Method Article
拟南芥种子高通量灭菌的标准化方法
Benson E. Lindsey III*1, Luz Rivero*1, Chistopher S. Calhoun1, Erich Grotewold1,2, Jelena Brkljacic1
1Arabidopsis Biological Resource Center, Center for Applied Plant Sciences, The Ohio State University, 2Department of Molecular Genetics, Center for Applied Plant Sciences, The Ohio State University
* 这些作者具有相同的贡献
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摘要
本研究的目的是确定漂白和氯气灭菌对不育培养基上的拟南芥种子萌发的影响。为防止微生物污染物的生长, 在满足种子存活的同时, 制定了优化的灭菌规程。
摘要
拟南芥(拟南芥) 幼苗往往需要在无菌培养基上生长。这就要求预先进行种子灭菌, 以防止种子表面存在的微生物污染物的生长。目前, 拟南芥种子使用两种截然不同的杀菌技术进行灭菌, 这种方法在实验室之间稍有不同, 而且没有标准化, 通常只会导致部分有效的灭菌或过度的种子死亡。大多数这些方法也不容易扩展到大量不同基因型的种子线。随着对拟南芥高通量分析技术的不断扩散, 对不同基因型的大量种子进行杀菌的标准化技术成为进行这些类型实验的关键。根据种子萌发率和微生物和其他病原菌对种子的污染程度, 对两种不同的拟南芥品系的反应进行了评价。这些治疗包括不同浓度的杀菌剂和暴露时间, 并结合确定拟南芥种子灭菌的最佳条件。为两种不同的杀菌方法: 漂白 (液相) 和氯 (Cl2) 气体 (蒸气相) 的优化协议, 两者都导致了高种子萌发率和极小的微生物污染。通过对野生类型和突变种子的试验, 说明了这些协议的实用性。我们的结果表明, 种子可以有效地消毒使用的任何一种方法, 没有过多的种子死亡, 虽然消毒的有害影响, 种子的萌芽潜力低于最佳。此外, 还建立了一个方程式, 使研究人员能够将标准的氯气灭菌条件应用于不同尺寸的密闭容器中。此处所述的协议允许对大量拟南芥线进行简单、高效和廉价的种子灭菌。
引言
拟南芥(拟南芥) 是植物生物学基础和应用研究的主要模型生物体1,2,3。虽然标准条件为拟南芥生长已经建立了良好的4, 种子杀菌对种子活力的影响没有经过严格的测试。在板或盒中的固体培养基通常用来促进拟南芥幼苗的生长, 许多实验应用, 如分离种群中的合合致死突变体的鉴定, 早期观察和根表型分期、无病原组织的分离、大量苗木组织的采集、转化或耐药植物的选育及发芽评价1,2,3,4.从温室或生长室种植的植物中收获的种子偶尔会受到微生物和灰尘的污染。拟南芥幼苗在不同类型的无菌培养基上的生长需要预先进行种子灭菌, 以清除种子表面存在的真菌和细菌等微生物污染物。有效的种子杀菌机制的使用对于平衡高发芽、最小污染和旺盛的植物生长至关重要。
用于拟南芥种子灭菌的两种最常用方法是基于商业漂白剂 (液相) 和氯气 (气相)。已采用各种程序进行液相灭菌1,4,5,6,7,8,9,10 和气相灭菌的拟南芥种子8,10,11,12131415 ,16。然而, 虽然这些程序是有效的完成种子灭菌的使用基因型, 详细分析了不同的杀菌处理对不同基因型种子的影响, 没有报告。因此, 需要对这些灭菌程序进行优化, 以确定有效杀菌与高萌发率相结合的条件。
拟南芥生物资源中心 (ABRC) 是唯一的定位到) 测试种子活力的各种不同的基因型在收集和 b) 利用的质量控制程序在内部应用, 并响应用户反馈关于种子发芽。本文的实验目的是确定各种灭菌方法对拟南芥基因型的种子萌发的影响。针对漂白和氯气灭菌, 提出了优化的灭菌程序, 使种子萌发率高, 同时保持最小的病原体污染。
研究方案
1. 制备1x 村和 Skoog (ms) 介质
- 将 MS 基盐混合物、10克蔗糖和0.5 克 2-(n-吗) 磺酸 (MES) 添加到含有0.8 升蒸馏水的烧杯中, 搅拌溶解。使用1米氢氧化钾 (KOH) 检查和调整 pH 值5.7。添加蒸馏水, 使1升.
