这项工作是由美国康奈尔大学农业试验站（CUAES）NYC - 125433哈奇格兰特和康奈尔大学的支持，启动拨款，批到OKV

第1部分。制备固体培养基上生长的植物。 我们通常集中Murasige和Skoog（MS）培养基上的文化植物辅以1％蔗糖和0.7％的琼脂。它包含了所有必要的成分，保持健康的植物。 准备介质pH值5.7的MS，1L <ol><li>在1.5 - 2.0L高压灭菌瓶，加入到800毫升的水的MS粉2.15克。 </li><li>放置一个搅拌棒装入瓶中，并解散MS粉搅拌，搅拌板介质。 </li><li>边搅拌，加，一滴一滴，1N氢氧化钾MS培养基的pH值调整到5.7。 </li><li>添加10克的蔗糖，不断搅拌。 </li><li>添加7克，琼脂* </li><li>调整的准备介质的最终体积1000毫升，并通过高压灭菌消毒.* </li><li>广场蒸压中等搅拌板，边搅拌冷却下来，因此，琼脂不会沉淀在瓶底。降温介质〜60 ° C。 </li><li>倒入100毫米的Petri板。 &#39;保持在4板° C。 </li></ol> *提示1：不要试图解散琼脂。它会溶解在高压灭菌。 *提示2：保持在高压灭菌过程中瓶中的搅拌棒，​​你会用到它。 *提示3：如果你能保持你的手10秒的灭菌瓶，这意味着中期有足够的冷却下来，你就可以开始浇筑板。 第2部分。植物材料和生长条件。 <ol><li>消毒拟南芥的种子<ol><li>的Eppendorf离心管放入种子100毫克，加1毫升70％的乙醇。充分混匀，孵育2分钟。自旋种子，吸出上清液。 </li><li>添加1.8％的漂白粉溶液1毫升含0.1％Tween - 20的。混合10分钟。离心种子和吸出的漂白粉溶液。 </li><li>这一步要在层流罩。添加1毫升无菌水的种子，拌匀。离心种子，吸​​水。重复此步骤5次。 </li></ol></li><li>传播到在步骤1片（100粒种子/盘）准备的种子。 </li><li>种子板块保持在4 ° 24 - C在暗分层。 </li><li>种植植物在生长室为14天8小时light/16 h黑暗光照（250μmol米-2 s - 1时光合作用光子通量密度）和75％，23 / 19 ° C光/暗温度政权的相对湿度。 </li></ol> *提示1：漂白溶液稀释一个家喻户晓的Clorox公司，含有6％次氯酸钠和Tween - 20的加入终浓度为0.1％。 第3部分。 拟南芥原生质体的分离。 <ol><li> 14日龄的幼苗在15毫升过滤灭菌电视线解决方案的新鲜刀片切片2克。一次性无菌培养皿中，我们砍的植物材料。 </li><li>切碎组织转移到200毫升烧杯中，加20毫升过滤灭菌的酶液，摇动烧杯，让组织与酶液混合，用封口膜，铝箔盖。 </li><li>摇植物组织，在黑暗中在35 RPM，在室温为16-18小时</li><li>通过8层纱布，在W5的解决方案预湿筛，收集到50毫升猎鹰管释放的原生质体。 </li><li>筛原生质体经水洗布​​W5的解决方案与15毫升的奶酪布更多的时间。 </li><li>小心覆盖W5的解决方案与10毫升的原生质体，不干扰糖梯度;离心7分钟100克。 </li><li>收集在10毫升原生质体的酶液和W5解决方案（图1B）接口，并转移到一个新的50毫升猎鹰管。 </li><li>添加15毫升W5的解决方案，离心5分钟，在60克去除上清。 </li><li>在60克resuspending15毫升的W5的解决方案，离心机原生质体原生质体酶液洗5min后</li><li>取出上清，悬浮颗粒的原生质体在13毫升W5的解决方案。 </li><li>评估与血球计数细胞原生质体的产量。 </li></ol>第4部分。试剂： 电视线：0.3 M山梨醇; 50毫米氯化钙2过滤消毒，并保存在-20 ° C 。 酶溶液：0.5 M的蔗糖，MES - ​​KOH pH值5.7，20毫米10毫米氯化钙 ，氯化钾40毫米，1％的纤维素酶（Onozuka R - 10），1％Macerozyme（R10） 。过滤，消毒和新鲜使用。 W5的解决方案：0.1％（W / V）葡萄糖，0.08％（W / V）的氯化钾，0.9％（W / V）氯化钠，1.84％（W / V），2毫米的MES KOH pH值5.7 氯化钙 。过滤，消毒和存放在室温下。 第5部分：代表性的成果： 拟南芥文化1和第2部分中所描述的条件，呈现健康苗，适合于分离原生质体（图1A）。原生质体纯化蔗糖密度梯度耳鼻喉科离心和W5和酶的解决方案（图1B）接口收集。使用第3部分中所描述的原生质体分离过程中，我们通常的收获从一克新鲜苗 5-10完整的原生质体10 6（图1C）。 <img src="/files/ftp_upload/1149/Fig1.jpg" alt="图1" /> 图1。拟南芥原生质体的分离 。 ，14日龄拟南芥幼苗组织收集，并转换为原生质体修改的过程（陈和Halkier，2000年），B，原生质体纯化蔗糖密度梯度离心法和酶液界面收集和W5的uffer（ 白色箭头 ），C，原生质体的明场显微镜。显微收集使用冷却CCD相机与蔡司Axioscope 2加显​​微镜接口。

