Maize ( Zea mays L.) is a highly adaptable crop grown worldwide across diverse climates and management practices, with uses across multiple sectors. Consequently, the traits prioritized in maize research and breeding programs vary depending on the specific objectives. Core traits, however, such as flowering time, plant and ear height, and stalk and root lodging, which are important for evaluating and improving the performance and stability of maize genotypes, are routinely evaluated across breeding programs, regardless of their goals. Standardized measurement of these core traits is essential to ensure data reliability and comparability, enabling the integration of phenotypic data across different experiments. Such efforts ultimately support better decision-making and accelerate the development of improved maize genotypes. This is particularly important in public sector programs, where large-scale evaluations, critical for assessing the value of specific genotypes, are often only feasible through collaboration across programs. Here, we provide a protocol for the standardized collection of phenotypic data, specifically focusing on how to measure core traits in maize field trials. These methods promote consistency and accuracy in the evaluation of these traits, and support communication and coordination among groups in the public sector and other research settings. Further, such standardization facilitates the integration and comparison of data across programs, enabling robust longitudinal and multienvironment analyses.
Maize ( Zea mays L.) is a highly versatile crop, grown worldwide under different climatic conditions and management practices. Its wide-ranging uses across multiple sectors, including food and feed production, biofuels, and as a source of raw materials for industrial processes, make the traits of interest highly variable, depending on research and breeding goals and the needs of commercial and public end users. The phenotype observed in these contexts reflects the combined effects of the genotype, the environment, and their interaction, making multienvironment trials crucial for identifying superior cultivars and predicting their performance across diverse conditions. Accurate and consistent trait measurements and recording are therefore essential to enable meaningful comparisons and integration of data across years, locations, and studies. This is particularly important in the public sector, where such multienvironment trails are often only feasible primarily through collaborative efforts. Here, we provide a brief overview of the importance of standardized trait measurements in maize research and breeding programs, and discuss their impact on enhancing the value and usability of publicly accessible phenotyping data. We also highlight how standardization helps overcome the challenges of conducting large-scale experiments across locations and years, particularly in resource-limited public programs, where multiinstitutional coordination is essential. Together, these approaches help advance reproducibility and data sharing across maize research programs.
Amino acids are important nutrients in maize grain used for food and feed. Because all 20 amino acids are required for growth and development, a deficiency in a single essential amino acid limits the utilization of dietary protein. In monogastric animals, 10 amino acids must be supplied by the diet and therefore are considered essential. The remaining amino acids can be made from the 10 essential amino acids. Lysine, tryptophan, and methionine are frequently limiting essential amino acids in grain-based diets. Therefore, increasing levels of limiting essential amino acids in grain is an important objective in crop improvement. Standard chromatographic methods for assessing levels of amino acids in grain are extremely accurate, but very expensive. Here, we present a protocol for high-throughput analysis of amino acids in grains, using microbial assays, conducted in 96 well plates, that can be carried out for a fraction of the cost of the standard chromatographic methods. We use Escherichia coli strains that have mutations in the biosynthetic pathway of the amino acid of interest. These strains are auxotrophic, so their growth is proportional to the amount of a specific amino acid in the media. The level of the amino acid of interest in a corn extract is determined by adding the corn extract to the microbial growth medium and measuring the growth of the culture as turbidity in a 96 well plate reader. This protocol is designed for analysis of methionine, but can be adapted for the analysis of any amino acid, by substitution of an appropriate auxotrophic strain of E. coli .
Phosphorus is an important nutrient for plants and animals. In maize seeds, phosphorus is stored in the form of phytate, which is the phosphorylated form of the sugar inositol. Monogastric animals lack the enzymes required to break phytate down, so it passes through their digestive systems, to create phosphorus-rich waste. This waste contaminates ground water and leads to water quality problems, such as eutrophication, or excessive concentration of nutrients. Phytate reduces the phosphorus available to animals from their feed, requiring animal feed to be supplemented with phosphate. In addition, phytate chelates nutritionally important metal cations, such as iron and zinc, contributing to globally important nutrient deficiencies in human diets. Development of low-phytate corn is an important breeding objective. To achieve this objective, it is crucial to be able to measure phytate, as well as available phosphorus, in maize seeds. Throughout, precision and cost are important considerations in plant breeding programs. This protocol describes methods for quantifying phytate and available phosphorus in maize seeds, in high-throughput 96 well plate assays, suitable for analysis of large-scale field studies and breeding efforts.
