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>

<ol>
	<li>Gepts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legumes+as+a+model+plant+family.+Genomics+for+food+and+feed+report+of+the+cross-legume+advances+through+genomics+conference.">Legumes as a model plant family. Genomics for food and feed report of the cross-legume advances through genomics conference.</a> Plant Physiol. 137 (4), 1228-1235 (2005).</li><li>Graham, P. H., Vance, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legumes:+importance+and+constraints+to+greater+use.">Legumes: importance and constraints to greater use.</a> Plant Physiol. 131 (3), 872-877 (2003).</li><li>Young, N. D., Udvardi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+Medicago+truncatula+genomics+to+crop+legumes.">Translating Medicago truncatula genomics to crop legumes.</a> Curr. Opin. Plant Biol. 12 (2), 193-201 (2009).</li><li>Young, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+genome+provides+insights+into+the+evolution+of+rhizobial+symbioses.">The Medicago genome provides insights into the evolution of rhizobial symbioses.</a> Nature. 480 (7378), 520-524 (2011).</li><li>Gallardo, K., Le Signor, C., Vandekerckhove, J., Thompson, R. D., Burstin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+Medicago+truncatula+seed+development+establishes+the+time+frame+of+diverse+metabolic+processes+related+to+reserve+accumulation.">Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation.</a> Plant Physiol. 133 (2), 664-682 (2003).</li><li>Verdier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+M.+truncatula+transcription+factors+identifies+putative+regulators+of+grain+legume+seed+filling.">Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling.</a> Plant Mol. Biol. 67 (6), 567-580 (2008).</li><li>Thompson, R., Burstin, J., Gallardo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-genomic+studies+of+developmental+processes+in+legume+seeds.">Post-genomic studies of developmental processes in legume seeds.</a> Plant Physiol. 151 (3), 1023-1029 (2009).</li><li>Wang, X. -. D., Song, Y., Sheahan, M. B., Garg, M. L., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+embryo+sac+to+oil+and+protein+bodies:+embryo+development+in+the+model+legume+Medicago+truncatula.">From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.</a> New Phytol. 193 (2), 327-338 (2012).</li><li>Kurdyukov, S., Song, Y., Sheahan, M. B., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+early+embryo+development+in+the+model+legume+Medicago+truncatula.">Transcriptional regulation of early embryo development in the model legume Medicago truncatula.</a> Plant Cell Rep. 33 (2), 349-362 (2014).</li><li>Mansfield, S. G., Briarty, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+Arabidopsis+thaliana.+2.+The+developing+embryo.">Early embryogenesis in Arabidopsis thaliana. 2. The developing embryo.</a> Can. J. Botany. 69 (3), 461-476 (1991).</li><li>Seefried, W. F., Willman, M. R., Clausen, R. L., Jenik, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+regulation+of+embryonic+patterning+in+Arabidopsis+by+microRNAs.">Global regulation of embryonic patterning in Arabidopsis by microRNAs.</a> Plant Physiol. 165 (2), 670-687 (2014).</li><li>Birnbaum, K. D., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slicing+across+kingdoms:+regeneration+in+plants+and+animals.">Slicing across kingdoms: regeneration in plants and animals.</a> Cell. 132 (4), 697-710 (2008).</li><li>Rose, R. J., Nolan, K. E., Bicego, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+highly+regenerable+seed+line+Jemalong+2HA+for+transformation+of+Medicagotruncatula+-+implications+for+regenerability+via+somatic+embryogenesis.">The development of the highly regenerable seed line Jemalong 2HA for transformation of Medicagotruncatula - implications for regenerability via somatic embryogenesis.</a> J. Plant Physiol. 155 (6), 788-791 (1999).</li><li>Nolan, K. E., Song, Y., Liao, S., Saeed, N., Zhang, X., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unusual+ABA+and+GA+synergism+increases+somatic+embryogenesis,+facilitates+its+genetic+analysis+and+improves+transformation+in+Medicago+truncatula.">An unusual ABA and GA synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.</a> PloS ONE. 9 (6), e99908 (2014).</li><li>Liu, C. M., Meinke, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+titan+mutants+of+Arabidopsis+are+disrupted+in+mitosis+and+cell+cycle+control+during+seed+development.">The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development.</a> Plant J. 16 (1), 21-31 (1998).</li><li>Nolan, K. E., Kurdyukov, S., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+SOMATIC+EMBRYOGENESIS+RECEPTOR-LIKE+KINASE+1+(SERK1)+gene+is+associated+with+developmental+change+in+the+life+cycle+of+the+model+legume+Medicago+truncatula.">Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula.</a> J. Exp. Bot. 60 (6), 1759-1771 (2009).</li><li>Iantcheva, A., Vlahova, M., Atanassov, A., Mathesius, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+embryogenesis+from+leaf+explants.">Somatic embryogenesis from leaf explants.</a> The Medicago truncatula handbook. , (2006).</li><li>Thomas, M. R., Johnson, L. B., White, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+interspecific+somatic+hybrids+of+Medicago+by+using+Agrobacterium+transformed+tissues.">Selection of interspecific somatic hybrids of Medicago by using Agrobacterium transformed tissues.</a> Plant Sci. 69 (2), 189-198 (1990).</li><li>Ochatt, S. J., Thorpe, T. A., Yeung, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+seeds+and+embryos+of+Medicago+truncatula+cultured+in+vitro.">Immature seeds and embryos of Medicago truncatula cultured in vitro.</a> Plant Embryo Culture: Methodsand Protocols, Methods in Molecular Biology. 710, 39-52 (2011).</li><li>Mantiri, F. R., Kurdyukov, S., Lohar, D. P., Sharopova, N., Saeed, N. A., Wang, X. D., VandenBosch, K. A., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+MtSERF1+of+the+ERF+subfamily+identified+by+transcriptional+profiling+is+required+for+somatic+embryogenesis+induced+by+auxin+plus+cytokinin+in+Medicago+truncatula.">The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.</a> Plant Physiol. 146 (4), 1622-1636 (2008).</li></ol>

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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

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 ...

protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Research

JoVE Journal

Developmental Biology

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

13.8K 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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

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 ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face 用于小鼠胚胎平面形态发生分析的心内膜垫制备

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...