Cold Spring Harbor protocols最新文献

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.

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.

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.

Maize is an important plant for both global food security and genetics research. As the importance of microorganisms to plant health is becoming clearer, there is a growing interest in understanding the relationship between maize and its associated microbiome; i.e., the collection of microorganisms living on, around, and inside the plant. The ultimate goal of this research is to use these microbial communities to support more robust and sustainable maize production. Here, we provide an overview of recent progress in the field of maize microbiome research. We discuss the major microbiome compartments (rhizosphere, phyllosphere, and endosphere) and known functions of the microbiome. We also review the methods currently available to study the maize microbiome and its functions, and discuss how to carry out maize microbiome experiments, including both a general workflow (suitable for most microbiome analyses) and maize-specific experimental considerations.

For most farmers, the production of maize grain is the ultimate goal of the entire field season. From the point of view of plant microbiome studies, seeds are particularly interesting in that they are the only avenue for vertical transmission of microbes from parent to offspring, though microbes can also enter maize seeds via wounds or silks. Although the presence of seed endophytes is well documented, their role, if any, in seed health and their effects on the next generation of plants are largely unknown. This protocol describes the isolation of seed endophytes. Its primary focus is properly sterilizing the seed surface, followed by grinding to release the endophytes. The end product is a cell suspension suitable for either culturing or DNA analysis.

One of the most common methods to survey bacterial communities is targeted amplification of the hypervariable regions of the 16s rRNA gene followed by sequencing. This protocol details Illumina library preparation of such amplicons from communities isolated from maize. We include both staggered PCR primers to improve Illumina base calling and peptide nucleic acids (PNAs) to reduce the presence of plant organelles. Primers are designed with Illumina adapter sequences for the addition of sample-specific indexes (barcodes). We also briefly discuss alternative primer sets, including ones that directly discriminate against plant organelles or that amplify different organisms (e.g., fungal internal transcribed spacer [ITS] sequences).

The microbiota of maize leaves can be beneficial or detrimental to the host. Foliar diseases are the most obvious detrimental impact of the leaf microbiome, though more subtle effects of the normal (nondisease) community are an active area of research. This protocol describes two specific methodologies to sample the maize leaf microbiome: one sampling the surface (epiphyte) microbiome and one sampling the interior (endophyte) microbiome. Each method begins with collected leaf tissue and finishes with a cell suspension suitable for either isolating live microbes or extracting DNA for sequencing.

The soil microbiome of maize shapes its fitness, sustainability, and productivity. Accurately sampling maize's belowground microbial communities is important for identifying and characterizing these functions. Here, we describe a protocol to sample the maize rhizosphere (including the rhizoplane and endorhizosphere) and root zone (still influential but further from the root) in a form suitable for downstream analyses like culturing and DNA extractions. Although this protocol is written with Zea mays as the focus, these methods can generally be applied to any plant with similar fibrous root systems.

Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an "intermediate diversity" treatment.