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?...

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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

35.7K 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

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 ...

科研

生物学

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 ...

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 ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

一种测量拟南芥花粉水合作用曲线的改进方法

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

作者聚焦：拟南芥的高分辨率、单粒、体内花粉水合生物测定  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

拟南芥的高分辨率、单粒、体内花粉水合生物测定

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...