The authors thank Davy Opdenacker for technical assistance and photography. We greatly thank Dr. Annick Bleys for helpful suggestions to improve the manuscript. This work was financed by the Interuniversity Attraction Poles Programme IUAP P7/29 'MARS' from the Belgian Federal Science Policy Office, by the FWO grant G027313N and by the Agency for Innovation by Science and Technology, IWT (IR).

1. 拟南芥 LRIS协议注:该文是指"小"或"大"规模的实验。小规模的实验，如标记线的分析和组织学染色6，14，只需要少数样品。大规模的实验，如定量实时定量RT-PCR，微阵列9-11或RNA测序，需要样品的量越大。这样，的〜1000苗每个样品，使用由Vanneste 等人 11的量到根段的解剖后执行微阵列实验。 第1天 <ol><li> 拟南芥种子消毒 注意:选择的种子消毒以下步骤之一。它们中的任何适合的下一步骤的协议。气体灭菌（1.1.2）给出了消毒液（1.1.1）两个主要优点:它处理大量麻木的时候花费较少的时间种子呃，因为没有移液必须发生对单个样品;和灭菌的种子可以贮存更长的时间。但是，它需要一个干燥器和小心处理。 <ol><li> 液体消毒 <ol><li>倾种子在微离心管的期望数量。在2 ml微离心管使用多达20毫克（〜800种子）在1.5ml管或40毫克（〜1600种子）。 </li><li>加入1毫升70％的乙醇。反转或轻轻摇动2分钟。 </li><li>更换70％的乙醇用1ml灭菌溶液15分钟。 （准备新鲜消毒液:3.85毫升次氯酸钠（次氯酸钠）股票（12％）（注意:使用通风橱），5微升吐温20，高达10毫升水倒置管几次，以确保所有的种子来。在与溶液接触。 </li><li>在层流气流柜，替换用1ml无菌蒸馏水灭菌溶液和冲洗种子5次，每次5分钟，通过反复LY替换用1ml新鲜无菌蒸馏水。 </li></ol></li><li> 气体消毒 <ol><li>倒在2 ml微离心管的种子的所需数目。有几个独立的线时，请使用单独的管子，并安排他们在一个塑料微型离心管中或支架打开。如果种子在一个管的数目超过500（12.5毫克），细分的种子在管的适当数量，以确保成功的灭菌。确保相邻的管帽不相互覆盖。配合在一起的盒子的盖子的底部，因为它需要在干燥器以及用于杀菌。 </li><li>放置箱在碗干燥器的，以及一个玻璃烧杯（小心:使用通风柜）。 </li><li> （:使用通风柜小心）用100毫升12％的NaOCl填充该烧杯。放置在干燥器的盖子，但留下一个小孔，其中烧杯放置。 </li><li>加入3毫升37％盐酸（发烟）（CAUTION:使用通风柜）的烧杯中通过小光圈和迅速关闭干燥器。该解决方案将泡了一段时间，由于氯2气的形成。离开系统封闭O / N（或至少8小时）。 </li><li>除去干燥器的盖子。取出盒子的盖子盖上，避免在运输过程中受到污染。 </li><li>把盒子中一个空气层流柜，取下盖子，并留下的种子进行约1小时，在室温，以允许气体释放。 注意:种子可以安全地储存在干燥，密闭微离心管长达2周4℃。 </li></ol></li></ol></li><li> 侧根诱导拟南芥 <ol><li> 侧根抑制（NPA处理） <ol><li>准备在体外生长培养基。培养基含有半强度Murashige和Skoog盐混合物15，0.1 g / L的肌醇，10克/升蔗糖，和缓冲用0.5克/升2-（N-吗啉代）乙磺酸（MES）。 </li><li>调节pH值至5.7使用1M KOH。添加植物组织琼脂8克/升和高压灭菌的培养基20分钟，在121℃。 </li><li>准备新人民军的25毫米原液在二甲基亚砜（DMSO）（注意:使用手套）。在一个空气层流柜，冷却高压灭菌介质下降到约65℃（这是在该瓶子可以用手保持的温度），并添加25mM的NPA原液，得到10μM的NPA的终浓度。 </li><li>轻轻摇动瓶子，持续10秒均质一旦加入。倾50毫升每平方培养皿介质（12厘米×12厘米）。 注:如遇大规模的实验，应用尼龙网（20微米），以确保轻松传输。准备一个目（9厘米×9厘米）高压灭菌（图1A）。当高压灭菌，应用网格，以使用无菌镊子（图1B）的生长培养基中。用STE网格轻轻地推向生长培养基rilized drigalsky，以确保它是在与生长培养基（图1B）的良好接触（注意:在无菌条件下工作）。 </li><li>母猪在NPA-板的种子。 注:如遇大规模的实验计划，母猪2致密，排列排50颗种子，以方便取样（见1.2.2.3）。 <ol><li>如果液体消毒，播种在用200微升吸管NPA-板的种子。切断3 - 为了4毫米的在无菌条件下（或尖端的灭菌前）的吸头端部，以便能够吸管种子。吸管上下几次重新悬浮的种子，然后吸管大约150微升无菌水，用10 - 20种子和等到种子沉积物在尖端的底部。 （注意:在无菌条件下工作） 注意:当轻轻与尖端接触琼脂或网格，一滴水与种子将被从它（图1C）释放。 </li> &lt;li&gt;在案件气体消毒，用高压灭菌toothpicks.Pinch牙签放入琼脂采取的种子，以保证表面发粘前播下种子。拿起使用牙签将种子逐个和分发的种子在平板上（图1D）。或者，无菌水添加到种子，和母猪中1.2.1.5.1中描述（注意:工作在无菌条件下）。 </li></ol></li><li>密封与透气胶带将板在黑暗中存储它们板在4℃下至少2天，最大一周。这是必要的分层，并确保同步，并更有效的发芽。 注意:可替换地，种子可以在播种前进行分层，浸渍在蒸馏水在微离心管中，其中需要较少的冰箱的空间。在这种情况下，储存管至少一周，在4℃，播种（见1.2.1.7）后，立即将板到生长室。 第3天 </LI&gt; <li>移动板向生长室（连续光（110μE米-2 -1），21℃）5天（2天的发芽和生长的3天）在一个几乎垂直的位置（80到90度）以确保根生长上，而不是在介质。 第八天 </li></ol></li><li> 侧根感应（NAA处理） <ol><li>在1.2.1.1和1.2.1.2所述准备相同的生长培养基。制备NAA的DMSO的50mM储备溶液（小心:使用手套）。冷却该高压灭菌介质下降到65℃，并添加NAA溶液在培养基中，以获得10微米的NAA的终浓度（注意:工作在无菌条件下）。 <ol><li>轻轻摇动瓶子，持续10秒均质一旦加入。倾50毫升每平方培养皿介质（12厘米×12厘米）。 </li></ol></li><li>从含有补充了10μM的NPA的那些SUP生长培养基平板转移苗执行完成与10μMNAA。 <ol><li>钩弯镊子的手臂下的幼苗子叶，轻轻地从板中删除。只有转移苗其根部增长完全与NPA-生长培养基，最好向下接触。 </li><li>脱脂根在含有NAA的生长介质表面上一个小的距离。确保植物根与生长介质接触良好。如果需要的话，轻轻按下根部到生长培养基表面与镊子。 注:如遇大规模的实验，删除不符合网和传输通过解除在网用镊子上部的两个角接触全苗。确保网格是在与含有NAA的培养基良好接触由掠过网格在琼脂表面上一个小的距离（图1E）。 </li></ol></li><li>密封新板的透气胶带，并将其放置在生长室（CON连续的光（110μE米-2 -1），21℃）中进行诱导直到采样后的希望的时间一垂直位置。 注意:如果一个大规模的实验中，使用解剖刀一次全部直接切根段上的网格和轻轻刮网的表面进行采样它们。 </li></ol></li></ol></li></ol> 2. LRIS玉米协议<ol><li> 玉米籽粒分层 <ol><li>在黑暗中温育的玉米籽粒在4℃下至少1周。 注:此协议一直使用B73玉米自交系发展。 </li></ol></li><li> 玉米籽粒灭菌 第1天 注意:虽然玉米幼苗不必在无菌条件下将要生长，建议来灭菌的玉米籽粒并用无菌材料的工作，以防止在纸卷真菌生长系统。 <ol><li>放玉米核的必要数量在无菌玻璃烧杯中。 </li><li>加入100毫升消毒液（6％次氯酸钠水溶液）（注意:使用通风橱）。 </li><li>添加磁性搅拌棒并将该烧杯放在磁力搅拌器上以低搅拌速度（约250转）在RT 5分钟。 </li><li>通过用100ml新鲜无菌水代替溶液冲洗五次5分钟。 </li></ol></li><li> 播种玉米籽粒 <ol><li>戴上手套，以减少污染的危险性，并取纸巾卷。 </li><li>撕下的手巾纸2延伸到两片（约92厘米×24厘米）的总长度，并把他们放在彼此顶部在清洁的表面（ 图2A）。 </li><li>折叠片材翻一番的长度（大约92厘米×12厘米）（ 图2A）。 </li><li>用镊子来自t相关的前10分发在内核约两厘米见方他的纸张整个长度，同时保持8厘米每个内核与8厘米之间的内空间自由两端（图2B）。确保内核的胚根朝下且朝向纸（ 图2B）。 </li><li>轻轻摇动纸张上的长度，同时保持种子到位（ 图2B）。为了便于滚动，它是可选的，以第一喷纸用无菌水。 </li><li>把纸卷在大约6厘米直径和14厘米高度 （例如，加入250毫升离心管）灭菌玻璃管并将其放置在机架（图2C）。 </li></ol></li><li> 侧根诱导玉米 <ol><li>准备一个50毫米NPA储备液（溶于DMSO）（注意:使用手套）。 </li><li>对于每个管，使50μM的NPA溶液中加入从50mM的NPA原液125微升至125毫升无菌水在无菌玻璃烧杯中。 </li><li>搅拌均匀，倒入125个毫升50μM的NPA溶液在纸卷在管。纸卷将吸收的解决方案，并成为完全湿透。 注意:当使用纸卷和管与其他尺寸，相应地调整音量。液体应达到中途的管，以确保有足够的摄取。 </li><li>将纸卷系统在一个生长室三天（27℃，持续光照，相对湿度70％）。确保纸卷仍然通过增加额外的50微米的不良资产解决方案浸泡，由于溶液蒸发随着时间的推移（ 图2D）。 第4天 </li><li>准备一个50毫米NAA储备液（溶于DMSO）（注意:使用手套）。 </li><li>对于每个管中，加入从50毫萘乙酸库存125微升至125毫升无菌水在一灭菌的玻璃烧杯中制备50μM的萘乙酸溶液。 </li><li>轻轻挤压了大部分剩余的不良资产解决方案从纸卷，然后将米在无菌水5分钟，以洗涤出来（小心:使用手套）。轻轻挤压出大部分的水从纸辊。重复此洗涤步骤三次。 </li><li>搅拌均匀，倒在纸卷在管的50μMNAA的解决方案。确保它们完全浸透。 </li><li>将纸卷系统中的生长室（27℃，持续光照，相对湿度70％）为诱导后所需的持续时间。 </li></ol></li><li> 侧根诱导玉米不定冠根 注:LRIS上述诱导侧根在主根。然而，在种植玉米等单子叶植物中，初生根和胚胎种子根，以及它们的侧根，是幼苗发育过程中主要是很重要的。在植物生长的稍后阶段，后胚胎不定根出现在杆和还开发侧根形成一个新的根系统 16。该LRIS也可使用D由下面的胚胎主根一些小的调整，LRIS促使这些胚胎后不定冠根侧根。 <ol><li>为了使纸卷，创建论文与两个分别在其外侧层的手巾纸，并萌发纸内的两个层的夹层。放置灭菌内核（见步骤2.1至2.2）在两片发芽纸之间。发芽纸相比，手巾纸，将是不容易通过根毛和侧根，这将使得纸张的开幕推出后更容易在穿透。另一方面，手毛巾纸的两个外层将有利于液体吸收。 </li><li>将700毫升的玻璃试管辊和对他们倒无菌水，无不良资产。确保它们完全浸透。 </li><li>在发芽生长柜中消毒内核（见步骤2.4.9）。 </li><li>成长的幼苗，直到不定冠根开始出现（6天为B73）。确保纸辊由必要时加入无菌水保持湿润，在任何时候。 </li><li>转移到4天（见步骤2.4.1至2.4.4）的25微米的NPA溶液，然后由一个类似的感应侧根开始的通过更换用清洗步骤之后的50微米的NAA溶液的NPA溶液（参见步骤2.4 0.5〜2.4.9）。 </li></ol></li></ol>

