This research was supported by the University of Missouri Research Board (Project CB000519) and the US National Science Foundation (IIA-1430428 to DMC). Nga T. Nguyen was supported by the Vietnam Education Foundation Training Program (Exchange visitor program No. G-3-10180). We also thank Roger Meissen (MU Bond Life Sciences Center) for his assistance and expertise during the video recording and editing sessions.

1.苗圃<ol><li> 拟南芥种子的气相消毒 <ol><li>倾种子（40-50毫克）到1.5毫升离心管中。 （参见图1适当的种子体积，约50微升）。标签用铅笔各管（墨水可能会在灭菌过程中消失）。将每个标管帽开放，进入干燥器13。 </li><li>放置在干燥器中的积极通风柜并关闭干燥器的阀门。 </li><li>等分试样百毫升漂白的（次氯酸钠6.15％）到250ml烧杯中，然后将其放置在干燥器中。 </li><li>迅速加入3毫升12M盐酸，使用移液管的漂白剂。迅速关闭干燥器的盖作为反应迅速进行。允许灭菌进行4小时（标记用墨水的管，看到墨水消逝有助于可视化已经生成的氯气的足量）。 注意:氯气是有毒的;处理其残基在功能通风柜额外的安全预防措施。与当地政府联系，或访问环境健康与安全部门的网页-密苏里大学（ESH-MU）14化学品安全和指南使用通风橱:https://ehs.missouri.edu/chem/。 </li><li>十五分钟之前灭菌完成（3.75小时），打开层流罩和清洁用70％的乙醇的表面。 </li><li>灭菌后的4小时开启阀门，简要地除去干燥器的盖通风柜内，取出漂白剂，并且根据机构程序处理它。这个步骤将释放氯气烟雾的很大一部分。密封消毒室，并把它的层流罩。广泛打开盖子和曝气消毒的种子大约40分钟。在此之后，立即用种子或在干燥的地方存放。 注:建议种子气相灭菌但是其它方法ス在Alatorre，科沃斯等2所描述与乙醇，漂白剂和水通道作为候补洗涤液同样有效。 </li></ol></li><li> 种子萌发培养基 注意:在此步骤中制备的培养基是四分之一维生素15 Murashige和Skoog（MS）。 <ol><li>添加450毫升去离子水（DI水），0.55克MS培养基加维生素将0.3g MES（4-吗啉乙磺酸水合物）和磁性搅拌棒放入一个1升玻璃烧杯中。 </li><li>溶解，用NaOH调节pH值至5.7，然后加入3.5克植物琼脂。不断搅拌溶液5分钟。 </li><li>倾整个溶液置于量筒，并添加去离子水至500毫升。高压灭菌这家医院500 ml溶液，与内部的磁力搅拌棒，用1升高压灭菌瓶中。 </li><li>该解决方案已被高压灭菌后，用搅拌在瓶子的磁力搅拌器搅拌7-10分钟的解决方案。 </li><li>之后，媒体已经冷却下来至50-60℃，倒入媒体到平板在无菌条件下，让它凝固。板可存放在冷室以后使用。 </li></ol></li><li> 电镀种子 <ol><li>打开使用和清洗，用70％的乙醇的表面之前的层流罩15分钟。下列项目是必需的:无菌种子，滤纸，牙签，胶带微孔和1/4 MS板。 </li><li>放置在无菌滤纸无菌种子。无菌牙签（用无菌水或戳¼MS培养基）的微湿一端。使用此滋润月底挑选从滤纸上的种子，然后躺在他们到介质表面。 </li><li>分布在该板的种子以每厘米2大约1种子（ 见图2）的密度。然后用微孔胶带保持附着于盘体的板盖。这种类型的带的帮助，以防止污染，同时允许空气和insid小气候之间的气体交换Ë板。 </li><li>发芽前，通过保持所述板在冷室2天分层种子从光覆盖。 </li><li>分层后，将种子在生长室或在具有最佳生长条件（23℃，16小时光照/ 8小时黑暗，60％为拟南芥相对湿度）的地方。幼苗将准备发芽后10-12天水耕。 注意:在萌发有可能在板的盖下显著缩合，防止溺水，多余的水应在层流罩在无菌条件下被丢弃。 </li></ol></li></ol> 2.水培的安装和移植过程<ol><li> 水培液 注意:如在引言中提到的，植物可以具有特定营养要求拟南芥已成功地生长与表1 16中所示的营养液根据不同的供应商，该。此处列出的盐可具有不同的水含量（水合），并使用这样的替代，只要摩尔浓度保持恒定，不影响营养液的性质。 <ol><li>准备在不同瓶（ 表1）和在无菌瓶除铁EDTA所有微量营养素每大量营养素的储备溶液（用0.22微米的膜过滤除菌）。总是在最后的混合解决方案时，添加铁EDTA。制备在4℃下预先实验，但高压釜和存储的10倍的营养液。使用或改变，只有当营养物溶液达到室温，营养。 </li></ol></li><li> 移植 <ol><li> 准备厂房架和集装箱水培 <ol><li>使在泡沫的切口，沿着用剃刀刀片其长度延伸（ 见图3）。准备每株一个插头。 </li><li>液釜泡沫管塞在去离子水中浸泡。&lt;/ LI&gt; <li>切割所述泡沫面板成更小的板，确保泡沫板的宽度和长度是0.5-1.0厘米小于容器的大小（ 见图4）。 </li><li>用软木钻孔以创建在泡沫板的通孔。植物的密度应均匀分布，每10cm 2理想地1植物。这种密度将保持彼此截然分开植物;然而更高的密度是可能的，并且不排除在实验的成功。确保孔的尺寸将插头的尺寸相匹配（参见图4）。 </li><li>装满营养液的容器。确保溶液的深度是足够的根发育（至少5厘米）。然后小心地将泡沫板到解决方案的表面。 </li><li>设置空气泵系统，以提供氧气进入溶液（参见图5）。 注意:在水耕容器营养液萨姆填ê天幼苗被移植。从光覆盖容器双方将有助于防止藻类生长。 </li></ol></li><li> 从板块幼苗转移到水培系统 <ol><li>用小镊子每株秧苗轻轻向外拉出中板和沿发泡管塞的切口打下了根。小心将发泡管抱着苗成泡沫板然后将板回水培容器。参见图6适当操作。 </li></ol></li></ol></li></ol> 3.水培试验<ol><li> 营养液的更换和处理 <ol><li> 营养液更换 <ol><li>要更换营养液，如步骤2.1中所述准备新鲜的水培液。从水培容器中取出含有植物泡沫板，并将其放置在一个充满临时容器水培或解决方案。 </li><li>丢弃旧溶液，短暂地冲洗该容器用DI水三次。新配制的水培液添加到这个容器中，轻轻地放在泡沫板与植物放回容器中水培。每周两次更换水培液。 </li></ol></li><li> 改变水培液的营养成分 <ol><li>调整表1所示的修改感兴趣的元素的最终浓度的水培液的组成。例如，为了诱导铁（Fe）缺乏症，修改水培溶液以降低的Fe-EDTA浓度。包括一组上生长充分（或充满）水耕溶液对照植物的，没有任何修改，以进行比较。 </li><li>能够操作与有毒元素的营养液，先准备好所需的有毒元素的一个独立的原液，最好是1000倍浓。用一个吸管穗与使用1000倍浓储备所需最终浓度的有毒元素的水培液。 </li><li>例如，为了使3升含有镉的20μM的水培溶液中，制备0.5M的氯化镉的库存，并加入120微升的0.5M 氯化镉库存的入3升的水培溶液。包括水培种植无氯化镉比较植物的控制集。 注意:有毒元素如镉，砷和铅对人体健康和环境非常危险。请与当地政府联系，或访问EHS-MU的网页（https://ehs.missouri.edu/train/chemical.html）14 事先进行各种实验环境和健康安全准则。 </li></ol></li></ol></li><li> 器械灭菌明年实验 <ol><li>由于几乎所有使用的材料准备水培设置即可重复使用，请用稀释的漂白剂的不同部分（次氯酸钠0.6％）。 </li><li>用漂白粉漂洗后，用去离子水彻底冲洗所有材料。保持容器，泡沫板和泡沫水族馆石头以备将来使用干燥的地方。泡沫塞去除根和被高压灭菌后准备重用。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

