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>苜蓿</em
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.
Materials
| P4 medium | Sigma-Aldrich | Use Sigma-Aldrich Chemicals or other analytical grade supplier | |
| Major salts | |||
| Minor salts | |||
| Vitamins | |||
| Agar | Bacto Laboratories | 214010 | Bacto agar |
| Plant hormones | |||
| 1-Naphthaleneacetic acid | Sigma-Aldrich | N0640 | Dissolve in small amount of 1 M NaOH |
| Abscisic acid | Sigma-Aldrich | A1049 | Dissolve in small amount of 1 M NaOH |
| 6-Benzylaminopurine | Sigma-Aldrich | B3274 | Dissolve in MQ water with heating and few drops 1N HCl |
| Gibberellic Acid | Sigma-Aldrich | G7645 | Dissolve in small amount of ethanol |
| Equipment | |||
| Stereo dissecting microscope | Leica | MZFLIII | Or similar |
| Light microscope | Zeiss | Axiophot | Or similar, with suitable optics |
| Digital camera | Zeiss | AxioCam HRc | Or similar |
| Sterilising leaves | |||
| 250 mL screw cap polycarbonate container with polypropylene lid | SARSTEDT | 75.9922.519 | Autoclavable |
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