- 将介质分成两个1升的瓶子, 每瓶500毫升。每瓶加5克的琼脂。把盖子松开.
- 20 分钟121° C 和 #176 的高压釜, 15 psi, 瓶中有磁性搅拌棒.
- 灭菌后, 将瓶子放在低转速的搅拌盘上, 使 MS 介质冷却到45-50 和 #176; C (直到瓶子可以用手握住).
- 从该步骤开始, 在层流罩中执行无菌条件下的所有步骤。添加500和 #181; Gamborg 和 #39 的维生素溶液每瓶和搅拌 MS 培养基均匀分布的维生素溶液.
- 将足够的介质倒入板中, 以覆盖板材深度的大约一半.
- 允许平板在室温下冷却约1小时, 以使琼脂凝固.
注: 如果不立即使用, 包装在塑料和存储在4和 #176; C (冰箱温度)。固化琼脂的盖板、盒或管可在4和 #176 中存放数周; C 在密闭容器内.
2。用漂白剂灭菌拟南芥种子
- 根据议定书1节准备 MS 板。高压釜100毫升蒸馏水与 MS 介质同时进行。使用此后, 作为冲洗水和帮助暂停种子, 以协助电镀.
注: 如果需要, 0.8% 琼脂混合 (w/v) ( 如 phytagar) 也可以在这一步蒸压。在电镀过程中, 琼脂混合可代替蒸馏水 (步骤2.5.3。琼脂混合的额外粘度使它更容易在盘子上的空间种子或植物在必要的行. - 准备 50% (v/v) 漂白溶液, 用于消毒种子。稀释漂白剂, 加入100毫升的漂白剂至100毫升蒸馏水。添加50和 #181; 吐温20的洗涤剂到漂白剂溶液.
注: 只要只在无菌条件下打开, 准备好的漂白剂溶液可贮存长达一个月. - 分100种子放入1.5 毫升离心管中.
- 使用50% 漂白溶液对种子进行消毒.
- 在层流罩中, 添加500和 #181; 50% 漂白溶液中的 L 离心管含有种子。点击底部的管, 以暂停在漂白溶液的种子.
注: 或者, 转子或平台振动筛可用于保持种子暂停.
- 在层流罩中, 添加500和 #181; 50% 漂白溶液中的 L 离心管含有种子。点击底部的管, 以暂停在漂白溶液的种子.
- 从管道中漂洗漂白溶液。
- 在10分钟后, 使用吸管或末端装有吸管端的器从离心管中取出漂白剂溶液.
- 添加500和 #181; 在试管中加入无菌蒸馏水。关闭管和倒置混合。允许种子沉淀到管子的底部。一旦种子已落户到底部的管小心删除漂白溶液的移。重复此冲洗过程6次.
- 在试管中加入1毫升的蒸压蒸馏水以暂停种子.
- 将已灭菌的种子放在 MS 板上.
- 在层流罩中, 用股票名称和当前日期标记 MS 板的底部.
- 将种子从离心管上倒入 MS 板上。使用无菌、一次性接种循环或无菌吸管尖端在 MS 板上散布种子.
注: 如果种子被播种在排, 一个200和 #181 的吸管; L 尖可用于在所需位置单独放置种子。为了改善种子的流动, 吸管尖端的末端可以用剪刀修剪3-5 毫米。任何错位的种子或种子簇, 然后可以使用无菌一次性接种循环重新定位或分离. - 将 MS 板放在层流罩的背面, 盖子半封闭。允许多余的水从 MS 板上蒸发.
- 将盖子盖在 MS 板上。通过用微孔纸外科胶带包裹板来封住 MS 板 (请参见 材料表 ).
3。拟南芥籽的氯气灭菌
- 根据和 #160 准备 MS 板; 《议定书》1节。高压釜100毫升蒸馏水与 MS 介质同时;这将被用来帮助暂停种子, 以协助在电镀.
