Cold Spring Harbor protocols最新文献_第5页

Corrigendum: Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants. 更正：农杆菌介导的利用幼苗叶片轮毂外植体的热带玉米转化。

Cold Spring Harbor protocols Pub Date : 2025-03-20 DOI: 10.1101/pdb.corr108644

Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang

引用次数: 0

Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants. 利用农杆菌介导的热带玉米幼苗叶轮外植体转化。

Cold Spring Harbor protocols Pub Date : 2025-03-20 DOI: 10.1101/pdb.prot108596

Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang

{"title":"Agrobacterium-Mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants.","authors":"Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang","doi":"10.1101/pdb.prot108596","DOIUrl":"10.1101/pdb.prot108596","url":null,"abstract":"Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cassette that drives the expression of the morphogenic transcription factors (MTFs) Baby boom (Bbm) and Wuschel2 (Wus2), which have been shown to greatly enhance transformation efficiency. We outline here the steps required for the preparation of seedling leaf whorl explants and subsequent Agrobacterium infection, and describe the tissue culture regimen that results in transgenic plant regeneration. Because constitutive expression of Bbm and Wus2 prevents normal plant regeneration and the production of fertile plants, the cassette containing these genes must be excised. As such, we include the steps for the Cre/loxP-mediated excision of the MTF gene cassette. The protocol outlines a year-round, more affordable, and efficient approach for carrying out maize transformation for crop improvement.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281403","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 : 2025-03-12 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":"https://doi.org/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":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","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

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 : 2025-03-12 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":"https://doi.org/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":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","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

Testing AuxInYeast Synthetic Biology Strains via Fluorescence Flow Cytometry. 荧光流式细胞术检测生长素酵母合成生物学菌株。

Cold Spring Harbor protocols Pub Date : 2025-03-12 DOI: 10.1101/pdb.prot108635

Britney L Moss, Amy Lanctot, Román Ramos Báez

{"title":"Testing AuxInYeast Synthetic Biology Strains via Fluorescence Flow Cytometry.","authors":"Britney L Moss, Amy Lanctot, Román Ramos Báez","doi":"10.1101/pdb.prot108635","DOIUrl":"https://doi.org/10.1101/pdb.prot108635","url":null,"abstract":"Understanding how the auxin hormone signaling pathway components come together to orchestrate cellular responses is key to engineering the growth and development of maize. Although a variety of techniques exist to measure auxin activities in plants, many are time- and resource-intensive or do not easily allow for high-throughput quantitative measurement of component libraries. The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the auxin hormone signaling pathway from essentially any plant. AuxInYeast uses Saccharomyces cerevisiae yeast as a heterologous expression platform for auxin signaling pathway components with fluorescent tags that facilitate measurement of auxin perception, repression, and activation. This protocol describes how to use fluorescence flow cytometry for these AuxInYeast experiments. As a case study, we focus on AuxInYeast strains built to measure maize auxin perception (i.e., those that express receptors and fluorescently tagged repressors that degrade upon auxin exposure). This protocol describes two different types of cytometry assays. The Steady-State Assay measures the extent of auxin-induced repressor degradation at one or two time points across many AuxInYeast strains and is particularly useful for initial assessment of whether auxin-induced degradation occurs and for dose response assays. The Time-Course Assay is used to measure auxin-induced repressor degradation dynamics over 2-3 h in a smaller number of strains. It is most useful for assessing the range of degradation rates across sets of repressors or receptors, and to precisely determine the impact of mutations and natural variation on degradation rate.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143613789","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

Doubled Haploid Technology: Generation of Doubled Haploid Maize Lines Using Haploid Inducers. 双单倍体技术：使用单倍体诱导剂生成双倍单倍体玉米品系。

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.prot108624

Vencke K Grüning, Thomas Lübberstedt, Ursula K Frei

{"title":"Doubled Haploid Technology: Generation of Doubled Haploid Maize Lines Using Haploid Inducers.","authors":"Vencke K Grüning, Thomas Lübberstedt, Ursula K Frei","doi":"10.1101/pdb.prot108624","DOIUrl":"10.1101/pdb.prot108624","url":null,"abstract":"Doubled haploid (DH) technology allows for the development of completely homozygous lines from heterozygous plants in only two generations. This approach has been widely adopted in maize breeding programs, as it expedites the generation of inbred lines compared to traditional methods. The DH approach is based on the use of maize genotypes that have the ability to induce haploid seeds when used as the pollen parent. The most common method for producing maize haploid plants for the generation of DH lines is in vivo maternal haploid induction. The process involves pollination with a haploid inducer maize line to generate haploid seeds. Then, haploids are screened for and identified (typically via the expression of a particular marker gene), germinated, treated with an exogenous doubling agent to induce genome duplication, and transplanted to the field. Following successful self-pollination, seeds harvested from the ear represent fully homozygous lines. The seed set at this stage, however, is often low, necessitating one or two additional rounds of self-pollination to increase the number of fully homozygous inbred lines. Here, we describe a protocol for the generation of maize DH lines using maternal haploid-inducing maize lines. We outline the steps for setting up the donor material, performing induction crosses, selecting haploids based on two different marker alleles, treating seedlings with colchicine to double the genome, transplanting the treated seedlings to the field, and self-pollinating the treated plants.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108624"},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459795","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

