Name: protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula
Uploaded: 2015-06-09T13:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1.合子胚胎发育<ol><li>植物材料<ol><li>成长的苜蓿野生型Jemalong或者其近等基因，高度重可生成基因型Jemalong 2HA 13（被称为2HA）与一个14小时光照温室中和23℃/ 19℃，白天/夜间温度。 <ol><li>皮尔斯种皮的表面（与23政针）播种前种子，这样的水被允许进入种子浸泡在水中过夜。加入足够的水来完全覆盖种子。 </li><li>播种3种子在每个直径为15cm锅（共10个盆）在盆栽混合物（粗砂，珍珠岩，椰壳-泥炭（1:1:1）加5克的Osmocote精确标准:缓释肥料），并转移到温室。保留发芽后的健康幼苗所以每盆和环绕1工厂通过网格为植物的茎爬上去。 </li></ol></li></ol></li><li>收获豆荚<ol><li>收集来自瓦特豆荚ILD型Jemalong或2HA，以获得适当的早期胚胎阶段，因为首先描述9。 注意:不同荚阶段示于图1和相应的胚胎阶段示于图2。 </li><li>检查不同荚阶段收获时使用标准按表1，测量从6段所经过的时间，以帮助独立的阶段6和7（ 表2）。 </li></ol></li><li>从豆荚解剖胚珠和检查胚胎阶段<ol><li>隔离单独使用细镊子豆荚或立体解剖显微镜和镊子胚珠是必要的。如果需要使用标准化合物显微镜（ 图3B）胚胎阶段可以快速检查。 </li><li>制备改性含7.5克阿拉伯胶100克水合氯醛，加入5ml甘油60毫升去离子蒸馏水霍尔的溶液。搅拌3-5小时或过夜溶解。 </li><li>欲了解更多细化ð镜清除修改霍耶的解决方案15,16解剖胚珠。仔细挑选了完整的胚珠和地方在小体积的霍耶的解决方案（足以覆盖胚珠）在室温下，直到清晰。 </li><li>查看样品用微分干涉对比（DIC）光学器件。捕捉图像iwth数码相机9（ 图2）。 </li><li>从荚的中心区域获得最均匀胚珠和积聚所需的数目。一个例子示于图3A。收集用于进一步分析在冰上和存储胚珠在所需的温度（-80℃，核酸）。 注:有关分析组织切片，透射电子显微镜的细节，并在原位杂交请参阅文件8,9。 </li></ol></li></ol> 2.体细胞胚胎发育的影响<ol><li>使用M.苜蓿品种牛逼帽子是能够容易地形成在培养胚胎。对于这个协议叶植使用2HA植物13,17是2-4个月。 </li><li>以从茎伸长的最新近完全展开叶枳传单。 </li><li>保持枳叶在潮湿的纸巾在锅里的文化，以避免脱水。 注意:一旦三叶叶子收获它们需要以最小的延迟利用。 </li><li>培养基的制备<ol><li>使用P4 10:4:1:在琼脂5培养基（0.8％（重量）:体积）中9厘米培养皿。 注:P4 10:4:1:5介质由在P4基础介质18加10μM的1-萘乙酸（NAA），4μM的6-苄氨基嘌呤（BAP），1μM的脱落酸（ABA）和5μM的赤霉的酸（GA）。使用GA + ABA的加速胚胎形成，并导致胚胎数14实质性的增加。 </li><li>弥补在1L的P4的基础培养基;由主要盐（使calcium，最高分别），微量盐和维生素（ 表3），螯合铁（补分开），酪蛋白氨基酸（酪蛋白水解物），30克蔗糖，将8g琼脂。 </li><li>商店原液主要的盐，钙，酪蛋白氨基酸，次要盐和维生素的在-20℃下在合适的等分试样。存储螯合铁在4℃。 </li><li>补螯合铁（200倍）通过在搅拌的同时约900毫升去离子蒸馏水中溶解7.44克的的Na 2 EDTA•2H 2 O，然后使溶液至98-99℃，同时加入1.853克的FeSO 4•7H 2 O.最后补到1升时，成分溶解。储存在琥珀色的瓶4℃。 </li><li>弥补与储备溶液在P4基础培养基如表4中。在培养基中的激素是10微米的NAA，4μM的BAP，1μM的ABA，5μM的GA（ 见表4和具体材料表 ）。添加NAA和以前的BAP以高压灭菌和高压灭菌并冷却至约55℃，用过滤灭菌以附连到注射器一个0.22微米的过滤器后添加的ABA和赤霉素。轻轻旋转拌匀。 </li><li>调节高压灭菌前将培养基至pH 5.8，用1M的KOH。高压釜在121℃下进行20分钟。 </li><li>后高压灭菌添加过滤灭菌的ABA和GA，并倒在一个层流罩或生物危害柜大约25毫升的媒体到无菌9厘米培养皿。清凉让琼脂集与盖掉。然后把盖子上，并确保有盖子上没有冷凝水。根据需要，并储存于4℃（在一个硬纸板箱，以防止盖子缩合物）的标签。 </li></ol></li><li>叶片组织的消毒<ol><li>在紫外线消毒的层流罩或生物危害柜的工作。 </li><li>叶放置到一个以前蒸压春茶输液的网格球。 </li><li>浸入含有的叶子70％（体积:体积）茶输液乙醇在一个CULTURE壶30秒。 注:有关此过程中的所有步骤使用250毫升螺钉蒸压帽聚碳酸酯文化盆。 </li><li>漏，然后转移并浸入叶组织在1:8（体积比）稀释漂白剂（0.5％氯）10分钟。轻轻晃动，不时。 </li><li>沥干漂白剂和文化火锅含有灭菌去离子蒸馏水转移茶包。轻轻旋转，沥干多余的水分。重复一次用新的文化锅灭菌去离子蒸馏水。 </li><li>取出树叶从茶包用无菌镊子到一个新的文化锅灭菌去离子蒸馏水。拧上瓶盖的无菌及反相和漩冲洗。离开叶浮在无菌水，直到准备准备植。 </li></ol></li><li>准备和电镀植<ol><li>利用无菌操作技术，从修剪用手术刀边缘个别消毒传单，切剩下的长方形分成两个或THRee值矩形外植体（8-10 x 3-5毫米）与中间静脉中每个外植体（ 图5A）的中心。 注:外植体的大小是在发起第一愈合组织相当重要。进行切割无菌盖从外卖食品容器。 </li><li>板6的外植体在直径9厘米的培养皿（ 图5B）的琼脂固化的培养基中。板背面植一面朝下，保持平常叶的方向。 </li><li>密封围绕盘拉伸培养皿用Parafilm（ 图5C）。该组织正准备孵化。 </li></ol></li><li>组织孵化<ol><li>在整个培养期间的控制温度的房间或生长柜孵育在黑暗中的板在28℃。 注:植将启动分化，进行细胞分裂，增殖（骨痂形成）和胚胎（差异化）。 </li><li>亚文化差异expla3周后NTS到新鲜培养基。在此传代培养转用无菌不锈钢刮刀和镊子的整个外植体，在层流罩或生物危害柜。作为愈合组织发生和愈伤组织超过最大的一个在图5C的尺寸，传代培养至新鲜的培养基中，当个体的愈伤组织转移50％。 </li><li>观察第一胚在大约4周。虽然有一定程度的同步性，在收集一个星期4-8潜伏期不同胚胎阶段（ 图6A，B）。根据实验目标解剖显微镜和进程所收集。 </li><li>传代培养每3周，并定期取出胚胎，使外植体将继续产生的胚胎为3个月。 </li></ol></li></ol>