注: 如果需要, 0.8% 琼脂混合 (w/v) ( 如 phytagar) 也可以在这一步蒸压。在电镀过程中, 琼脂混合可代替蒸馏水 (步骤3.6.2。琼脂混合的额外粘度使它更容易在盘子上的空间种子或植物在必要的行. - 在开始灭菌之前, 计算产生氯气所需的漂白剂和盐酸 (HCl) 的数量.
- 计算用于生成灭菌所需的 6.1% Cl 2 所需的 HCl 量.
- 使用以下公式:
以7000毫升作为灭菌容器的体积, 6.1 作为%Cl 2 ; HCl 的体积被计算为3毫升。 注: 为执行对容器的不同卷和%Cl 2 的计算而编写的电子表格作为 补充表 1 提供。
- 分100种子到0.5 毫升离心管中。关闭每个瓶子的瓶盖, 把瓶子放在塑料架上, 放在一旁.
注: 只要储存在适当的条件下, 种子可以在这一点长时间储存。Rivero 和同事 4 可以在协议的3.3.2 节中找到存储条件。96-井板格式也可以使用. - 准备执行氯气灭菌所需的材料.
- 从其存储位置获取漂白剂和 HCl.
- 剪切一条大的石蜡薄膜 (请参阅 材料表 ) 以在步骤3.5.3 中使用. 密封灭菌容器.
- 将带有盖子的塑料容器放在通风罩内进行灭菌。打开所有种子瓶上的瓶盖, 将整个种子架放在塑料容器内.
- 在室温下执行氯气灭菌.
注意: 分别使用漂白剂和酸。不要留下任何一瓶上限, 以减少溢出的风险。使用适当的个人防护装备 (PPE), 包括手套和实验室大衣。如果漂白剂或酸溅在手套上, 在处理其他材料之前更换手套。在漂白剂或酸性污染的情况下, 一定要把手套放在油烟罩里。- 在容器内放置一个250毫升的烧杯, 并添加100毫升的漂白剂.
注: HCl 和漂白剂之间的反应至少需要22卷多余的漂白剂。反应将使用氯化钠 (氯化钠) 和水作为副产品释放 Cl 2 气体, 从而消耗 HCl 和漂白剂。使用大量过量的漂白剂可以消耗额外的 HCl d期间的排气时间, 这减少了碳酸氢钠 (NaHCO 3 ) 的数量, 以抵消处置解决方案.
注意: 烧杯应至少是漂白剂 + HCl 的总液体量的两倍。这可以防止溅出的烧杯在下一个步骤, 这可能损坏种子, 漂白衣服, 或烧伤裸露的皮肤. - 在含有漂白剂的烧杯中添加3毫升的 HCl.
注意: 最初的反应会产生气泡, 特别是在气体浓度高于6.1% 的时候。这一步需要长袖的实验室大衣. - 关闭灭菌容器并立即将其与石蜡膜密封.
- 在灭菌时间内监控灭菌容器, 以确保气体积聚; 氯气的积聚应在容器内可见为微弱的黄色薄雾.
注意: 定期检查灭菌容器, 确保内部的压力没有把盖子或石蜡膜盖上。如果盖子已经松动, 或石蜡薄膜已经松散, 关闭盖子, 仔细包装的容器与一层额外的石蜡薄膜. - 经过1小时灭菌期后, 打开容器, 取出石蜡膜, 在一个拐角处开盖子。允许容器排出3小时以完成反应并消除氯气.
- 关闭种子架中所有离心管的顶盖.
注意: 只要在干燥的情况下储存, 灭菌的种子可以储存到电镀时间. - 卸下种子架并将其放置在层流罩中.
- 中和氯气反应
- 在包含漂白剂/HCl 溶液的烧杯中缓慢添加1.5 克 NaHCO 3 粉末, 并用玻璃棒搅动以将 NaHCO 3 溶解到溶液中。继续添加 NaHCO 3 直到二氧化碳 (CO 2 ) 气体气泡停止形成.
