我们感谢Sankalpi Warnasooriya和艾米洪堡的手稿的批判性阅读和编辑。这项工作是由能源部（DE-SC0008769）和美国国家科学基金会（IOS-1127017）资助。

1。 S的 ​​成长贻贝 <ol><li>将生长室条件，以31°C/22°C（昼/夜），12小时light/12小时黑暗，50％与黎明前治疗15°C从上午8:30至9:00的相对湿度PM。 （ 图1）。在这些条件下，S.贻贝 A10.1大约需要21-22天从种子播种到开花。 </li><li>填写单位（4×9细胞）与新城混合360和移除一个细胞浇水。 </li><li>轻轻薄雾水在土壤表面。 </li><li>播种的土壤，每单元1种子的表面上的种子。 </li><li>覆盖种子用一层土（约0.5cm） </li><li>薄雾从底部土壤和水的单位的表面。留种，播种后从底部浇水。 </li><li>对于将至少有三家工厂执行的每个交叉将用于母本和九厂作为将被用于男性父母。播种应错开，因为所有的植物不会花同一天。种子sowi纳克建议每天超过三天的时间。 </li><li>植物浇水一天，这是依赖于植物和土壤条件的尺寸的1-2倍。在土壤中的过量的水是不利于植物的最佳生长，并且可能导致在土壤和叶组织的破坏真菌的生长。 </li><li>每周两次施肥的植物，使用Jack的15-16-17在100ppm的浓度存在。 </li></ol> 2。修剪和穗的标签去雄前 - 第1天<ol><li>在之前的授粉下午或晚上，将植物从生长室到实验室（温度（T）：24.06°C±0.13°C，相对湿度（RH）21.79％±1.15％）。 </li><li>选择具有1花的花1/3上已经开花的穗初级圆锥花序。 </li><li>在这项研究中穗内的四个结构域的定义如下（ 图2A;尖，远端中间，近端和中间基）。 </li><li>理想情况下，Choo鞋子EA穗与已开花1-5花，切断穗（远端）的尖端，并用弹簧剪刀或细镊子取出所有的鲜花在近端中间和底座部分。 </li><li>如果一个圆锥花序具有约1/10至已经开花的花的1/5，切断尖端和从穗基部修剪花，只保留远端中间和近端中间上部区段的下部分，用于进一步修整。 </li><li>如果一个圆锥花序具有大约1/4的已开花的花的1/2，切断前端中间部，并仅保留近端中间部分，用于进一步修整。 </li><li>用手术剪刀轻轻修剪鬃毛。 </li><li>删除已开花和所有不成熟的花朵留下约20-30花/穗被均匀地分布在区域（ 图3）所有的花朵。练习注意保持在该地区的刷毛，将为了保护小花从可能的热损伤授粉。对于每个小穗，保留了上最为成熟的小花。最上部的小花将有开花的可能性较高时授粉（ 图2B）的时间。 </li><li>标记每朵花的一侧有一个红色记号笔和记录小花对穗（ 图2B）的数量。 </li><li>旗穗包含以下信息：去雄，持续时间和在哪个去雄进行温度的日期。 </li><li>一旦穗修剪，删除每个工厂的所有分蘖，以确保更多的资源重新分配至主穗的发展。 </li></ol> 3。阉割用温水，浸泡 - 第1天<ol><li>将水浴至48℃±0.1℃的热处理。 </li><li>轻轻地弯曲的修剪穗都蘸温水中在48℃下3-6分钟。请小心不要折断穗茎。 </li><li>约3-4圆锥花序，可蘸山口疗法在同一时间，同时保持圆锥花序的热量均匀分布之间有足够的空间。它是必不可少的热处理过程中，以淹没整个穗的温水中。旗叶（叶级联，它提供了养分的穗穗）不应该与温水接触，以防止热损伤叶组织。 </li><li>一旦所需的持续时间进行了阉割，从水浴中取出穗，轻轻地从穗抖落的水。 </li><li>放置一个定制的微穿孔面包袋完全覆盖阉割穗和一拧领带将其固定。微穿孔​​面包袋可以根据穗的大小被定制（包括视频）。 </li><li>放置植物放回生长室中，直到第二天。 </li></ol> 4。去雄后隧道/授粉 - 第2天与第3天<ol><li>上午9时，第2天与第3天，将女性（阉割）和MALE从生长室家长实验室（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％）执行十字架。 </li><li>观察并添加另一个红点已经开花或使用红色记号笔盛开的花朵。记录每穗开花的花的总数。 </li><li>记录授粉的日期和父本的标记的名称来跟踪哪些十字架被执行的。 </li><li>观察开花的高峰时间在父本。根据我们的室内条件下（ 图1）上父本的圆锥花序花朵开始同步开口周围10:10 AM。从开口的花的时间，20分钟后，将发生的花药和花粉的释放完全外露。 </li><li>授粉理想阶段是在该父本的花粉被释放阶段。从花粉释放20分钟后的花将关闭。因此，持续时间为开花期是从开口流动的时间约40分钟雇员再培训计划。从黄白色花粉变化的花后的颜色棕色历时约30分钟关闭。 </li><li>立即开始授粉，一旦黄白色花药都在父本花可见。授粉是通过使用三种技术中的一个在第2天与第3天进行： <ol><li> 穗至穗授粉 ：使用一个分离穗为男性，轻轻地用阉割穗搓穗在一起，方便授粉并丢弃男性穗，以避免污染。重复授粉过程，直到每一个被阉割的穗已经授粉2-3圆锥花序。 </li><li> 花药到柱头授粉 ：用钳子挑选那些绽放着黄白色花药花或挑花药和身体接触对母本花的柱头。使用来自父本一花授粉一朵花的阉割穗。重复授粉process直到阉割穗每个柱头已授粉由少数（约2-3）鲜花/花药。戴护目镜放大镜会提供更好的可见性来进行授粉。 </li><li>不同父本的授粉之间，蘸，然后用擦的Kimwipes，以避免污染，由于结转不同杂交组合间的花粉在95％乙醇的镊子。 </li><li> 穗绑定 ：去雄后，选择3-4个花序上的父本，将盛开的第二天。它们与母本用扭扣的阉割穗绑定在一起。定位略低于父本穗穗阉割。将玻璃纸套在穗和保护袋在基地与回形针。这可能是第2天完成一次父本的圆锥花序开始开花。 </li><li>在2日和3日，轻弹或摇晃玻璃纸袋子，以使几日早上盛开的时间itate授粉。继续在整个花期在上午的整个持续时间（9:00 AM-11之间：上午00点）轻弹或摇晃圆锥花序，每15分钟。授粉后取出父本穗和标签上的阉割穗花开花的对面。记录的花朵绽放在每个母本的穗的数量。 </li></ol></li><li>一旦授粉已经完成，放置一个定制的微穿孔面包袋覆盖母本的穗，并在该基地采用授粉后扭领带，直到收获种子安全。 </li></ol> 5。种子收获<ol><li>从授粉的每天2周（14-16天）收获后种子。将标签在同一个袋子的种子。具有两侧红色标记代表种子，导致从所述受控的遗传杂交的种子。这些预计将异型杂交后代。 </li><li>因为它们代表放弃任何种子没有红色标记那个阉割后导致新开发的花的种子。种子用红色标记只在一侧可能会代表从自花授粉产生的，因为他们不晕染的时候被执行的控制十字架时的种子。 </li><li>干种子在30℃下，在一个机种2天。商店干燥的种子在实验室（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％），或在种子室（T：4.0°C±1.0°C，相对湿度：20％±1％）。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