用于水培苗的健康是造成水培实验成功的主要因素之一。仪器仪表，种子和培养基灭菌在减少污染的风险也扮演着重要的角色，并提供一个良好的开端植物它们被移植到水培系统之前。为一个良好的实验设置与设施，如高压釜，通风柜，冷室（4℃），并用受控条件（光强度和温度）生长空间A的工作环境是必要的。 营养液的新鲜度也决定了植物健康，进而确定水培试验的成功。因为水在直接照明蒸发更快，盐的浓度将由于减少总溶液体积的改变;因此它是最好的一个星期，以改变它的水培液的至少两倍。但是，如果大，深集装箱配备了一个空气泵系统中使用它可能没有必要更换该短持续时间实验的营养液。注意，在拟南芥的情况下，我们使用品红容器（77宽×77毫米长×97毫米高度），但也可以使用其它的，较大的容器，以容纳更大的植物。 对于有兴趣的植物营养素的研究人员，水培试验提供了测试植物的表型和应对不同的养分供应17一个独特的环境。通过操纵感兴趣的元素的浓度，研究人员可以设置不同的实验，以测试充分性，缺乏，或者必需和非必需的营养物质的毒性浓度的影响。相比于土壤为基础的系统中，水培系统提供了更均匀的营养培养基的植物土传病害的风险较小。此外，根和芽组织可以收获和易于分离对具体的植物组织进一步的分析。 中的代表性部分中，我们介绍了在将其使用于对植物营养更详细的研究一简单的水培系统中的两个例子。在第一个例子中，通过在锌浓度梯度生长的植物，我们能够说明可在营养组合物使用本水耕系统来实现的控制水平。 7μM锌种植的植物生长更加积极相比，50μM锌种植的植物，而植物相比，7μM锌种植的植物生长，无需额外添加的锌分别发育不良。这部分是由于时间的植物被允许充分的条件下生长的长度;较早从媒体去除锌很可能诱导强锌缺乏症状。应用相同的原理，我们能够使用非必要的金属，镉，这是众所周知的损害植物的生长以诱导毒性。 在第二例如，用20μM的镉处理72小时的Col-0根和枝条的元素组成，通过ICP-OES测定。我们发现在根与芽之间的所有检测到金属的差异。宏观元件在相对于所述根枝条较高浓度被发现，而铁和锌，发现根更丰富。镉其次类似于铁和锌的模式，是多集中在根部较笋。这些数据强化了叶和根提供有关核电厂的ionome状态不同的信息，因此需要单独分析，以了解矿质营养和成分在全厂级这两个组织中的想法。除了​​ICP-OES几个光谱方法如原子吸收光谱（AAS）或电感耦合等离子体质谱法（ICP-MS）也可以用于测量植物的元素组成（ionome）组织中18-20。 在hydroponiÇ实验，症状和植物响应不同营养条​​件的表型表现的是什么可以扩展到更多的开始详细制定分析，如基因表达（转录）和蛋白质丰度（蛋白质组学）。这些-omic技术是键通过考虑在一个组织特异性方式过程，以植物代谢集成。