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The authors declare that they have no competing interests.

所描述的协议是相对简单的，让豆类胚胎与所有的现代细胞和分子技术调查。我们认识到，有优势和在体内和体外方法的缺点。两者都允许更专注于早期胚胎发育比较成熟的种子19的文化。 在体内研究的情况下，所描述主要是从它适合于许多胚胎研究荚果胚珠的分离。它当然可以通过切片关闭"钩"区（ 图3B）中以进一步富集胎儿组织。它难以分离胚胎发育的非常早的阶段作为disection期间胚胎常常损失，因为它不能很好地附着在相邻组织，使得利用胚珠有利的。采用吊舱形态消除了鲜花的标签，并具有时间Course制度每朵花。此外，还有潜在的变异性发育时间的方法，除非环境非常严格的控制。使结荚不胁迫条件下发生的最佳植物的生长是很重要的。熟悉荚果发育和轻微的调整可以定义形态阶段，以匹配特定的胚胎发育阶段。 在体细胞胚胎发生的情况下，它必须被认识到，将胚无性而不是来自于一个受精卵。另外，前述营养源是不同的合子胚。在情况下，体细胞胚，这是培养基中和周围的愈伤组织，并在所述合子胚的情况下，它是胚乳。这意味着柄好得多开发的合子胚胎的情况下（ 图4A，B）。 分枝的特定值蒺藜系统是响应于所述异常的4性激素大量的胚胎Ë制度。如果不发生大的数字，然后检查构成该激素和其除了所述介质的细节会被检查。与外植体组织的任何培养方法无菌无损伤（进行小的调整，以乙醇和次氯酸钠杀菌时间，如果这是一个问题，定时）与护理一起重要期间所有的制备步骤保持无菌。 我们所发现的是，它是有价值的体内和体外方法来补充。从我们自己的研究的实例是转录因子MtSERF1（体胚相关因子1），它是一种乙烯反应转录因子基因的调查。 MtSERF1首次发现使用RNA干扰，启动子GUS融合和原位hybridisations体细胞胚;然后显示被表达在合子胚20。 豆科植物胚胎发育的研究是用于h重要UMAN营养，并使用M。苜蓿现已提供可以提高这方面的细胞和分子技术结合起来的胚胎。洞察可以得到成如何胚胎大小并因此产量可以与所需要的碳水化合物，脂肪和蛋白质的组合物进行优化。这些因素在很大程度上是坐落在火车早期胚胎发育8,9。

豆类是高等植物有大约20,000种和豆科（豆科或）家族的第三大家族的第二个收获面积和总产量1谷物。大豆是全球第三大种植作物。谷物豆类提供约三分之一的膳食蛋白质和三分之一的植物油用于人类消费2。豆类与他们的N 2固定能力也有助于可持续的农业系统。 苜蓿 ，大豆一样，商店蛋白质和脂肪在其种子的子叶，并具有相当的遗传和基因组资源3,4遗传和基因组豆类模型。虽然M.蒺藜已启用的进步在了解豆科植物-根瘤菌共生4已越来越多地用于研究豆科植物种子生物学5-7和胚胎发育8,9。拟南芥胚胎已被广泛研究10,11，但它，我山非豆科和胚胎的细节不是完全相同紫花8,10。在M.合子胚胎苜蓿有有趣的功能，具有鲜明的多脑垂体，一个endoployploid柄和基底细胞转移8。 体细胞胚胎发生（SE）通常用于再生植物12。在豆科植物模型M.苜蓿 ，种子线Jemalong 2HA（2HA）已经制定了从母公司Jemalong有体细胞胚13的比例很高。产生的胚胎的数量最近已实质上通过增加两个赤霉酸（GA）和脱落酸（ABA）的长期建立的介质14。在这种情况下，GA和ABA协同作用，这是不寻常鉴于GA和ABA平时行事拮抗14。从愈伤组织中产生的胚胎发育的表面，它允许胚胎的阶段容易地确定visuall上y和收获很容易。具有近等基因系是胚（2HA）和非胚（Jemalong）有利于体细胞胚的调查和在体内和体外系统提供了不同的实验的可能性有。 理解胚胎发育的细胞和分子机制对于理解种子和植物发展至关重要。在豆类，如在其他双子叶植物，它是存储了用于人类营养产品胚胎的子叶。早期胚胎发育涉及细胞迅速分裂，并正确胚胎图案。在受精后约8天，M。苜蓿胚胎早期达到子叶阶段。形态特征是不完全的天施肥温室条件后表示。因此，一个高效的标准化的方法来表示胚胎发育的阶段，是研究遗传REGUL有价值通货膨胀的早期胚胎合子。 在本文中，我们提供了两个标准化的协议，收集胚胎发育生物学研究胚胎发育中的豆类模型M.蒺藜 。第一个是，而第二种是通过培养叶片为外植体细胞胚提供轻松访问大量胚胎数胚胎发育关联和POD形态收集合子胚。