Grain quality is defined as the suitability of grain for a particular use. It is usually designated by chemical composition or physical properties of the grain. The ability to measure grain quality is important for identity preservation of specialty grain market classes, for development of new varieties with improved quality through breeding, and for basic scientific studies on the genetic or biochemical control of grain quality traits. This review introduces official methods for measuring maize compositional traits, including protein, starch, oil, amino acid, phytate, and phosphorus content. Additionally, we discuss two nonofficial methods: measuring phytate and available phosphorus levels, and assessing amino acid balance. Phytate and available phosphorous impact the mineral nutrition of grain, while amino acid balance reflects the value of grain as a protein source and the bioavailability of protein. We also describe the use of near-infrared spectroscopy (NIRS) to assess levels of various compounds in maize. NIRS relies on the fact that compounds with differing molecular properties uniquely interact with the near-infrared region (750-2500 nm) of the electromagnetic radiation spectrum, and thus, generate spectral information that can be used to develop calibration models/equations for predicting the concentration of the compounds in grain samples. We discuss how sensitivity, accuracy, precision, throughput, and cost influence the choice of assay used to assess grain quality. Furthermore, we discuss how appropriate experimental design and data analysis can improve analytical outcomes when assessing grain quality.
The human immune system evolved to defend against the panoply of microbial threats. By harnessing such ability, vaccines have cumulatively saved hundreds of millions of lives. Despite such tremendous success, there have also been remarkable failures, such as the lack of a clinically proven vaccine against Staphylococcus aureus (SA), which continues to pose an urgent public health threat. In practice, it has proven challenging to identify the molecular basis for relevant epitopes for this pathogen. Here, we summarize our experience implementing an integrated approach using phage display technology for the identification of B-cell epitopes of microbial virulence factors, which we developed with a focus on SA. This approach was used to define minimal B-cell epitopes of the staphylococcal leucocidin family of pore-forming toxins (PFTs) that have been implicated in staphylococcal clinical infection. Our methodology provides proof of principle for an approach well suited for the rapid and efficient generation of modular protein-based vaccines for protection from clinical infection, which can be used to target pathogens for which no vaccine is currently available.
To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein–protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody–epitope binding interactions.
Phage display technology is enabled by genetic fusion of a foreign protein domain to a phage coat protein, without interfering with the phage's ability to replicate by infecting bacterial host cells. The displayed domain is exposed on the phage particle (virion) surface, where it can interact with molecules or other substances in the surrounding medium; in this regard, it acts like a normal protein. However, it possesses a superpower that is unavailable to ordinary proteins: It is easily replicated in great abundance because it is attached to a replicating virion whose genome includes its coding sequence. The main way this technology is exploited is construction of huge phage display “libraries,” comprising billions of phage clones, each displaying a different protein domain, and each represented by thousands, millions, or billions of genetically identical virions—all mixed together in a single vessel. Surface display allows exceedingly rare virions whose displayed protein domains happen to bind a user-defined molecule or other substance—generically called the “selector”—to be isolated from such libraries by an affinity selection process. The yield of selector-binding virions is much too low to be of practical use, but their number is readily increased by many orders of magnitude by propagating the virions in host bacteria in culture. This overview is a critical review of recent developments of this technology. It does not review the entire arena of contemporary phage display; there is special emphasis on phage display's most prominent application, phage antibodies, in which the displayed domain is an antibody domain, and the selector is an antigen of interest.
