Name: a flexible low cost hydroponic system for assessing plant responses to small molecules in sterile conditions
Uploaded: 2018-08-25T13:30:00.000+00:00
Duration: 11 min 27 s
Description: Watch this Scientific Journal Video about A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions at JoVE.com

这项工作得到圣保罗研究基金会 (FAPESP) 的支持;格兰特 12/19561-0) 和普朗克社会。Araújo (FAPEMIG 14/30594), 卡罗莱纳 c. 蒙特卡罗 (FAPESP;赠款 14/10407-3), Valéria 马夫拉 (FAPESP;赠款 14/07918-6) 和薇薇安妮 (海角/CNPEM 24/2013) 感谢研究金。作者感谢 Bourgin (INRA, 凡尔赛, 法国) 的基督教迈耶, 慷慨地为 RPS6 提供抗体。作者感谢室温硫化 UNICAMP 和 Aparecido de 索萨 Manoel 在录音过程中的技术支持。

1. 液体和固态培养基的制备
<ol><li>用半强力 Murashige 和 Skoog (MS) 培养基与维生素 [0.0125 毫克/升氯化钴五水合物, 0.0125 毫克/升铜 (ii) 硫酸盐五水, 18.35 毫克/升的 ethylenediaminetetraacetate 铁钠, 3.10 毫克/升硼酸, 0.415 毫克/升的碘化钾, 硫酸锰的8.45 毫克/升, 钼酸钠的0.125 毫克/升, 硫酸锌水合物的4.30 毫克/升, 氯化钙的166.01 毫克/升, 磷酸二氢钾85毫克/升, 950 毫克/升硝酸钾, 硫酸镁90.27 毫克/升, 825 毫克/升的硝酸铵, 1 毫克/升的甘氨酸, 50 毫克/升的肌醇, 0.25 毫克/升的烟酸, 0.25 毫克/升的盐酸哆, 和0.05 毫克/升的盐酸硫胺胺] 补充0.25 克/升的MES, 并调整 pH 值为5.8 与10米 KOH。</li>
	<li>添加10克/升的琼脂, 使一个半强度的 MS 固体培养基。蒸压釜的介质在121°c 20 分钟前使用。</li>
</ol>2. 水培系统组装
注: 应严格遵循这些步骤, 建立水培系统。
<ol><li>
材料灭菌
	<ol>
<li>包装在蒸压袋的吸管尖端机架 (无盖), 将用作 minitanks。蒸气压机架在121°c 20 分钟, 15 psi。 注: 我们使用的聚丙烯吸管尖架有以下尺寸: 120 毫米 (长) x 89 毫米 (宽) x 55 毫米 (高度)。吸管尖端平坦的表面必须有一个区域, 以增加培养基。其他刀尖架可以使用 (见材料表)。 注: 在整个水培系统的组装过程中, 有必要使用一个层流罩, 必须在使用前用70% 乙醇清洗和消毒。实验者必须穿上实验室的外衣, 洗手和暴露的皮肤, 用70% 乙醇消毒。手套是可选的, 除了药物应用。</li>
		<li>在进入层流罩之前, 用70% 乙醇清洁所有上面描述的附件 (一次性塑料盒、胶带、吸管、剪刀和镊子)。如果引擎盖允许, 在水培系统的组装之前打开紫外线10分钟, 以保持工作区域被净化。</li>
	</ol>
</li>
	<li>
Minitank 装配
	<ol>
<li>用胶带封住吸管尖端的上表面 (图 1B)。如有可能, 将其留在紫外线照射下10分钟。</li>
		<li>使用多通道吸管 (图 1C), 在每个井中添加180µL 的熔融固体 MS 培养基 (稍暖)。 注: 在准备许多坦克时, 使用热板防止 MS 介质凝固。</li>
		<li>允许介质完全凝固 (约30分钟)。 注: 在凝固期间, UV 光可以打开。</li>
		<li>完全用液体 MS 培养基 (图 1D) 填满吸管尖端架, 确保固体和液体介质之间有密切的接触。</li>
		<li>卸下吸管尖端平面上表面的胶带, 仔细地将其安装在机架上。水培系统现在已经准备好接收被灭菌的种子。</li>
	</ol>
</li>
</ol>3. 种子杀菌
<ol><li>在1.5 毫升的微细中放置 500拟南芥种子。根据实验所需的植物数量, 尽可能多地使用管内。</li>
	<li>用70% 乙醇洗净种子, 用温和的搅拌2分钟。让种子安定下来, 然后小心地去除乙醇。</li>
	<li>添加1毫升的10% 次氯酸钠溶液含有2µL 的吐温20洗涤剂。搅动解决方案为5分钟. 仔细删除解决方案。</li>