黎明前的治疗的重要性要获得异交后代的高频，必须有男性和女性家长的高度同步开花。最初，我们循环温度和光照强度（31°C/22°C，日/夜）和使用的S。贻贝加入A10.1所有授粉。在这种情况下，最开放的花朵在清晨前的温度上升到31℃，虽然少数花开放之间随机8:00 AM-8：00 PM。以往的研究在酿酒谷子表明一天的时间，地点和季节的贡献开花7,15,16会有变化。锡莱斯等 6指出，花期用的温度和湿度的迅速变化相关联，但不与低温和高湿度，本身作为总结在以往的研究7,15。因此，我们采用了黎明前的冷处理的15℃30分钟（上午8:30 - 9：上午12时），以模仿自然黎明前的条件。这黎明前的冷处理辅助在父母的同步开花。我们观察到，尽管设置了腔室的相对湿度50％的水平，该腔室内部的相对湿度提高到一个电平在70％以上，随着温度从22℃降温至15℃，并继续留在70％以上，直到温度逐渐上升到31℃，之后9:30 AM。在上午9:00，阉割母本和父本植物被带到实验室（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％）。父本花开始四处上午10:10开放，和授粉可以在10:30 AM-11来进行：上午00点。在表2中提供的所有试剂和设备的详细清单。 穗准备阉割的重要性下腔室条件，在此研究中（ 图1）中，初级圆锥花序开花时间介于2-3天。典型地，位于花序的远端中间（ 图2A）开放的第一开放之前向近侧和远侧的花朵。鲜花被保留在穗理想的数字是20-30。应注意去除不成熟的鲜花时要采取这样只有发达的花朵（上小穗17最上面的花或最大）被保留。标签上的花朵两侧红色标记可以帮助去雄后，能够有效地辨别公认的异型杂交后代来自新开发的花卉种子。此外，重要的是要离开的圆锥花序刷毛去雄前保护小花从广泛的热损伤，但是，作为一种选择，它们可以被阉割后使用弹簧剪刀，以促进授粉除去，特别是当一个穗结合技术时（ 图2C和2D）。 优化的温度D处理时间为温水处理的基础上，通过温水浸泡去雄是花粉热比柱头表面更加敏感。然而，持续时间和热处理温度可能狗尾草物种和种质各不相同。在一般情况下，较高的温度下用较短的处理时间或较低的温度下具有较长的处理时间将不会对去雄相同的效果。已经报道了42℃的热处理20分钟，10或47℃下进行10分钟14呈现S.谷子花粉不存活，但是这些治疗的效率并没有确定。我们已经开发了协议后的假设，即优化处理温度和持续时间下，所有花朵的花粉保留在母本将为非可行的，而柱头仍将接受。然而，比较和分析不同温度的影响后治疗周期（ 表1），我们发现，对热处理的敏感性之间留存在每个圆锥花序的花而变化，因而难以完全消除从自花授粉产生的种子。我们的结论是生产异型杂交后代的效率是最大的，当治疗是在48℃下在S进行3-6分钟贻贝加入A10.1。 花和异花授粉的高峰时间当鲜花达到成熟阶段，并即将开放，花药黄白色的颜色和花粉是尽快从花药花8 exsert棚。花药逐渐变成褐色后，花粉脱落的花朵开始关闭。一旦释放，花粉的生活力是未知的。因此，要尽快用花粉的父本打开或刚刚打开的花朵是至关重要的。根据我们的室内条件下（ 图1），多数flowe的男性家长RS开始打开在上午10:10和花朵开在棚10:30花粉。因此，理想的窗口进行授粉10:30和11:00之间。柱头后保持密切花的颖片外，可能是接受花粉。这先前已经观察到的和在S证实谷子的柱头是接受约48小时后花开放了西莱斯等。6，所以它是下列热处理重要的包穗和执行控制杂交后。正如上面所讨论的，我们建议双方第2天与第3天后期去雄授粉表现。 方法授粉我们比较了三种授粉技术的效率。如果花开不是限制性的，穗至穗授粉是最有效的技术，并且将产生一个数字异型杂交后代的更高。如果圆锥花序开花都限制，邻频率较高当采用花药到柱头方法utcross子代会产生。我们已获得异交后代的两种方法成功。在“绑定穗”的方法已经在穿越S.被使用谷子 6，但我们发现这种方法是最有效的方法为时间窗口父本散粉是短暂的。另外，有较少的控制授粉和S上的刷毛贻贝穗数也可能阻碍花粉的运动到柱头表面。 种子采收，干燥和储存收获后，种子应在30-33℃下干燥2天。 Anthecium应为GUS染色被除去，如果需要的话。种子应存放在干燥，阴凉的位置（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％），用于短期存储（不超过两年），或在种子室（T：4.0 10°C±1.0°C，相对湿度：20％±1％），用于长期存储。可怜的储存环境也可能导致低生存能力率8。我们观察到S的发芽率贻贝 A10.1约为5％时，收获后4天播种，但可提高到90-96％，保存后在实验室（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％）为110天收获后接着进行为期三天的分层在-80°C至打破种子休眠。对于分层在-80℃，干燥的种子可以被放置在一个密闭的容器（ 如微离心管中或硬币包络在一个密封的塑料袋），并保持在-80℃下在播种前3天。存储后在实验室16个月（T：24.06°C±0.13°C，相对湿度：21.79％±1.15％），90〜95％发芽率仍然可以得到。此外，打破休眠，种子可在30-33℃干燥收获后第2天，然后再给予3天的分层，在-80℃，接着除去anthecium前种植。这些处理后，高达33％的种子发芽率可以达到。 优势，局限性和可能的​​修改在这里，我们提供了第一个标准协议在S进行杂交贻贝 A10.1采用的去雄热处理。相较于物理阉割，这个协议是创伤小，比较容易建立一个实验室。它通常需要大约15分钟，修整一穗约15穗可修剪和交叉/人/天。假设平均3-5异型杂交后代/穗可在优化条件下被回收，总共45-75异型杂交后代可以由一个人在一天之内产生的。此外，这种技术可应用于跨越在其他狗尾草物种，尽管额外的优化将是可能的。如果生长室空间有限，植物可以在温室或生长室中生长没有一个黎明前的珍稀tment直到穗出现他们被转移到优化室内环境之前。

