Name: an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)
Uploaded: 2022-09-07T14:15:00
Description: Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

作者感谢乐杰博士和宋秀芬博士分别对微分干涉对比显微镜和常规清除方法的有益建议。这项研究由中国国家自然科学基金（31870299）和中国科学院青年创新促进会资助。图 2 是使用 BioRender.com 创建的。

1. 溶液的制备
<ol><li>通过在 50 mL 离心管中加入 2.5 mL 37% 甲醛、2.5 mL 冰醋酸和 45 mL 70% 乙醇来制备 FAA 固定剂。涡旋并将其储存在4°C。 使用前新鲜制备FAA固定剂。 注意：37%的甲醛具有腐蚀性，如果暴露或吸入，可能会致癌。固定剂必须在通风橱中进行，同时穿戴适当的个人防护设备。</li>
	<li>通过在用锡箔包裹的 100 mL 玻璃瓶中加入 5 mL 100% 甘油、40 g 水合氯醛和 10 mL 蒸馏水来制备...

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与机械切片相比，清除技术对于三维成像更有利，因为它保留了植物组织或器官的完整性16。由于化学溶液更容易渗透，传统的澄清方案通常仅限于小样品。番茄种子是组织清除的一个有问题的样品，因为它的大小比拟南芥种子大约 70 倍，并且具有更多的渗透屏障。拟南芥种皮由四到五个活细胞层组成，这些活细胞层来自外皮和内皮24。然而，与<e...

