Cold Spring Harbor protocols最新文献_第2页

How to Monitor Growth and Identify Developmental Stages of Maize (Zea mays). 如何监测玉米（Zea mays）的生长和发育阶段识别。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108637

Miriam Nancy Salazar-Vidal, Melissa A Draves, Sarah L Fitzsimmons, Zachary B Traylor, William F Tracy, Sherry Flint-Garcia

引用次数: 0

High-Throughput Fluorescence Microscopy Using Aniline Blue Staining to Study the Maize-Exserohilum turcicum Pathosystem. 应用苯胺蓝染色的高通量荧光显微镜研究玉米-黄茎枯病菌的病理系统。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108643

Rashmi Pokhrel, Alexander Mullens, Peyton Sorensen, Santiago Mideros, Tiffany Jamann

{"title":"High-Throughput Fluorescence Microscopy Using Aniline Blue Staining to Study the Maize-Exserohilum turcicum Pathosystem.","authors":"Rashmi Pokhrel, Alexander Mullens, Peyton Sorensen, Santiago Mideros, Tiffany Jamann","doi":"10.1101/pdb.prot108643","DOIUrl":"https://doi.org/10.1101/pdb.prot108643","url":null,"abstract":"Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused by Exserohilum turcicum, is an important fungal foliar disease of maize that is highly prevalent and causes yield losses globally. Microscopy can be used to visualize plant-fungal interactions on a cellular level, which enables pathology and genetics studies. Host resistance and isolate aggressiveness can be characterized at different stages of disease development, which enables a more detailed understanding of the pathogenesis process and host-pathogen interactions. Our protocol outlines an efficient, cost-effective method for staining E. turcicum tissue on inoculated maize leaves and visualizing samples using a compound fluorescence microscope. This protocol uses KOH treatment followed by aniline blue staining, which stains glucans present in plant and fungal cell walls, and samples are visualized using fluorescence microscopy. Quantitative data about fungal structures including the conidia, hyphal structures, and appressoria, the structures formed to push through the plant leaf surface after conidia have germinated, can be obtained from the images generated using this technique. Visualization of these structures can help pathologists understand plant-pathogen interactions for maize and E. turcicum This method has advantages over other methods because the stain is less toxic than other available stains, samples can be processed in a more high-throughput manner than other protocols, and the required supplies are relatively inexpensive.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

How to Pollinate Corn (Zea mays). 如何给玉米授粉（玉米）。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108638

Sarah L Fitzsimmons, Miriam Nancy Salazar-Vidal, Zachary B Traylor, Melissa A Draves, Sherry Flint-Garcia

引用次数: 0

Uses and Opportunities for Ethyl Methanesulfonate Mutagenesis in Maize. 甲基磺酸乙酯诱变在玉米中的应用与机遇。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.top108504

Rajdeep S Khangura, Norman B Best, Brian P Dilkes

{"title":"Uses and Opportunities for Ethyl Methanesulfonate Mutagenesis in Maize.","authors":"Rajdeep S Khangura, Norman B Best, Brian P Dilkes","doi":"10.1101/pdb.top108504","DOIUrl":"https://doi.org/10.1101/pdb.top108504","url":null,"abstract":"Creating mutations in maize has provided key foundational information for our mechanistic understanding of genetics, evolution, and even the role of chromosomes as units of inheritance. Chemical mutagenesis is used in biological research to create novel genetic variation. Ethyl methanesulfonate (EMS) is an alkylating agent and a highly potent and frequently used mutagen. EMS mutagenesis can be used to identify genes based on phenotypes induced by mutagenesis (forward genetics) and to validate the functions of genes by independently creating multiple mutant alleles in known genes (reverse genetics). Due to our ability to collect huge quantities of maize pollen and to easily apply pollen to the silks of maize ears to conduct pollination and achieve hundreds of fertilization events, pollen EMS mutagenesis is uniquely facile in maize. While pollen EMS mutagenesis is commonly performed, treatment of maize seeds with EMS is also highly effective, and can be used for certain research objectives that are difficult to achieve with pollen mutagenesis, such as recovering mutant sectors. The alkylation of guanine residues by EMS primarily results in G > A or C > T transitions in the DNA, making the molecular profiling of mutations caused by EMS easy, with an extremely low false positive rate. EMS is hydrophilic, has a moderate half-life in water, and is sensitive to light and high temperatures. With appropriate precautions in research settings, EMS can be relatively safe to handle. Here, we provide an introduction to chemical mutagenesis via EMS, including some history on its use in maize and the considerations for the effective and safe design of mutagenesis experiments with EMS in maize.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inoculation of Maize Ears with Fusarium graminearum to Study Gibberella Ear Rot. 接种玉米穗镰孢菌研究赤霉素穗腐病。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108641