狗尾草是NADP-ME的C亚型4草密切相关的生物能源原料的柳枝稷（NAD-ME亚型），粮食作物谷子和农业杂草羊草5。的狗尾草，S的栽培形式的510 MB的基因组谷子，最近被测序1,2和杂草相对，S的25倍覆盖基因组序列贻贝 ，正在进行中（未发表）。S。贻贝有许多特点，使得它包括一个快速的生成时间，小身材，简单的生长要求，而且多产的种子生产3潜在的很好的模型遗传系统。重要的是，方法最近已被开发用于S的瞬态和稳定转化贻贝 ，提供了机会，创造转基因植物4。然而，在遗传工具为S的发展的一个主要瓶颈贻贝是其重新计算itrance到异交。迄今为止，还没有标准的协议已经公布，将允许快速生产的种子从控制的十字架。 在S遗传杂交谷子通常由阉割通过去除花药从花6,7,8或通过母本9-11鲜花热处理进行。下面的这些治疗，花药或未经处理的穗花/父本轻轻打磨柱头对花粉释放。在去雄，花药都用细镊子小花打开后，立即和花药也开始exsert删除，但仍未摆脱花粉6-8。其中后一种方法所面临的挑战是在花的开口和花粉脱落之间的窄的时间间隔进行去雄的难度。一花独放的开幕式和闭幕式的持续时间将视加入和环境条件而异，可以从7分钟&lt;SUP&gt; 6至2.5小时12 S。谷子 。自脱落花粉的发生是由于花药从小花exsert，花药需要仔细和迅速地除去，因此，很难以避免污染，由于自花授粉。此外，作为授粉被阉割后立即进行，也可以为每个人进行/每天杂交的数量是有限的。 作为一种替代方法，成熟的圆锥花序，可蘸温水加热，杀死发展花粉粒9,10,13,14与大量圆锥花序开展杂交的优势。然而，热处理的温度和持续时间从不同的实验（ 例如，47℃，10分钟14至 42℃下进行20分钟10）广泛变化。此外，没有对热介导的emasculations疗效系统的分析已经出版。因此，对于在S进行遗传杂交的标准和简单的方法viridis将大大加快遗传资源的开发S.和建立贻贝作为研究界的一个模型系统。 我们报告的标准协议的开发和优化的S中进行遗传杂交贻贝 。植物在可控环境中进行同步花的发育和增加的程序的再现性生长。十字架使用的是转基因学执行贻贝线作为含有GUS报告基因4，以促进成功控制遗传杂交鉴定父本。 GUS的转基因是通过水稻泛素启动子使F1种子的收获进球后，立即，从而提供了一种简单的定性分析，以确定去雄和授粉的效率驱动。我们采用用温水简单的热处理去雄伴随着已emascula花卉标签特德以消除去雄后新开发的花产生种子。测试了一系列的热处理在许可的温度和不同浸在温水的时间之后，我们已经建立了48℃的最佳温度和持续时间3-6分钟。黎明前冷处理，发现以促进父母双方同步开花，以便于交叉授粉。我们还讨论了协议，这种方法的未来应用到其它狗尾草种质的关键步骤。