<table><tbody><tr><td>P4 medium</td><td>Sigma-Aldrich</td><td></td><td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td></tr><tr><td>Major salts</td><td></td><td></td><td></td></tr><tr><td>Minor salts</td><td></td><td></td><td></td></tr><tr><td>Vitamins</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>Bacto Laboratories</td><td>214010</td><td>Bacto agar</td></tr><tr><td>Plant hormones</td><td></td><td></td><td></td></tr><tr><td>1-Naphthaleneacetic acid</td><td>Sigma-Aldrich</td><td>N0640</td><td>Dissolve in small amount of 1 M NaOH</td></tr><tr><td>Abscisic acid</td><td>Sigma-Aldrich</td><td>A1049</td><td>Dissolve in small amount of 1 M NaOH</td></tr><tr><td>6-Benzylaminopurine</td><td>Sigma-Aldrich</td><td>B3274</td><td>Dissolve in MQ water with heating and few drops 1N HCl</td></tr><tr><td>Gibberellic Acid</td><td>Sigma-Aldrich</td><td>G7645</td><td>Dissolve in small amount of ethanol</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Stereo dissecting microscope</td><td>Leica</td><td>MZFLIII</td><td>Or similar</td></tr><tr><td>Light microscope</td><td>Zeiss</td><td>Axiophot</td><td>Or similar, with suitable optics</td></tr><tr><td>Digital camera</td><td>Zeiss</td><td>AxioCam HRc</td><td>Or similar</td></tr><tr><td>Sterilising leaves</td><td></td><td></td><td></td></tr><tr><td>250 mL screw cap polycarbonate container with polypropylene lid</td><td>SARSTEDT</td><td>75.9922.519</td><td>Autoclavable</td></tr></tbody></table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

早期胚胎发生从单细胞受精卵开始经过快速细胞分裂和形态发生，和形态学特征在于预球状，球状，心脏，鱼雷和子叶阶段。这种渐进的发展是一个下复杂的分子网络的严格监管。在类似发展阶段收获足够早期胚胎是用于研究的早期胚胎发育的细胞和分子调节必需的。这并不简单，因为早期胚胎发育经历快速的形态一会儿例如，8天苜蓿达到早期子叶期。在这里，我们用两种方法解决这个问题。第一个建立在帮助表示合子胚胎阶段的胚胎发育和形态荚之间的联系。这是特别基于荚果螺旋和刺的发展的数量。以补充体内秒的另一种方法案例研究是通过培养叶植产生体细胞胚。该介质包括一个不寻常的激素组合 - 生长素（1-萘乙酸），细胞分裂素（6-苄基氨基嘌呤），脱落酸和赤霉酸。不同阶段可以辨别生长出愈伤组织，而不清扫。