番茄（番茄 ）是世界上最重要的蔬菜作物之一，2020年产量为1.868亿吨肉质水果，面积达510万公顷1。它属于大型茄科，约有2，716种2，包括许多具有重要商业意义的作物，如茄子，辣椒，马铃薯和烟草。栽培番茄是一种二倍体物种（2n = 2x = 24），基因组大小约为900 Mb3。长期以来，通过从野生茄属中选择理想的性状，在番 茄 驯化和育种方面做出了巨大努力。番茄遗传学资源中心列出了 5，000 多种番茄种质，全球储存了 80，000 多种番茄种质4.番茄植株在温室中多年生，通过种子繁殖。成熟的番茄种子由三个主要隔室组成：一个成熟的胚胎、残留的细胞型胚乳和一个坚硬的种皮5，6 （图 1A）。双重受精后，细胞型胚乳的发育先于受精卵的发育。在开花后~5-6天（DAF），当胚乳由6至8个细胞核7组成时，首先观察到双细胞前胚。在 茄子松中，胚胎在20 DAF后接近其最终大小，种子在32 DAF8后可以发芽。随着胚胎的发育，胚乳逐渐被吸收，种子中只剩下少量胚乳。残留胚乳由胚根尖端周围的小胚乳和种子其余部分的侧胚乳组成9，10。外种皮由外皮增厚木质化的外表皮发育而来，与外皮残余物的死层形成硬壳以保护胚胎和胚乳5。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64445/64445fig01.jpg"> 图1： Solanum lycopersicum 和 拟南芥中 成熟种子的示意图。 （A）成熟番茄种子的纵向解剖结构。（B）成熟 拟南芥 种子的纵向解剖。成熟的番茄种子的大小大约是 拟南芥 种子的 70 倍。比例尺 = （A） 400 μm， （B） 100 μm。 <a href="https://www.jove.com/files/ftp_upload/64445/64445fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
高质量番茄种子的生产取决于胚胎、胚乳和母体种子成分之间的协调11.剖析种子发育中的关键基因和网络需要对突变种子进行深入和完整的表型记录。传统的包埋切片技术，如半薄切片和石蜡切片，被广泛用于番茄种子，以观察胚胎的局部和更精细的结构12，13，14，15。然而，从薄片分析种子发育通常很费力，并且缺乏z轴空间分辨率。相比之下，组织清除是一种快速有效的方法，可以查明最有可能发生的胚胎缺陷的发育阶段16。清除方法通过用一种或多种生化剂均质化折射率来降低内部组织的不透明度16。全组织清除可以在不破坏其完整性的情况下观察植物组织结构，并且清除技术和三维成像的结合已成为获取植物器官形态和发育状态信息的理想解决方案17，18。多年来，种子清除技术已用于各种植物物种，包括拟南芥、大麦和普通贝塔19、20、21、22、23。其中，全镶样胚珠清除技术因其体积小、种皮细胞4-5层、核型胚乳24、25等特点，成为研究拟南芥种子发育的有效方法。随着不同清除混合物的不断更新，例如Hoyer溶液26的出现，尽管大麦胚珠的胚乳占种子的大部分，但大麦胚珠的内部结构以高度清晰的方式成像。甜菜的胚胎发生可以通过清除结合真空处理和盐酸软化来观察19。尽管如此，与上述物种不同，尚未报道通过清除番茄种子协议进行的胚胎学观察。这阻碍了对西红柿胚胎和种子发育的详细调查。
水合氯醛通常用作澄清溶液，其允许浸没的组织和细胞在不同的光学平面上显示，并基本上保留细胞或组织成分27，28，29。基于水合氯醛的清除方案已成功用于种子的全安装清除，以观察拟南芥21，28的胚胎和胚乳。然而，这种清除溶液在清除番茄种子方面效率不高，番茄种子比拟南芥种子更不透水。物理屏障包括：（1）番茄外皮在3至15 DAF 30，31处有近20个细胞层，（2）番茄胚乳是细胞型，而不是核型32，（3）番茄种子的大小约为70倍33，34和（4）产生大量的种皮粘液，这阻止了清除试剂的渗透并影响了胚胎细胞的可视化。
因此，本报告提出了一种优化的基于水合氯醛的清除方法，用于在不同阶段对番茄种子进行整体安装清除，该方法可以对胚胎发育过程进行深度成像（图2）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,000 &#181;L pipette</td>
			<td>GILSON</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 &#181;L pipette tips</td>
			<td>Corning</td>
			<td>T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>DAMAO</td>
			<td>685-2013</td>
			<td></td>
		</tr>
		<tr>
			<td>5,000 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3120000275</td>
			<td></td>
		</tr>
		<tr>
			<td>5,000 &#181;L pipette tips</td>
			<td>biosharp</td>
			<td>BS-5000-TL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol</td>
			<td>BOYUAN</td>
			<td>678-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle glass</td>
			<td>Fisher</td>
			<td>FB800-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral Hydrate</td>
			<td>Meryer</td>
			<td>M13315-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Leica</td>
			<td>384200</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager A1</td>
			<td>10x, 20x and 40x magnification</td>
		</tr>
		<tr>
			<td>Disinfectant</td>
			<td>QIKELONGAN</td>
			<td>17-9185</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Bioroyee</td>
			<td>17-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Flower nutrient soil</td>
			<td>FANGJIE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>HAIOU</td>
			<td>4-94</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>BOYUAN</td>
			<td>676-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Solarbio</td>
			<td>G8190</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RET basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Tom</td>
			<td>Tomato Genetics Resource Center</td>
			<td>LA3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>QILINBEIER</td>
			<td>QB-206</td>
			<td></td>
		</tr>
		<tr>
			<td>Seeding substrate</td>
			<td>PINDSTRUP</td>
			<td>LV713/018-LV252</td>
			<td>Screening:0-10 mm</td>
		</tr>
		<tr>
			<td>Single concave slide</td>
			<td>HUABODEYI</td>
			<td>HBDY1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Leica</td>
			<td>3800381</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>S8 APO</td>
			<td>1x to 4x magnification</td>
		</tr>
		<tr>
			<td>Tin foil</td>
			<td>ZAOWUFANG</td>
			<td>613</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>SHIDING</td>
			<td>SHB-III</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex meter</td>
			<td>Silogex</td>
			<td>MX-S</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)

番茄（Solanum lycopersicum L.）是全球主要的经济作物之一。番茄种子是研究植物繁殖过程中遗传学和发育生物学的重要模型。番茄种子内更精细的胚胎结构的可视化通常受到种皮粘液、多细胞分层外皮和厚壁胚乳的阻碍，这需要通过费力的包埋切片来解决。更简单的替代方案是采用组织清除技术，使用化学试剂使种子几乎透明。虽然传统的清除程序可以深入了解具有较薄种皮的较小种子，但清除番茄种子在技术上仍然具有挑战性，尤其是在发育后期阶段。
这里介绍的是一种快速且省力的清除方案，用于观察开花后 3 至 23 天胚胎形态接近完成时的番茄种子发育。该方法将广泛用于 拟南芥 的水合氯醛澄清溶液与其他修饰相结合，包括省略福尔马林 - 乙酰醇（FAA）固定，添加次氯酸钠处理种子，去除软化的种皮粘液以及洗涤和真空处理。该方法可用于不同发育阶段番茄种子的高效清除，有助于全面监测突变种子的发育过程，具有良好的空间分辨率。该清除协议也可以应用于茄科其他商业重要物种的深度成像。

当使用拟南芥等常规方法清除番茄种子时，致密的胚乳细胞在3 DAF和6 DAF下阻止了早期番茄胚胎的可视化（图3A，B）。随着胚胎总体积的增加，球状胚胎在9 DAF处几乎无法区分（图3C）。然而，随着种子尺寸的不断增加，其渗透性降低，导致12 DAF处的心脏胚胎模糊（图3D）。从13 DAF开始，种子粘液和种皮逐渐致密?...

Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

番茄种子是研究植物繁殖过程中遗传学和发育生物学的重要模型。该协议可用于清除不同发育阶段的番茄种子以观察更精细的胚胎结构。

一种用于研究番茄种子发育的有效清除方案（Solanum lycopersicum L.）

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

这种方法可以帮助研究快速轻松地找到番茄胚胎发育的异常时期。该方案的主要优点是用次氯酸钠处理番茄种子，这为观察番茄胚胎提供了基础。从开花或DAF后3至23天开始化学制备和收获茄子果实。立即将果实放在冰上，并根据开花后的天数将它们分为早果、中果和晚果。将早期的水果打碎，放在载玻片上，在体视显微镜的1倍至4倍放大镜下用精密镊子仔细收集新鲜种子。对于中后期水果，将水果掰开，用精密镊子直接收集新鲜种子。在传统方法中，将新鲜收集的种子立即转移到含有1.5毫升FAA固定剂的两毫升离心管中，并在室温下以5rpm的速度将管放在轨道振荡器上四小时。然后将种子在70%95%和100%乙醇中以5rpm的速度在轨道振荡器中脱水一小时。完成后，将种子放入载玻片上存在的三到五滴透明溶液中，并用盖玻片轻轻覆盖。使用单个凹面载玻片进行中后期种子。根据种子的发育阶段，在室温下孵育它们以进行清除。孵育后，用微分干涉对比观察样品，或配备数码相机的DIC显微镜以10倍，20倍和40倍放大倍率观察样品。实时调整和优化每个样品和捕获图像的透射光亮度、DIC滑块和聚光镜孔径。将从破碎的水果中收集的新鲜种子转移到装有1.5毫升消毒剂溶液的两毫升离心管中。在室温下将样品在轨道振荡器上以30rpm孵育3至50分钟。一旦最里面的种皮轮廓清晰可见，就丢弃消毒剂溶液。对于中后期种子，将种子转移到载玻片上。使用镊子和解剖针在体视显微镜的1倍至4倍放大镜下去除种子粘液。使用镊子将种子转移回原始离心管中。在1.5毫升去离子水中洗涤五次，每次10秒，然后丢弃去离子水。接下来，将双倍体积的澄清溶液添加到种子中。根据种子阶段，进行0至50分钟的间歇真空处理，每次真空处理10分钟后间隔10分钟。真空处理后，更换澄清溶液。为了便于清除种子，请将离心管置于室温下避光30分钟至7天。对于晚期胚胎，每天用新鲜的透明溶液替换溶液，然后将种子进行真空处理10分钟。将清除的种子放在载玻片或单个凹面载玻片上。使用配备数码相机的DIC显微镜，实时调整和优化每个样品的透射光亮度，DIC滑块和聚光镜孔径并捕获图像。在清除石蒜种子的传统方法中，致密的胚乳细胞在3和6 DAF处阻断了早期胚胎的可视化。种子生长过程中渗透率降低导致9 DAF后图像模糊。种子粘液和种皮逐渐变密，从13 DAF开始阻止清除剂的渗透。尽管延长了7天的治疗时间，但14至19个DAF种子中的胚胎轮廓非常模糊。22 个 DAF 种子的内部结构完全看不见。由种皮表皮细胞产生的剥离种皮粘液从13 DAF开始突出。次氯酸钠处理能够清晰地识别内种皮和大部分粘附粘液的分离，而不会损坏种子。在优化的清除方案中，种子在所有测试的发育阶段都表现出令人满意的透明度。在3 DAF处观察到种皮的不同细胞层，在5 DAF下可区分胚乳细胞，在7 DAF下观察到棍状胚胎，在9 DAF处观察到球状阶段，在11 DAF下观察到心脏阶段。在优化方案中，在胚胎发育的各个阶段很容易捕获子叶和枝顶端分生组织的卷曲程度。灭菌可去除种子粘液和种皮色素，从而增强种子渗透和可视化。真空处理可以加速清除溶液的渗透。

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