Department of Plant and Microbial Biology and Zurich-Basel Plant Science Center, University of Zurich.
Department of Plant and Microbial Biology and Zurich-Basel Plant Science Center, University of Zurich;
J Vis Exp. 2023 Feb 24(192). doi: 10.3791/65156.
In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo. A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is often reported, which severely confounds such analyses. Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.
在开花植物中，花粉管（雄性配子体）在雌蕊内的生长和引导以及花粉管被雌性配子体接收，对于双受精和随后的种子发育是必不可少的。在花粉管接收过程中，雌雄配子体之间的相互作用最终导致花粉管破裂，释放出两个精子细胞，从而实现双受精。由于花粉管的生长和双受精深深地隐藏在花的组织中，因此在体内观察这个过程是很困难的。已经开发并实施了一种半离体（SIV）方法，用于对拟南芥等模式植物的受精进行活细胞成像研究。这些研究有助于阐明受精过程在开花植物中发生的基本特征，以及雌雄配子体相互作用过程中发生的细胞和分子变化。然而，由于这些活细胞成像实验涉及到单个胚珠的切除，它们在每个成像会话中只能进行有限数量的观察，因此这种方法繁琐且非常耗时。除了其他技术困难之外，体外花粉管受精失败的情况经常被报道，这严重干扰了此类分析。在这里，提供了一个详细的视频协议，用于以自动化和高通量的方式对花粉管接收和受精进行成像，每个成像会话最多可以观察 40 次花粉管接收和破裂。结合使用遗传编码的生物传感器和标记线，这种方法可以在减少时间投入的情况下生成更大的样本量。该技术的细微差别和关键点，包括花的分期、解剖、培养基的准备和成像，都以视频格式清楚地详细说明，以促进花粉管导向、接收和双受精动力学的未来研究。