植物是指能合成由无机离子，水和CO 2利用来自太阳1捕获的能量所需的所有代谢物的少数生物体之间。水耕是生长植物的方法，通过提供所有的营养的利用了这一事实，在其无机形式，有或没有固体介质的液体溶液。水培系统已经探索的营养要求，并在拟南芥的一些元素和其他植物物种2-5的毒性被广泛使用的科学家。例如，Berezin 等 3，Conn 等4和Alatorre-科沃斯等 2用于水培系统和几种植物物种，包括番茄和烟草，以产生用于矿物分析2-4足够量的植物生物质。水耕的工业应用也已经开发了用于作物，如番茄和莴苣6。在这里，我们Øutline在研究，在可用的方法可能的变化背景下使用水栽法，最后提出一个系统，它可以很容易地扩展性和可用于对研究植物矿质营养研究实验室。 水培系统允许根组织容易分离和养分有效性精确控制 水耕过土壤为基础的系统提供了若干优点。当从土壤除去，根组织往往机械剪切引起组织或损害的损失。这是细根结构，如侧根和根毛尤其如此。不利用惰性粒状介质水耕系统允许根的微创分离和拍摄的组织。 在土壤系统，在整个土壤基质的养分养分有效性的变化结合创建土壤中的微环境中的土壤颗粒。这^ heterogeneity可以在需要养分或其他分子的外部浓度的精确控制实验的复杂性增加了额外的水平。与此相反，水培溶液是均匀的，并且可以在整个实验过程中很容易地更换。 水培系统的变种 所有的水培培养依靠营养液传送到工厂的基本要素。除了营养物，根部也需要氧气的稳定供应。当根系缺氧成为他们无法承担运输代谢产物的植物体7的其余部分。由饱和空气（经典水耕）的溶液中，在任何时候都不会淹没根部氧气输送，或者让根被完全暴露在:水培系统可以根据他们如何提供氧气和其他营养物质的根源进行分类空气（aeroponics）8。在水培，营养液可以与之前其使用空气是饱和的和频繁地改变，或者空气可以在溶液在工厂9的生命周期中连续供给。备选地，植物也可在惰性介质（ 例如 ，岩棉，蛭石，或粘土粒料）培养，并进行由通过媒体滴落溶液或周期性地浸没在营养液10在基板湿法干燥循环。在aeroponics，根都与营养物溶液喷雾以防止干燥。 水培系统的缺点 虽然水培文化土壤上为基础的系统具有明显的优势，有一定解释数据时承认一些注意事项。例如，水培系统暴露的植物可被视为非生理条件。因此，表型或植物响应使用水培系统在大小磨片可能会有所不同检测N组植物生长在替代系统（ 例如，土壤或基于琼脂培养基）。这些考虑并不适合水培系统唯一的;如果植物在不同类型的土壤11,12的生长，也可观察到差的反应。 下面的协议提供了如何建立一个水培系统，在实验室中一步一步的指示。此协议为拟南芥 （ 拟南芥 ）进行了优化;然而，类似的或在某些情况下，相同的步骤可用于种植其他物种。

