这项工作是由粮食和农业的美国农业部国家研究所，农业与食品研究计划补助2010-65108资助 - 20582到KMJ我们感谢Brian K.沃什伯恩对稿件的严格审查。

在执行步骤1，2，4，并在无菌的层流罩6被推荐。 1。 M的制备苜蓿幼苗A17 注:M。在这些研究中使用苜蓿种子A17冷却温室条件〜22℃，或维持在22-26℃，〜150-400毫摩尔/米-2 -1光植物生长室下产生的。 <ol><li>倒入种子发芽板:采用100平方毫米板（15毫米深），倒蒸压1.1-1.2％w / v的纯化琼脂10〜3-5毫米的深度。每块板将被用于发芽〜0.25-0.5克种子（相当于63-125粒/板）。 注意:使用植物细胞培养测试，纯化琼脂在本协议中所述的所有板块，这一点至关重要。为琼脂的厂商和产品目录号信息列特定的试剂和设备的表中。 </li><li>划破/消毒M。苜蓿种子A17S:称取足够的种子，种苗所需的号码。 （ 蒺藜苜蓿 A17种子〜4毫克每）地方的种子在无菌125毫升瓶用无菌铝箔盖（0.25-0.5克种子/瓶，这是〜63-125种）和吸管20集中（96％）毫升H 2 SO 4沿烧瓶壁。 注:酸蚀将消毒的种子。此外，通过移液沿烧瓶壁酸，它会重新消毒已经接触到的未经消毒的种子接触的烧瓶内。 </li><li>分手种子团块用无菌玻璃吸管。经常摇动烧瓶各为10-12分钟。小，褐色坑将成为种子可见。这些都是在种皮11小孔。 注:用浓H 2 SO工作4时，戴上护目镜和手套。 </li><li>除去H 2 SO 4和冲洗种子:当种子至少10％有褐色的小凹点，或是12分钟过去了，取至上，从烧瓶中除去所有的酸，用无菌玻璃吸管。 （地点废酸到含有至少为300毫升自来水1-2升烧杯中，这会冲淡酸，防止过热。）倾斜烧瓶收集在烧瓶中的曲线的剩余酸和使用小玻璃吸管来删除所有剩余的酸。如果残留的酸依然存在，它会与冲洗水反应，加热的种子，并杀死他们。 <ol><li>通过快速浇筑百毫升无菌超纯水到每个烧瓶中漂洗种子。水的过量体积将稀释的酸，防止酸与水的反应过程中的过热和种子的损伤。倒出的水和火焰灭菌的烧瓶内的唇。 </li><li>冲洗种子8至10倍，用50ml灭菌超纯水。每次冲洗过程中摇动烧瓶。烧瓶中，应考虑无菌此时，将烧瓶的唇部应该每次漂洗之前将熄火。 </li></ol></li><li>让种子吸收过夜:在最后一次漂洗，倒入50毫升无菌超纯水到每个烧瓶，更换无菌铝箔盖在黑暗中过夜每个烧瓶中放于4℃。 </li><li>在催芽板瓜子地点:后让种子吸收过夜，倒​​掉水，冲洗用2倍〜25毫升超纯水，然后加入20毫升无菌超纯水，漩涡悬浮种子和从他们倒到1.1-1.2％的琼脂平板发芽步骤1.1。如果种子留在烧瓶中，加入更多的水，并再次倾。分发种子从每个烧瓶中一板（63-125粒/板）。 <ol><li>旋流板分发种子。种子应在板的一端均匀地分布在一个4-5厘米波段。这将是该板的"顶部"。 "底"5-6厘米应保持自由的种子（ 图2A）。 </li><li>抽出的水用无菌吸管。倾斜板以45°角，以使剩余的水漏到板的底部。重新装上盖子上直到特德板和避光。板应允许排出10-20分钟。 （ 图2B）。 </li></ol></li><li>成立发芽:删除已收集的倾斜板的底部，用无菌吸管所有的水。 <ol><li>重新装上盖子，并用封口膜密封板。戴上手套，并保持无菌（ 图2C）。 </li><li>堆栈发芽板，然后开启所有的人都在向上90°角。这些种子将趴在琼脂的垂直表面。充分吸胀的种子应该很容易附着在琼脂。根将向下生长（ 图2D）。 </li><li>包裹发芽板在箔的叠层挡住光并保持在25℃3天。 </li></ol></li></ol>注意:如果发芽所得不足3天，根部可能太短达到琼脂缩影。