这项工作是由瑞士国家科学基金会资助和日内瓦州的支持。

一旦空气呼吸器被组装时，胚胎的生长被监控超过数天。因此，空气呼吸器的种子解剖过程和组装之前，需要消毒的种子，以避免将来的污染，可以防止种皮材料对胚胎生长的影响，正确的评估。 1。种子消毒<ol><li>倒入50-60微升成熟和拟南芥干种子在1.5 ml离心管中，并准备一个70％的乙醇溶液。 </li><li>加入1毫升70％的乙醇溶液至含有种子的微量离心管中，并在1,200 rpm下在涡流混合器摇动在室温下搅拌10分钟。 </li><li>离心机的离心管中，持续3秒在4000×g离心浓缩种子在管的底部。 </li><li>用真空吸嘴小心而使种子在微量离心管的底部吸出70％的乙醇溶液。 </li><li>加入1毫升无菌蒸馏水至吨<html>他离心管中仍含有的种子。 </li><li>摇在室温下在1200rpm 10分钟。 </li><li>离心机的离心管中，持续3秒在4000×g离心浓缩种子在管的底部。用真空吸嘴小心而使种子在管的底部抽吸水上清液。 </li><li>加入1毫升无菌水蒸馏水至仍含有种子的微量离心管中。确保水的流动均匀挂起管内的种子。这样做的目的是为了避免进一步的晃动，从而保持种子的完整性。 </li><li>让种子定下来通过重力在该管的底部由仍留在管30秒。用真空吸嘴小心而使种子在管的底部抽吸水上清液。 </li><li>重复四次步骤1.8 - 1.9。整体灭菌过程大约需要30分钟。 </li></ol> 2。种子电镀ontent"&gt;所有后续步骤都在层流柜中进行，以保持无菌条件。 <ol><li>准备陪替氏培养皿盘（100毫米直径20毫米高）制备通过高压灭菌含有4.3克/ L的Murashige和Skoog培养基中2.5毫米2的溶液中装有30ml萌发培养基 - （N-吗啉）乙磺酸（MES-KOH ; pH值5.7）和0.8克/升琼脂。设在50℃水浴中冷却液倒在培养皿板浇筑前。 </li><li>加入200μl的无菌蒸馏水到含种子的试管中。重悬的种子，并用标准移液管与1毫升尖萌发培养基的表面上传送。 </li><li>除去周围用真空吸嘴的萌发培养基的表面上的种子的水分。 </li><li>离开含种子没有它的盖培养皿中的层流柜内2小时为进一步干燥。关闭培养皿的盖子，让它经得起层下流柜约90分钟，使约4小时，自该种子灭菌过程（步骤1.2）的启动通过。 </li></ol> 3。种子解剖下面的步骤必要用立体显微镜放置在层流罩柜内工作。一个杜蒙钳＃5于截断（ 即钝）提示极大地方便了种子的处理（ 图1A）。 <ol><li>准备装有30ml萌发培养基的新培养皿板。这里的介质2并列的矩形灭菌的Whatman 3MM纸（1.5厘米×1.0厘米; 图1B，步骤1）的表面上。的Whatman 3MM纸被高压灭菌。 </li><li>传送上使用无菌镊子（ 图1B，步骤2）中的Whatman 3MM纸种子。 </li><li>通过使用截断钳的并列钝头（轻轻按压种子仇视的Whatman 3MM纸单粒种子<st荣&gt;图1B，步骤3）。 </li><li>切割用注射器针头种子的种皮（外种皮和胚乳）（ 如 U100胰岛素注射器+ 1ml注射器，0.33毫米（29 G）×12.7毫米）（ 图1B，步骤4）。 拟南芥种子的形状是椭圆形的，因此建议削减种皮沿尽可能接近的地方子叶接合到胚根（ 即远离基端部）的位置上最长的半主轴。这有助于在清扫过程的下一步骤中胚胎的安全释放。 </li><li>使用截短的镊子（ 图1B，步骤5）的并置的钝头推压的Whatman 3MM纸上的种子。此步骤会释放出胚种皮通过在步骤3.4（ 图1B，步骤6）中创建的开口。它建议申请在该处的子叶的顶端是在最靠近胚根的尖端处的种子（ 即相差fr处的推OM开放）。 </li></ol> 4。大会的种皮床品含量（SCBA） 为种皮和胚胎的数量的适当的选择看讨论。 <ol><li>准备装有30ml萌发培养基的新培养皿板。地方的介质的表面上的无菌一块长方形的尼龙网片（3.5厘米×5.0厘米）（ 图2，步骤1）。 </li><li>为避免损坏，胚胎和种皮可以通过使用注射器针头（ 图2，步骤2）轻轻推他们移动到钳子的金属边缘的表面上。转移的胚胎和种皮上的尼龙筛网（ 图2，步骤3）。 </li><li>组装种皮的使用钝钳和针确保种子外套是在尽可能多的靠近，尽可能地（ 图2，步骤4）的圆形和单层。尝试以暴露尽可能由所产生的种皮开口针向上。这一步是没有系统地测试。但是，它可能会增加空气呼吸器的效率，因为可扩散物质抑制了胚胎的生长预期直接扩散出来对胚胎而不是远离它。 </li><li>将胚胎作为组装在步骤4.3种皮床中心的环状单层（ 图2，步骤5）。确保胚胎是在尽可能多的更接近越好。 </li><li>在20-21℃下孵育下连续光照（40微摩尔/米2秒 EC）的SCBAs在帧中继脉冲用来逮捕发芽，组装空气呼吸器正常光线下，与实际情况相符的FR脉冲9，离开培养皿在黑暗的情况下。 </li></ol>