<table>
	<tbody>
		<tr>
			<td>For seed sterilization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>The Clorox Company</td>
			<td>NA</td>
			<td>The regular bleach</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>www.cloroxprofessional.com</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator body</td>
			<td>Nalgene</td>
			<td>D2797 SIGMA</td>
			<td>Marketed by Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Desiccator plate</td>
			<td>Nalgene</td>
			<td>5312-0230</td>
			<td>Marketed by Thermo Scientific</td>
		</tr>
		<tr>
			<td colspan="2">For one quarter MS medium preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Acros Organics</td>
			<td>172591000</td>
			<td>4-Morpholineethanesulfonic acid hydrate</td>
		</tr>
		<tr>
			<td>Murashige and Skoog (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M0404-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytoagar</td>
			<td>Duchefa Biochemie</td>
			<td>P1003.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Square plate</td>
			<td>Fisher Scientific</td>
			<td>0875711A</td>
			<td>Disposable Petri Dish With Grid</td>
		</tr>
		<tr>
			<td>For seed plating&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1004090</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Jarden Home Brands</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds Wrap</td>
			<td>NA</td>
			<td>Standard aluminum foil</td>
		</tr>
		<tr>
			<td>Micropore tape</td>
			<td>3M Health Care</td>
			<td>19-898-074</td>
			<td>Surgical tape; Marketed by Fisher Scientific</td>
		</tr>
		<tr>
			<td>For hydroponic solution preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP368-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P386-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca(NO3)2</td>
			<td>Acros Organics</td>
			<td>A0314209</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>Sigma</td>
			<td>B9645-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M7634-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z0251-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4</td>
			<td>Aldrich</td>
			<td>737-860-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl2</td>
			<td>Fisher Scientific</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl</td>
			<td>Sigma-Aldrich</td>
			<td>232696-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>FeEDTA</td>
			<td>Sigma</td>
			<td>E6760-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;Stericup &#38; Steritop&#8221; bottle&#160;</td>
			<td>Milipore Corporation</td>
			<td>SCGVU02RE</td>
			<td>Micronutrient container</td>
		</tr>
		<tr>
			<td>For root wash buffer preparation</td>
			<td></td>
			<td></td>
			<td>www.milipore.com</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Acros Organics</td>
			<td>A0305456</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>For hydroponic set up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable foam tube plug</td>
			<td>Jaece Industries Inc.</td>
			<td>L800-A</td>
			<td>Identi-Plugs fit to holes with 2R=6-13mm</td>
		</tr>
		<tr>
			<td>Foam Board</td>
			<td>Styrofoam Brand&#160; Dow</td>
			<td>ESR-2142</td>
			<td>Thickness is 1/2 inches</td>
		</tr>
		<tr>
			<td>Cork borer</td>
			<td>Humboldt</td>
			<td>H-9662</td>
			<td>Cork Borer Sets with Handles, , Plated Brass Set of 6, 3/16&#34; to 1/2&#34; OD Size</td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Aqua Culture</td>
			<td>MK-1504</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">Marketed by Wal-mart Stores, Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Airline tubing and aquarium bubble stones</td>
			<td>Aqua Culture</td>
			<td>Tubing: 928/25-S</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Marketed by Wal-mart Stores, Inc.</td>
			<td>Stone: ASC-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A995-4</td>
			<td>Reagent Alcohol</td>
		</tr>
		<tr>
			<td>Cadmium Chloride (CdCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>10108-64-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

hydroponics: a versatile system to study nutrient allocation and plant responses to nutrient availability and exposure to toxic elements