注意: 加慢以防止飞溅。使用适当的 PPE, 包括手套和实验室大衣. - 使用 ph 值试纸或 ph 计测试溶液的 ph 值。如果需要, 请添加其他 NaHCO 3 , 直到溶液的 ph 值为中性 (ph 7.0)。在这一点上, 解决方案可以从油烟罩中删除, 并根据所有适用的处置准则处理.
注意: 如果在处理过程中发现有异味, 则应立即将溶液送回油烟罩.
- 在包含漂白剂/HCl 溶液的烧杯中缓慢添加1.5 克 NaHCO 3 粉末, 并用玻璃棒搅动以将 NaHCO 3 溶解到溶液中。继续添加 NaHCO 3 直到二氧化碳 (CO 2 ) 气体气泡停止形成.
- 在容器内放置一个250毫升的烧杯, 并添加100毫升的漂白剂.
- 将灭菌的种子放在 MS 板上.
- 在层流罩中, 用股票名称和当前日期标记 MS 板的底部.
- 添加500和 #181; 在每个离心管中加入灭菌蒸馏水, 以暂停种子。将种子撒在 MS 板上, 用无菌的、一次性接种的循环或无菌的吸管尖在盘子周围均匀地播撒种子.
- 将 MS 板放在层流罩的背面, 盖子半封闭。允许多余的水从 MS 板上蒸发.
- 将盖子盖在 MS 板上。用微孔纸外科胶带将钢板包裹起来, 封住 MS 板.
4。MS 板上拟南芥的生长
- 将带有盖子的板放置在三和 #160 上;d 总是在4和 #730; C 和环境湿度.
注意: 这个过程被称为分层, 用来同步个体种子的萌发. - 将板转移到增长环境.
- 将温度保持在23和 #176; C 和光照强度在 120-150 和 #181; 摩尔/m 2 与 16 h 光/8 小时暗光周期。把盘子平放到生长环境中, 盖子放在顶端, 这样根部就会长成培养基.
- 让板上的幼苗生长8天.
注: 8 天的生长期允许发芽的种子发芽。如果所有的种子都发芽了, 盘子可以比8天更早地得分. - 对萌发率进行评分.
- 记录已发芽且尚未发芽的种子数量。通过将发芽的种子数量除以板上种子的总数来计算发芽率.
注意: 发芽是计算的, 当胚根在种子外衣外投射, 并且二子叶是可看见的. - 还要计算受霉菌影响的种子数量, 以确定灭菌条件的有效性.
- 记录已发芽且尚未发芽的种子数量。通过将发芽的种子数量除以板上种子的总数来计算发芽率.
结果
从野外、温室或生长室收集的拟南芥种子有时受到真菌和细菌等各种微生物的污染1,4。因此, 在无菌培养基上发芽的种子可能特别具有挑战性, 因为板的污染, 特别是当种子供应有限。漂白和氯气灭菌的优化协议, 其结果如下所示, 最大限度地减少了这一问题, 并保留了高吞吐量应用程序所需的种子的生存能力。
漂白杀菌对拟南芥 Col-0 种子萌发的影响
漂白剂是众多植物种中最常用的种子杀菌剂。杀菌剂的最佳浓度和曝光时间各不相同。使用漂白剂进行拟南芥种子灭菌的一些协议1,4,5,6,7,8,9,10. 对四种不同浓度的漂白进行了测试, 并在 图 1中显示了五不同暴露时间段的结果。该疗法适用于哥伦比亚野生型 (Col-0) 种子。漂白剂浓度对 Col-0 种子萌发的影响随灭菌时间的不同而变化 (图 1、 P和 #60; 0.001、方差分析。
在实验以绝育时间在5和 10 min 之间, 处理与所有漂白的集中导致同样高发芽率 Col-0 种子 (图 1)。在所有灭菌时间, 家庭漂白剂浓度为40% 和 50%, 也观察到高发芽率。与较短浸泡时间 (P和 #60; 0.01, 方差分析) 相比, 80% 和100% 漂白时间长于10分钟的处理导致发芽率显著降低。此外, 对于80% 和100% 漂白处理20分钟, 与相应的40% 和50% 漂白处理 (P和 #60; 0.001, 方差分析) 相比, 发芽率显著降低。