<table border="1">
		<tbody>
		 <tr>
			<td>Name</td>
			<td>Company</td>
			<td>Cat. Number</td>
 <td></td>
		 </tr>
		 <tr>
			<td>Scissors, spring</td>
			<td>World Precision Instruments, Inc</td>
			<td>14126</td>
		<td></td>	
		 </tr>
		 <tr>
			<td>Forceps, Dumostar Biology Polished</td>
			<td>SPI Supplies</td>
			<td>TD5BP-XD</td>
		<td></td>	
		 </tr>
		 <tr>
			<td>Surgical Scissors</td>
			<td>F.S.T (Fine Science Tools)</td>
			<td>14005-12</td>
		<td></td>	
		 </tr>
		 <tr>
			<td>Europack Clear Polypropylene Micro-Perforated Crusty Bread Bags 6"x28"</td>
			<td><a href="http://www.pjpmarketplace.com" target="_blank">http://www.pjpmarketplace.com</a></td>
			<td>361001</td>
 <td></td>
		 </tr>
		 <tr>
			<td>Flats</td>
			<td>T.O. Plastics</td>
			<td>715401C </td>
			<td></td>	
		 </tr>
		 <tr>
			<td>metro mix 360</td>
			<td>Hommert International</td>
			<td>10-0356-1</td>
			<td></td>	
		 </tr>
		 <tr>
			<td>Jack's 15-16-17</td>
			<td>Hommert International</td>
			<td>07-5925-1</td>
			<td></td>	
		 </tr>
		 <tr>
			<td>Kimwipes</td>
			<td>VWR International</td>
			<td>34120</td>
			<td></td>	
		 </tr>
		 <tr>
			<td>Sharpie Ultra Fine Point Permanent Markers, Red</td>
			<td>Staples</td>
			<td>37002</td>
 <td></td>
		 </tr>
		 <tr>
			<td>Donegan DA-10 OptiVisor Headband Magnifier, 3.5x Magnification, 4" Focal Length</td>
			<td>Amazon</td>
			<td>DA-10, B0015IP380</td>
 <td></td>
		 </tr>
		 <tr>
			<td>12"24/7 Packaging Hand Impulse Sealer Heat Seal Machine Poly Sealing Free Element Grip </td>
			<td>Amazon</td>
			<td>N/A</td>
 <td></td>
		 </tr>
		 <tr>
			<td>water bath</td>
			<td>VWR scientific</td>
			<td>Model: 1166</td>
			<td></td>	
		 </tr>
		 <tr>
			<td>BDW walk in plant growth chamber</td>
			<td>Conviron</td>
			<td>BDW 40</td>
 <td></td>
		 </tr>
		</tbody>
 </table>