	<li>用无菌蒸馏水冲洗种子, 直到全部漂白残渣全部清除 (大约 5x)。 注: 在表面杀菌后, 种子浸泡在无菌蒸馏水中, 在黑暗中分层4摄氏度, 以同步发芽。 注:狗尾草贝(加入 A10.1) 的种子在浓硫酸中 preincubated 15 分钟 (打破物理休眠), 在无菌蒸馏水中彻底冲洗, 然后用5% 次氯酸钠溶液杀灭。包含0.1% 吐温20为5分钟以温和的鼓动21。其余的灭菌步骤与所描述的拟南芥种子相同。</li>
</ol>4. 种子应用
<ol><li>用一把不育的手术刀, 稍微切开200µL 尖端的肢体。</li>
	<li>将拟南芥种子吸进吸管尖平面上表面的固体培养基中。注意介质不从单位松开;否则, 种子将被着色, 幼苗不会正常生长 (图 1E)。 注: 对狗尾草种子使用无菌镊子 (胚向上定位)。</li>
	<li>储存尽可能多的 minitanks 在一次性塑料盒内保持高湿度和保持环境免受微生物 (图 1F)。</li>
	<li>用胶带密封一次性塑料盒, 避免污染。</li>
	<li>将水培系统放入生长室中, 适当的生长条件为感兴趣的植物。 注: 在这项工作中, 使用了以下条件: 75% 的湿度, 150 µmol m-2 s-1的辐照度和春分条件 12 h 光 (21 °c)/12 h 暗 (19 °c) 为拟南芥, 或300µmol m-2 s-1的狗尾草的辐照度和 12 h 光 (28 °c)/12 h 暗 (25 °c) (图 1G和1H)。</li>
</ol>5. 验证使用该水培系统抑制雷帕霉素激酶的靶向性
注: 这一水培系统最初是为了促进对植物的化学管理而开发的, 一般来说, 在大型实验中应用是非常昂贵的。作为一个概念的证明, atp 竞争抑制剂 AZD-8055, 这是已知的专门针对 atp 结合部位的 tor 蛋白激酶22, 被用来跟踪抑制 tor 活动的种子, Columbia-0 幼苗 ( 诺丁汉拟南芥股票中心, NASC ID: N22681)。这里简要描述了所使用的协议。
<ol><li>根据上文所述的气候条件, 将种子培至1.04 级, BBCH23级 (约 11 d)。用含有0.05% 亚砜 (控制)、2µM AZD-8055 (TOR 抑制剂) 稀释的亚砜中的新鲜培养基取代营养液, 或在夜间 (EN) 结束时不进行治疗 (模拟)。</li>
	<li>在治疗后, 在不同的时间点收获一些幼苗, 并将它们分离成根和芽。将样品冷冻在液氮中, 将其研磨成机械磨床中的细粉 (见材料表), 并将粉末贮存在-80 摄氏度, 直至使用。</li>
	<li>应用免疫印迹对40S 核糖体蛋白 S6 (RPS6) 的磷酸化和非磷酸化形式进行了研究。24。</li>
	<li>漂白完整的幼苗为样品脱色, 洗涤他们在蒸馏水, 浸泡他们在碘溶液中5分钟25, 并且相片显微镜的幼苗 (0.63X 目标, 20x 近似, 和7.5x 大小) 为淀粉含量的定性评价。</li>
	<li>在酶降解和释放葡萄糖 spectrophotometrically 的测定后, 将其与 NADP+ 26、27的还原量相耦合, 量化淀粉。</li>
	<li>进行总 RNA 提取、cDNA 合成和定量 rt-pcr 检测, Caldana等。28评估与不同类型的应力相关的基因的表达水平。</li>
	<li>可选, 在类似气候条件下, 在 0.1 L 容量的塑料罐中种植园艺基质的幼苗 [60% 的湿度, 150 µmol m-2 s-1的辐照度, 和春分条件 12 h 光 (21 °c)/12 h 暗 (19 °c)] 为了比较它们与幼苗生长的培。 注: 用于基因表达测定的靶基因为ABF3 (At4g34000)、 ASN1 (At3g47340) 和TPS5 (At4g17770), 其表达水平采用增量 Ct 法29 ACT2 (At3g18780) 或PDF2 (At4g04890) 作为内部参考基因, 假设100% 的 PCR 放大效率横跨所有样品。用于定量 PCR 的寡核苷酸对: ABF3 (GTTCTCAACCTGCAACACAGTGC;TCCAGGAGATACTGCTGCAACC), ASN1 (AGGTGCGGACGAGATCTTTG;GTGAAGAGCCTTGATCTTGC), TPS5 (CTGCTCTGATGCTCCTTCTTCC;AAGCTGGTTTCCAACGATGATG), ACT2 (CGTACAACCGGTATTGTGCTGG;CTCTCTCTGTAAGGATCTTCATG) 和PDF2 (TAACGTGGCCAAAATGATGC;GTTCTCCACAACCGCTTGGT)。</li>
</ol>

作者没有什么可透露的。

这种优化的培水结构能够成功地培养植物的体外培养。种子在吸管尖端平坦表面的固体培养基上发芽良好, 与种子浸泡在营养液中的系统相比, 有相当大的增益。该系统的一个很好的优点是在幼苗发育过程中, 根直接接触液体培养基, 而不需要迁移。此外, 化学处理可以很容易地应用于液体介质中, 减少体积。湿度保持高, 避免了养分溶液的蒸发和补给。此外, 在幼苗建立过程中, 可以很容易地?...