在使用高漂白剂浓度15分钟或更长的时间内, 种子显示出不同程度的漂白和萎缩。除了相对较高 (高达 32%) 的种子死亡率, 发芽种子在这些条件下的灭菌往往显示生长缺陷, 反映在失败的子叶和胚展开和拉长, 导致发育停止。大多数治疗 (14 出 20) 完全无模, 导致整体模具水平平均0.21% ± 0.003 (表 1)。
以50% 漂白水和浸泡时间10分钟为最佳杀菌方法, 结合高发芽率, 对表面病原体生长有较好的抑制作用。这项治疗被选中, 以测试漂白消毒对不同的突变株线的影响, 如下所述。
氯气杀菌对 Col-0 种子萌发的影响
为了优化氯气灭菌条件, 用三种不同浓度的氯气对 Col-0 种子进行两段时间的消毒 (表 2)。根据浓缩 HCl 体积和灭菌容器容积计算出气体浓度, 采用以下公式:
该方程采用理想气体定律, 假定为 12.3 M HCl, 温度为23° c, 标准大气压为101.3 帕斯卡。
氯气体浓度对 Col-0 种子萌发的影响取决于时间和浓度因子之间的显著相互作用 (图 2A、 P和 #60; 0.01、方差分析).氯气浓度对 1 h 灭菌 Col-0 种子萌发无显著影响。随着灭菌时间的推移, 所有浓度的氯气也促进了类似的高萌发率, 高于 85% (P和 #62; 0.05, 方差分析)。另一方面, 3 h 长的灭菌导致了最高浓度氯气的发芽率显著降低, 与两个较低的气体浓度 (P和 #60; 0.05, 方差分析) 相比较。这些结果表明, 对拟南芥种子与任何经测试的氯气体浓度为1小时, 或气体浓度低于 16.5% 3 h, 是同样有效的保存种子活力, 因为发芽率总是更大超过82%。然而, 用16.5% 的气体对 3 h 的种子进行杀菌, 对种子萌发有害。
霉菌的发生率也取决于气体浓度和曝光时间。霉菌生长被有效地抑制了以相对地高浓度的6.1% 和16.5% 氯气体为 1 h 长的治疗和与所有气体集中为 3 h (图 2B)。
根据这些结果, 选择气体浓度为6.1% 的 1 h (表 2) 作为最佳气相灭菌条件, 以测试气体杀菌对不同突变株系的影响, 因为它结合了高萌发率 (85%), 模具污染程度极低 (0.02%)。
漂白和氯气杀菌对不同发芽势种子的影响
统计分析表明, 对灭菌方法的萌发反应取决于品系的萌发电位 (图 3A、 P和 #60; 0.01、方差计算)。漂白和氯气灭菌均不降低种子萌发率高 (组4和 5)。两种处理方法对低发芽率组的发芽率均有影响潜力 (组 1)。相比之下, 氯气的杀菌效果显著降低了大约 12-18% (P和 #60; 0.01, 方差分析) 在种子萌发的中间萌发电位 (组2和 3)。漂白消毒也降低了2组的13% 的发芽率, 但并没有降低3组的萌发率。虽然在任何萌发组 (图 3A、 P和 #62 中的漂白和氯气处理的发芽率之间没有显著差异; 0.442,方差分析), 使用漂白剂进行灭菌的种子确实略高萌发率高于所有萌发组中的气体灭菌种子。
灭菌处理明显改变 (P和 #60; 0.001, 方差分析) 受霉菌影响的种子百分比 (图 3B)。氯气和漂白剂的灭菌导致霉菌生长减少 (P和 #60; 0.05, 方差分析), 与无灭菌相比。在任何一个组 (图 3B, P & #62; 0.4, 方差分析) 中, 在气体和漂白剂之间检测到的霉菌水平没有差别。
图 1: 漂白剂浓度和灭菌时间对拟南芥 Col-0 种子萌发的影响.值是指从实验的5独立副本获得的。* 在漂白浓度范围 (P和 #60; 0.01, 方差分析) 中, 表示与5分钟浸泡时间有关的显著差异。#表示80% 和100% 漂白剂浓度在20分钟时间段 (P和 #60; 0.01, 方差分析) 的显著差异。请单击此处查看此图的较大版本.