Root lodging, the agronomic term for plant mechanical failure, causes yield loss in crops, including maize. Brace roots can provide structural support and assist in preventing root lodging. While the mechanics of brace roots (e.g., stiffness and strength) can play a role in their ability to prevent root lodging, there has been limited characterization of individual brace root mechanical properties. Methods to quantify root mechanics can thus be useful for characterizing maize mechanical traits and breeding new varieties with improved root anchorage and lodging resistance. Here, we describe a protocol for evaluating mechanical properties of maize brace roots. Specifically, we outline the steps necessary to perform three-point bend mechanical testing of maize brace roots using an Instron Universal Testing Stand. We describe root preparation, instrument setup, method establishment, testing, and data analysis. While we exemplify the protocol using maize brace roots, the approach can be adapted for assessing the mechanics of other plants or root types.
Root lodging due to wind is common in maize production worldwide, and can reduce photosynthetic capacity as well as nutrient uptake, resulting in significant yield loss and seed quality reduction. Lodging also causes harvesting problems, and ultimately increases production costs. Evaluating maize resistance to lodging is thus important for both breeders and researchers, to optimize agricultural practices, enhance breeding strategies, and ultimately develop new maize varieties with improved resilience. Here, we describe a novel procedure to accurately and quantitatively assess the resistance of maize plants to root lodging in the field. In this approach, users measure mechanical properties of maize root systems and estimate the magnitude of the wind force acting on the maize plants to ultimately derive an antilodging index, a measure that thus considers the balance between internal and external forces acting on the plants in the field. The procedure, which focuses on the plant as a whole and not only on the root system, has been successfully used to evaluate lodging resistance throughout the entire growth period, from the V8 growth stage to plant maturity, in different maize genotypes. We also compare the procedure to others in the literature, and discuss its applicability for assessing crop root lodging resistance in breeding and cultivation programs.
The mechanical properties of individual roots and entire root systems play key roles in essential root functions such as water and nutrient acquisition, defense against soil microorganisms, and plant anchorage. However, relatively few studies have quantified the mechanics (e.g., stiffness and strength) of individual and entire root systems, or explored the link between root mechanics and root functions. This limitation is likely due to a lack of standardized methods for quantifying root mechanical properties, and has created a gap in our understanding of how root mechanical traits contribute to root functions. To date, most of our knowledge comes from studies in maize, where mechanical failure (i.e., root lodging) has detrimental impacts on crop yield. Here, we review the importance of root mechanics for maize production and discuss methods used to measure individual and entire root system mechanics.
Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.
Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.
Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei . The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR–Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.
Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR–Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.
Understanding how the auxin hormone signaling pathway components come together to orchestrate cellular responses is key to engineering the growth and development of maize. Although a variety of techniques exist to measure auxin activities in plants, many are time- and resource-intensive or do not easily allow for high-throughput quantitative measurement of component libraries. The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the auxin hormone signaling pathway from essentially any plant. AuxInYeast uses Saccharomyces cerevisiae yeast as a heterologous expression platform for auxin signaling pathway components with fluorescent tags that facilitate measurement of auxin perception, repression, and activation. This protocol describes how to use fluorescence flow cytometry for these AuxInYeast experiments. As a case study, we focus on AuxInYeast strains built to measure maize auxin perception (i.e., those that express receptors and fluorescently tagged repressors that degrade upon auxin exposure). This protocol describes two different types of cytometry assays. The Steady-State Assay measures the extent of auxin-induced repressor degradation at one or two time points across many AuxInYeast strains and is particularly useful for initial assessment of whether auxin-induced degradation occurs and for dose response assays. The Time-Course Assay is used to measure auxin-induced repressor degradation dynamics over 2–3 h in a smaller number of strains. It is most useful for assessing the range of degradation rates across sets of repressors or receptors, and to precisely determine the impact of mutations and natural variation on degradation rate.
