Cold Spring Harbor protocols最新文献_第4页

CRISPR-Cas-Directed Genome Editing in Maize. crispr - cas在玉米基因组编辑中的应用

Cold Spring Harbor protocols Pub Date : 2026-07-01 DOI: 10.1101/pdb.top108448

Bing Yang, Kan Wang

{"title":"CRISPR-Cas-Directed Genome Editing in Maize.","authors":"Bing Yang, Kan Wang","doi":"10.1101/pdb.top108448","DOIUrl":"10.1101/pdb.top108448","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108448"},"PeriodicalIF":0.0,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145243830","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 Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot. 用最后一种球孢根接种玉米根腐病研究。

Cold Spring Harbor protocols Pub Date : 2026-06-01 DOI: 10.1101/pdb.prot108640

Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros

{"title":"Inoculation of Maize Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot.","authors":"Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros","doi":"10.1101/pdb.prot108640","DOIUrl":"10.1101/pdb.prot108640","url":null,"abstract":"Maize is a globally important field crop for food and fuel production. Yield can be affected early in the growing season by oomycete and fungal pathogens that cause root rot or prevent seed germination. The diseases caused by these pathogens are referred to as seedling blights, root rots, or damping off. Pythium root rot is one of the most significant of these diseases. The disease is caused by multiple species of the oomycete genera Globisporangium and Pythium and results in significant yield losses due to reduced seed germination and reduced vigor of surviving seedlings. In this protocol, we mimic the natural infection process by mixing the inoculum into the potting media in which seeds are planted. Then, we flood the seeds daily for several days in large plastic totes to induce flooding conditions. Disease severity is assessed using stand counts and measuring root mass and length. This protocol allows researchers to investigate quantitative differences in disease symptoms, isolate aggressiveness, as well as levels of host resistance. This protocol was developed for the pathogen Globisporangium ultimum var. ultimum, but it can be adapted for other species.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108640"},"PeriodicalIF":0.0,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215157","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

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 : 2026-06-01 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":"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":"pdb.prot108643"},"PeriodicalIF":0.0,"publicationDate":"2026-06-01","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

Corrigendum: Inoculation of Maize Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot. 勘误：用最后一种球孢菊接种玉米根系研究牙周病。

Cold Spring Harbor protocols Pub Date : 2026-06-01 DOI: 10.1101/pdb.Corr108654

Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros

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 : 2026-06-01 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":"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":"pdb.prot108641"},"PeriodicalIF":0.0,"publicationDate":"2026-06-01","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

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 : 2026-06-01 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":"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":"pdb.prot108642"},"PeriodicalIF":0.0,"publicationDate":"2026-06-01","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

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 : 2026-06-01 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":"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":"pdb.top108447"},"PeriodicalIF":0.0,"publicationDate":"2026-06-01","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

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 : 2026-05-01 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

How to Harvest and Store Corn (Zea mays). 如何收获和储存玉米（玉米）。

Cold Spring Harbor protocols Pub Date : 2026-05-01 DOI: 10.1101/pdb.prot108639

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

{"title":"How to Harvest and Store Corn (Zea mays).","authors":"Miriam Nancy Salazar-Vidal, Melissa A Draves, Sarah L Fitzsimmons, Zachary B Traylor, Sherry Flint-Garcia","doi":"10.1101/pdb.prot108639","DOIUrl":"10.1101/pdb.prot108639","url":null,"abstract":"Harvest scheduling, seed drying, and good storage practices are essential for maize research to avoid negative impacts on the quality of the seeds and to maximize seed viability. Embryo growth and accumulation of energy reserves in the endosperm are completed ∼40 days after pollination, of which the last 10-20 days are devoted to maturation and desiccation. Seed maturity is affected by many factors including temperature, day length, humidity, and soil moisture. Once seeds are mature, they must be harvested. Hand harvesting, which allows for greater control and minimizes ear damage, is primarily used in genetic nurseries and general research because each genotype is represented by a small number of plants. Hand harvesting is also used where there is a mix of manually pollinated and undesired open pollinated ears, and can be a selective harvest depending on the research objectives. However, hand harvesting is more labor-intensive and time-consuming compared to combine harvesting. If harvesting a yield trial, where the purpose is to collect yield data and identify promising genotypes, the use of a combine (not described in this protocol) is critical to consistently capture grain weight, moisture, and test weight for each plot. Following harvest, materials must be dried to the appropriate moisture content before storage. Corn is typically stored using the \"active collection\" model, with temperatures set between 5°C and 10°C and low relative humidity. This harvest protocol is intended to assist laboratories that are new to maize research and may be modified based on project goals, genetic material, equipment, or available space.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108639"},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215152","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 : 2026-05-01 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