Cold Spring Harbor protocols最新文献_第5页

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 : 2026-05-01 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":"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":"pdb.top108444"},"PeriodicalIF":0.0,"publicationDate":"2026-05-01","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

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

Cold Spring Harbor protocols Pub Date : 2026-05-01 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

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 : 2026-04-01 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":"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":"pdb.top108504"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","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

A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards. 蜥蜴卵母细胞注射手术方法及CRISPR-Cas9诱变。

Cold Spring Harbor protocols Pub Date : 2026-04-01 DOI: 10.1101/pdb.prot108652

Christina E Sabin, Sukhada P Samudra, Anna L Iouchmanov, Amber L Rittgers, James D Lauderdale, Douglas B Menke

{"title":"A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards.","authors":"Christina E Sabin, Sukhada P Samudra, Anna L Iouchmanov, Amber L Rittgers, James D Lauderdale, Douglas B Menke","doi":"10.1101/pdb.prot108652","DOIUrl":"10.1101/pdb.prot108652","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108652"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144759366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","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 : 2026-04-01 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":"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":"pdb.prot108650"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","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

Ethyl Methanesulfonate Treatment of Maize Pollen for Development of Segregating Mutant Populations or Targeted Mutagenesis. 玉米花粉甲基磺酸乙酯处理对分离突变群体发育或定向诱变的影响。

Cold Spring Harbor protocols Pub Date : 2026-04-01 DOI: 10.1101/pdb.prot108651

Rajdeep S Khangura, Norman B Best, Brian P Dilkes

{"title":"Ethyl Methanesulfonate Treatment of Maize Pollen for Development of Segregating Mutant Populations or Targeted Mutagenesis.","authors":"Rajdeep S Khangura, Norman B Best, Brian P Dilkes","doi":"10.1101/pdb.prot108651","DOIUrl":"10.1101/pdb.prot108651","url":null,"abstract":"In maize, abundant pollen production and easy controlled pollination permit the direct mutagenesis of pollen to produce populations of independent mutant lines. Pollen can be treated with alkylating agents, such as ethyl methanesulfonate (EMS), to induce point mutations. The ease of applying and decontaminating this mutagen after the mutagenesis application and the advantages provided by the mutation spectra for subsequent bioinformatic analysis make EMS an attractive mutagen. We provide a maize pollen mutagenesis protocol with a list of critical supplies, a step-by-step procedure, and troubleshooting tips. Pollen is freshly collected and suspended in an emulsion of EMS and paraffin oil. The slurry of pollen, oil, and EMS is then directly placed on prepared maize silks to perform pollinations. Mutations result during embryogenesis due to replication-dependent mispairing at alkylated residues contributed by sperm nuclei. Thus, each seed bears an independent set of mutations. These progenies can be analyzed directly, as is the case in targeted mutagenesis experiments or the exploration of dominant genetic variation. Alternatively, the progenies of self-pollinated plants can be screened in the next generation to discover novel recessive mutations. In addition to the dose of EMS and contact time, the genetic background of maize can significantly influence outcomes, and some optimization of dose and contact time may be required for a genetic background and specific use case. Although we outline good practices for safe handling of EMS and waste, researchers should consult their local safety officers to ensure safe handling, decontamination, and disposal of EMS, which is toxic.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108651"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215148","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

Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution. 蜥蜴作为爬行动物发育和进化中基因功能研究的模式系统。

Cold Spring Harbor protocols Pub Date : 2026-04-01 DOI: 10.1101/pdb.top108535

Christina E Sabin, James D Lauderdale, Douglas B Menke

{"title":"Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution.","authors":"Christina E Sabin, James D Lauderdale, Douglas B Menke","doi":"10.1101/pdb.top108535","DOIUrl":"10.1101/pdb.top108535","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108535"},"PeriodicalIF":0.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144759365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sterol and Brassinosteroid Hormone Quantification by LC/MS of Picolinyl Ester Derivatives. Picolinyl酯衍生物的LC/MS定量分析甾醇和油菜素类固醇激素。

Cold Spring Harbor protocols Pub Date : 2026-03-02 DOI: 10.1101/pdb.prot108646

Brian P Dilkes, Norman B Best

引用次数: 0

Building AuxInYeast Synthetic Biology Strains for Biochemical Characterization of Maize Auxin Hormone Signaling Components. 构建玉米生长素信号成分生长素酵母合成生物学菌株。

Cold Spring Harbor protocols Pub Date : 2026-03-02 DOI: 10.1101/pdb.prot108634

Román Ramos Báez, Amy Lanctot, Britney L Moss

{"title":"Building AuxInYeast Synthetic Biology Strains for Biochemical Characterization of Maize Auxin Hormone Signaling Components.","authors":"Román Ramos Báez, Amy Lanctot, Britney L Moss","doi":"10.1101/pdb.prot108634","DOIUrl":"10.1101/pdb.prot108634","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108634"},"PeriodicalIF":0.0,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613786","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

Synthetic Biology Approaches to Study Maize Signaling Pathways. 合成生物学方法研究玉米信号通路。

Cold Spring Harbor protocols Pub Date : 2026-03-02 DOI: 10.1101/pdb.top108450

Amy Lanctot, Román Ramos Báez, Britney L Moss

{"title":"Synthetic Biology Approaches to Study Maize Signaling Pathways.","authors":"Amy Lanctot, Román Ramos Báez, Britney L Moss","doi":"10.1101/pdb.top108450","DOIUrl":"10.1101/pdb.top108450","url":null,"abstract":"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.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108450"},"PeriodicalIF":0.0,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613787","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