methods for performing crosses in setaria viridis, a new model system for the grasses

狗尾草是对C 4草一个新兴的模型系统。它是密切相关的生物能源原料柳枝稷和粮食作物谷子。近日，谷子，S的510 MB的基因组谷子，已被测序1,2和杂草相对S的25倍覆盖基因组序列贻贝正在进行中。S.贻贝有许多特点，使得它包括一个快速的生成时间，小身材，简单的生长要求，多产的种子生产3和开发系统瞬态和稳定转化4潜在的很好的模型遗传系统。然而，S的遗传学贻贝在很大程度上是未开发的，部分是由于缺乏详细的方法进行杂交。迄今为止，还没有标准的协议已被采纳，将允许快速生产的种子从控制的十字架。 该协议的压力ented这里是在S进行遗传杂交优化贻贝 ，加入A10.1。我们已经修剪穗保留20-30朵，花标识，以消除去雄后，新开发的花卉种子造成就业后用温水简单的热处理阉割。测试了一系列的热处理在许可的温度和浸泡不同的时间后，我们已经建立了一个最佳的温度和48℃的时间范围为3-6分钟。通过使用这种方法，至少15杂交可以通过每天单个工人进行和平均每穗3-5异型杂交的后代可以被回收。因此，平均45-75异型杂交后代可以由一个人在一天之内进行制造。广泛实施这一技术将有利于S的重组自交系群体的发展贻贝新时贻贝或S。贻贝新时谷子 ，通过批量映射本身的突变gregant分析和建立更高阶的突变体进行基因分析。