水培系统已被用作用于植物生物学研究的标准方法之一，并在商业生产也可用于多种作物，包括莴苣和番茄。在植物研究界，无数的水培​​系统已被用于研究生物和非生物胁迫植物响应。在这里，我们提出了一个水培协议，可以在热衷于追求植物矿质营养研究实验室可以轻松实现。 本协议描述了水培系统的详细设置和植物材料的成功实验的准备工作。大多数本协议中所述的材料都可以科学供应公司以外找到，使得设置了水培试验更便宜和方便。 使用水耕生长系统的是的情况下最有利的，其中所述营养培养基需要被很好的控制，并当完好滚装OTS需要被收获下游应用。我们还证明浓度如何养分可被修改以诱导对两者必需的营养物质和有毒的非必需元素植物反应。

在本节中，两种类型的实验中，使用此处描述的水培系统的结果，现介绍。在第一个实验中，营养液被修改，以得到不同浓度的锌。我们还通过添加有毒元素镉非致死浓度（ 图7）改性的营养液。在第二个实验中，我们使用电感耦合等离子体光学发射光谱法（ICP-OES）1来测量根和在水培含有溶液镉（ 图8）中生长的植物的叶子的元素组成。这个实验说明分别获得根和叶的优势。 实验1 拟南芥秧苗（Col-0中）在公关描述的水培系统中生长otocol步骤1和2植物被允许与不同锌浓度（ 图7A-B）或镉的非致死浓度（ 图7C）处理前生长，共3周。六天后处理，植物生长在高锌浓度（&gt; 42微米）显示延迟由于锌毒性的增长，而无需额外的锌植物相比具有7微米的Zn 2+生长的植物加入也显示延迟生长， 图7还示出了减少枝条生长，根系生长，并且褪绿叶症状典型暴露于镉（ 图7C）的植物。 实验2 如在步骤1和2描述了两个星期之后的Col-0植物生长，未改性（充满）溶液用80毫升含有20μM的镉水培溶液替换。 72小时后S，根部组织通过整个泡沫板与植物转移到含有80 ml的三的20mM（pH 8.0）中和5mM EDTA的新血管洗涤。该解决方案将消除结合于根表面的重金属。植物在5分钟旋转振荡器含EDTA的溶液中温育。 EDTA溶液然后通过80毫升DI水置换和植物一起孵育的旋转振荡器额外5分钟。重复用DI水二次漂洗步骤。用DI水漂洗植物后，叶和根组织中独立地收集和用于ICP-OES 1进行处理。 图8显示了叶子的元素组成是从根，不同的地方大量营养素（钙，钾，和Mg）的叶组织中目前在高浓度相比根源。另一方面，微量营养素如Zn和Fe中根优先积累。非必需元素镉的浓度被发现是喜<html> gher根相比芽。 <img alt="图1" src="/files/ftp_upload/54317/54317fig1.jpg" /> 图1. 气相灭菌拟南芥种子。（A）每1.5个ml离心管拟南芥种子的数量。 （B）含用盖在管架子保持部准备灭菌开放种子，一个管与油墨标在帽包括管。 （C）灭菌设置了干燥器，内盖和阀门关闭。 （D）包括在之前和灭菌后的墨水标记的色彩浓厚的种子消毒过程管的盖子的墨水标记。 <a href="https://www.jove.com/files/ftp_upload/54317/54317fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 文件/ ftp_upload / 54317 / 54317fig2.jpg"/&gt; 图2. 种子电镀步骤。（A）种子电镀前放置在消毒纸。也需要这一步消毒牙签。 （B）的微湿与在介质板的一侧的介质或水中的牙签的末尾。 （ 三 ）种子移到¼MS板。 （ 四 ）种子的理想密度为≈1籽/厘米2。 <a href="https://www.jove.com/files/ftp_upload/54317/54317fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54317/54317fig3.jpg" /> 图3. 用于保存在营养液苗泡沫塞 。在泡沫管塞半切口可帮助从板块移栽水培期间举行的苗子。<a href="https://www.jove.com/files/ftp_upload/54317/54317fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54317/54317fig4.jpg" /> 图4. 泡沫板做准备。（A）大批量制备泡沫板前检查模板泡沫板与容器尺寸的大小。在泡沫板的中心由两个小穿孔使其更容易保持和用镊子处理泡沫。 （B-C）打孔器用于创建对泡沫板的孔。 （ 四 ）检查发泡管塞和孔的泡沫板之间建立适当的配合。 <a href="https://www.jove.com/files/ftp_upload/54317/54317fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5.空气泵设置为从顶视图（A）和侧视图（B）的水培试验中的数字表示:1 -泵供给空气; 2 - 塑料管连接用阀系统来控制空气流动的空气泵; 3 - 阀系统; 4和5​​ - 塑料管材连接用石头泡曝气阀系统; 6和7 -泡泡石（鱼缸销售） <a href="https://www.jove.com/files/ftp_upload/54317/54317fig5large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54317/54317fig6.jpg" /> 图6. 传输苗水培系统。（A）使用镊子取苗出来的中厚板。 （ 二 ）将苗袋鼠沿着泡沫管塞切口吨。 （C）将发泡管插头插入泡沫板。