作者想感谢克里斯泰勒博士（美国俄亥俄州立大学）毛状根技术引入到我们的实验室和博士。 Senthil Subramanian（南达科他州州立大学），娟（中国，山东鲁东大学）为提高协议。我们也感谢帮助这个视频成浩博士（南京农业大学）。这项工作是支持部分由美国能源部（DE - SC0001295），美国国家科学基金会（MCB - 0923779）和美国农业部（2010-65116-20514）的赠款，以OY，

下面的协议已被用来产生毛状根的复合植物模型豆科植物M. truncatula。类似的协议已被改编为至少8个植物物种 1-4 。我们用M。 truncatula毛状根复合植物研究在根和根瘤发育的基因功能。该协议分为四个部分：1）植物材料的准备; 2）产生毛状根复合植物; 3） 共生根瘤菌感染; 4）转基因root的身份。我们使用的是二进制向量含有绿色荧光蛋白（GFP）基因作为筛选转基因复合植物根3记者。以抗生素为基础的选择相比，绿色荧光蛋白为基础的筛查是快速，简便，价格低廉。在我们的构造，优化ER 表达绿色荧光蛋白基因是由超级ubiquitin启动子，具有很强的构的GFP转基因根源信号驱动，允许转基因和非转基因根源之间很容易区别。 1。植物材料的准备<ol><li> M. truncautla种子收获在温室中种植的植物（相对湿度50％，16 / 8 h光/暗，22/18 ° C昼/夜温度） 。成熟的种子可以保存在4 ° C </li><li>大约100粒种子scarified定期摇晃10分钟10毫升浓硫酸（95-98％，Sigma - Aldrich公司，MO）。 </li><li>种子轻轻摇动无菌水冲洗5-7次。 </li><li>种子在室温下在水中浸泡6小时，并保存在4 ° C为36-48小时同步发芽。 </li><li>大约20-30种子置于培养皿中的湿滤纸上传播。他们都保持在22 ° C，16 / 8 h光/暗周期，40μmol.m-2。-1光的强度，为5-6天。 </li><li>发芽的幼苗被转移到土壤和温室放置一个月。 </li></ol> 2。产生毛状根复合植物<ol><li>携带基因的二元结构转化成A 。使用标准协议的发根。我们已经设计了一套二进制向量含有绿色荧光蛋白基因作为标记，以促进转基因组织的筛选。这些载体含有不同的促销员都过度表达或沉默靶基因 3,5网关克隆网站（Invitrogen公司，卡尔斯巴德，CA） 。 </li><li> 答：发根培养50毫升选择适当的抗生素的LB培养基在28 ° C时16-24小时。 </li><li>这种细菌的收集和再悬浮外径600至终浓度氮气的免费植物营养液（表1和6）= 0.3。 </li><li>为了支持毛状根再生，消毒的支持矩阵（“岩棉”，Hummert国际地球城，密苏里州），切成约3厘米3插头。我们放入一个培养皿中，4个插头。 </li><li>顶部每个插件使用枪头戳一个洞，以方便插入植。A.发根 （5毫升）被添加到每个插件。 </li><li>一个2-3个腋芽的拍摄部分切除一个斜斜切。外植体，然后插入插头，生长在22 ° C约三个星期。我们无需浇水保持苜蓿外植体为第10天，那么，水5毫升无氮的解决方案，必要时。我们发现，消除根尖分生组织，可能会减少不定根的形成。 </li></ol> 3。共生根瘤菌（ 苜蓿根瘤菌 ）感染<ol><li>三个星期后，会出现一些不定根岩棉。复合植物毛状根转移到沙或蛭石（消毒），岩棉带或不带。它们是生长在温室22℃，16 / 8 h光/暗周期，和300μmol.m-2 -1光照强度为一个星期。 </li><li>此前根瘤菌感染，S.根瘤菌 1201是在50毫升酵母中提取甘露醇在30 ° C（表2和6,7）7天的中期培养。 </li><li>根瘤菌细胞重新悬浮稀释至终浓度的外径600 = 0.08使用无氮营养液。一个10毫升的新鲜暂停的 S. 根瘤菌是适用于每个复合植物诱导根瘤的形成。 </li></ol> 4。转基因root的身份， <ol><li>根瘤菌接种后两个星期，复合植物根深蒂固的水冲洗。根可以很容易地分开砂或珍珠岩水。 </li><li>我们置于紫外显微镜（尼康SMZ1500，激发460 - 500nm的，无障碍比510nm，505nm分色）毛状根。转基因根源GFP阳性和不定根的GFP负。然后，我们收集基层accordin克GFP信号，计数的结节，测量根长，并计算侧根密度。 GFP负根被用作控制。 </li></ol> 5。代表性的结果在我们的实验中，新的发根再生答 ：在2-3周后，从植发根 innoculation。在紫外显微镜，携带绿色荧光蛋白基因的转基因根表现出较强的绿色荧光（图1） 。再生根和GFP阳性根部分的金额取决于植和复合plants.On平均生长环境的条件下产生的根源，25％是转基因毛状根。为了提高转化效率， 1）塑料盖的厄运，如视频所示，就足以保持增长托盘的最初几天的湿度，植物只需要在头几天少量的水。过多的浇水，不利于形成毛状根; 2）农杆菌innoculant的浓度是非常重要的转型。过量的细胞是不是有利于毛状根形成根瘤的形成。根瘤的形成，浇水的解决方案的需求无氮，否则，将形成几个结节。 可以为起始原料，使用或entact幼苗的子叶产生毛状根的复合植物。上述协议的只有轻微的修改，是ncessary从8其他组织产生毛状根。更重要的是，每个毛状根是一个独立的转化事件。因此，在一个复合植物中观察到的表型的几个transforamtion事件，需要重复确认在多个复合植物的总和<img src="/files/ftp_upload/2633/2633fig1.jpg" alt="图1" /> 图1。 A.转基因根源，可以整理出毛状根 。 B.结节形成复合植物毛状根 ，我们把4 周龄 M. truncatula毛状根在紫外显微镜和GFP阳性根植物可以很容易识别。 （红色箭头：GFP的根;黄色箭头：非GFP的根） <table border="1"><tbody><tr><td>股票</td><td> g/200ml </td><td>毫升股票/升</td></tr><tr><td>硫酸镁4 .7 H 2 O </td><td> 12.3 </td><td> 2 </td></tr><tr><td>氯化钙2 .2 H 2 O </td><td> 14.7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 .3 H 2 O </td><td> 6.8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td>铁CL 3 .6 H 2 O </td><td> 0.49 </td><td> 2.5 </td></tr><tr><td>微量营养素</td><td>见下文</td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td>微量营养素</td><td>克每升</td></tr><tr><td> H 3 BO 3 </td><td> 0.142 </td></tr><tr><td> H 2 O的 MnSO 4。 </td><td> 0.077 </td></tr><tr><td>硫酸锌4 .7 H 2 O </td><td> 0.1725 </td></tr><tr><td>硫酸铜4 .5 H 2 O </td><td> 0.037 </td></tr><tr><td> NaMoO 4。 H 2 O </td><td> 0.024 </td></tr><tr><td> CoCl 2。 H 2 O </td><td> 0.0025 </td></tr><tr><td> NISO 4 </td><td> 0.001 </td></tr></tbody></table> 表1。Nitogen免费营养液<table border="1"><tbody><tr><td> K 2 HPO 4 </td><td> 0.5克</td></tr><tr><td>氯化钠</td><td> 0.1克</td></tr><tr><td>硫酸镁4 · 7H 2 O </td><td> 0.2克</td></tr><tr><td>酵母提取物</td><td> 0.4克</td></tr><tr><td>甘露醇</td><td> 10克</td></tr><tr><td> pH = 6.8时</td><td></td></tr></tbody></table> 表2。酵母提取甘露醇培养基（每升）

