About

Robert Stupar is a professor in the Department of Agronomy and Plant Genetics at the University of Minnesota. His educational background includes a Ph.D. in Plant Breeding and Plant Genetics from the University of Wisconsin, obtained in 2005. He is a member of the graduate faculty in Applied Plant Sciences, Plant and Microbial Biology, and Bioinformatics and Computational Biology. His research focuses on the molecular genetics and genomics of legume crop species, including soybean and pea. His primary interests include understanding the genetic basis of natural and induced phenotypic variation, developing novel genetic resources, and applying genomic strategies for trait improvement. Stupar teaches courses such as Agro 3660 Plant Genetic Resources and Agro 8241 Chromosomal and Molecular Genetics of Plant Improvement. He has received numerous honors and awards, including the CFANS Distinguished Teaching Award for Graduate Faculty in 2025, the UMN Executive Vice President and Provost Award for Excellence in Academic Service in 2024, and the Crop Science Society of America Fellow in 2020. His contributions include patents related to methods for genetic transformation and genome modification in legumes and the development of haploid inducer lines for accelerated genome editing.

Research topics

• Biology
• Genetics
• Computational biology
• Computer science
• Biotechnology

Selected publications

• Targets and strategies to design soybean seed composition traits

  The Plant Genome · 2025-09-26 · 2 citations

  reviewOpen accessSenior authorCorresponding

  Breeders and geneticists have put great effort into enhancing soybean seed composition and developing elite varieties with desired traits. However, diverse end-uses and changing consumer preferences present new challenges and opportunities to develop desired compositional profiles for the market. Recent advances in genetics and novel technologies have allowed researchers to characterize genes impacting seed composition and provide a means for addressing these shifting consumer needs. Some of the desired compositional traits for soybean [Glycine max (L.) Merr.] include increased levels of protein, oil, sucrose, sulfur-containing amino acids, and omega-3 fatty acids, and reduced allergens, raffinose family oligosaccharides, or saponins. This comprehensive review considers the current status of key components of soybean seed composition and forecasts opportunities based on candidate genes/pathways that may be targeted to add seed value. The review addresses a holistic view on interactions between the genome, transcriptome, proteome, and epigenome, along with metabolic flux analysis to gain insights into biological underpinnings governing seed composition.

  PublisherDOI

• Molecular polymorphisms that underlie trait variation in crops: Lessons learned from soybean

  The Plant Genome · 2025-12-30

  articleOpen accessSenior author

  Genetic variation within a germplasm is important in crop improvement, providing a foundation for breeders to develop new varieties. Traits of agronomic and economic importance are often mapped to identify the genetic basis of observed phenotypes, oftentimes using quantitative trait locus (QTL) analysis. While some mapped QTLs can be directly utilized in breeding programs, others benefit from a deeper understanding of the causal genes and sequence polymorphisms for effective deployment. In this review, we examine the factors that differentiate QTL where causal DNA sequence polymorphisms can be readily identified from those where causation remains difficult to resolve. We present a case study for soybean (Glycine max (L.) Merr.), in which we surveyed the literature to find the sequence variants underlying QTL in this crop. We cataloged causal variants by type, including variations within coding and regulatory regions, transposable element insertions and deletions, epigenetic modifications, and gene content variants (e.g., copy number variants). Additionally, we discuss the impediments to gene discovery, including challenges in phenotyping, the nature of sequence polymorphisms, and the difficulty of functional validation. Finally, we highlight future opportunities in the field of gene discovery, emphasizing how advances in high-resolution mapping, near-gapless genome assemblies, pangenome resources, and genome engineering will help overcome existing barriers and accelerate the discovery of causal genes and variants underlying complex traits.

  PublisherDOI

• Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing

  PLANT PHYSIOLOGY · 2025-12-10 · 4 citations

  articleOpen access

  Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.

  PublisherOA PDFDOI

• A synthetic transcription cascade enables direct in planta shoot regeneration for transgenesis and gene editing in multiple plants

  Molecular Plant · 2025-11-06 · 12 citations

  articleOpen access
  PublisherOA PDFDOI

• Soybean RIN4 represents a mechanistic link between plant immune and symbiotic signaling

