Cold Spring Harbor protocols最新文献_第2页

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

GFP and FAP Imaging of Neuropeptide Release in Drosophila. 果蝇神经肽释放的 GFP 和 FAP 成像。

Cold Spring Harbor protocols Pub Date : 2025-03-03 DOI: 10.1101/pdb.prot108069

Edwin S Levitan, Dinara Bulgari, Markus K Klose

引用次数: 0

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction. 果蝇幼虫神经肌肉接头处突触传递的电压钳分析

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108132

Bing Zhang, Bryan Stewart

{"title":"Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108132","DOIUrl":"10.1101/pdb.prot108132","url":null,"abstract":"Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a \"ceiling effect,\" because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108132"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189538","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

Electrophysiological Recording from a "Model" Cell. 模型 "细胞的电生理记录

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108130

Bing Zhang, Bryan Stewart

{"title":"Electrophysiological Recording from a \"Model\" Cell.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108130","DOIUrl":"10.1101/pdb.prot108130","url":null,"abstract":"The muscle cell or neuron membrane is functionally equivalent to a resistor-capacitor (RC) circuit with the membrane resistance and capacitor in parallel. Once inserted inside the membrane, an electrode introduces a serial resistance and small capacitance to the RC circuit. Through a narrow opening at its tip (∼0.1-μm), current can pass through the electrode, into the cell, and back to the outside (ground) across the membrane to complete the circuit. This arrangement enables a voltage difference between the outside and inside of the cell membrane to be recorded. To determine cell membrane properties, a current can be injected into the cell through the electrode. One complication with this approach, however, is that the voltage difference measured with the electrode includes the voltage drop across the cell membrane and that across the electrode. Furthermore, a small amount of current is drawn by the electrode capacitor, thereby slowing the current flow across the membrane. Fortunately, most amplifiers are equipped with bridge balance and capacitance compensation functions so that the effects of the electrode on cell membrane properties can be canceled out or minimized. This protocol describes the basics of setting up and conducting electrophysiological experiments using a model cell. For the novice, a model cell provides a way to learn the operation of electrophysiology equipment and software without the anxiety of damaging living cells. This protocol also illustrates passive membrane properties such as the input resistance, capacitance, and time constant.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108130"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189533","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

Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording in Drosophila. 用于果蝇电生理记录的微电极、抽吸电极和病灶电极的制作。

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108134

Bing Zhang, Bryan Stewart

{"title":"Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording in Drosophila.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108134","DOIUrl":"10.1101/pdb.prot108134","url":null,"abstract":"Electrophysiological recording is a group of techniques used to record electrical field potentials generated by cells. These techniques rely on several types of electrodes, which can be manufactured in the laboratory. In intracellular recording, glass microelectrodes are used to pierce the cell membrane, and then to measure the electrical potential difference between the inside and the outside of the cell. Another technique, called loose patch or focal recording, is similar to intracellular recording but the electrode tip does not pierce into the cell membrane. Rather, the electrode tip is placed near a nerve or the postsynaptic side of the neuromuscular junction (NMJ) to record extracellular changes in local potentials. A third technique involves a suction electrode, which is used to draw part of the motor nerve into the electrode so that electrical pulses can be applied to elicit action potentials of the nerve. Suction electrodes are specifically used to evoke synaptic potentials at the Drosophila larval NMJ. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108134"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189534","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

Synaptic Electrophysiology of the Drosophila Neuromuscular Junction. 果蝇神经肌肉接头的突触电生理学

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.top107820

Bing Zhang, Bryan Stewart

{"title":"Synaptic Electrophysiology of the Drosophila Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.top107820","DOIUrl":"10.1101/pdb.top107820","url":null,"abstract":"Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal experiences an influx of calcium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neurotransmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SVs at presynaptic terminals. The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic features of synaptic transmission, as well as the molecular mechanisms regulating the SV cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ is an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Here, we describe the theory and practice of electrophysiology as applied to the Drosophila larval NMJ preparation. We introduce the basics of membrane potentials, with an emphasis on the resting potential and synaptic potential. We also describe the equipment and methods required to set up an electrophysiology rig.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107820"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189537","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