Name: high-throughput, robust and highly time-flexible method for surface sterilization of arabidopsis seeds
Uploaded: 2021-10-04T14:15:00
Description: Watch this Scientific Journal Video about High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds at JoVE.com

这项研究由特伦托自治省通过E. Mach基金会生态基因组学小组的核心资金资助。

1. 试剂和培养基制备
<ol><li>制备70%乙醇溶液：将737毫升95%工业乙醇加入263毫升蒸馏水中。彻底混合。 注意：在非无菌工作台上准备70%乙醇溶液。 注意：乙醇高度易燃，会对眼睛造成严重刺激。远离火焰和热源。如果接触眼睛，请用大量清水冲洗。</li>
	<li>准备5%漂白剂溶液：将5毫升家用漂白剂（含有约3.5%次氯酸钠，NaClO）加入95毫升无菌蒸馏水中。加入几滴非离子洗涤剂（例如吐温20）并彻...

<ol>
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S., Niu, Q. -. W., Chua, N. -. 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Arabidopsis Protocols.</a> Methods in Molecular Biology. 82, 27-30 (1998).</li><li>Li, M., Cappellin, L., Xu, J., Biasioli, F., Varotto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+in+planta+characterization+of+VOC+biosynthetic+genes+by+PTR-ToF-MS.">High-throughput screening for in planta characterization of VOC biosynthetic genes by PTR-ToF-MS.</a> Journal of Plant Research. 133 (1), 123-131 (2020).</li><li>Li, 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=In+planta+recapitulation+of+isoprene+synthase+evolution+from+ocimene+synthases.">In planta recapitulation of isoprene synthase evolution from ocimene synthases.</a> Molecular Biology and Evolution. 34 (10), 2583-2599 (2017).</li><li>Li, 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=Evolution+of+isoprene+emission+in+Arecaceae+(palms).">Evolution of isoprene emission in Arecaceae (palms).</a> Evolutionary Applications. 14, 902-914 (2020).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiologia Plantarum. 15 (3), 473-497 (1962).</li><li>Bent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+thaliana+floral+dip+transformation+method.">Arabidopsis thaliana floral dip transformation method.</a> Methods in Molecular Biology. 343, 87-104 (2006).</li><li>Lundberg, D. 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种子灭菌是拟南芥功能研究的基本步骤。虽然它经常用于许多不同的目的，但对拟南芥高通量种子表面灭菌的研究有限。
到目前为止，吞吐量最高的方法之一是使用漂白剂与浓缩HCl混合产生的氯气。虽然这种方法需要有限的动手时间，但它使用的气体对人类有剧毒27.此外，必须仔细控制要进行表面灭菌的种子数量和表面种子灭菌的持续时间。如果种子太多?...