如果发芽转移到缩影板前超过4天进行，苗木成活率会是穷人。如果M。苜蓿生态型或突变与慢发芽率要进行比较，慢慢发芽生态型应允许发芽超过3天。 2。个别板块缩影准备<ol><li>准备Jensen的介质12（见表1组成），允许〜70毫升培养基为每个缩影。准备在投手或瓶是方便快速浇注（不超过1-2升，按每投手）和高压灭菌过程中留在投手磁力搅拌棒。 <ol><li>中等后冷却至55-65〜°C，补充已被添加，并且pH值已经被选中（见表1），倒入圆形直径3.94英寸，15毫米深的板块。板应浇〜0.9-1厘米深（〜70毫升/板）。 </li><li> Jensen的介质的部件可以形成混浊沉淀物，所以它的投手定期返回磁力搅拌板，以防止部件从沉淀出来是很重要的。沉淀物可能是相在板IBLE它们凝固后，只要它们被均匀地分布在板之间不影响性能。 </li><li>浇注，以防止结露的盘子上眼睑形成过程中堆叠板。 </li></ol></li><li>之后Jensen的板块都凝固了，陷两个板和盖子与已弯成U形5（ 图3A）称重铲。热锅铲与燃烧器弯曲，直到红热，缺口5-6片，然后再加热。当缺口盖子被放置在缺口板，这将创建一个椭圆形的门户，通过该苗的枝条将增长（ 图3B）。如果缺口板将被储存起来，单独包装用石蜡膜.. </li></ol> 3。 苜蓿的制备根瘤菌文化前两天植物接种，启动所有S的文化根瘤菌菌株被接种到幼苗。丘尔RES应生长在LBMC辅以2.5毫米硫酸镁4，2.5毫米氯化钙2，和适当的抗生素（TY培养基也效果很好）（LB培养基[米勒]）介质13。少至2-3毫升每种培养物就足够了。 <ol><li>成长在30℃振荡2天，直到细胞密集。 </li><li>对于大多数植物接种，在S的生长期根瘤菌不是关键的。通常情况下，登录后期或早期静止期细胞使用。 </li></ol> 4。铺设了M。对个体缩影苜蓿幼苗<ol><li> 3天之后，打开一个发芽板（在步骤1中制备）和紧接在洪水灭菌超纯水。钳浸在乙醇和简要火焰消毒。用无菌镊子轻轻推全苗的根尖下的水，以防止干燥。罗茨将范围从2-6厘米长。多数为3-4厘米。苗木选择与直根系并没有明显的缺陷。 </li><li>检查Jensen的介质微观的盖子的积聚凝结。轻弹盖子，除去冷凝或用新的缺口盖更换。如果有过量的缩合的盖子，幼苗根可能会附着在盖子，然后死亡的缩合干燥后。 </li><li>使用镊子，轻轻的挑子叶幼苗从发芽板打下根在Jensen的媒介缩影。确保根尖是在与琼脂接触。 </li><li>温和地去除种皮，如果它仍然存在。在水中浸泡5-10分钟后，种皮要宽松。轻轻挑逗种皮的钳子，直到它让路，并丢弃。芽的子叶和大约0.5厘米应该伸出的U形凹口的在板（ 图4A）。小心用U型缺口的盖子在幼苗更换板的盖子，创建一个门户网站的幼苗（ 图4B）。在水平堆叠搁置板，直到所有的苗都被放在缩影。 </li><li>使用全苗从外观上可行，并有直每根发芽板。 （如果可能的话，让苗未开封作为替代品刚接种前萎蔫苗使用的一个板块。） </li><li>铺设全苗之后，让他们坐在水平Jensen的缩影，而S。根瘤菌接种悬浮液正在准备。 </li></ol> 5。 S的 ​​制备根瘤菌接种物悬浮液<ol><li>检查S。接种后定期苜蓿根瘤菌的文化，以确保它们正在日益增强。经过2天的增长，外径600应该是2和4之间。 </li><li>吸取0.5毫升每一种文化的进入无菌1.5mL管中，离心沉淀。除去上清液。 </li><li>无论是0.85％无菌氯化钠或无菌超纯水洗涤细胞两次。重悬细胞于0.5ml 0.85％输注液激怒NaCl或超纯水。 </li><li>衡量一个1/10的稀释悬浮S的外径600 苜蓿根瘤菌从步骤5.3文化。