Doubled Haploid Technology: Opportunities and Challenges for the Rapid Generation of Maize Homozygous Lines. 双单倍体技术：快速生成玉米同源系的机遇与挑战。

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.top108437

Vencke K Grüning, Thomas Lübberstedt, Ursula K Frei

引用次数: 0

Quantifying Nitrogen Uptake Rates of Maize Roots Using Stable Isotopes. 利用稳定同位素量化玉米根系的氮吸收率

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.top108436

Findimila Dio Ishaya, Amanda Rasmussen

{"title":"Quantifying Nitrogen Uptake Rates of Maize Roots Using Stable Isotopes.","authors":"Findimila Dio Ishaya, Amanda Rasmussen","doi":"10.1101/pdb.top108436","DOIUrl":"10.1101/pdb.top108436","url":null,"abstract":"Nitrogen is an essential element for plant growth and development; however, application of nitrogen (N)-based fertilizers comes with a high environmental cost. This includes the energy required for production, volatilization from fields, and runoff or leaching to waterways triggering algal blooms. As such, a key goal in plant breeding programs is to develop varieties that maintain yield while requiring less fertilization. Central to this goal is understanding how roots take up nitrogen and finding traits that represent improvements in the net uptake. Maize, one of the most widely produced crops in the world, has seminal, crown, and brace root types, each under independent developmental control. Recent evidence suggests that these independent developmental patterns may result in different nutrient uptake characteristics. As such, understanding the uptake dynamics of each root type under different environmental conditions is an essential aspect for the selection of new maize varieties. A key method for tracking nitrogen uptake is the use of the 15N stable isotope, which is naturally less abundant than the main 14N isotope. This method involves replacing the 14N in nutrient solutions with 15N, exogenously providing it to the plant tissues (roots in this case), and then measuring the 15N content of the tissues after a fixed amount of time. Here, we provide a brief overview of nitrogen uptake and remobilization in maize, and discuss current techniques for measuring nutrient uptake, with a focus on methods using stable isotopes of nitrogen.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108436"},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140915945","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

Imaging Neuropeptide Release from Drosophila Clock and Motor Neurons. 果蝇时钟和运动神经元神经肽释放成像

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.top107798

Edwin S Levitan, Dinara Bulgari, Markus K Klose

引用次数: 0

Methods for Measuring Nutrient Uptake in Maize Using Nitrogen Stable Isotopes. 利用氮稳定同位素测量玉米养分吸收的方法

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.prot108562

Findimila Dio Ishaya, Amanda Rasmussen

{"title":"Methods for Measuring Nutrient Uptake in Maize Using Nitrogen Stable Isotopes.","authors":"Findimila Dio Ishaya, Amanda Rasmussen","doi":"10.1101/pdb.prot108562","DOIUrl":"10.1101/pdb.prot108562","url":null,"abstract":"Nitrogen is a key nutrient for plant growth and development, and understanding nutrient uptake is central to improving nitrogen use efficiency in crops, including maize. Reducing the need for fertilizer without reducing yield is extremely important, as nitrogen fertilizers come with a high environmental cost, in terms of both emissions from manufacturing and losses to waterways or volatilization off fields. Maize develops multiple different root types, including primary, seminal, crown, and brace roots. Part of improving efficiency in maize involves understanding the differences in nutrient uptake via each distinct root type, but these differences have been largely ignored to date. Here, we describe a protocol that uses stable isotopes for determining nitrogen uptake rates by maize root types. We describe the steps both for intact roots, for which we use rhizoboxes with openable front windows that allow access to the roots without disturbing the rest of the plant, and for field-grown plants, for which intact analysis is not feasible and requires excising the roots. The methods described here can also be modified to measure uptake kinetics and for monitoring nutrient translocation between roots and shoots. Advancing our understanding of root physiology and nutrient dynamics will improve breeding opportunities for efficient nutrient uptake varieties, reducing the need for fertilizer additions.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108562"},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140915932","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