<ol>
	<li>Debeaujon, I., Leon-Kloosterziel, K. M., Koornneef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+testa+on+seed+dormancy,+germination,+and+longevity+in+Arabidopsis.">Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis.</a> Plant Physiol. 122, 403-414 (2000).</li><li>Baroux, C., Spillane, C., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+origins+of+the+endosperm+in+flowering+plants.">Evolutionary origins of the endosperm in flowering plants.</a> Genome Biol. 3, reviews1026 (2002).</li><li>Debeaujon, I., Lepiniec, L., Pourcel, L., Routaboul, J. -. M., Bradford, K., Nonogaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seed development, dormancy and germination. , 25-43 (2007).</li><li>Finch-Savage, W. E., Leubner-Metzger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+the+control+of+germination.">Seed dormancy and the control of germination.</a> New Phytol. 171, 501-523 (2006).</li><li>Bethke, P. 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=The+Arabidopsis+aleurone+layer+responds+to+nitric+oxide,+gibberellin,+and+abscisic+acid+and+is+sufficient+and+necessary+for+seed+dormancy.">The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy.</a> Plant Physiol. 143, 1173-1188 (2007).</li><li>Lee, K. P., Piskurewicz, U., Tureckova, V., Strnad, M., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+seed+coat+bedding+assay+shows+that+RGL2-dependent+release+of+abscisic+acid+by+the+endosperm+controls+embryo+growth+in+Arabidopsis+dormant+seeds.">A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19108-19113 (2010).</li><li>Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M., Chory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochrome+A+and+Phytochrome+B+Have+Overlapping+but+Distinct+Functions+in+Arabidopsis+Development.">Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development.</a> Plant Physiol. 104, 1139-1149 (1994).</li><li>Shinomura, T., 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=Action+spectra+for+phytochrome+A-+and+B-specific+photoinduction+of+seed+germination+in+Arabidopsis+thaliana.">Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 8129-8133 (1996).</li><li>Lee, K. P., 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=Spatially+and+genetically+distinct+control+of+seed+germination+by+phytochromes+A+and+B.">Spatially and genetically distinct control of seed germination by phytochromes A and B.</a> Genes Dev. 26, 1984-1996 (2012).</li><li>Lee, K. P., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+seed+germination+in+the+shade.">Control of seed germination in the shade.</a> Cell Cycle. 11, 4489-4490 (2012).</li><li>Piskurewicz, U., 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=The+gibberellic+acid+signaling+repressor+RGL2+inhibits+Arabidopsis+seed+germination+by+stimulating+abscisic+acid+synthesis+and+ABI5+activity.">The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.</a> Plant Cell. 20, 2729-2745 (2008).</li><li>Piskurewicz, U., Tureckova, V., Lacombe, E., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-red+light+inhibits+germination+through+DELLA-dependent+stimulation+of+ABA+synthesis+and+ABI3+activity.">Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity.</a> EMBO J. 28, 2259-2271 (2009).</li><li>Debeaujon, I., Koornneef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellin+requirement+for+Arabidopsis+seed+germination+is+determined+both+by+testa+characteristics+and+embryonic+abscisic+acid.">Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid.</a> Plant Physiol. 122, 415-424 (2000).</li><li>Sun, T. P., Kamiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+GA1+locus+encodes+the+cyclase+ent-kaurene+synthetase+A+of+gibberellin+biosynthesis.">The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.</a> Plant Cell. 6, 1509-1518 (1994).</li><li>Alonso, J. M., 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=Genome-wide+insertional+mutagenesis+of+Arabidopsis+thaliana.">Genome-wide insertional mutagenesis of Arabidopsis thaliana.</a> Science. 301, 653-657 (2003).</li><li>Rook, F., 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=Impaired+sucrose-induction+mutants+reveal+the+modulation+of+sugar-induced+starch+biosynthetic+gene+expression+by+abscisic+acid+signalling.">Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.</a> Plant J. 26, 421-433 (2001).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里所描述的种皮床上用品法（SCBA）程序原则上适用于其中拟南芥种子发芽受阻（或延迟），并在那里胚乳被怀疑实施这一逮捕任何情况下。后者可通过除去种皮（外种皮和胚乳），并观察到胚胎生长前进速度相对于所观察到的，当胚胎是由种皮包围得以证实。发芽可能被阻止响应于特定环境的物理参数（ 例如 ，水或潜在的光质）或在特定的遗传背景（ 例如，不存在GA合成?...