在这项研究中，我们使用了序列S。贻贝加入A10.1为母本，与转基因S。贻贝线（也A10.1）含有GUS报告基因4作为父本为所有授粉。一个优化的生长机制被用作如图1所示的同步开花。我们已经测试了温度去雄热处理和时间的几种组合，并指出，约40％-80％的保留在阉割穗花开花的第 2天，其余盛开在第 3天（ 图2）。然而，如果热处理是太严重，举一个例子，49℃，4分钟，很少或无花开花的第 2天。异花授粉是在第2 次或第 3天去雄后，这是依赖于父本的开花高峰时间进行。 这是observ编着，年轻的花朵去雄继续以后的发展，成熟，自花授粉后3-6天控制授粉。后7-10天交授粉，花是白色的都在穗可见。这些白色的花是未受精的，由于无论是热损伤或授粉不良。与此同时，已成功授粉的花朵开始承受黑暗的种子，并开始14天后，拆东墙补西墙。黑暗的种子，收获授粉后第 14天红色标记可以从种子中明确区分没有红色标记的仍然是绿色的，并没有动摇。如果收割延迟，种子带和不带红色标记两者的anthecium转暗，这使得它很难迅速分辨的种子有红色标记的收获池。 以优化被执行的交叉协议几个热处理实验总结在表1中 。平均0-10粒/穗从各种热处理实验回收。收获后，种子在30℃下干燥的种子干燥器2天，然后通过除去使用anthecium去除的anthecium的。除去anthecium后，假定F1种子沾满GUS染色解决方案，以及异型杂交后代在1-2小时（ 图2F）染蓝的颜色。基于这些结果，我们推荐的48°C加热处理3-6分钟。此外，我们建议将十字架上都第2天及3天以下去雄执行。 为了检验这种技术进行杂交与其他狗尾草加入或物种的有效性，十字架之间S。贻贝 A10.1和八个不同的S。贻贝种质和一个S。荔加入了执行。虽然天至开花的材料间不同的，我们发现，所有八个S的花朵贻贝种质和一个S。黎明前的冷处理后荔入世开始上午10:00-11:00之间开放，峰值开放时间为上午10:20-11:00。这表明黎明前冷处理可应用于多种狗尾草种质。 48℃的热处理5-6分钟是用来阉割S。贻贝 A10.1这是作为母本进行执行的所有十字架。我们从一个恢复至12种子从A10.1和三个不同的学之间的交叉贻贝加入和之间的距离S的单交6种子贻贝 A10.1和单个S。荔加入。然而，在这个时候我们还没有决定，如果这些种子源于一个成功的测交或者是自我污染的结果。无论如何，这些初步结果表明，用S的生长条件贻贝 A10.1可用于产生杂交与不同的S。贻贝种质和其他可能的种狗尾草的。 <tablë边境=“1”&gt; <tbody><tr><td> 温度热处理（℃） </td><td> 时间热处理（分钟） </td><td> ＃十字架 </td><td> 平均。第修剪后花 </td><td> 当天授粉（天emasc后） </td><td> 平均。 ＃种子（红）/穗（平均） </td><td> 平均。 ％格斯（+）/种子（红） </td><td> 平均。 ＃HYB种子（GUS +） </td><td> 闵＃HYB种子 </td><td> 马克斯＃HYB种子 </td></tr><tr><td> 47 </td><td> 10 </td><td> 2 </td><td> 27 </td><td>第2天</td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr><td> 48 </td><td> 3 </td><td> 3 </td><td> 36 </td><td>第2天</td><td> 5 </td><td> 60 </td><td> 3 </td><td> 1 </td><td> 5 </td></tr><tr><td></td><td> 4</td><td> 3 </td><td> 25 </td><td>第3天</td><td> 10 </td><td> 50 </td><td> 5 </td><td> 3 </td><td> 7 </td></tr><tr><td></td><td> 5 </td><td> 8 </td><td> 25 </td><td>第2天</td><td> 5 </td><td> 60 </td><td> 3 </td><td> 1 </td><td> 6 </td></tr><tr><td></td><td> 6 </td><td> 3 </td><td> 25 </td><td>第2天</td><td> 4 </td><td> 75 </td><td> 3 </td><td> 0 </td><td> 4 </td></tr><tr><td></td><td> 7 </td><td> 4 </td><td> 24 </td><td>第2天</td><td> 3 </td><td> 33 </td><td> 1 </td><td> 0 </td><td> 4 </td></tr><tr><td></td><td> 7 </td><td> 1 </td><td> 25 </td><td>第3天</td><td> 4 </td><td> 75 </td><td> 3 </td><td> N / A </td><td> Ñ​​/一</td></tr><tr><td> 49 </td><td> 1 </td><td> 3 </td><td> 23 </td><td>第2天</td><td> 5 </td><td> 25 </td><td> 1 </td><td> 