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Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

Genetically modified (GM) organisms and crops have been a feature of food production for over 30 years. Despite extensive science-based risk assessment, the public and many politicians remain concerned with the genetic manipulation of crops, particularly food crops. Many governments have addressed public concern through biosafety legislation and regulatory frameworks that identify and regulate risks to ensure human health and environmental safety. These domestic regulatory frameworks align to international scientific risk assessment methodologies on a case-by-case basis. Regulatory agencies in 70 countries around the world have conducted in excess of 4400 risk assessments, all reaching the same conclusion: GM crops and foods that have been assessed provide no greater risk to human health or the environment than non-GM crops and foods. Yet, while the science regarding the safety of GM crops and food appears conclusive and societal benefits have been globally demonstrated, the use of innovative products have only contributed minimal improvements to global food security. Regrettably, politically-motivated regulatory barriers are currently being implemented with the next genomic innovation, genome editing, the implications of which are also discussed in this article. A decade of reduced global food insecurity was witnessed from 2005 to 2015, but regrettably, the figure has subsequently risen. Why is this the case? Reasons have been attributed to climate variability, biotic and abiotic stresses, lack of access to innovative technologies and political interference in decision making processes. This commentary highlights how political interference in the regulatory approval process of GM crops is adversely affecting the adoption of innovative, yield enhancing crop varieties, thereby limiting food security opportunities in food insecure economies.

Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions. Supplementary Information The online version contains supplementary material available at 10.1007/s11248-021-00257-8.

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

The USDA’s Animal and Plant Health Inspection Service (APHIS) is one of three agencies that govern the importation, interstate movement, or environmental release of certain genetically engineered (GE) organisms. APHIS regulations are in 7 CFR part 340, originally issued in 1987 (https://www.aphis.usda.gov/biotechnology/downloads/7_cfr_340.pdf) and based on the premise that a GE crop could pose a plant pest risk. A major revision was proposed June 6, 2019 (https://www.aphis.usda.gov/brs/fedregister/BRS_20190606.pdf), with public comments accepted for 60 d. Members of the Society for In Vitro Biology (SIVB)—especially within the plant section—are impacted by the current rules and will be impacted by changes. Therefore, the public policy committee studied the changes and prepared a response that was reviewed by the board of directors and submitted to APHIS. APHIS received 6186 comments overall. In general, the SIVB welcomes the proposed revisions, which recognize established scientific guidelines and principles for plant pest risk assessment. In addition, they recognize the safety record accumulated over multiple decades of current GE mechanisms of action (MOA), so the proposed rules include an exemption for new events with established MOA’s in a previously reviewed crop. Further, the proposed rules focus on DNA function and impact rather than its source organism with regulatory review triggered by potential for increased plant pest risk. Most importantly, the proposed revisions codify a light regulatory approach for many applications of genome editing, because these applications result in plants that could otherwise have been developed through traditional breeding techniques or found in nature. If implemented appropriately in the final rule, we find these changes remove many current barriers, and thus are likely to stimulate university and business “startup” innovation. The SIVB recognizes the need for exemption for further model species and gene delivery organisms. Such concerns were adequately expressed by the submitted response. Remaining hurdles for innovative plant incorporated protectants are still problematic. Nevertheless, the SIVB applauds the efforts to update APHIS rules in a scientifically defensible manner. The detailed response is shared below.

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single-nucleotide variants. The accumulation of new single-nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN-treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 to 6 bp, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss-of-function mutations that impact single genes.

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cis-regulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

