SDS) [31]. Using the pathogenic toxin Tox1 as a target, soybeans were transformed with an antibody gene encoding scFv an‐ ti-FvTox1 to create transgenic lines with enhanced foliar SDS resistance compared to control plants. Their results suggest that FvTox1 is a pathogenicity factor for the development of SDS and that expression of a soybean plant scFv antibody can reduce a toxin-induced plant disease. This biotechnology approach may be translational in fighting other plant diseases

To date there are no commercially available transgenic soybeans that confer resistance to disease, including fungal pathogens. In 2011 DuPont, Pioneer Hi-Bred, and Evogene an‐

mediated process.

feasibility of this approach.

that are induced by pathogenic toxins.

*3.3.3. Fungal*

Relationships

454

infection.

Plant parasitic nematodes are a significant agricultural problem causing major limitations on crop yield and quality. It is estimated that plant parasitic nematodes cause approximately \$157 billion [USD] in damage worldwide. Current approaches used to combat agricultural losses include the use of nematicides, cultivation techniques, and varieties with natural re‐ sistance. Nematicides include some of the most hazardous compounds used in agriculture and alternative control is required due to health and environmental concerns over their use. In soybean, the majority of yield loss can be attributed to infection by nematodes of the ge‐ nus *Meloidogyne* and *Heterodera* commonly referred to as root knot nematodes (RKN) and soybean cyst nematodes (SCN), respectively. RKN and SCN infect plant roots and induce the formation of specialized feeding sites. The establishment and maintenance of feeding sites are crucial to the survival of nematodes making them an obvious target of interest for novel control strategies. One approach that has emerged in recent years is the use of *in plan‐ ta* RNA interference (RNAi) to target genes of feeding nematodes. Through biotechnology, plants can be engineered to produce dsRNAs that silence essential nematode genes. Inges‐ tion of plant-derived dsRNAs by the feeding nematode would trigger the RNAi process thereby inactivating targeted genes and preventing or limiting nematode infection. There are numerous genes known to be essential for nematode survival, and they have been the subject of past reviews [33, 34]. Many of these appear to be candidates for use in an *in planta* RNAi strategy to control nematode infection.

Steeves et al., was one of the first to demonstrate efficacy of an RNAi-based strategy to con‐ trol SCN [35]. Transgenic soybeans were generated following transformation with an RNAi expression vector containing inverted repeats of a cDNA clone of the SCN major sperm pro‐ tein (MSP). RNA silencing was elicited in the cyst nematode following ingestion of dsRNA molecules, and resulted in ~75% suppression of reproductive capabilities. Several years later Li et al., used RNAi to test potential gene targets known to be involved with nematode re‐ production and fitness [36]. Soybean roots expressing small interfering RNAs against the SCN genes Cpn-1, Y25, and Prp-17 showed a significant reduction in transcript levels in nematode feeding sites. Furthermore, nematode suppression levels were similar to those ob‐ served with conventional resistance. Recently RNAi was used to disrupt genes involved with RKN gall formation [37]. Genes encoding tyrosine phosphatase (TP) and mitochondrial stress-70 protein precursor (MSP) were stably expressed in soybean roots, and following in‐ fection with RKN the number of galls was decreased by >90%. Nematode growth within roots was measured and the diameter of nematodes inside transformed soybean roots was reduced 5-fold over that of nematodes inside control roots.

Although there are a few cultivars of soybean that have natural resistance to some species of nematode, there are currently no commercially available soybean varieties that offer geneti‐ cally modified resistance to nematodes. Over the past 10 years, there have been numerous

candidate genes found within the nematode-plant interaction that hold potential for the de‐ velopment of novel genetically modified soybeans using an RNAi-based strategy. Results from the above studies show the potential of RNAi technology for reducing gall formation, limiting nematode reproduction and infection, and ultimately broadening soybean resist‐ ance to SCN and RKN. The production and eventual commercialization of nematode resist‐ ant soybean will benefit both producers and consumers by decreasing dependence on hazardous nematacides and increasing overall soy grain yield.