在此基础上的阅读，使每个文化在无菌超纯水中的悬浮液在OD 600 = 0.05。如果细胞密度过高，结瘤可能受到抑制。使稀悬浮液足以使100微升可以接种到各幼苗。外径600 = 0.05悬挂的云应该只是用肉眼几乎难以察觉。 </li></ol> 6。接种和缩影的密封<ol><li>前已开始接种，检查幼苗枯萎，但没有存活转移到Jensen的媒体缩影。取代那些幼苗从发芽片新鲜的苗。 </li><li>打开每个缩影，接种100μL的适当的学苜蓿根瘤菌悬挂到幼苗根系。重新装上盖子，并预留水平STAC6-10缩影KS。对于每个S。要比较的根瘤菌菌株，接种至少15家工厂。对于具有共生生产力细微的差别株，接种30-35植物。至少留出10株未接种作为阴性对照。接种30-35植物与S根瘤菌 1021或其他适当的野生型菌株作为阳性对照。 </li><li>之后，S。根瘤菌悬浮有时间来吸附在根表面和所述悬浮液已经浸透到琼脂（至少20分钟），单独包装每板用石蜡膜。板应包裹起来，以缺口的边缘，但石蜡膜不应该阻止秧苗（ 图4C）的生长。 </li><li>在包装时，使对于根部附着在板盖内最后的检查。从盖子除去附着的根，奠定了板式平在板凳上，然后点击或轻拂盖子回落敲根到琼脂苏rface。 </li><li>排队每个堆栈6-10板的幼苗门户。与苗朝上（ 图4D）放置在堆栈成90°角。石蜡膜的粘性将持有的板块在地方足够长的时间来包装箔整个堆栈，留下1-2厘米带解开其中的幼苗出现（ 图4E）。箔将保护根部由轻。 </li><li>将铝箔包裹堆在一个被点燃的生长室或生长室在21-25°C，60-70％的相对湿度和100-175微摩尔/平方米-2 -1光在16小时光照/ 8小时黑暗周期（ 图1A）。在这些生长条件下，延长培养在光照水平高于200微摩尔/米-2 -1可具有对植物生长产生不利影响。 </li></ol> 7。考试根系共生表型的定量植物可以保持在微生态长达9个星期。 <ol><li>根瘤数目可以在一系列时间点由每一堆块解缠箔和计数结节进行量化。开发植物根瘤接种S.根瘤菌 1021野生型是显而易见的接种后10-14天。 </li><li>共生的生产率，也可在每个时间点，通过测量出现芽的长度测量。 </li><li>通常是由分离的拍摄和测量拍摄的鲜重进行7周接种后生产率共生的最后定量。这一步可以迟9周进行，则可较长时间的增长是需要的。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+production+of+the+exopolysaccharide+succinoglycan+enhances+Sinorhizobium+meliloti+1021+symbiosis+with+the+host+plant+Medicago+truncatula.">Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula.</a> J Bacteriol. 194, 4322-4331 (2012).</li><li>Queiroux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+genomics+screen+identifies+a+Sinorhizobium+meliloti+1021+sodM-like+gene+strongly+expressed+within+host+plant+nodules.">A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules.</a> BMC Microbiol. 12, 74 (2012).</li></ol>