对应于不同胚胎阶段合子胚不同荚结构示于图1A - f当不同胚胎阶段示于图2A - F。在相同阶段选择豆荚，是胚珠样品相当均匀，可以得到（ 图3A）。通过利用RT-qPCR的胚胎特异性基因可容易地检测和时程研究评价9。一些额外的清扫将允许胚胎（ 图3B）的进一步富集。加工用于原位杂交策略可以容易地进行，以及涉及光任何互补研究（ 图4A，B）和电子显微镜8。在这个系统中特定值是垂体和柄（ 图4A，B）的独特性。 对于体细胞胚胎发生的C从个体folioles初始外植体的utting示于图5A。外植体，然后放置背轴面朝上与琼脂（ 图5B）紧密接触。愈伤组织后形成胚开始后3-4周，通过7周有无数的胚胎在子叶阶段（ 图5C）的出现。通过胚胎周4和7之间在独立的阶段之后的板可位于（ 图6A，B）。不同的胚胎阶段可以很容易地观察到，只是摘下进行分析。这可以是一个相当快的方式来获得某些类型的信息。例如图7示出了如何胚胎早期子叶阶段表现出的一种类型的MtOLEOSIN基因，但不另一种表达。感兴趣的基因的时程研究然后可使用合子或体细胞胚检查。在体细胞胚体系的特殊优点是callu的这种转变S可以是进行而不在胚胎发育的不同阶段再生整株植物和基因表达可视化使用的GUS（β葡糖醛酸酶），或荧光标记物。一个例子示于图6C。 <img alt="图1" src="/files/ftp_upload/52635/52635fig1.jpg" /> 图1:布丁形态和胚胎发育阶段 ，在豆荚发展与胚胎分离的阶段不同阶段。阶段2-7所示。 （A）和（B）是非常早期和早期预球状（C）的第4阶段早期球状（D）的第5阶段晚球形（E）级6心脏和（F）级7晚鱼雷阶段2和3。酒吧为2毫米。第一阶段是开花（未示出）。 <img alt="图2" src="/files/ftp_upload/52635/52635fig2.jpg" /> 图2:胚胎发育阶段。 &lt;/在不同发展阶段STRONG&gt;清除胚胎。 （ 一 ）第3阶段的早期预球状，（B）第4阶段早期球状，（C）阶段5晚球形，（D）阶段6心脏，和（E）级7晚鱼雷。酒吧是60微米。 <img alt="图3" src="/files/ftp_upload/52635/52635fig3.jpg" /> 图3:隔离胚珠胚珠与第5阶段的胚胎（A）组。标准复式显微镜下（B）胚珠观察显示胚胎可在"挂钩"到底被切除。酒吧为1毫米（A）和200微米（B）。 <img alt="图4" src="/files/ftp_upload/52635/52635fig4.jpg" /> 图4:通过极早期胚胎显示早期胚胎的形态（a）第荷兰国际集团胚胎适当的（四个单元），垂体（后四个单元），柄（接下来的四个细胞具有大液泡）和关系胚珠组织。 （ 二）通过早期鱼雷期胚（E）与著名的柄（S）组。甲苯胺蓝染色。酒吧是10微米（A）和50微米（B）。 <img alt="图5" src="/files/ftp_upload/52635/52635fig5.jpg" /> 图5:培养外植体，以产生体细胞胚 （A） 的枳叶放映其中来自foliole植体服用。镀上琼脂（B）外植体。 （C）从7周后培养的外植体产生的体细胞胚。酒吧IS10毫米<img alt="图6" src="/files/ftp_upload/52635/52635fig6.jpg" /> 图6:体细胞胚胎阶段，并确定基因expressio的位置N使用转化和GUS。（A）体细胞胚在球形（G），心脏（H）和鱼雷（T）阶段。 （B）的体细胞胚在早期子叶阶段。 （C）的球形阶段的体细胞胚表示为MtWOX9基因在胚胎适当（E）的强GUS表达并降低染色垂体（H）和柄（S）。酒吧（A，B）为100微米。杆（C）是50微米。 <img alt="图7" src="/files/ftp_upload/52635/52635fig7.jpg" /> 图7:使用定量聚合酶链反应（定量PCR）OLEOSIN3（A）和OLEOSIN4（B）在从胚性愈伤组织胚胎早期切除子叶期胚胎的基因表达胚胎史稿基因表达在体外 。表达是相对的牛逼Ø叶。标准误差表示。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 舞台号码 </td><td> 胚胎阶段 </td><td> 结荚期 </td></tr><tr><td>第1阶段</td><td>花开花时</td><td></td></tr><tr><td>第2阶段</td><td>很早就预球形胚</td><td>吊舱带有一个或两个螺旋</td></tr><tr><td>第3阶段</td><td>早期的预球形胚</td><td>荚有三个完整的螺旋</td></tr><tr><td>第4阶段</td><td>早期的球状</td><td>波德有五个完整的螺旋和刺不可见</td></tr><tr><td>第5阶段</td><td>晚球形</td><td>波德六螺旋和不成熟的刺不超过荚宽</td></tr><tr><td>第6阶段</td><td>心脏阶段</td><td>波德六螺旋和拉长的刺成熟荚超过宽度</td></tr><tr><td> 7期</td><td>鱼雷舞台</td><td>波德六螺旋，厚成熟刺更长，增加肚带</td></tr></tbody></table> 表1:收获豆荚阶段的标准中识别对应于定义的胚胎阶段荚阶段使用形态学标准。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 花阶段1（S1） </td><td> 荚阶段2（S2）的 </td><td> 吊舱3阶段（S3） </td><td> 结荚期4（S4） </td><td> 结荚期5（S5） </td><td> 结荚期6（S6） </td><td> 结荚期7（S7） </td></tr><tr><td> 天 </td><td> 0 </td><td> 1.5-2 </td><td> 0.5-1 </td><td> 0.5 </td><td> 0.5-1 </td><td> 1 </td><td> 1-1.5 </td></tr></tbody></table> 表2.时间过程POD集合。从t时间他以前的胚胎发育阶段，显示在天。