Type I Diacylglycerol acyltransferase (DGAT1) catalyzes the final step of the biosynthesis process of triacylglycerol (TAG), the major storage lipids in plant seeds, through the esterification of diacylglycerol (DAG). To characterize the function of DGAT1 genes on the accumulation of oil and other seed composition traits in soybean, transgenic lines were generated via trans-acting siRNA technology, in which three DGAT1 genes (Glyma.13G106100, Glyma.09G065300, and Glyma.17G053300) were downregulated. The simultaneous downregulation of the three isoforms in transgenic lines was found to be associated with the reduction of seed oil concentrations by up to 18 mg/g (8.3%), which was correlated with increases in seed protein concentration up to 42 mg/g (11%). Additionally, the downregulations also influenced the fatty acid compositions in the seeds of transgenic lines through increasing the level of oleic acid, up to 121 mg/g (47.3%). The results of this study illustrate the importance of DGAT1 genes in determining the seed compositions in soybean through the development of new potential technology for manipulating seed quality in soybean to meet the demands for its various food and industrial applications.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single nucleotide variants. The accumulation of new single nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis, rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 – 6 base pairs, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss of function mutations that will impact single genes.Significance StatementIrradiation mutagenesis is commonly viewed as a method to induce large structural variants in genomes. We also find enrichment in small insertion and deletion (indel) variants. The radiation-mutagenized lines averaged 32 indels per line, far exceeding the number estimated to occur by spontaneous processes, indicating that these arose from the irradiation treatment. Nevertheless, naturally-occurring standing variation among soybean accessions is still four orders of magnitude higher than the level of diversity introduced by mutagenesis. Induced mutations from any source are likely to constitute a relatively small portion of the genetic variation present in crop species. However, irradiation mutagenesis is useful for altering genomes by introducing small indels into single genes or disrupting gene clusters by creating structural variants.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, themPingelement from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences inmPingresulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version ofmPingcalledmmPing20. Transgenic soybean events carryingmPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of themmPing20Factivation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the originalmPingelement, improving the overall effectiveness of the mutagenesis system.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP‐TF, an isoform of 5‐enolpyruvylshikimate 3‐phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP‐TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP‐induced substitution of 142nd amino acid (PtrEPSP‐TFD142E) dramatically impairs the DNA‐binding and transcriptional activity of PtrEPSP‐TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA‐binding helix‐turn‐helix (HTH) motif is absent, the PtrEPSP‐TF, but not PtrEPSP‐TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP‐TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP‐TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

BackgroundSwitchgrass (Panicum virgatumL.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method usingAgrobacterium tumefacienswith subsequent plant regeneration.ResultsRegenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates.Agrobacterium tumefaciensstrain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and apporRFPorange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines.ConclusionThe new cell suspension-based protocol enables high-throughputAgrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.

The advent of genome-edited products that are nearing commercialization in agriculture has highlighted that the US biotechnology regulatory system has not kept pace with technological advances. Of the three agencies that regulate engineered crops and animals for agriculture, only one has indicated how it will regulate edited plants. The Food and Drug Administration can regulate any plant, but has not indicated if it will single out edited plants. The US Department of Agriculture currently has no authority over edited plants when the edit is a deletion or does not contain any added DNA from a plant pest. Depending on how the statutes are interpreted, the Environmental Protection Agency might be able to regulate plants edited to tolerate pests and diseases. Labeling requirements also remain undefined. Regardless, sectors of the industry and some consumer groups are uneasy over editing technology, and may be the ultimate arbiters of whether edited products make it to market.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Quantitative trait loci (QTLs) E and M are major soybean alleles that confer resistance to leaf-chewing insects, and are particularly effective in combination. Flavonoids and/or isoflavonoids are classes of plant secondary metabolites that previous studies agree are the causative agents of resistance of these QTLs. However, all previous studies have compared soybean genotypes that are of dissimilar genetic backgrounds, leaving it questionable what metabolites are a result of the QTL rather than the genetic background. Here, we conducted a non-targeted mass spectrometry approach without liquid chromatography to identify differences in metabolite levels among QTLs E, M, and both (EM) that were introgressed into the background of the susceptible variety Benning. Our results found that E and M mainly confer low-level, global differences in distinct sets of metabolites. The isoflavonoid daidzein was the only metabolite that demonstrated major increases, specifically in insect-treated M and EM. Interestingly, M confers increased daidzein levels in response to insect, whereas E restores M’s depleted daidzein levels in the absence of insect. Since daidzein levels do not parallel levels of resistance, our data suggest a novel mechanism that the QTLs confer resistance to insects by mediating changes in hundreds of metabolites, which would be difficult for the insect to evolve tolerance. Collective global metabolite differences conferred by E and M might explain the increased resistance of EM.