作者什么都没有透露。

有几个步骤是成功的关键协议:1）用纯化，植物细胞培养的必要性测试琼脂怎么强调也不过分。这不是对苜蓿幼苗关键的，但它是为M的增长的关键蒺藜苜蓿 A17。 2）在步骤1中所述的发芽幼苗上垂直琼脂平板上是很重要的。在一个15毫米深板7萌发的幼苗在一个倒置的水平面上的广泛使用的技术是不建议在这个协议，因为幼苗根系将是太短和/或不直。苗2-4厘米长的直线增长的根...

豆科植物寄主植物蒺藜苜蓿 A17和根瘤菌菌苜蓿根瘤菌 1021之间的互动是最听话的模型系统根瘤发育和固氮共生的研究之一。两者共生伙伴的基因组已被测序1,2和厂房及细菌都是服从遗传操作3,4。植物和细菌突变体两者的表型分析需要观察根瘤发育的阶段，量化的共生生产率随着时间的能力。在这里，我们描述了一种观察M的根瘤发展苜蓿品种。 Jemalong A17接种S。根瘤菌 1021从标准，圆形直径3.94英寸，15毫米深的实验板（ 图1A）提出个人的缩影中。在拍摄时，通过在该板的一侧的切口的门户（曝光和生长FIGURES 1B，1C和1E）。根都包含在微观范围内，并保持无菌，同时通过板（ 图1D）的盖允许观察。由于拍摄访问，其增长是不受限的，它可以以周期性的间隔进行测量而不会影响根的无菌性。最初开发创建缺口板微观的方法由雷， 等。5种植苜蓿植物，但它并没有被广泛采用，尽管其比其他方法的许多优点。这种方法的变化也被开发为植物根系和菌根6之间的相互作用的分析。现在，我们已经适应和优化这种方法M的增长苜蓿植物。此协议的过较常用的方法的优点如下所述。 有几种方法，这些方法目前广泛使用的分枝结瘤的研究苜蓿 inoculated与S。根瘤菌 7。最常用的方法大规模制备根瘤的根是生长在气雾沉箱7。在这种方法中，植物被悬挂在一个大的容器中，并根通入S的混合物根瘤菌和营养液7。此方法是实用的，如果只有一个基因型的S。根瘤菌是进行测试。因为它要求高浓度的细菌的雾化，有沉箱接种不同菌株之间的交叉污染的可能性很高。经常被用于制备大量接种的植物的另一种方法是生长在浴盆或珍珠岩，蛭石，沙子或输注用的营养液并接种S.煅烧粘土花盆根瘤菌 7。此方法需要使用开放桶或盆，并需要浇水的营养液和补充。盆的另一个缺点是，植物必须从这样的颗粒基质进行检查根和根瘤中除去。这种方法的另一个缺点是，大面积的培养箱空间时，需要许多不同的S。根瘤菌的基因型进行比较，因为一个单独的锅必须用于每个细菌基因型。在"伦纳德罐子"是这个方法8,9的变化。甲伦纳德罐子是由一个堆叠在另一个之上，并通过油绳连接的两个消毒容器。生长培养基被放置在下部容器，并通过油绳的毛细管作用进入的珍珠岩，蛭石，沙子或在上部容器煅烧粘土生长基质绘制。秧苗（s）被放置在生长基质和接种S.根瘤菌 。此方法不需要浇水，但检查根和根瘤要求苗从颗粒生长基质除去。 有几种方法，并允许易检查的袋鼠TS。其中之一是透明的塑料"成长袋"7。这种方法的一个缺点是，经常浇水，因为仅仅≤的10ml液体培养基是最佳的生长分枝需要苜蓿中袋7。小袋实验也通常限制在〜2周7，由于在袋内部的纸芯的击穿。这是常用的两种其它方法类似于我们的方法，所述植物生长在琼脂上和根是可见的，但这些方法也有我们的程序避免了缺点。在这些方法中，植物被完全包含在24.5厘米周围的多孔外科胶带的顶部密封或塞住用棉塞或塑料盖7琼脂斜面培养试管x24.5厘米琼脂平板上。这两种方法都允许容易检查根和可以保持无菌状态。但是，琼脂斜面管通常生长仅20毫升琼脂培养基上7和需要浇水和养分一个ddition植物的长期增长。 24.5 CM中生长的植物x24.5厘米琼脂平板微观有足够的水分和养分的长期增长，但增长的快速拍摄变得内的板子缩影和乙烯气体可以建立抑制结瘤7约束。在这里描述的过程中，拍摄暴露，并自由地生长，其中包含〜70毫升媒体，允许长期增长的缩影之外。 这里描述的过程可能是有用的，不仅为豆科植物结瘤的研究，同时也为其他中型植物的根表型的研究。这些微观和传统的聚碳酸酯植物组织培养罐之间的区别是，在这里所描述的板微生态根上的琼脂的垂直表面上​​生长，而不是分解成琼脂在罐的底部的水平层。这允许根被抬离琼脂表面以最小的D豪悦国际为根毛和琼脂的根表面，它通过显微镜促进根毛的考试最小的附着力。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Agar purified, plant cell culture-tested</td>
			<td>Sigma</td>
			<td>A7921</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