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> 主要盐/ L </td><td colspan="2"> 微量盐/ L </td><td colspan="2"> 维生素/ L </td></tr><tr><td></td><td> 毫克 </td><td></td><td> 毫克 </td><td></td><td> 毫克 </td></tr><tr><td> KNO 3 </td><td> 1875年</td><td>硫酸锰4•H 2Ø </td><td> 10 </td><td>肌醇</td><td> 100 </td></tr><tr><td>的NH 4 NO 3 </td><td> 600 </td><td> H 3 BO 3 </td><td> 3 </td><td>盐酸硫胺</td><td> 10 </td></tr><tr><td> 磷酸二氢钾 </td><td> 131 </td><td>硫酸锌4•7H 2 O </td><td> 2 </td><td>烟酸</td><td> 1 </td></tr><tr><td> KCL </td><td> 225 </td> &lt;TD&gt; KI </td><td> 0.75 </td><td>盐酸吡哆醇</td><td> 1 </td></tr><tr><td>硫酸镁4•7H 2 O </td><td> 225 </td><td>娜2的MoO 4•2H 2 O </td><td> 0.25 </td><td></td><td></td></tr><tr><td colspan="2"> 单独的钙 </td><td>硫酸铜4•5H 2 O </td><td> 0.025 </td><td></td><td></td></tr><tr><td>氯化钙2•2H 2 O </td><td> 300 </td><td>氯化钴2•6H 2 O </td><td> 0.025 </td><td></td><td></td></tr><tr><td></td><td></td><td colspan="2"> 螯合铁分离 </td><td></td><td></td></tr><tr><td></td><td></td><td>硫酸亚铁4•7H 2 O </td><td> 9.267 </td><td></td><td></td></tr><tr><td></td><td></td><td>的Na 2 EDTA•2H 2 O </td><td> 37.2 </td><td></td><td&gt; </td></tr></tbody></table> 表3:P4介质 P4的介质的组分。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 零件 </td><td> 量/ L </td></tr><tr><td> 10×各大盐</td><td>百毫升</td></tr><tr><td> 100×钙</td><td> 10毫升</td></tr><tr><td> 1000×微量盐</td><td> 1毫升</td></tr><tr><td> 200×铁</td><td> 5毫升</td></tr><tr><td> 100×酪蛋白氨基酸</td><td> 10毫升</td></tr><tr><td> 1000×维生素</td><td> 1毫升</td></tr><tr><td>蔗糖</td><td> 30克</td></tr><tr><td>洋菜</td><td> 8克</td></tr><tr><td> 激素 </td><td></td></tr><tr><td> 1000μMNAA </td><td> 10毫升</td></tr><tr><td> 1000μMBAP </td><td> 4毫升</td></tr><tr><td> 1000μMABA </td><td> 1毫升</td> &lt;/ TR&gt; <tr><td> 1000μMGA </td><td> 5毫升</td></tr></tbody></table> 表4:制作了含激素的P4媒体制作与现货的解决方案激素P4媒体。

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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Nanyang Technological University

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Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

13.9K 次观看.  University of Newcastle, Callaghan, New South Wales, Australia. The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided. 

视频: Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

科研

遗传学

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

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科

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此视频中的章节

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

表征在拟南芥胚方法

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

从小鼠胚胎植入前胚胎干细胞系的推导

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

一种简单有效的斑马鱼胚胎移植装置

Preparation of Single-Somite Explants from Zebrafish Embryos

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束