  eLife · 2025-06-24 · 1 citations

  preprintOpen access

  Abstract The legume-rhizobium symbiosis represents a unique and beneficial interaction between legumes and nitrogen-fixing soil bacteria, called rhizobia. The initiation and development of this symbiosis is complex and begins with recognition of key molecular signals, produced by the plant and its symbiont, which determine symbiotic compatibility. Current data suggest that the invading symbiont initially triggers plant immune responses that are subsequently suppressed. Hence, there is growing evidence that features of plant immunity may be relevant to symbiotic establishment. RIN4 is a key immune regulator in plants, regulating basal immunity and it is also targeted by pathogen effector proteins that either confer susceptibility or resistance, depending on the presence of the appropriate resistance protein. Surprisingly, we found that RIN4 was rapidly phosphorylated upon rhizobial inoculation of soybean root hairs. RNAi silencing and mutant studies indicate that RIN4 expression is essential for effective nodulation of soybean. RIN4 phosphorylation occurs within a fifteen amino acid motif, which is highly conserved within the Fabales (legumes) and Rosales orders, which comprise species capable of nitrogen-fixing endosymbiosis with rhizobia. RIN4 proteins mutated in this conserved phosphorylation site failed to support efficient soybean nodulation. Phosphorylation of this site is mediated by the symbiotic receptor-like kinase, SymRK, a well-studied member of the symbiotic signaling pathway. The data implicate RIN4 phosphorylation as a key mediator of rhizobial compatibility, interconnecting symbiotic and immune signaling pathways.

  PublisherDOI

• Author response: Soybean RIN4 represents a mechanistic link between plant immune and symbiotic signaling

  2025-06-24

  peer-reviewOpen access

  The legume-rhizobium symbiosis represents a unique and beneficial interaction between legumes and nitrogen-fixing soil bacteria, called rhizobia. The initiation and development of this symbiosis is complex and begins with recognition of key molecular signals, produced by the plant and its symbiont, which determine symbiotic compatibility. Current data suggest that the invading symbiont initially triggers plant immune responses that are subsequently suppressed. Hence, there is growing evidence that features of plant immunity may be relevant to symbiotic establishment. RIN4 is a key immune regulator in plants, regulating basal immunity and it is also targeted by pathogen effector proteins that either confer susceptibility or resistance, depending on the presence of the appropriate resistance protein. Surprisingly, we found that RIN4 was rapidly phosphorylated upon rhizobial inoculation of soybean root hairs. RNAi silencing and mutant studies indicate that RIN4 expression is essential for effective nodulation of soybean. RIN4 phosphorylation occurs within a fifteen amino acid motif, which is highly conserved within the Fabales (legumes) and Rosales orders, which comprise species capable of nitrogen-fixing endosymbiosis with rhizobia. RIN4 proteins mutated in this conserved phosphorylation site failed to support efficient soybean nodulation. Phosphorylation of this site is mediated by the symbiotic receptor-like kinase, SymRK, a well-studied member of the symbiotic signaling pathway. The data implicate RIN4 phosphorylation as a key mediator of rhizobial compatibility, interconnecting symbiotic and immune signaling pathways.The nitrogen fixing legume-rhizobium symbiosis is a cornerstone of sustainable agriculture, with ongoing efforts to transfer this unique ability to non-leguminous crop plants. Plants are surrounded by a myriad of microbes in the soil, and, therefore, require constant surveillance in order to distinguish between a pathogen or symbiont. Plants monitor for specific molecular signals that indicate pathogen or symbiont presence. We show that RIN4, a key immune regulator, plays an essential role in promoting the development of the symbiotic nitrogen-fixing relationship between soybean and its compatible symbiont Bradyrhizobium japonicum. Therefore, RIN4 is likely a key player in mediating the appropriate response upon infection by friend or foe.

  PublisherDOI

• Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-03-07 · 2 citations

  preprintOpen access

  Abstract Soybean transformation remains challenging and has not kept pace with the rapid advancement of genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 ( WUS2 ) and isopentenyltransferase ( IPT ) achieved higher transformation efficiencies (15.2% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produces heritable transgenic events within 9-11 weeks and successfully delivers CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2 / IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Together, our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation efficiency, reduces tissue culture requirements, and offers a promising genome editing platform for soybean improvement.

  PublisherOA PDFDOI

• Shoot at Site: Advancing <i>in planta</i> transformation, regeneration and gene-editing through a cascade of wounding-mediated developmental regulators

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-08 · 4 citations

  preprintOpen access

  ABSTRACT Developing transgenic and/or gene-edited plants largely depends on tedious, lengthy, and costly in vitro regeneration protocols. While plants have remarkable regeneration ability, not all species, genotypes or even explants exhibit the same transformation and regeneration potential under in vitro conditions. To tackle this bottleneck, we have developed a seamless and user-friendly system to induce transgenic and gene-edited de novo meristems via a synthetic cascade comprising a wound-induced regeneration pathway, plant developmental regulators (DRs) and gene-editing reagents. WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) is used as a transcriptional regulator to control the expression of various DR genes through ENHANCER OF SHOOT REGENERATION 1 (ESR1) promoter. This cascade was strategically applied in planta to the non-meristematic internode of N. benthamiana to induce meristematic activity and regenerate de novo shoots with knock-out mutations of the phytoene desaturase ( PDS ) gene. This synthetic toolkit was further applied successfully to tomato and soybean. This methodology offers a transformative approach to overcome barriers in plant biotechnology, potentially accelerating the generation of transgenic and gene-edited plants without reliance on conventional tissue-culture intermediates.