0 </td><td> 2 </td></tr><tr><td></td><td> 2 </td><td> 7 </td><td> 26 </td><td>第2天</td><td> 8 </td><td> 25 </td><td> 2 </td><td> 0 </td><td> 4 </td></tr><tr><td></td><td> 3 </td><td> 5 </td><td> 22 </td><td>第2天</td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 1 </td></tr><tr><td></td><td> 4 </td><td> 4 </td><td> 25 </td><td>第2天</td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr></tbody></table> 表1中。优化去雄热处理。改变热处理的温度和时间的结果。授粉以下去雄和成功交配s执行一次（第2天），两天（ 第 3天）芯与GUS染色<img alt="图1" fo:content-src="/files/ftp_upload/50527/50527fig1highres.jpg" fo:content-width="5in" src="/files/ftp_upload/50527/50527fig1.jpg" /> 图1。优化的生长室中的条件与黎明前冷处理，温度和花卉生产的最佳生长和同步的时间段被示出。 ，绿色和红色箭头显示的最佳窗口，进行杂交和去雄分别。 <img alt="图2" src="/files/ftp_upload/50527/50527fig2.jpg" /> 图2。对于交叉和异型杂交后代的GUS染色穗准备。的狗尾草 A10.1（A）修剪，显示出大约的花儿开了1/10前穗。四节和方向性沿穗开花的显示过程。 <strong去雄与刷毛保留前&gt;（B）修剪穗（C）去除刷毛经过阉割穗（D）的第2天后期去雄，显示出争艳（留影10:30花阉割穗AM）（E）的父本（转基因狗尾草 A10.1线携带β-葡萄糖醛酸酶（GUS的穗）报告基因）是经过黎明前的治疗（图片拍摄于10:30）绽放， 一个通过E，比例尺= 1毫米（F）的 GUS染色2小时，接着distaining在70％（V / V）乙醇后推定的异型杂交后代。从右边的2种子是阴性和阳性对照，分别为。 <img alt="图3" src="/files/ftp_upload/50527/50527fig3.jpg" /> 图3。流程图协议的 M的示意图参与表演S. ajor步骤贻贝跨越。

Watch this Scientific Journal Video about Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses at JoVE.com

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

我们已经开发了用于执行狗尾草 （ 狗尾草 ）杂交的方法。该方法包括修剪穗前一个热水处理，以杀死可行的花粉。十字架执行下列一个良好控制的增长机制，通常导致1〜7异花授粉的种子/ s的每穗的恢复。

方法中进行杂交<em&gt;狗尾草</em&gt;，新模型体系的草

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

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Research

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Environment

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

21.6K 次观看.  Donald Danforth Plant Science Center. 我们已经开发了用于执行狗尾草 （ 狗尾草 ）杂交的方法。该方法包括修剪穗前一个热水处理，以杀死可行的花粉。十字架执行下列一个良好控制的增长机制，通常导致1〜7异花授粉的种子/ s的每穗的恢复。 

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

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

科研

环境科学

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

土壤重采样的方法监控森林土壤的化学浓度的变化

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

蚂蚁视觉系统解剖学研究方法

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

利用水下立体视频工具量化鱼类密度的新方法研究进展

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

一种用于气溶胶取样的新型便携式体外曝光盒

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...