用苗准备（D）已完成的泡沫板设置放置在营养液。 <a href="https://www.jove.com/files/ftp_upload/54317/54317fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54317/54317fig7.jpg" /> 图7. 养分溶液可以被修改，以测试缺乏或元件的毒性作用 -4周老水耕治疗后6天生长的拟南芥 :生长具有（AB）植物0，7，14，21，28，35，42，和50μM锌。在与具有种植的植物高锌含量（&gt; 42μM）显示生长迟缓（毒性），而没有锌厂加入也表明生长迟缓（营养缺乏）种植的植物<html> 7μM锌离子 。 （C）的植物中的不存在（左）或20μM的镉的存在下，在营养液生长（照片拍摄之后镉暴露6天）。镉暴露引起萎黄，并降低增长。 <a href="https://www.jove.com/files/ftp_upload/54317/54317fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54317/54317fig8.jpg" /> 从植物根与芽的 图8. 元素组成水耕种植。芽含有比较多根营养素（钙，钾，镁），而必需的微量营养素锌和铁多集中于根。同样地，非必要的元件镉是根优先积累。误差棒代表95％置信区间（14，芽和n = 9，根）。_upload / 54317 / 54317fig8large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table><tbody><tr><td> 营养类型 </td><td> 盐/试剂 </td><td> 浓度的水培液 </td><td> 单元 </td></tr><tr><td>营养素</td><td> KNO 3 </td><td> 1.250 </td><td>毫</td></tr><tr><td>营养素</td><td> KH 2 PO 4 </td><td> 0.625 </td><td>毫</td></tr><tr><td>营养素</td><td> 硫酸镁 </td><td> 0.500 </td><td>毫</td></tr><tr><td>营养素</td><td>的Ca（NO 3）2 </td><td> 0.500 </td><td>毫</td></tr><tr><td>微量营养素</td><td> H 3 BO 3 </td><td> 17.500 </td><td> μM </td></tr><tr><td>微量营养素</td><td>氯化锰2 </td><td> 5.500 </td><td> μM </td></tr&gt; <tr><td>微量营养素</td><td> 硫酸锌</td><td> 0.500 </td><td> μM </td></tr><tr><td>微量营养素</td><td> 的 Na 2的MoO 4 </td><td> 0.062 </td><td> μM </td></tr><tr><td>微量营养素</td><td>氯化钠2 </td><td> 2.500 </td><td> μM </td></tr><tr><td>微量营养素</td><td> 氯化钴</td><td> 0.004 </td><td> μM </td></tr><tr><td>微量营养素</td><td>的FeEDTA </td><td> 12.500 </td><td> μM </td></tr></tbody></table> 表1中的水培液的营养成分的有效浓度。

Watch this Scientific Journal Video about Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements at JoVE.com

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培:一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

hydroponics-versatile-system-to-study-nutrient-allocation-plant

Research

JoVE Journal

Biology

31.4K 次观看.  University of Missouri, Columbia. Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

视频: Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

海洋微生物的趋化反应的微观尺度的营养层

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

在靶标和标记下使用裂解分析植物中的H3K4me3修饰

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

结合透明和荧光显微镜，可视化基因表达的变化和对芸苔疟原虫的生理反应

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

激光消融和活体显微镜研究肠道重塑

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...