Commercial-scale plant breeding is a complex process in which new crop varieties are continuously being developed to improve yield and agronomic performance over current varieties. A wide array of naturally occurring genetic changes are sources of new characteristics available to plant breeders. During conventional plant breeding, genetic material is exchanged that has the potential to beneficially or adversely affect plant characteristics. For this reason, commercial-scale breeders have implemented extensive plant selection practices to identify the top-performing candidates with the desired characteristics while minimizing the advancement of unintended changes. Selection practices in maize (Zea mays L.) breeding involve phenotypic assessments of thousands of candidate lines throughout hundreds of different environmental conditions over many years. Desirable characteristics can also be introduced through genetic modification. For genetically modified (GM) crops, molecular analysis is used to select transformed plants with a single copy of an intact DNA insert and without disruption of endogenous genes. All the while, GM crops go through the same extensive phenotypic characterization as conventionally bred crops. Data from both conventional and GM maize breeding programs are presented to show the similarities between these two processes.

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

The effects of 11 different auxins and one cytokinin-like compound were tested at four concentrations for their ability to induce primary and repetitive somatic embryos from mature, dry peanut (Arachis hypogaea L.) epicotyls of genotype AT120. Treatment with picloram and centrophenoxine at 83.0 and 124.4 μM resulted in the greatest number of embryos per explant and the highest percentage of explants responding. In a follow-up experiment, picloram, centrophenoxine, and dicamba were tested at 83.0 and 124.4 μM on four peanut genotypes (AT120, 59-4144, GK7, and VC1). Picloram and centrophenoxine induced similar numbers of globular-stage and total embryos from each genotype, while dicamba was less effective. Similar results were observed with percentage of responding axes. Genotypes AT120 and VC1 yielded more clusters of repetitive embryos than GK7 and 59-4144. After 5 months, embryos derived from repetitive embryogenic cultures were converted into mature plants.

Embryogenic cultures of soybean [Glycine max (L.) Merr. cv. Jack and Asgrow A2872] were established in liquid Finer and Nagasawa medium, maintained by transfer to fresh medium at biweekly intervals, and subjected to microprojectile bombardment over time. Cultures were not amenable to transformation until they were at least 6 months old. Over time, different cell lines of the same genotype acquired very different culture phenotypes. Histological analysis of cell lines differing in transformation ability showed that the most transformable cultures had cytoplasmic-rich cells in the outermost layers of the tissue. In contrast, the outer layers of less transformable cultures contained cells with prominent vacuoles. Although fresh weight accumulation of the cultures was curvilinear during the 2-week subculture period, a burst of mitotic activity was evident shortly after transfer to fresh medium. This activity usually lasted from the 2nd to the 6th day following subculture, and peaked on the 4th day. Tissues at or near this stage always produced more transient expression of a reporter gene than did bombardments at other times. In addition, the cell lines most amenable to transformation also exhibited the highest mitotic index. Thus any treatment to increase the mitotic index, especially when the cell lines are less than 6 months old, may facilitate the transformation of cell lines from which efficient recovery of transgenic plants is still possible.

Soybean, Glycine max (L.) Merr., has been grown as a forage and as an important protein and oil crop for thousands of years. Domestication, breeding improvements and enhanced cropping systems have made soybeans the most cultivated and utilized oilseed crop globally. Soybeans provide a high-quality protein source for livestock and aquaculture, oil for industrial uses and a valued component of human diets. Originating in China and Eastern Asia, today 80–85% of the world’s soybeans, approximately 88 million ha, are grown in the Western Hemisphere. United States soybean breeding and development efforts for over 80 years have transitioned from primarily universities and United States Department of Agriculture (USDA) programs to private company-led investments in commercial cultivar development. Soybean breeders continuously adapt tools and technologies that encompass classical breeding, mutation breeding and marker-assisted selection, biotechnology and transgenic approaches, gene silencing, and genome editing. In addition to breeding technologies, improved agronomics, precision agriculture and digital agriculture have advanced soybean production and profitability. The primary goals of soybean breeding and cropping systems advances include yield improvement, increased seed protein and oil composition and quality, and yield preservation through weed, pathogen, insect pest and abiotic stress resistance and management. This chapter primarily describes the introduction and improvement of soybeans in the United States. Contributing authors describe classical and molecular breeding, biotechnology, biotic and abiotic stress management, and soybean agronomics and cropping systems improvements that maximize soybean productivity, profitability and sustainability to supply a continually increasing world demand for protein and oil for feed, fuel and food.