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Biol. 1, 212-225 (2007).</li><li>Parizot, B., Beeckman, T., Crespi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+of+root+development.">Genomics of root development.</a> Root Genomics and Soil Interactions. , 3-28 (2012).</li><li>Bargmann, B. O., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+activated+cell+sorting+of+plant+protoplasts.">Fluorescence activated cell sorting of plant protoplasts.</a> Journal of visualized experiments : JoVE. , (2010).</li><li>Nakazono, M., Qiu, F., Borsuk, L. A., Schnable, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection,+a+tool+for+the+global+analysis+of+gene+expression+in+specific+plant+cell+types:+identification+of+genes+expressed+differentially+in+epidermal+cells+or+vascular+tissues+of+maize.">Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize.</a> Plant Cell. 15, 583-596 (2003).</li><li>Ludwig, Y., Hochholdinger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+plant+cells.">Laser microdissection of plant cells.</a> Methods Mol Biol. 1080, 249-258 (2014).</li><li>Beeckman, T., Engler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+technique+for+the+clearing+of+histochemically+stained+plant+tissue.">An easy technique for the clearing of histochemically stained plant tissue.</a> Plant Mol. Biol. Rep. 12, 37-42 (1994).</li><li>Ditengou, F. A., 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=Mechanical+induction+of+lateral+root+initiation+in+Arabidopsis+thaliana.">Mechanical induction of lateral root initiation in Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. USA. 105, 18818-18823 (2008).</li><li>Lucas, M., Godin, C., Jay-Allemand, C., Laplaze, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin+fluxes+in+the+root+apex+co-regulate+gravitropism+and+lateral+root+initiation.">Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation.</a> J. Exp. Bot. 59, 55-66 (2008).</li><li>Wang, S., Taketa, S., Ichii, M., Xu, L., Xia, K., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+root+formation+in+rice+(Oryza+Sativa.+L.):+differential+effects+of+indole-3-acetic+acid+and+indole-3-butyric+acid.">Lateral root formation in rice (Oryza Sativa. L.): differential effects of indole-3-acetic acid and indole-3-butyric acid.</a> Plant Growth Regul. 41, 41-47 (2003).</li><li>Martinez-de la Cruz, E., Garcia-Ramirez, E., Vazquez-Ramos, J. M., Reyes de la Cruz, H., Lopez-Bucio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxins+differentially+regulate+root+system+architecture+and+cell+cycle+protein+levels+in+maize+seedlings.">Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.</a> J Plant Physiol. 176, 147-156 (2015).</li><li>Beyer, E. M., Morgan, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethylene+on+uptake,+distribution,+and+metabolism+of+indoleacetic+acid-1-14C+and+-2-14C+and+naphthaleneacetic+acid-1-14C.">Effect of ethylene on uptake, distribution, and metabolism of indoleacetic acid-1-14C and -2-14C and naphthaleneacetic acid-1-14C.</a> Plant Physiol. 46, 157-162 (1970).</li><li>Delbarre, A., Muller, P., Imhoff, V., Guern, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mechanisms+controlling+uptake+and+accumulation+of+2,4-dichlorophenoxy+acetic+acid,+naphthalene-1-acetic+acid,+and+indole-3-acetic+acid+in+suspension-cultured+tobacco+cells.">Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells.</a> Planta. 198, 532-541 (1996).</li><li>Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgenomics:+genome-scale,+cell-specific+monitoring+of+multiple+gene+regulation+tiers.">Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers.</a> Annu Rev Plant Biol. 64, 293-325 (2013).</li><li>Perez-Torres, C. A., 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=Phosphate+availability+alters+lateral+root+development+in+Arabidopsis+by+modulating+auxin+sensitivity+via+a+mechanism+involving+the+TIR1+auxin+receptor.">Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor.</a> Plant Cell. 20, 3258-3272 (2008).</li></ol>