拟南芥是一种二倍体植物，属于芸苔科。其相对较短的生命周期（在长日生长条件下每代两个月），植物体积小，并且每株植物产生数百个种子的自花授粉使其成为第一个基本植物模型物种1，2。此外，其基因组被完全测序3，广泛的反向遗传学工具（饱和的T-DNA、转座子和化学诱变的群体）都可用4、5、6，并且有效的农杆菌介导的转化是建立起来的，以获得足够的转基因品系，用于进一步的下游工作7.因此，在过去的二十年中，使用拟南芥作为模型物种在分子水平上剖析植物生物学的各个方面方面取得了巨大进展，包括自然，遗传和表型变异8，9。
为了在功能上表征拟南芥感兴趣的基因，种子表面灭菌以消除真菌和细菌污染物是许多需要轴线培养的下游方案的先决条件步骤。遗传转化对于过表达10、敲低（RNA-I11）或敲除（基因组编辑12、13）的基因功能、亚细胞定位14、启动子活性15、16、蛋白-蛋白17和蛋白-DNA相互作用18，仅举出最常见的应用，都需要种子表面的灭菌步骤。因此，尽管相对简单，但种子表面灭菌在许多功能分析中起着重要作用。
到目前为止，已经开发了两大类基于气相或液相灭菌的种子表面灭菌方法19。虽然气相种子表面灭菌的通量为中到高，但使用有害试剂氯气作为表面杀菌剂阻碍了其广泛应用。相反，基于液相灭菌的方法依赖于较温和的化学品，如乙醇和漂白剂溶液进行表面灭菌，并且尽管它们的吞吐量本质上低于氯熏蒸，但它们的使用范围更广。通常，通常使用两种使用液体试剂的不同方法。一种广泛使用的方法是基于用乙醇和漂白剂以不同的浓度洗涤不同的时间20，21。另一种方法是基于漂白剂的应用只有21、22。这两种方法主要应用于小规模的种子表面灭菌。然而，在许多实验中，需要筛选许多源自一次转化的拟南芥转基因品系15、23或平行筛选由不同转化产生的许多转基因品系24、25。据我们所知，尚未发表基于液体的高通量种子表面灭菌方法，尽管鲜为人知，但这构成了功能基因组学方法的重要瓶颈。因此，开发安全、稳健和高通量的种子表面灭菌方法是同时成功实现许多基因功能表征的必要和关键步骤。
为此，在目前的研究中，提出了一种改进的拟南芥种子表面灭菌方法。这种方法安全、低成本、高稳健性和高通量，允许从种子表面灭菌开始到培养皿中种子播种结束的一小时内处理96条独立生产线。所展示的方法依赖于广泛使用的基本实验室仪器，如真空泵，消耗性玻璃器皿和塑料器皿。这种改进的方法为科学界提供了一种安全，简单且负担得起的方法来简化种子表面灭菌，其吞吐量足以满足拟南芥和其他非模型植物物种的现代功能基因组学方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquarium valve</td>
			<td>Amazon</td>
			<td>B074CYC5SD</td>
			<td>Kit including 2 valves and thin-walled tubings. The valve prevents the liquids to go back to the sterile tip</td>
		</tr>
		<tr>
			<td>Arabidopsis Col-0 wild-type seeds</td>
			<td>Nottingham Arabidopsis Stock Center</td>
			<td>N1093</td>
			<td>Wild type seeds (sensitive to kanamycin)</td>
		</tr>
		<tr>
			<td>Arabidopsis transgenic line AdoIspS-79 seeds</td>
			<td>NA</td>
			<td>NA</td>
			<td>Transgenic line overexpressing an isoprene synthase gene from Arundo donax transformed in the Col-0 background, resistant to kanamycin (Li et al. (2017) Mol. Biol. Evol., 34, 2583&#8211;2599). Available on request from the authors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>EP022628188</td>
			<td>Benchtop microcentrifuge used for spinning down the seeds</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa</td>
			<td>M0222</td>
			<td>Standard medium for plant sterile culture</td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>Brand</td>
			<td>26300</td>
			<td>Used to operate the serological pipette</td>
		</tr>
		<tr>
			<td>Polyethylene tube 1</td>
			<td>Roth</td>
			<td>9591.1</td>
			<td>Tube for connection from vacuum pump to decantation bottle (inner diameter: 7 mm; outer diameter: 9 mm)</td>
		</tr>
		<tr>
			<td>Polyethylene tube 2</td>
			<td>Roth</td>
			<td>9587.1</td>
			<td>Tube for connection from decantation bottle to the aquarium valve&#160; (inner diameter: 5 mm; outer diameter: 7 mm)</td>
		</tr>
		<tr>
			<td>Screw cap with connectors</td>
			<td>Roth</td>
			<td>PY86.1</td>
			<td>2-way dispenser screw cap GL45 in polypropylene for decanting bottle</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Brand</td>
			<td>27823</td>
			<td>Graduated glass (reusable) serological pipette. Disposable pipettes can be used instead</td>
		</tr>
		<tr>
			<td>Shakeret al.</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>TissueLyser II bead mill used normally for tissue homogenization. Without the addition of beads to the tubes it works as shaker.</td>
		</tr>
		<tr>
			<td>Technical ethanol</td>
			<td>ITW Reagents (Nova Chimica Srl)</td>
			<td>212800</td>
			<td>Ethanol 96% v/v partially denatured technical grade</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck Millipore</td>
			<td>655205</td>
			<td>Non-ionic detergent acting as surfactant</td>
		</tr>
		<tr>
			<td>Universal tubing connectors</td>
			<td>Roth</td>
			<td>Y523.1</td>
			<td>Can be used to improve/simplify tubing connections</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Merck Millipore</td>
			<td>WP6222050</td>
			<td>Used for making the suction device</td>
		</tr>
	</tbody>
</table>

high-throughput, robust and highly time-flexible method for surface sterilization of arabidopsis seeds

拟南芥是迄今为止最广泛用于功能研究的植物模型物种。拟南芥种子的表面灭菌是实现这一目标所需的基本步骤。因此，建立高通量拟南芥种子表面灭菌方法以同时处理数十至数百个样品（例如，转基因品系，生态型或突变体）至关重要。本研究提出了一种基于用普通真空泵构成的自制抽吸装置有效消除管中液体的种子表面灭菌方法。通过大大减少劳动密集型的动手时间，这种方法可以在一天内处理数百个样品，而几乎不费吹灰之力。系列时间过程分析进一步表明，通过保持高发芽率，表面灭菌的时间范围高度灵活。该方法可以很容易地适应于其他种类小种子的表面灭菌，只需根据种子大小和消除液体所需的速度对抽吸装置进行简单的定制。

为了评估整个种子灭菌过程所需的时间，计算了当前方案中96个样品的液体处理时间差，并与传统移液方法进行了比较。结果表明，当前协议节省了时间，将液体处理时间缩短到传统协议的四分之一（表1）。该表进一步强调，与传统方法相比，当前方案中的液体去除时间节省了更多时间，总体减少了八倍。
种子灭菌时间范围的选择 较长的灭菌步骤...

Watch this Scientific Journal Video about High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds at JoVE.com

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

提供用于 拟南芥（Arabidopsis） 种子表面灭菌的高通量方案，通过带有真空泵的简单抽吸装置优化液体处理步骤。数百个种子样品可以在一天内进行表面灭菌。

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 ...