Overexpression of a native gene can cause expression of both introduced and native genes to be silenced by posttranscriptional gene silencing (PTGS) mechanisms. PTGS mechanisms rely on sequence identity between the transgene and native genes; therefore, designing genes with mutations that do not cause amino acid changes, known as synonymous mutations, may avoid PTGS. For proof of concept, the sequence of acetyl-coA carboxylase (ACCase) from creeping bentgrass (Agrostis stolonifera L.) was altered with synonymous mutations. A native bentgrass ACCase was cloned and used as a template for the modified gene. Wild-type (WT) and modified genes were further modified with a non-synonymous mutation, coding for an isoleucine to leucine substitution at position 1781, known to confer resistance to ACCase-inhibiting herbicides. Five-hundred calli of creeping bentgrass 'Penn A-4' were inoculated with Agrobacterium containing either the WT or modified genes, with or without the herbicide-resistance mutation. Six herbicide-resistant-transgenic events containing the modified gene with the 1781 mutation were obtained. Transcription of the modified ACCase was confirmed in transgenic plants, showing that gene-silencing mechanisms were avoided. Transgenic plants were confirmed to be resistant to the ACCase-inhibiting herbicide, sethoxydim, providing evidence that the modified gene was functional. The result is a novel herbicide-resistance trait and shows that overexpression of a native enzyme with a gene designed with synonymous mutations is possible.

Creeping bentgrass (Agrostis stolonifera L.) is a fine-textured, cold-hardy turfgrass used by the golf industry in the transition zone and farther north. Grassy weeds are a recurrent problem for seed producers and golf course managers. Her- bicide-resistant creeping bentgrass would pro- vide the seed producer and golf course manager with an additional tool to control grassy weeds. In the present study, in vitro selection was used to obtain mutants of creeping bentgrass via somaclonal variation that were resistant to the acetyl coenzyme A carboxylase (ACCase) inhibitor, sethoxydim {2-(1-(ethoxyimino) butyl)- 5-(2-(ethylthio)propyl)-3-hydroxy-2-cyclohexen- 1-one}. Selection was imposed on creeping bentgrass calli (total of 13,725) by growing them on induction medium containing 10 µM sethoxydim. Four independent sethoxydim- resistant calli were identified and subsequently sequenced toconfirm the presence of an A to C mutation in the first position of the 1781 amino acid codon causing an isoleucine to leucine substitution. Whole plant dose response experi- ments showed the sethoxydim-resistant event, SR1, was resistant to sethoxydim at rates of >3200 g a.i. ha -1 . SR1 also showed cross resis- tance to the ACCase-inhibitor herbicide, fenoxa- prop at rates of > 800 g a.i. ha -1 .

QTL-M and QTL-E enhance soybean resistance to insects. Pyramiding these QTLs with cry1Ac increases protection against Bt-tolerant pests, presenting an opportunity to effectively deploy Bt with host-plant resistance genes. Plant resistance to leaf-chewing insects minimizes the need for insecticide applications, reducing crop production costs and pesticide concerns. In soybean [Glycine max (L.) Merr.], resistance to a broad range of leaf-chewing insects is found in PI 229358 and PI 227687. PI 229358's resistance is conferred by three quantitative trait loci (QTLs): M, G, and H. PI 227687's resistance is conferred by QTL-E. The letters indicate the soybean Linkage groups (LGs) on which the QTLs are located. This study aimed to determine if pyramiding PI 229358 and PI 227687 QTLs would enhance soybean resistance to leaf-chewing insects, and if pyramiding these QTLs with Bt (cry1Ac) enhances resistance against Bt-tolerant pests. The near-isogenic lines (NILs): Benning(ME), Benning(MGHE), and Benning(ME+cry1Ac) were developed. Benning(ME) and Benning(MGHE) were evaluated in detached-leaf and greenhouse assays with soybean looper [SBL, Chrysodeixis includens (Walker)], corn earworm [CEW, Helicoverpa zea (Boddie)], fall armyworm [FAW, Spodoptera frugiperda (J.E. Smith)], and velvetbean caterpillar [VBC, Anticarsia gemmatalis (Hübner)]; and in field-cage assays with SBL. Benning(ME+cry1Ac) was tested in detached-leaf assays against SBL, VBC, and Southern armyworm [SAW, Spodoptera eridania (Cramer)]. In the detached-leaf assay, Benning(ME) showed the strongest antibiosis against CEW, FAW, and VBC. In field-cage conditions, Benning(ME) and Benning(MGHE) suffered 61 % less defoliation than Benning. Benning(ME+cry1Ac) was more resistant than Benning(ME) and Benning (cry1Ac) against SBL and SAW. Agriculturally relevant levels of resistance in soybean can be achieved with just two loci, QTL-M and QTL-E. ME+cry1Ac could present an opportunity to protect the durability of Bt genes in elite soybean cultivars. These results should assist the development of effective pest management strategies, and sustainable deployment of Bt genes in soybean.

