Cold Spring Harbor protocols最新文献

The rolled towel assay (RTA) is a soil-free method to evaluate juvenile phenotypes in crops such as maize and soybean. Here, we provide an updated RTA-based protocol to phenotype maize seedling responses to chemicals of interest. We exemplify the protocol with two synthetic auxin herbicides (2,4-dichlorophenoxyacetic acid and picloram), an auxin precursor (indole-3-butyric acid), and an auxin inhibitor (N-1-naphthylphthalamic acid), but the method can be used with other hormones or plant growth regulators that are soluble in growth media. We also include instructions on how to annotate root traits and analyze primary root length trait data. The protocol can be scaled up for use in genetic screens, preparing tissue for gene expression analyses, carrying out genome-wide association studies (GWASs), and quantitative trait locus (QTL) identification.

Plant hormones have key functions in plant morphology, physiology, and stress responses. Studies on the biology of hormones and their effect on plant physiology and metabolism are greatly facilitated by the exogenous application of these compounds. In general, methods for exogenous hormone application are easy and fast, and provide useful information about their effects in planta. Although hormone effects have been studied in several plant species, the used methods need to be tailored specifically to each species to get robust data. Maize is an established model for basic and applied research, and an excellent system for studying the effects of hormones on developmental and stress responses in a cereal crop. Different methods have been reported for the exogenous application of plant growth regulators in maize, including watering, spraying, immersion, and application to the apical whorl. These various methods are useful to analyze hormone responses at different developmental stages, in specific organs, and within tissues. As with all exogenous application assays, suitable experimental design and the inclusion of proper controls are critical factors in these methods, to obtain reliable and reproducible results. Here, we provide an overview of various methods for hormone exogenous application in maize, and technical considerations to get successful results.

Exogenous application of hormones in plants is a valuable technique for studying and manipulating plant growth, development, and responses to environmental stimuli. The foliar spray method is one of the most common approaches for the exogenous application of hormones in plants due to its ease of use on aerial organs (such as leaves and inflorescences) and the rapid absorption of the treated tissue, facilitating subsequent analyses. Here, we provide a protocol to implement this method in maize. The approach consists of preparing dilutions of the hormones or plant growth regulators (PGRs) of interest, usually in an aqueous solution and at low concentrations, followed by application by foliar spraying using a defined treatment regimen. Users can then evaluate effects by measuring different parameters, such as stem size, flowering time, seed production, or others. The foliar spray method can easily be scaled up and automated in greenhouse and field settings, and can be used to treat plants at all developmental stages.

Plant embryogenesis encompasses the biological processes wherein the zygote (fertilized egg) undergoes cell division, cell expansion, and cell differentiation to develop histological tissue layers, meristems, and various organs comprising the primordial body plan of the organism. Studies of embryogenesis in the agronomically important maize crop advance our understanding of the fundamental mechanism of plant development, which, upon translation, may advance agronomic improvement, optimization of conditions for somatic embryogenesis, and plant synthetic biology. Maize embryo development is coordinated temporally and spatially and is regulated by interactive genetic networks. Single-cell RNA sequencing (RNA-seq) and spatial transcriptomics are powerful tools to examine gene expression patterns and regulatory networks at single-cell resolution and in a spatial context, respectively. Single-cell technology enables profiling of three-dimensional samples with high cellular resolution, but it can be difficult to identify specific cell clusters due to a lack of known markers in most plant species. In contrast, spatial transcriptomics provide transcriptomic profiling of discrete regions within a sectioned, two-dimensional sample, although single-cell resolution is typically not obtained and fewer transcripts per cell are detected than in single-cell RNA-seq. In this review, we describe the combined use of these two transcriptomic strategies to study maize embryogenesis with synergistic results.

Plant hormones play an essential role in both development and stress responses. These organic natural compounds have critical functions in plant-related processes, including but not limited to seed development, anther formation, root elongation, and responses to abiotic and biotic stress. One way to study the impact of hormones on these processes is by external application, followed by evaluation of parameters of interest. Here, we describe one such method for the exogenous application of hormones in maize: the apical whorl treatment approach, which is well suited for evaluating the role of these compounds in reproductive stages (e.g., when the target organ is the inflorescence meristem). This method involves direct application of a hormone solution to the apical part of the plants every 2 days until the tassel emerges, which takes 15-20 days, or until the treated plants show noticeable phenotypic changes for evaluation. This method is ideal for observing effects on the apical meristem, and it may be scaled up for analyzing large numbers of plants.

Maize is an important crop that contributes to the modern economy in various ways, including use for human consumption, as animal feed, and in industrial products. Research on maize is crucial for understanding plant development, which in turn provides valuable insight into improvement of maize crops to meet the food demands of a growing population. Maize embryogenesis, which is the primordial stage of the corn life cycle, determines the fundamental body plan and developmental programs that organize the tissue patterning and subsequent growth and reproduction of the corn plant. Investigating maize embryogenesis at high cellular resolution can enhance our understanding of the homology, ontogeny, and developmental genetic mechanisms of embryonic organ morphogenesis. However, until recently, no published studies have used methods for analyzing maize embryo development at single-cell resolution. This protocol describes single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic analyses, which are powerful, combinatorial tools that can be used to study maize embryogenesis at the single-cell level within a spatial context. These tools have the power to reveal transcriptomic relationships between tissues/organs, and to provide insight into the gene regulatory networks operating during embryogenesis. In this protocol, we describe a detailed procedure to prepare maize embryo samples for construction of scRNA-seq and Visium spatial transcriptomic libraries that are suitable for massively parallel sequencing. Our protocol borrows from prior published studies and manufacturer's instructions and is optimized for studies of the maize embryo.

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.

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.

Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.