在拟南芥成熟种子的种皮是由种皮，母体来源的死组织的外层，和胚乳，直接围绕胚胎1活组织的单细胞层。胚乳和胚胎均来自不同的施肥事件:胚乳是三倍体组织有两个产妇和一个父亲的基因组，而胚胎是一个产妇和一个父亲的基因组2二倍体组织。 传统上分配给该胚乳的主要功能是，营养组织。然而，它正变得越来越明显的是，胚乳也起着控制种子发芽的核心作用。这个概念在休眠的情况下成为了第一个明显的，由新产生的种子表现出的特质。休眠种子不能发芽，尽管有利发芽条件的存在。种子成熟期后失去他们的休眠，成为nondormanT， 即它们会发芽，当暴露在有利的条件下催芽。在许多植物物种，其中包括模式植物拟南芥 ，种皮是绝对必要的，以防止休眠种子的萌发，因为种皮去除触发胚胎生长和绿化3,4。在拟南芥中 ，贝思克等人观察到，除去种皮，同时保持周围的胚乳5胚乳萌发后仍然压抑。这些观察结果强烈表明，胚乳，种皮施加的镇压活动在胚胎内的组织。然而，种皮去除的实验并不一定有助于澄清发芽镇压活动由种皮所提供的性质，也不确定实现它的基因。 我们最近推出了种皮床上用品法（SCBA），其中种皮和胚在物理上分开，但保持着密切PROXIMITY使得由胚乳提供的发芽压制性的活性得以维持6。空气呼吸器允许组合使用休眠，nondormant，和转基因种皮和胚料。其结果是，在遗传途径控制发芽并在胚乳和胚具体操作可以被解剖。空气呼吸器被用于休眠的 ​​情况下，显示的胚乳释放激素脱落酸（ABA）对胚胎来抑制其生长6。此外，我们还可以使用空气呼吸器，以确定在胚乳操作的信号通路和胚胎组织，以促进休眠。 胚乳控制发芽的作用考虑承受远红（FR）的光脉冲nondormant种子的情况下得到进一步加强。在种子吸胀初期帧中继光脉冲是已知的抑制发芽7,8。当从种子中除去种皮FR光脉冲无法抑制发芽，强烈的建议，胚乳也可以抑制nondormant 9种子的萌发。值得注意的是，SCBA也可以用来概括发芽的FR-依赖性抑制。这可以表明，种子萌发的FR-依赖性抑制也有来自胚乳9阿坝释放的过程。此外，SCBA允许识别在胚乳和胚来控制nondormant种子萌发响应于光线索9,10操作不同的光信号转导途径。 因此，空气呼吸器似乎是一个可靠的技术来探索种子萌发的控制范围内胚乳的功能。这也是一个强大的工具， 在体外 ，以评估是否涉嫌基因控制发芽的胚乳，胚或两者的组织工作。下面我们详细组装一台空气呼吸器所需的各个步骤。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>To refer</td>
		</tr>
		<tr>
			<td>Thermomixer Comfort</td>
			<td>Eppendorf AG</td>
			<td>5355 000.011</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
		</tr>
		<tr>
			<td>Vacusafe Comfort</td>
			<td>INTEGRA Biosciences AG</td>
			<td>158 310</td>
			<td>Integra Biosciences AG, Zizers, Switzerland</td>
		</tr>
		<tr>
			<td>Petri dish plate (100 mm x 20 mm)</td>
			<td>Greiner Bio-One GmbH</td>
			<td>664 102</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
		</tr>
		<tr>
			<td>Murashige and Skoog</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>Agar (plant agar)</td>
			<td>Duchefa Biochemie B.V.</td>
			<td>P1001</td>
			<td>Duchefa Biochemie, Haarlem, Netherlands</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools GmbH</td>
			<td>11251-10</td>
			<td>Fine Science Tools GmbH, Heidelberg Germany</td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>BD Micro-Fine</td>
			<td>324827</td>
			<td>BD, Franklin Lakes, NJ USA</td>
		</tr>
		<tr>
			<td>Nylon mesh (SEFAR NYTEX)</td>
			<td>SEFAR AG</td>
			<td>03-50/31</td>
			<td>Sefar AG, Heiden, Switzerland</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>CLF Plant Climatics</td>
			<td>Percival I-30BLLX</td>
			<td>CLF plant Climatics, Wertingen, Germany</td>
		</tr>
		<tr>
			<td>Paclobutrazol</td>
			<td>Sigma-Aldrich</td>
			<td>46046</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
	</tbody>
</table>

a seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in arabidopsis thaliana

拟南芥胚乳由周边的成熟胚和播放，以防止休眠种子的萌发，或由远红光（FR）的光脉冲照射nondormant种子的重要作用单细胞层。为了进一步洞察底层由胚乳施加的发芽压制性活性的分子遗传机制，一个"种皮寝具"测定法（SCBA）被设计。空气呼吸器是一种剥离的过程实际上从种子，因此可以监视胚胎的生长种皮的下层上分离种皮和胚。值得注意的是，空气呼吸器重新组成种皮发芽镇压活动在种子休眠和萌发的FR-依赖的控制的情况下。由于空气呼吸器允许组合使用休眠，nondormant和转基因种皮和胚材料的遗传途径控制的萌发和专门运erating在胚乳和胚可以被解剖。下面我们详细的程序来组装一个自给式呼吸器。

以前的工作表明，突变体种子无法合成GA无法发芽，作为高ABA积累在种子11,12的结果。但是，不能发芽，需要种皮，因为它的去除触发胚胎生长13。这有力地表明，种子不能合成赤霉素的胚乳被释放ABA阻止胚胎发育。因此，我们预计种子大衣不能合成GA阻止胚胎在呼吸器的增长不像种皮不能合成赤霉素和脱落酸。这里我们使用GA1-21（Salk的插入线Salk_109115）携带一个T-DNA插入破?...

Watch this Scientific Journal Video about A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana at JoVE.com

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

我们目前的程序，从拟南芥种子组装种皮床上用品法（SCBA）。空气呼吸器被证明是一个强大的工具，基因和体外探索胚乳控制种子发芽的种子的休眠和反应轻线索如何。空气呼吸器是适用的原则，在胚乳被怀疑影响胚胎生长任何情况下。

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

a-seed-coat-bedding-assay-to-genetically-explore-vitro-how-endosperm

Research

JoVE Journal

Biology

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

13.4K 次观看.  Université de Genève. 我们目前的程序，从拟南芥种子组装种皮床上用品法（SCBA）。空气呼吸器被证明是一个强大的工具，基因和体外探索胚乳控制种子发芽的种子的休眠和反应轻线索如何。空气呼吸器是适用的原则，在胚乳被怀疑影响胚胎生长任何情况下。 

视频: A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

科研

环境科学

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

发育生物学

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

生物学

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

免疫与感染

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

一种省力和可重复的触摸力信号突变屏幕协议,用于研究阿拉伯植物示范植物的三角性发生

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

通过表皮表型评分 鉴定 参与气孔发育的基因

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

种皮床品检测到基因探索

摘要

探索更多视频

此视频中的章节

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

表征在拟南芥胚方法

原子力显微镜研究活拟南芥根表皮细胞的物理性质

印记基因的DNA甲基化的测定拟南芥胚乳

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

高效，快速的早期胚胎分离

拟南芥种子高通量灭菌的标准化方法

一种省力和可重复的触摸力信号突变屏幕协议,用于研究阿拉伯植物示范植物的三角性发生

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

通过表皮表型评分鉴定参与气孔发育的基因

种皮床品检测到基因探索

摘要

探索更多视频

此视频中的章节

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

表征在拟南芥胚方法

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

印记基因的DNA甲基化的测定拟南芥胚乳

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

高效，快速的早期胚胎分离

拟南芥种子高通量灭菌的标准化方法

一种省力和可重复的触摸力信号突变屏幕协议,用于研究阿拉伯植物示范植物的三角性发生

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

通过表皮表型评分 鉴定 参与气孔发育的基因

原子力显微镜研究活拟南芥根表皮细胞的物理性质

通过表皮表型评分鉴定参与气孔发育的基因