Summary Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 lg b carotene g � 1 dry seed weight with a desirable 12:1 ratio of b to a. The b carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated b-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the midmaturation b-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated b-carotene, oleic acid and protein traits in the b-carotene soya beans confer a substantial additive nutritional quality to soya beans.

Rhg4 is a major genetic locus that contributes to soybean cyst nematode (SCN) resistance in the Peking-type resistance of soybean (Glycine max), which also requires the rhg1 gene. By map-based cloning and functional genomic approaches, we previously showed that the Rhg4 gene encodes a predicted cytosolic serine hydroxymethyltransferase (GmSHMT08); however, the novel gain of function of GmSHMT08 in SCN resistance remains to be characterized. Using a forward genetic screen, we identified an allelic series of GmSHMT08 mutants that shed new light on the mechanistic aspects of GmSHMT08-mediated resistance. The new mutants provide compelling genetic evidence that Peking-type rhg1 resistance in cv Forrest is fully dependent on the GmSHMT08 gene and demonstrates that this resistance is mechanistically different from the PI 88788-type of resistance that only requires rhg1. We also demonstrated that rhg1-a from cv Forrest, although required, does not exert selection pressure on the nematode to shift from HG type 7, which further validates the bigenic nature of this resistance. Mapping of the identified mutations onto the SHMT structural model uncovered key residues for structural stability, ligand binding, enzyme activity, and protein interactions, suggesting that GmSHMT08 has additional functions aside from its main enzymatic role in SCN resistance. Lastly, we demonstrate the functionality of the GmSHMT08 SCN resistance gene in a transgenic soybean plant.

Summary GM crops are the most studied crops in history. Approximately 5% of the safety studies on them show adverse effects that are a cause for concern and tend to be featured in media reports. Although these reports are based on just a handful of GM events, they are used to cast doubt on all GM crops. Furthermore, they tend to come from just a few laboratories and are published in less important journals. Importantly, a close examination of these reports invariably shows methodological flaws that invalidate any conclusions of adverse effects. Twenty years after commercial cultivation of GM crops began, a bona fide report of an adverse health effect due to a commercialized modification in a crop has yet to be reported.

Contamination of newly planted bermudagrass (Cynodon spp.) varieties by undesirable off-type bermudagrass genotypes is an ever increasing concern for turf managers because selective control options are limited. In 2009, a sethoxydim {2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one} tolerant bermudagrass genotype (93-175) was identified during herbicide screening at the University of Georgia in Tifton. The objective of this research was to assess the tolerance of 93-175 to three Acetyl-CoA carboxylase (ACCase) herbicides in comparison to the susceptible genotypes Tifway and common bermudagrass. Greenhouse and field trials were performed between August 2011 and April 2013. Factors in the field experiment included ACCase herbicides, application rates, bermudagrass genotypes, and locations. Turfgrass injury ratings taken 42 days after treatment (DAT) and during greenup the following spring supported initial preliminary findings. At the 1x rate of sethoxydim (280 g a.i. ha–¹), 93-175 displayed 50 to 87% less injury in comparison to the susceptible genotypes. In the spring of 2013, 93-175 plots treated with a 1x rate of sethoxydim reached 100% recovery during the same time period as non-treated controls, while common and Tifway had only recovered to 48 and 60%, respectively. The tolerance mechanism of 93-175 to sethoxydim did not confer an appreciable reduction of clethodim {(E,E)-(6)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one} or fluazifop {(6)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid} herbicide treatment effects. 93-175 will continue to be studied to determine transferability of herbicide tolerance to progeny and the mechanism of the observed tolerance.