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

产生毛状根复合厂，是一个快速简便的的方法来获取许多双子叶植物物种的转基因材料的大量。这种方法虽然不能产生转基因植物种子，它可以在几个星期内生产的转基因材料。该方法尤其适合建立组织培养或生成稳定的转化有困难的植物。多年来，我们已经利用这种技术来研究基因的功能，促进功能，小分子RNA，根和侧根发育，国防和非生物胁迫的反应，结瘤和其他共生的过程，激素的反应，代谢分析，基因分析，蛋白组学分析，并其他生物过程。该协议是强大的和可复制。

generation of composite plants in medicago truncatula used for nodulation assays

发根农杆菌农杆菌tumerfaciens类似，可以转移到自治区根诱导质粒（RI）为基础的植物细胞外的DNA。 A。 发根 ，可引起植物组织毛状根形成和改造后形成的复合植物。在这些复合植物，一些再生根，转基因，携带野生型的T - DNA和工程的二进制向量，而芽仍非转基因，提供能源和增长的支持。这些毛状根复合植物不会产生转基因植物种子，但也有一些使这些非常有用的复合植物，在植物研究中的重要特征。首先，具有广泛的宿主范围，A.发根可以改变许多植物，尤其是双子叶植物，让各种物种的基因工程。其次，A.发根农杆菌感染组织和外植体直接，没有组织文化转型前是要取得复合的植物，使它们转化顽抗的植物物种的理想。此外，转基因根组织可以在几周内产生。 苜蓿truncatula，我们可以得到转基因根源，在尽可能短三个星期，比正常花香浸拟南芥转化速度更快。总体而言，毛状根复合厂技术是一种多用途的和有用的工具，研究基因功能和根相关的表型。在这里，我们演示了如何毛状根复合植物，可用于研究在难以转化的物种M.植物的根瘤菌的相互作用和结瘤truncatula。

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

我们演示了如何毛状根复合植物可以用来研究植物根瘤菌的相互作用和结瘤的难以转化的物种<em&gt;苜蓿truncatula</em&gt;。

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

generation-composite-plants-medicago-truncatula-used-for-nodulation

Research

JoVE Journal

Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

15.9K 次观看.  Donald Danforth Plant Science Center. 我们演示了如何毛状根复合植物可以用来研究植物根瘤菌的相互作用和结瘤的难以转化的物种苜蓿truncatula。

视频: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

科研

生物学

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM 微可可性能校准和使用在 22 T 的 Medicago 特伦卡图拉根上演示

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

基于AirID的植物蛋白质-蛋白质相互作用的邻近标记

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...