single-plant, sterile microcosms for nodulation and growth of the legume plant medicago truncatula with the rhizobial symbiont sinorhizobium meliloti

根瘤菌形成于兼容的主机豆科植物的根共生固氮根瘤。其中最发达的模型系统研究这些相互作用是植物蒺藜苜蓿品种。 Jemalong A17和根瘤菌菌苜蓿根瘤菌 1021。植物根系共生表型的得分反复成像需要的是无损要么植物或细菌的方法。一些植物和细菌突变体的表型共生变得明显增长后的时间都比较短，而且不需要长期观察主机/共生互动。然而，在共生效率和根瘤衰老表型不明显的结瘤过程的早期阶段，细微的差别需要相对较长的生长时期，他们可以拿下之前。几种方法已经发展为长期增长和观察这个主机/共生体对的。然而，许多这些方法都需要重复浇水，这增加了污染的其他微生物的可能性。其它方法需要对大量的植物的生长相对大的空间。该方法在这里描述的，M的共生增长苜蓿/ S。苜蓿根瘤菌在无菌，单植物微生态，具有几个优点。在这些微观植物有足够的水分和营养物质，以确保不要求浇水长达9周，防止浇水过程中的交叉污染。这使得表型进行量化，可能在短期内生长系统，如在根瘤发育和早期根瘤衰老微妙延迟错过。此外，在微观世界的根和根瘤通过板盖被容易地观看，从而向上生根观察的植物是不需要的。

相比，在引言中所述大多数其他方法的这些板微观的制备和接种是相对简单的。利用这些缩影中也允许延长植物生长（9周），无需浇水或营养补充，维护根不育的，既不易检查根和避光保护根系，厂房无约束增长笋，方便前往拍摄的长度测量，并去除植物从有根毛损伤极小的缩影。 图1A显示幼苗生长在微观世界中的孵化器。在图1B-1E显示的图像含有M缩影几种不同?...

Watch this Scientific Journal Video about Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti at JoVE.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

苜蓿植物的共生固氮细菌的苜蓿根瘤菌在个人，无菌微生态从标准实验室板制成的增长使得频 ​​繁的检查根系和根瘤的不妥协不育。植物可以保持在这些生长室长达9个星期。

单厂，无菌的缩影和结瘤的豆科植物生长<em&gt;苜蓿</em&gt;与根瘤菌共生体<em&gt;根瘤菌</em

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

single-plant-sterile-microcosms-for-nodulation-growth-legume-plant

Research

JoVE Journal

Environment

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

16.9K 次观看.  Florida State University.  苜蓿植物的共生固氮细菌的苜蓿根瘤菌在个人，无菌微生态从标准实验室板制成的增长使得频 ​​繁的检查根系和根瘤的不妥协不育。植物可以保持在这些生长室长达9个星期。 

视频: Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

科研

环境科学

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

成绩单及代谢物谱烟草树和杨树的评价作为原料的生物基工业

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

二斑叶螨的应用<em&gt;二斑叶螨</em&gt;植物病虫害的相互作用研究

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

单厂，无菌的缩影和结瘤的豆科植物生长

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