There is disagreement internationally across major regulatory jurisdictions on the relevance and utility of whole food (WF) toxicity studies on GM crops, with no harmonization of data or regulatory requirements. The scientific value, and therefore animal ethics, of WF studies on GM crops is a matter addressable from the wealth of data available on commercialized GM crops and WF studies on irradiated foods. We reviewed available GM crop WF studies and considered the extent to which they add to the information from agronomic and compositional analyses. No WF toxicity study was identified that convincingly demonstrated toxicological concern or that called into question the adequacy, sufficiency, and reliability of safety assessments based on crop molecular characterization, transgene source, agronomic characteristics, and/or compositional analysis of the GM crop and its near-isogenic line. Predictions of safety based on crop genetics and compositional analyses have provided complete concordance with the results of well-conducted animal testing. However, this concordance is primarily due to the improbability of de novo generation of toxic substances in crop plants using genetic engineering practices and due to the weakness of WF toxicity studies in general. Thus, based on the comparative robustness and reliability of compositional and agronomic considerations and on the absence of any scientific basis for a significant potential for de novo generation of toxicologically significant compositional alterations as a sole result of transgene insertion, the conclusion of this review is that WF animal toxicity studies are unnecessary and scientifically unjustifiable.

Alfalfa (Medicago sativa L.) production is dra- matically reduced in acidic, Al-rich soil. Trans- genic plants of several species overexpress- ing organic acid synthesis and/or organic acid transporter genes have shown enhanced toler- ance to Al. The objective of this research was to evaluate the effect of the citrate synthase (CS) and the Daucus carota L. plasma membrane H + -transporting adenosine triphosphatase (H + ATPase) (DcPA1) transgenes on Al tolerance in alfalfa. Stem cuttings from a full-sib T 2 popu- lation including four isogenic groups (plants with neither transgene, CS only, DcPA1 only, and both transgenes) together with nontrans- formed check genotypes were evaluated for Al and acid soil tolerance in a greenhouse assay in limed and unlimed soil. Aluminum and acid soil tolerance was assessed by measuring the ratios of root and shoot dry weight in unlimed soil compared to limed soil. The three transgenic populations, CS, DcPA1, and CS plus DcPA1, all showed higher Al and acid soil tolerance and lower levels of Al in shoot tissue than the non- transgenic isogenic population or the nontrans- genic parental genotypes. This suggests that an Al-exclusion mechanism could be driving Al and acid soil tolerance in this study. We observed no advantage of combining both transgenes in the same genetic background. These transgenes offer an efficient method to achieve enhanced Al and acid soil tolerant alfalfa cultivars, but more information is needed on their stability across generations and genetic backgrounds and their performance under field conditions.

Plant breeding has a long history of developing varieties with desirable traits in response to the needs of both growers and consumers. Although the bases for most of these traits are not known genetically or biochemically, conventional breeding combines these multiple traits to create new hybrids

Current regulatory regimes for genetically engineered crops fail to use a scientifically defensible approach or tailor the degree of regulatory review to the level of actual hazard or risk. We describe a rational way forward.

Benning(M) and Benning(MGH) are near-isogenic lines (NILs) of the soybean cultivar Benning, which contain insect-resistance quantitative trait loci (QTLs) from the soybean accession PI 229358. Benning(M) contains QTL-M, which confers antibiosis and antixenosis. In addition to QTL-M, Benning(MGH) contains QTL-G, which confers antibiosis, and QTL-H, which confers antixenosis. Soybean meal was produced from Benning and the NILs. Nutritional composition, digestible amino acid content, and nitrogen-corrected true metabolizable energy (TMEN) were equivalent among soybean meals. A 21-day broiler feeding trial was carried out to determine if the QTLs affect soybean meal quality. Weight gain and feed-to-gain ratio were evaluated. No biologically significant differences were detected for broilers fed Benning, Benning(M), and Benning(MGH). This demonstrates that soybean meal produced from the insect-resistant NILs is equivalent to soybean meal produced from their non-insect-resistant parent cultivar for broiler weight gain.