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

与康奈尔大学的科技企业和商业化中心（CCTEC），2008年6月已提交临时专利，其在植物基因功能分析的使用说明此过程。文案没有50341/015001：Vatamaniuk，确定，和翟，Z快速的植物基因功能分析的方法。

为了确保完整的原生质体的产量高，它是非常重要的启动与健康的植物。使用过滤消毒隔离原生质体解决方案。记住，原生质体是脆弱的。因此，当你正在处理的原生质体时，不要混用，吸管或涡他们大力，因为这将制动他们。相反，混合，通过缓慢旋转或胶带的离心管中的原生质体。所描述的过程产量完整的原生质体是可行的至少96小时。因此，原生质体，可用于研究各种细胞过程，如蛋白质?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5524</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1296</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dishes (100x15mm)</td>
<td>&nbsp;</td>
<td>Kreckeler Scientific</td>
<td>82-4001</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase (Onozuka R&#x2010;10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>C32200</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme (R10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>M22010</td>
<td>&nbsp;</td>
<tr>
<td>Cheesecloth wipes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>06-665-29</td>
<td>&nbsp;</td>
<tr>
<td>Lab Rotator</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1167152Q</td>
<td>&nbsp;</td>
<tr>
<td>Allegra X&#x2010;15R Centrifuge</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>392932</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop2 Plus Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

原生质体是植物细胞有细胞壁酶去除的。超过40年前首次报道从不同植物组织的原生质体的分离<sup&gt; 1</sup&gt;，并一直适应研究各种细胞过程，如蛋白质的亚细胞定位，完整的细胞器的隔离和有针对性的双链RNA干扰（RNAi）基因失活，<sup&gt; 2-5</sup&gt;。原生质体分离协议大多采用成熟的拟南芥叶组织（例如35天的老厂）<sup&gt; 2-4</sup&gt;。我们采用14日龄的拟南芥幼苗修改现有的协议。在此过程中，一克14日龄的幼苗产生5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt;原生质体至少96小时保持不变。从幼苗原生质体的产量是准备成熟的拟南芥叶相媲美，但原生质体的分离，而不是35-36天，在15天完成。这使得减少的时间和需要隔离时，使用成熟的植物原生质体的生长室空间，并加快下游研究，需要完整的原生质体。

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

这个视频展示了从14日龄拟南芥幼苗组织中分离完整的原生质体的过程。此过程加快，原生质体保持不变的至少96小时，是从，而不是一月龄成熟的植物幼苗孤立的，需要完整的原生质体的检测。

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

isolation-protoplasts-from-tissues-14-day-old-seedlings-arabidopsis

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

35.9K 次观看.  Cornell University. 这个视频展示了从14日龄拟南芥幼苗组织中分离完整的原生质体的过程。此过程加快，原生质体保持不变的至少96小时，是从，而不是一月龄成熟的植物幼苗孤立的，需要完整的原生质体的检测。

视频: Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.