Sarah Lipps, Peyton Sorensen, Mara Krone, Santiago Mideros, Tiffany Jamann

{"title":"Inoculation of Maize Ears with Fusarium graminearum to Study Gibberella Ear Rot.","authors":"Sarah Lipps, Peyton Sorensen, Mara Krone, Santiago Mideros, Tiffany Jamann","doi":"10.1101/pdb.prot108641","DOIUrl":"https://doi.org/10.1101/pdb.prot108641","url":null,"abstract":"Maize is an important food and fuel crop globally. Ear rots, caused by fungal pathogens, are some of the most detrimental maize diseases, due to reduced grain yield and the production of harmful mycotoxins. Mycotoxins are naturally occurring toxins produced by certain fungal species that can cause acute and chronic health issues in humans and animals that consume mycotoxin-contaminated grain. Pathogens can infect the developing ear through silks, or through wounds in the ears produced by pests. Plants naturally develop genetic resistance to pathogens. The maize genes involved in resistance to the pathogen may be different, depending on whether the ear was infected via silks or wounds. To differentiate between these two forms of resistance, natural infections can be reproduced by injecting inoculum through the silk channel, or by producing wounds using a needle, and introducing inoculum directly onto developing ears. Our protocol describes a technique used to inoculate developing maize ears with Fusarium graminearum, one of the fungal species that causes ear rot. We describe both silk channel and side needle inoculation techniques. Our protocol uses a backpack inoculator for both methods of infection, allowing for high-throughput inoculations, which are necessary for large field experiments. After harvest, the ears are visually rated on a percentage of disease scale. The protocol results in quantitative data that can be used for research on elucidating genetic resistance to fungal pathogens to assist breeding selections, and to understand plant-pathogen interactions of ear rots in maize.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Planting, Pollinating, Harvesting, and Monitoring Growth in Maize (Zea mays) for Research. 研究用玉米（Zea mays）的种植、授粉、收获和生长监测。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.top108444

Zachary B Traylor, Sarah L Fitzsimmons, Melissa A Draves, Miriam Nancy Salazar-Vidal, William F Tracy, Sherry Flint-Garcia

{"title":"Planting, Pollinating, Harvesting, and Monitoring Growth in Maize (Zea mays) for Research.","authors":"Zachary B Traylor, Sarah L Fitzsimmons, Melissa A Draves, Miriam Nancy Salazar-Vidal, William F Tracy, Sherry Flint-Garcia","doi":"10.1101/pdb.top108444","DOIUrl":"https://doi.org/10.1101/pdb.top108444","url":null,"abstract":"Zea mays, also known as maize or corn, is a staple crop as well as a classical model organism for plant genetic studies and research. To conduct maize research, plants must be properly cultivated in field or greenhouse conditions to ensure reproductive success and safeguard genetic identity through controlled pollinations. Genetic studies require knowing which alleles or genetic combinations (genotype) are present in an individual so the geneticist can create new combinations or select the desired genotypes. In order to determine and maintain the genetic identity of a corn plant and make precise selections of male and female plants, reproductive structures must be covered and isolated prior to silking and anthesis, or pollen shed. Doing so allows experimenters to make controlled pollinations to produce the desired genotype. Successful pollination of corn requires proper field design and preparation, careful planting to maintain distinct genetic families, and careful monitoring of growth and husbandry followed by proper harvest and seed storage. These activities have been optimized over the past 100 years. In this review, we summarize each step needed to produce a generation of corn from planting to harvest.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inoculation of Maize Leaves with the Bacterial Foliar Pathogen Clavibacter nebraskensis. 玉米叶片细菌病原菌内布拉斯加大杆菌的接种研究。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108642

