Name: single-plant, sterile microcosms for nodulation and growth of the legume plant medicago truncatula with the rhizobial symbiont sinorhizobium meliloti 
Uploaded: 2013-10-01T09:00:00
Description: 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.

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...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+Biochemical+Comparisons+of+Plant+Induced+Ineffective+Nodules.">Histological and Biochemical Comparisons of Plant Induced Ineffective Nodules.</a> Phytopathology. 71, 884 (1981).</li><li>Vance, C. P., Johnson, L. E. 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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>

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?...

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 ...

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

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