图 2: 氯气体浓度和灭菌时间对拟南芥 Col-0 种子的影响.(A) 萌发率和 (B) 模具级别。误差线代表的意思是从5生物和5技术复制的实验获得的± SD。不共享字母的方法明显不同 (P和 #60; 0.05, 方差分析)。请单击此处查看此图的较大版本.
图 3: 漂白剂和氯气杀菌对不同发芽潜能种子的影响.(A) 萌发率和 (B) 模具级别。在无任何杀菌剂的情况下, 将100索尔克 T DNA 线根据其萌发潜能定义为五组。根据发芽率的组: 组 1 (0-20%), 组 2 (21-50%), 组 3 (51-70%), 组 4 (71-90%) 和组 5 (91-100%)。随机选择这些品系, 其萌发潜能不依赖于基因型。值是指从实验的三独立副本获得的。每个值上面的字母 ("a"、"ab"、"c" 等) 表示分类方法的统计分组。不共享字母的方法明显不同 (P和 #60; 0.05, 方差分析)。请单击此处查看此图的较大版本.
| 灭菌时间 (分钟) | 漂白剂浓度 (%) | |||
| 40 | 50 | 80 | 100 | |
| 5 | 0.00% | 0.00% | 0.00% | 2.13% |
| 8 | 0.00% | 0.00% | 0.00% | 0.00% |
| 10 | 0.00% | 0.50% | 0.00% | 0.36% |
| 15 | 0.00% | 0.00% | 0.00% | 0.68% |
| 20 | 0.19% | 0.28% | 0.00% | 0.00% |
表 1: 漂白消毒 Col-0 种子的模具水平。
| 漂白剂 | 盐酸 | 时间 | % 氯气 (摩尔 Cl2/摩尔总气体) |
| 毫升) | 毫升) | h) | |
| 25 | 1 | 1 | 2。1 |
| 25 | 1 | 3 | 2。1 |
| 100 | 3 | 1 | 6。1 |
| 100 | 3 | 3 | 6。1 |
| 200 | 9 | 1 | 16。5 |
| 200 | 9 | 3 | 16。5 |
表 2:氯 (Cl2) 在哥伦比亚野生型种子上使用7升容器进行气体灭菌处理.
补充表 1:为容器和%Cl2的不同卷进行计算而编写的电子表格。请单击此处下载此文件.
讨论
当在无菌培养基上生长拟南芥种子时, 必须采用某种形式的灭菌。漂白剂和氯气的杀菌处理均导致相似的发芽率和霉菌生长抑制。两种灭菌方法均不能显著降低萌发潜能种子的发芽率;然而, 对于具有较低发芽电位的线 (20-70%), 由于小的, 虽然显著, 与气体灭菌相比, 发芽率的提高 (图 3A), 建议使用漂白剂灭菌。
将拟南芥种子从40-100% 的漂白剂中消毒10分钟, 可获得满意的发芽率和有效的防霉效果。虽然漂白剂浓度低于40% 为大多数种子地段提供了足够的消毒, 使用浓度为40% 或更高的保证有效杀菌的甚至严重污染的种子地段。在使用漂白剂浓度等于或高于80% 的情况下, 不超过10分钟的灭菌是很重要的, 以避免高种子死亡和幼苗发育的缺陷。
用6.1% 或16.5% 的氯气浓度对拟南芥种子进行1小时的处理, 会导致高发芽率和适当的霉菌消除。低氯气体浓度 (2.1%) 可通过将灭菌时间增加到 3 h 来成功使用。
当需要消毒的线路很少时, 建议在溶液中用50% 漂白水进行10分钟的液体灭菌。对于更多的线, 气体的浓度为6.1% 的气体消毒1小时是一个更好的选择, 因为许多线路可以迅速和容易地消毒, 减少操作。
我们的研究结果为对不同基因型的大量种子和具有较低发芽潜能的种子提供了标准化的杀菌条件。这些杀菌技术的唯一限制是, 它们不能用于种子发芽率小于20% 由于广泛的种子死亡率。替代方法, 如超声17, 在没有灭菌的情况下提高发芽率可能对这些病例有益。
披露声明
作者没有什么可透露的。
致谢
我们要感谢歌达塔在准备实验材料方面的帮助。我们也感谢贝蒂娜 Wittler 和詹姆斯. 曼对手稿的评论。这项工作得到了 NSF 赠款 DBI-1049341 和 MCB-1143813 的支持。
材料
| Name | Company | Catalog Number | Comments |
| 0.65 mL Microcentrifuge tubes | GeneMate | C-3260-5 | Tube in which Arabidopsis thaliana seeds are placed to perform sterilization |
| 1.7 mL Microcentrifuge tube | GeneMate | C-3262-1 | Tube in which Arabidopsis thaliana seeds are placed to perform sterilization |
| 7 L plastic container | Sistema | 1016265438 | Container in which gas sterilization is performed |
| Concentrated HCl | Sigma-Aldrich | 320331 | Chemical used in the process of creating chlorine gas |
| Disposable sterile inoculating loop | Fisher Scientific | 22-363-603 | Loop is used to spread or position Arabidopsis seeds on MS plates |
| Gamborg’s vitamin solution | Sigma-Aldrich | G1019 | Vitamin solution used in the process of making MS media |
| Household bleach | Clorox | Regular-Bleach | Chemical used in the process of creating chlorine gas and liquid sterliziation |
| MES hydrate | Sigma-Aldrich | M2933 | Chemical used in the process of making MS media |
| Micropore surgical tape | 3M | 1530-1 | Microporous surgical paper tape used to seal MS plates |
| Murashige and Skoog basal salt mixture (MS) | Sigma-Aldrich | M5524 | Chemical used in the process of making MS media |
| Parafilm M | Bemis Company | #PM996 | Parraffin film used to seal sterilization container |
| Petri dish 100 X 15 mm | Fisher Scientific | FB0875713 | Petri dishes in which MS media is poured for the purpose of growing Arabidopsis thaliana |
| pH indicator strips | Whatman | 2613991 | Used to check pH of neutralizied chlorine and sodium bicarbonate solution |
| Phytoagar | Fisher Scientific | 50-255-212 | Used to aid in the suspension of Arabidopsis seeds in the process of plating seeds |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | Chemical used in the process of neutralizing chlorine gas reaction |
| Sucrose | Sigma-Aldrich | S0389 | Chemical used in the process of making MS media |
| Tween 20 | Fisher BioReagents | BP337-100 | Chemical used in the process of liquid sterilization |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | CS70000 (Col-0) | Arabidopsis wild-type seeds |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_037606C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_041402C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_059101C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_063470C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_072048C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_072240C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081989C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_084124C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085049C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_089717C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_107354C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110111C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111322C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113109C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114702C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114872C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115657C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_116803C | Arabidopsis seeds used in testing as a part of Group 