作者什么都没有透露。

在拟南芥 LRIS协议，它只能传输已经变得完全在与含NPA生长培养基接触的幼苗是重要的。这确保侧根开始被阻止在整个根长度。为了防止伤人传输过程中的小植株，弯曲钳子的臂能下的幼苗的子叶钩住。转让时，请确保幼苗根在与含NAA琼脂培养基充分接触。这可以通过掠过根在琼脂表面上一个小的距离来实现。这将确保一个有效的同步感应侧根萌生过的总根长。的根源可以种植没有他们的?...

根系统是植物生长的关键，因为它确保锚固的水分吸收和养分从​​土壤。因为根系统的扩展主要是依赖于生产侧根，其起始和形成已被广泛研究。侧根开始在柱鞘细胞的特定子集，称为创始人电池 1。在大多数双子叶植物，如拟南芥 ，这些细胞都位于原生木质部极2，而在单子叶植物，如玉米，它们被发现在韧皮部磁极3。创办人细胞通过增加生长素应答4标记，接着通过特定的细胞周期基因表达（例如， 细胞周期蛋白B1; 1 / CYCB1; 1），之后，它们经历了第一轮的不对称背斜司5。经过一系列的协调背斜与平周分裂，一侧根原基形成的最终将成为一个一utonomous侧根。的位置和侧根起始定时是但是不可预见的，因为这些事件既不丰富也不同步。这妨碍了使用分子的方法，例如转录研究这一过程。 为了解决这个问题，一个侧根诱导系统（LRIS）已经开发6,7。在这个系统中，幼苗首先用N - 1-naphthylphthalamic酸（NPA），其抑制生长素运输和积累，从而阻断侧根开始8。通过随后转移至苗含有合成植物生长素-1-萘乙酸（NAA）培养基中，整个柱鞘层响应该升高的生长素水平从而大规模诱导侧根启动细胞分裂6。因此，这一制度导致了快速，同步和广泛的侧根灌顶，方便收集富含晚期的特定阶段根样本拉尔根系发育。随后，这些样品可以被用来确定在侧根形成的全基因组表达谱。该LRIS取得了约侧根开始在拟南芥和玉米9-13，但需要已经显著的知识，这个系统应用到其他植物物种变得更基因组被测序更加明显，并且有越来越多的兴趣将知识传授给经济的重要种类。 这里， 拟南芥和玉米LRISs的详细方案给出。接着，使用该系统的一个例子是提供，通过说明如何从玉米LRIS获得转录数据可用于识别具有在不同的植物物种侧根发起期间的保守功能功能同系物。最后，指导方针，以优化LRIS其他植物物种提出了建议。