该协议改善了种子表面灭菌，这是拟南芥模型物种功能基因组学的基本步骤。该技术的主要优点是简单易用且通量高，每天可处理数百个样品。通过一些简单的修改，这种方法可以应用于许多其他模型和非模型植物物种。对于首次使用的用户，请注意培养基的温度和离心速度，因为它们对种子存活都至关重要。首先，通过将95%工业乙醇加入蒸馏水中并充分混合来制备70%乙醇。接下来，通过将5毫升家用漂白剂加入95毫升无菌蒸馏水中来制备5%漂白剂溶液。然后，在漂白剂溶液中加入几滴非离子洗涤剂，并充分混合。接下来，通过在800毫升蒸馏水中加入2.2克MS培养基粉末（包括维生素和10克蔗糖）来制备半强度的Murashige和Skoog培养基。使用一摩尔氢氧化钾将培养基的pH值调节至5.7，然后使用蒸馏水将体积调高至一升。将500毫升培养基等分到一升瓶中，并加入四克琼脂以制备固体培养基。高压灭菌后，让培养基在水浴中冷却至50至53摄氏度。然后将其倒入层流罩下的培养皿中。要制备选择性培养基，将卡那霉素加入培养基中，并如前所述将其倒入培养皿中。要设置吸气器，请将真空泵入口连接到合适尺寸的聚乙烯管的一端。然后将管的另一端连接到倾析瓶双向盖的出口。用密封膜紧紧包裹管道的连接处，以确保气密连接。接下来，将第二个聚乙烯管连接到倾析瓶上螺旋盖的入口。然后将管子的另一侧连接到装有薄聚乙烯管的水族箱阀的出口。如有必要，用密封膜包裹连接处，以消除漏气。在用渐进式数字标记两批48个1.5毫升微量离心管后，将100至200个拟南芥种子添加到96个无菌微量离心管中的每一个中，大约在管锥形末端的底部上方一到两毫米。接下来，使用10毫升无菌血清学移液器，向每个管中加入约1毫升70%乙醇，并小心地合上盖子。在振荡器中以八赫兹的振荡频率摇动管三分钟。然后从摇摇杯中取出适配器，并将其转移到台式微量离心机的篮子中，以使用脉冲功能旋转种子。将管子转移到机架后，打开层流罩下的所有管子。请勿触摸盖子中适合管子的部分，以免污染。然后，在层流罩下，将无菌的200微升黄色尖端安装到自制吸气器的水族箱阀门入口处，然后打开泵。将尖端插入种子水平上方，以避免在吸吮液体时接触种子。接下来，使用10毫升无菌血清学移液管，将一毫升5%漂白剂溶液等分到每个管中。在将管子放回摇摇杯适配器之前，请先盖上盖子，然后如前所述摇动管子。使用台式离心机的脉冲功能旋转种子后，将新的无菌200微升黄色尖端安装到水族箱阀上，然后打开泵。将尖端插入种子水平上方，以避免在吸入漂白剂溶液时接触种子。接下来，使用10毫升无菌血清学移液管，将一毫升无菌水等分到每个管中。将新的无菌200微升黄色尖端安装到水族箱阀门上后，打开泵。将尖端插入种子水平上方，以避免在吸水时接触种子。最后，将500微升无菌水等分到每个管中，并关闭层流罩中的所有盖子。种子现在已经准备好播种了。如果需要，将管子保持在室温下长达几个小时或四摄氏度过夜。使用一毫升移液器，将种子与300至400微升无菌水转移到培养皿中。转移10管后，将1.5至2毫升融化的半强度Murashige和Skoog培养基（不含抗生素）倒入每个板中。快速旋转板以分配种子，并将板粘在相对的两侧。用塑料或铝箔包裹盘子，并在黑暗中将其放入冰箱中三天，以获得均匀发芽。在第二天、第三天、第四天和第七天进行的发芽分析表明，用70%乙醇灭菌10至40分钟之间没有显着差异。然而，当灭菌时间超过40分钟时，发芽率下降。相应地，绿色子叶的出现率也有所下降。根据该研究，选择3分钟用于70%乙醇和3分钟用于5%漂白剂作为灭菌种子的最短时间。为了证明最初使用的种子被微生物污染，将非无菌种子直接播种在盘子上。与无菌种子相比，非无菌种子在两天后显示出真菌外观，在发芽七天后扩散到整个盘子上。使用单个无菌移液器尖端处理不同种子样品的交叉污染测定表明，在用卡那霉素播种的Columbia-0基因型种子中未观察到绿色子叶。同时，在Columbia-0种子中播种在没有卡那霉素的盘子中，所有子叶在发芽后都呈现绿色。这些结果表明，尽管使用单个移液器吸头去除无菌溶液，但样品之间没有残留的污染物。该程序是许多其他功能基因组学方法的基础，如基因过表达，基因组编辑，亚细胞定位，启动子活性以及蛋白质 - 蛋白质和蛋白质 - DNA相互作用。

Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

科研

生物学

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

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

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

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 ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...