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_039445C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_039782C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_043037C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_045828C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_048556C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049514C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049725C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080816C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081176C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081770C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082262C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082289C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082702C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_083630C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_084635C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085337C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085656C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_093049C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_103332C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_105336C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_105704C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_106388C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_109575C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110580C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110617C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111424C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111584C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_112097C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113339C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115837C | Arabidopsis seeds used in testing as a part of Group 2 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_019535C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_026478C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_046565C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049258C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049339C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_056307C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081292C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081597C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_083488C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110573C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_112793C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113658C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113904C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114673C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114709C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115455C | Arabidopsis seeds used in testing as a part of Group 3 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_013186C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_018261C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_062509C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080639C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_088586C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_096651C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_106900C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110131C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111051C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111245C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113223C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_121391C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_125097C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_201905C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_210001C | Arabidopsis seeds used in testing as a part of Group 4 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_000662C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_029335C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_047760C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_071275C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080530C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_103881C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110864C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_120294C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_124390C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_132808C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_137036C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_139519C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_140643C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_142288C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_143304C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_147597C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_209076C | Arabidopsis seeds used in testing as a part of Group 5 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081081C | Arabidopsis seeds used in testing as a part of Groups 5 and 1 |
| Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_107487C | Arabidopsis seeds used in testing as a part of Groups 5 and 2 |
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