  PublisherDOI

• A tritrophic plant-insect-pathogen system used to develop a closely linked Rag2 and Rsv1-h recombinant haplotype in double-resistant soybean germplasm

  BMC Genomics · 2025-05-28 · 1 citations

  articleOpen access

  BACKGROUND: The colocalization of two resistance (R) genes on chromosome 13 of soybean (Glycine max (L.) Merrill) that confer resistance against the soybean aphid (Aphis glycines) and soybean mosaic virus (SMV) gives rise to a very unique R-avr tritrophic incompatible interaction system that goes across biological kingdoms. In this tritrophic system, the insect is the only natural vector of the virus and soybean is a host-plant for both pests/pathogen. The almost unavoidable co-evolution of pathogen-vector with that of the R-genes in soybean plants through an endless arms race to avoid each other's defense-attack mechanisms raises interesting questions. The objectives of this work were to (i) develop double-resistant recombinant inbred lines (RILs) with a Rag2-Rsv1-h gene haplotype in coupling phase using resistance alleles from two different genetic sources (PI 243540 (Rag2) and Suweon 97 (Rsv1-h)), (ii) confirm phenotypically the resistant reaction against both pests in double-resistant RILs, and (iii) dissect the Rag2-Rsv1-h region with molecular markers and investigate the potential for structural variation. RESULTS: RILs in a region of chromosome 13 ca. 21 kb long (between positions 30,297,227 and 30,318,949 in Wm82.a2.v1) that lies between the reported locations of the Rsv1-h and Rag2 genes (29,815,463--29,912,369 and 30,412,581--30,466,533 intervals, respectively, based on Wm82.a2.v1), indicating the double-resistant haplotype is in coupling phase. The tight LD estimates obtained between haplotype markers underscored the physical proximity of the two resistance genes. Only 10 recombinant haplotype classes (excluding double heterozygotes) were observed among the 51 that were possible with a four loci haplotype. The 10 recombinant classes represented 15 out of 192 screened individuals. A joint SMV-aphid phenotypic greenhouse screen allowed us to identify the best aphid biotype 1 and SMV-G1, double resistant haplotype class in recombinant progeny. Our molecular marker results agree with previous fine-mapping reports and preclude the presence of resistance genes other than Rag2 and Rsv1-h in double-resistant RILs. A comparative genomic hybridization analysis revealed no obvious structural variants in the region. CONCLUSIONS: To our knowledge, this is the first report of double-resistant Rag2-Rsv1-h soybean RILs that used a plant-insect-pathogen tritrophic system for germplasm enhancement. The co-occurrence of Rag and Rsv genes in a region that clusters resistance genes on chromosome 13 may be a unique feature of domesticated soybean. The recombinant genotypes will be useful in breeding to develop soybean cultivars with resistance to both the vector and the virus. The parental and recombinant genotypes may be helpful in future studies to elucidate interesting evolutionary questions regarding vector, host, and virus tritrophic systems.

  PublisherOA PDFDOI

• Oregon Wolfe barley genetic stocks – Research and teaching tools for next generation scientists

  Journal of Plant Registrations · 2025-09-01

  articleOpen access

  Abstract The Oregon Wolfe Barley (OWB) mapping population (Reg. no. MP‐4, NSL 554937 MAP) is a resource for genetics research and instruction. The OWBs are a set of doubled haploid barley ( Hordeum vulgare L.) lines developed at Oregon State University from the F 1 of a cross between Dr. Robert Wolfe's dominant and recessive marker stocks. Exhibiting a high level of genetic and phenotypic diversity, the OWBs are used throughout the world as a research tool for barley genetics. To date, these endeavors have led to 56 peer‐reviewed publications, as well as three reports in the Barley Genetics Newsletter. At the same time, the OWBs are widely used as an instructor resource at the K–12, undergraduate, graduate, and professional levels. They are currently used at universities and/or institutes in German, Italy, Norway, Spain, and the United States and are currently being developed further for educational use in other countries. Genotype and phenotype data, lesson plans, and seed availability information are available herein and online.

  PublisherOA PDFDOI

Frequent coauthors

• Carroll P. Vance

  74 shared

• William J. Haun

  69 shared

• Wayne Wenzhong Xu

  67 shared

• Yung‐Tsi Bolon

  University of Minnesota

  52 shared

• David Grant

  Iowa State University

  52 shared

• David L. Hyten

  University of Nebraska–Lincoln

  48 shared

• Adrian O. Stec

  University of Minnesota

  47 shared

• Daniel J. Gerhardt

  Infinera (United States)

  42 shared

Awards & honors

• CFANS Distinguished Teaching Award: Graduate Faculty (2025)
• UMN Executive Vice President and Provost Award for Excellenc…
• Richard Bernard Mid-Career Award (2023)
• Fellow - Crop Science Society of America (elected in 2020)
• UMN Emerging Leaders in the Plant Sciences Award (2019)