种植植物使用水培的优势已被广泛认可, 在生产大型和均匀的植物, 使可再生的实验1,2,3。在该系统中, 营养液的组成可以在植物生长发育的各个阶段得到适当的控制和循环利用。此外, 根系不受非生物胁迫, 如土壤生长的植物, 如养分饥饿和缺水4。随着植物的生长培呈现出与土壤中培养的形态和生理特征相当相似的生物, 该系统在研究中得到了广泛的应用, 因为它允许监测根/芽的生长及其收获, 而不伤害2,5。
由于有可能改变营养液的成分和浓度, 大多数使用水培条件的研究已经完成, 以表征微-和营养素1,3 的功能。,6,7,8。然而, 该系统已证明是非常有用的广泛的应用于植物生物学, 如阐明激素和化学物质在植物中的作用。例如, 发现 strigolactones 作为一个新的类激素9和加速生长表型触发 brassinosteroid 应用10是在水培条件下进行的。此外, 该系统还可以进行标记同位素的实验 (例如, 14n/15n 和13CO2)11,12 , 以评估其纳入蛋白质和代谢物通过质谱分析。
考虑到该系统在植物研究中的重要性, 在过去几年中设计了大量的水培技术, 包括使用 (i) 将幼苗从盘子转移到水培容器3的系统, 13;(二) 西斯尔, 限制进入根系发育的早期阶段2、14、15;(iii) 聚乙烯颗粒为浮动体, 使小分子/处理的均匀应用困难16;或 (iv) 植物数量减少9,17。其中许多协议中描述的水培池体积通常很大 (体积从 1-5 升到32升)18, 这使得化学品的应用极为昂贵。虽然很少有研究表明在无菌条件下的水培栽培8,19, 系统的装配通常是相当费力的, 包括完美的调整尼龙网格成塑料或玻璃容器5,8,17,20。
由于拟南芥作为模型植物的重要性, 大多数的水培系统是为这个物种1,2,8,14,18,19,20. 尽管如此, 还有几项研究报告了其他植物的水耕生长特性, 并对种子进行预处理, 以改善它们在体外8、16 的发芽和同步速率..为了进行大规模的工作, 我们制定了一项协议, 建立一个简单和低成本的维修水培系统, 使种植植物的无菌条件, 包括一. 芥和其他物种, 如草狗尾草贝。本方法适用于不同的实验, 因为幼苗生长可以最大化, 同步, 易于监测。此外, 该系统具有以下优点: (一) 其装配简单, 可重复使用;(二) 使不同的化学品易于在液体介质中应用;(iii) 幼苗在培养基中发芽并直接生长, 不需要迁移到水培系统;(iv) 可密切监督苗根发育/生长, 并无损伤地收获幼苗;并且 (v) 它使有可能大规模地工作, 保持生理条件。

<table><tbody><tr><td>Ethanol</td><td>Merck</td><td>100983</td><td></td></tr><tr><td>Sodium hypochlorite solution</td><td>Sigma-Aldrich</td><td>425044</td><td></td></tr><tr><td>Polysorbate 20&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P2287</td><td></td></tr><tr><td>Murashige and Skoog (MS) medium including vitamins&amp;nbsp;</td><td>Duchefa Biochemie</td><td>M0222</td><td></td></tr><tr><td>2-(&lt;em&gt;N&lt;/em&gt;-morpholino)ethanesulfonic acid (MES) monohydrate</td><td>Duchefa Biochemie</td><td>M1503</td><td></td></tr><tr><td>Agar&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A7921</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>484016</td><td></td></tr><tr><td>Laminar flow hood</td><td>Telstar</td><td>BH-100</td><td></td></tr><tr><td>Hotplate</td><td>AREC</td><td>F20510011</td><td></td></tr><tr><td>Growth chamber</td><td>Weiss Technik</td><td>HGC 1514</td><td></td></tr><tr><td>Glass Petri dish (150 mm x 25 mm)</td><td>Uniglass</td><td>189.006</td><td></td></tr><tr><td>200 &amp;mu;L pipette tip racks&amp;nbsp;</td><td>Kasvi</td><td>K8-200-5 *</td><td></td></tr><tr><td>300 &amp;mu;L multichannel pipette</td><td>Eppendorf</td><td>3122000060</td><td></td></tr><tr><td>300 &amp;mu;L pipette tips</td><td>Eppendorf</td><td>30073088</td><td></td></tr><tr><td>200 &amp;mu;L pipette&amp;nbsp;</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>200 &amp;mu;L pipette tips</td><td>Eppendorf</td><td>30000870</td><td></td></tr><tr><td>Scissors</td><td>Tramontina</td><td>25912/108</td><td></td></tr><tr><td>Tweezer</td><td>ABC Instrumentos</td><td>702915</td><td></td></tr><tr><td>Scalpel blade</td><td>Sigma-Aldrich</td><td>S2771</td><td></td></tr><tr><td>Adhesive transparent tape (45mm x 50m)</td><td>Scotch 3M</td><td>5803</td><td></td></tr><tr><td>Disposable plastic boxes, external dimensions: 353 mm (L)x 178 mm (W) x 121mm (H)</td><td>Maxipac</td><td>32771</td><td></td></tr></tbody></table>