Synthetic biology approaches merge the tenets of engineering with established biological techniques to answer fundamental questions about living systems and to engineer biological forms and functions. Following the engineering principle of design–build–test–iterate, this review serves as a guide to applying synthetic principles and approaches in maize. We outline strategies for (1) choosing the optimal model organism to serve as a heterologous chassis for maize signaling pathways, (2) designing and building biological parts and devices to express pathway components, (3) choosing an analytical technique to measure pathway function, and (4) optimizing and troubleshooting the designed system. Auxin is a hormone that is essential for plant growth and development, regulating cellular proliferation and differentiation. Considering the importance of auxin for maize development in aerial and underground tissue, it was an obvious starting point for synthetic biology approaches. We use the maize nuclear auxin response recapitulated in yeast (AuxInYeast) system to showcase the power of heterologous expression approaches for testing fundamental attributes of the evolution, genetics, and biochemistry of signaling pathways that may be challenging to assay in planta. This approach involves co-expression of maize auxin signaling components in Saccharomyces cerevisiae coupled with fluorescence flow cytometry to quantify signaling activity. We and others have used this system to interrogate the dynamics of pathway signaling, interactions between paralogous components, and the adaptation of auxin signaling over large evolutionary distances. Thus, the AuxInYeast system is a fast, high-throughput, hypothesis-generating platform that can be readily adapted by the maize community to creatively answer questions about fundamental maize biology and to drive development of novel tools for breeding and plant engineering.
The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the plant auxin hormone signaling pathway. As a plant synthetic biology chassis, Saccharomyces cerevisiae yeast offers rapid growth, well-established genetic and biochemical tools, and core eukaryotic cellular machinery compatible with heterologous plant gene expression. The AuxInYeast system for maize consists of yeast cells containing the minimal necessary set of plant auxin signaling parts: a receptor (ZmTIR1/AFB), repressor (ZmIAA), corepressor (REL2), transcription factor (ZmARF), and auxin response cis-element (auxRE). In plants, auxin binding to a receptor:repressor complex triggers ubiquitination and degradation of the repressor, preventing it from binding to transcription factors on auxin response elements. Thus, auxin-induced repressor degradation allows for the activation of auxin-inducible transcriptional responses. Tagging various auxin signaling components with fluorescent protein reporters then enables quantitative measurement of signaling dynamics via high-throughput approaches such as flow cytometry. As these signaling proteins each belongs to large gene families, AuxInYeast users can build strains with defined components to study their behaviors in isolation or various combinations. Such strains enable researchers to dissect auxin sensitivity, the dynamics of auxin repressor degradation and transcriptional activation, and promoter architecture. It also allows a head-to-head comparison of maize components with orthologs from other plant species to test the evolutionary conservation of component interactions. This protocol describes the construction of such strains. Finally, this protocol and the AuxInYeast approach can also be adapted to assay other multicomponent maize biochemical pathways in yeast.
Seed mutagenesis using alkylating chemical agents such as ethyl methanesulfonate (EMS) can generate somatic and germinal mutations in many plant species. In monoecious plants like maize, the sperm- and egg-producing reproductive germlines are derived from distinct cell lineages in the embryo. This separation results in independent mutations inherited via the egg and sperm lineages and prevents the recovery of recessive mutant phenotypes in diploid progeny after the first round of self-pollination. Thus, two generations of self-pollination are required to screen for recessive mutations when conducting seed mutagenesis. The additional time and manual self-pollination make this approach laborious. However, a high mutation rate and the ability to screen for somatic sectors in heterozygous mutant plants and other defined genetic backgrounds make seed mutagenesis an effective but underutilized mutagenesis tool for maize research. This protocol provides the directions and optimization steps to perform effective seed mutagenesis in maize. A high frequency of somatic mutations from seed mutagenesis can be achieved, but comes at the expense of poor and disordered growth, failure to form reproductive structures, and low or no seed production at high EMS concentrations or long contact times. In experiments where germinal mutations are a goal, an optimum dose of EMS is required in the first generation. Maize genetic backgrounds vary in their sensitivity to EMS, requiring some pilot testing in new genetic backgrounds. Researchers using this protocol can carry out seed mutagenesis safely and effectively to develop libraries of mutants or alleles for various experiments.