Alexander Mullens, Peyton Sorensen, Julian Cooper, Tiffany Jamann

{"title":"Inoculation of Maize Leaves with the Bacterial Foliar Pathogen Clavibacter nebraskensis.","authors":"Alexander Mullens, Peyton Sorensen, Julian Cooper, Tiffany Jamann","doi":"10.1101/pdb.prot108642","DOIUrl":"https://doi.org/10.1101/pdb.prot108642","url":null,"abstract":"Maize significantly contributes to food and fuel production. Yields can be reduced due to foliar diseases, which reduce photosynthetic leaf area. The bacterial foliar disease Goss's wilt (caused by Clavibacter nebraskensis) can cause significant yield losses in susceptible maize varieties. C. nebraskensis can infect leaves through wounds and colonize the vascular tissue of the leaf. We present a protocol that replicates this process with the use of a \"clapper\" with pins on one end to create wounds and a sponge soaked in inoculum on the other end, which allows for efficient field inoculations of maize leaves. Disease severity is then rated on a percentage scale multiple times over the season to generate an area under disease progress curve (AUDPC). Genetic host resistance is one of the most effective forms of foliar disease control in maize, as there are few effective forms of chemical control for bacterial diseases that affect maize. Screening for resistance in diverse germplasm, or for fine mapping a specific resistance gene, requires inoculating large populations in the field for obtaining phenotypic data. Our high-throughput protocol allows for large-scale disease evaluations and is useful for finding forms of genetic resistance or to understand plant-pathogen interactions of bacterial foliar pathogens.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Ethyl Methanesulfonate Treatment of Maize Seed for Recovery of Vegetative Mutant Sectors and Segregating Germinal Mutations. 用甲基磺酸乙酯处理玉米种子恢复营养突变区段和分离萌发突变。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108650

Rajdeep S Khangura, Norman B Best, Brian P Dilkes

{"title":"Ethyl Methanesulfonate Treatment of Maize Seed for Recovery of Vegetative Mutant Sectors and Segregating Germinal Mutations.","authors":"Rajdeep S Khangura, Norman B Best, Brian P Dilkes","doi":"10.1101/pdb.prot108650","DOIUrl":"https://doi.org/10.1101/pdb.prot108650","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Field Preparation and Planting Corn (Zea mays). 大田准备和种植玉米（玉米）。

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.prot108636

Melissa A Draves, Zachary B Traylor, Miriam Nancy Salazar-Vidal, Sarah L Fitzsimmons, Sherry Flint-Garcia

引用次数: 0

Pathogen Inoculation and Rating Strategies for Studying Maize Diseases. 玉米病害研究的病原接种与分级策略

Cold Spring Harbor protocols Pub Date : 2025-06-03 DOI: 10.1101/pdb.top108447

Peyton Sorensen, Santiago Mideros, Tiffany Jamann

{"title":"Pathogen Inoculation and Rating Strategies for Studying Maize Diseases.","authors":"Peyton Sorensen, Santiago Mideros, Tiffany Jamann","doi":"10.1101/pdb.top108447","DOIUrl":"https://doi.org/10.1101/pdb.top108447","url":null,"abstract":"Maize is a globally important staple that is used as food for human and animal consumption, fuel, and other industrial applications. Pathogens affect all stages of the plant life cycle and every plant organ, and lead to significant yield losses. An integrated strategy incorporating cultural and chemical management practices, as well as development of resistant plant varieties, is needed to prevent yield losses due to plant diseases. Large numbers of breeding material must be screened to develop pathogen-resistant maize varieties. Inoculation methods must be high-throughput to accommodate the large screening experiments. Additionally, there needs to be an extensive understanding of the plant-pathogen interaction to use a targeted biotechnology-based approach, which takes advantage of knowledge of the system to engineer resistance. To evaluate germplasm for breeding and biotechnology approaches, inoculation methods must replicate natural infection, and disease severity must be rated consistently to accurately screen germplasm or gather data on pathogens of interest. Here, we review inoculation and rating methods for Gibberella ear rot, seedling blight caused by Globisporangium ultimum var. ultimum, and Goss's wilt that are efficient and high-throughput. We also introduce fluorescence microscopy techniques for leaf samples infected with Exserohilum turcicum, the causal agent of northern corn leaf blight. These pathogens all cause significant yield losses, and in particular, Gibberella ear rot is associated with the accumulation of harmful mycotoxins. Understanding how pathogens cause disease and how plants defend against attack is a major goal of maize pathology studies and critical for developing integrated management strategies.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0