a flexible low cost hydroponic system for assessing plant responses to small molecules in sterile conditions

植物生物学的广泛研究是利用水培培养进行的。在这项工作中, 提出了一种用于评估植物对化学物质和其他物质的反应的体外培水生长系统。该系统高效地获得了 c3和 c4型拟南芥和狗尾草贝的均匀健康苗。无菌栽培避免了藻类和微生物的污染, 这是植物正常生长和栽培发展的已知限制因素。此外, 该系统具有可伸缩性, 能够大规模地收获植物材料, 具有较小的机械损伤, 而且如果需要, 还能收获植物的各个部分。详细的协议表明, 该系统有一个简单和低成本的组装, 因为它使用吸管架作为种植植物的主平台, 提供。采用拟南芥苗对该系统的可行性进行了验证, 以评价雷帕霉素 (TOR) 激酶靶化学抑制剂 AZD-8055 药物的作用。在根和芽 AZD-8055 治疗后, 早期30分钟有效检测 TOR 抑制。此外, AZD-8055-treated 植物显示预期淀粉过剩表型。我们建议这种水培系统作为植物研究人员的理想方法, 目的是监测植物诱导剂或抑制剂的作用, 以及使用同位素标记化合物评估代谢通量, 一般来说, 需要使用昂贵的试剂。

TOR 激酶是一种主要的调节剂, 它集成了营养和能量信号, 促进了所有真核生物的细胞增殖和生长。在植物中阐明 tor 功能的努力包括通过 RNA 干涉或人工 microRNA28、30、31, 产生含有 TOR 条件压制的拟南芥转基因线。考虑到 TOR 击倒植物的胚胎致死表型32,33<s...

Watch this Scientific Journal Video about A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions at JoVE.com

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

一种简单、多功能、低成本的体外水培系统成功地进行了优化, 使得在无菌条件下进行大规模实验。该系统促进了化学物质在溶液中的应用, 并为分子、生物化学和生理学研究提供了有效的根系吸收。

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 ...

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10.5K 次观看.  Brazilian Center for Research in energy and materials (CNPEM). 一种简单、多功能、低成本的体外水培系统成功地进行了优化, 使得在无菌条件下进行大规模实验。该系统促进了化学物质在溶液中的应用, 并为分子、生物化学和生理学研究提供了有效的根系吸收。

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

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

方法中进行杂交<em&gt;狗尾草</em&gt;，新模型体系的草

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

科研

环境科学

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

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

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

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

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

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 ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

生物工程

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

生物学

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

植物与环境相互作用实时生理表型的遥测重力平台

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

免疫与感染

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

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential 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 ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

半水培玻璃罐系统，用于多种植物物种的渗出物分析

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

作者聚焦:在新型生长系统中探索植物与微生物通过根系分泌物的相互作用 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

用于半水培根系渗出物分析的多功能玻璃罐系统

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...

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

摘要

探索更多视频

此视频中的章节

方法中进行杂交

评估乌西拉戈病原性的快速高效方法，可在玉米和特奥辛特线上使用

单厂，无菌的缩影和结瘤的豆科植物生长

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

植物激素在应激反应监测

植物与环境相互作用实时生理表型的遥测重力平台

在浮动水培系统中监测阿拉伯菌的殖民化与维护

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

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

用于半水培根系渗出物分析的多功能玻璃罐系统

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

摘要

探索更多视频

此视频中的章节

方法中进行杂交

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

单厂，无菌的缩影和结瘤的豆科植物生长

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

植物激素在应激反应监测

植物与环境相互作用实时生理表型的遥测重力平台

在浮动水培系统中监测阿拉伯菌的殖民化与维护

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

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

用于半水培根系渗出物分析的多功能玻璃罐系统

评估乌西拉戈病原性的快速高效方法，可在玉米和特奥辛特线上使用