<table border="0" cellpadding="0" cellspacing="0" width="1258">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ARABIDOPSIS LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Arabidopsis seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>Col-0 ecotype</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Gas sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 1.5 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 125.215</td>
			<td>Eppendorf microtubes 3810X, PCR clean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 2 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 120.094</td>
			<td>Eppendorf Safe-Lock microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">hydrochloric acid</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1,003,171,000</td>
			<td>37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">glass desiccator</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;"></td>
			<td>Pyrex</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">plastic micro-centrifuge tubes box or holder</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ethanol</td>
			<td style="width:228px;">Chem-Lab nv</td>
			<td style="width:119px;">CL00.0505.1000</td>
			<td>Ethanol, abs. 100% a.r. dilute to 70%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Tween 20</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth medium</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Murashige and Skoog salt mixture</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M0221-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">myo-inositol</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">I5125-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M1503.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sucrose</td>
			<td style="width:228px;">VWR, Internation LLC</td>
			<td style="width:119px;">27483.294</td>
			<td>D(+)-Sucrose Ph. Eur.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">KOH</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1050211000</td>
			<td>pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Plant Tissue Culture Agar</td>
			<td style="width:228px;">LabM Limited</td>
			<td style="width:119px;">MC029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Making a mesh for transfer</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">nylon mesh</td>
			<td style="width:228px;">Prosep byba</td>
			<td style="width:119px;"></td>
			<td>Synthetic nylon mesh 20 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">square petri dish plates</td>
			<td style="width:228px;">GOSSELIN</td>
			<td style="width:119px;">BP124-05</td>
			<td>12 x 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">50 ml DURAN tubes</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">CLS430304</td>
			<td>Corning&#160;50 mL centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">drigalski</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">K732.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">pipette</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">cut pipette tips</td>
			<td style="width:228px;">Daslab</td>
			<td style="width:119px;">162001X</td>
			<td>Universal 200, cut off 5 mm of tip before autoclaving</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">breathable tape&#160;</td>
			<td style="width:228px;">3M Deutschland GmbH</td>
			<td style="width:119px;">cat. no. 1530-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">growth room</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>21 &#176;C, continuous light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Materials</td>
			<td style="width:228px;">Company</td>
			<td style="width:119px;">Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">MAIZE LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Maize kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>B-73</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stirrer&#160;</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">C267.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>50 &#181;M (maize primary root), 25 &#181;M (maize adventitious root)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>50 &#181;M (maize)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">paper hand towels</td>
			<td style="width:228px;">Kimberly-Clark Professional*</td>
			<td style="width:119px;">6681</td>
			<td>SCOTT Hand Towels - Roll / White; sheet size (24 x 46 cm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">seed germination paper</td>
			<td style="width:228px;">Anchor Paper Company</td>
			<td style="width:119px;"></td>
			<td>10 X 15 38# seed germination paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">250 ml (centrifuge) tubes</td>
			<td style="width:228px;">SCHOTT DURAN</td>
			<td style="width:119px;">2160136</td>
			<td>approx. 5.6 cm diameter and 14.7 cm height&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">700 ml tubes</td>
			<td style="width:228px;">DURAN GROUP</td>
			<td style="width:119px;">213994609</td>
			<td>cylinders, round foot tube, D 60&#160; x 250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rack</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>for maize tubes, home made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">growth cabinet</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>27 &#176;C, continuous light, 70% relative humidity</td>
		</tr>
	</tbody>
</table>

lateral root inducible system in arabidopsis and maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

执行侧根启动过程的LRIS中的应用比较转录 在LRIS的一个应用是在侧根形成不同物种的比较和基因表达谱的关系。比较转录方法创建查明参与了不同种类的侧根发育过程中的同源基因的可能性。侧根开始，它由从包含在一个已形成的根轴的细胞的子集形成一个新的器官，是由被子植物共享，以控制它们的根构造的主...

Watch this Scientific Journal Video about Lateral Root Inducible System in Arabidopsis and Maize at JoVE.com

拟南芥和玉米侧根诱导系统生物学 |视频

Lateral Root Inducible System in Arabidopsis and Maize

侧根诱导系统<em&gt;拟南芥</em&gt;和玉米

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

lateral-root-inducible-system-in-arabidopsis-and-maize

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Lateral Root Inducible System in Arabidopsis and Maize

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13.6K 次观看.  Ghent. The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

视频: Lateral Root Inducible System in Arabidopsis and Maize

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

科研

生物学

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

用于映射植物根系架构特征的简单协议

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...