离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。     

被子植物受精作用的分子和细胞生物学机制

被子植物双受精包括精-卵、精子-中央细胞两个融合过程。由于双受精深藏于母体组织中进行, 长期以来一直是植物有性生殖研究中的难点。近年来, 随着各种植物配子体cDNA文库的构建, 各种离体研究系统的建立和突变体分析的兴起, 极大地推动了被子植物受精作用研究的快速发展, 增进了人们对被子植物受精过程的分子和细胞生物学机制的深入了解。本文着重讨论受精作用的若干重要发育事件, 包括受精前卵器细胞对花粉管向胚珠定向生长的近距离引导信号, 精子的靶向运动,精、卵细胞相互作用和配子融合后卵细胞的激活与中央细胞发育的启动等。     

被子植物受精作用的分子和细胞生物学机制

被子植物双受精包括精-卵、精子-中央细胞两个融合过程。由于双受精深藏于母体组织中进行,长期以来一直是植物有性生殖研究中的难点。近年来,随着各种植物配子体cDNA文库的构建,各种离体研究系统的建立和突变体分析的兴起,极大地推动了被子植物受精作用研究的快速发展,增进了人们对被子植物受精过程的分子和细胞生物学机制的深入了解。本文着重讨论受精作用的若干重要发育事件,包括受精前卵器细胞对花粉管向胚珠定向生长的近距离引导信号,精子的靶向运动,精、卵细胞相互作用和配子融合后卵细胞的激活与中央细胞发育的启动等。     

阿拉伯半乳糖蛋白在被子植物有性生殖中的作用

阿拉伯半乳糖蛋白(arabinogalactan-proteins,AGPs)是一类主要分布在细胞表面和胞外基质中的糖蛋白.它们在植物的雄性器官(花粉、花粉管、精细胞)、雌性器官(柱头、花柱、子房)和胚胎(合子胚和体细胞胚)等组织和细胞中均有大量的表达.大量研究表明AGPs在被子植物有性生殖过程中起着非常重要的作用,既可能参与花粉管粘附、营养、传导或提供信号的作用,也可能参与受精过程中配子识别和受精后胚胎的发育与分化等过程.该文就其分子结构、特性以及在植物有性生殖过程中各种器官和组织内的表达和功能研究进展做了较为全面的概述.     

被子植物的离体受精与早期胚胎发生

就近年来有关被子植物的离体受精、合子的离体发育以及受精与早期胚胎发育过程中的基因表达的研究情况作了介绍。     

高等植物离体受精研究进展

高等植物的卵细胞深藏在子房内的胚珠体细胞组织中,形成了对高等植物受精过程研究的技术障碍。以前采用超微结构观察研究受精过程已取得了一定的结果,但用固定切片技术研究受精机理需将卵细胞杀死,并且不能进行定点追踪观察。将高等植物的精、卵细胞分离出来在体外诱导其融合的离体受精技术可在很大程度上克服这些技术障碍,对雌、雄配子的识别和融合,合子开始胚胎发生等一系列的受精和胚胎发生机理进行研究。分离的雌、雄配子及合子使应用分子生物学方法研究这些细胞的结构和功能成为可能。将合子的二倍性和胚胎发生特性与外源DNA转入技术结合起来可使转基因植物研究的后期工作简单化。另外,异种植物离体精、卵细胞融合和杂种合子的培养也是进行远缘杂交的一条有潜力的途径。     

被子植物受精机制的研究进展

被子植物的受精是一个复杂而精巧的过程。花粉管到达子房,通过退化助细胞进入胚囊,释放出两个精细胞。原来在花粉管中相互联结的两个精细胞在退化助细胞中分开,一个与卵细胞融合,另一个与中央细胞融合,完成双受精。目前对双受精过程中有关雌、雄配子识别的机制还知之甚少。本文介绍了目前被子植物精、卵细胞融合前后的细胞周期变化、退化助细胞的功能、精细胞在退化助细胞中迁移的研究动态、精细胞的倾向受精和卵细胞的激活等被子植物受精生物学领域中的一些新的研究成果和发展趋势。     

被子植物的受精识别与糖蛋白

被子植物的受精识别与糖蛋白＊徐恒平＊＊曹宗巽（北京大学生命科学学院,１００８７１,北京关键词被子植物体外受精受精识别糖蛋白近年来,随着发育生物学的迅猛发展［１］,植物生殖生物学也成为一门国际性的热门学科［２,３］。由于被子植物与人类生存息息相关,研究其雌雄配子的识别机理及受精过程,自然是该学科的重点之一。该研究属于跨学科的国际前沿课题,对于阐明植物生殖过程的奥秘与服务农业遗传育种,具有理论与...     

禾本科植物离体受精技术

禾本科植物包含了世界上最重要的农作物,对其受精过程的机理研究一直是发育生物学的热点和难点.离体受精技术是将禾本科植物的精、卵细胞分离出来后使二者融合,并培养成完整植株,从而探讨雌雄配子间的相互识别及由合子开始的胚胎发生等一系列生殖生物学的机理问题.同时,离体受精技术在禾本科植物上的应用,还可将外源DNA转入卵细胞、合子培育新型转基因作物,也可将异种植物的配子进行融合,为远缘杂交培育新品种创造新途径.     

颈卵器植物颈细胞结构与功能的研究进展

颈卵器是苔藓、蕨类、裸子植物的雌性生殖器官,由卵细胞、腹沟细胞和颈细胞(颈沟细胞)构成。其中,颈细胞是这类植物雄配子进入颈卵器并完成受精作用的唯一通道,在颈卵器的发育和受精过程中发挥着重要作用。该文就近年来国内外有关对颈细胞的发生和发育、结构特点和功能等进行较为系统全面的分析和总结,并对未来的研究方向进行展望。     

Epigenetic reprogramming in plant reproductive lineages

Monoecious flowering plants produce both microgametophytes (pollen) and megagametophytes (embryo sacs) containing the male and female gametes, respectively, which participate in double fertilization. Much is known about cellular and developmental processes giving rise to these reproductive structures and the formation of gametes. However, little is known about the role played by changes in the epigenome in dynamically shaping these defining events during plant sexual reproduction. This has in part been hampered by the inaccessibility of these structures-especially the female gametes, which are embedded within the female reproductive tissues of the plant sporophyte. However, with the recent development of new cellular isolation technologies that can be coupled to next-generation sequencing, a new wave of epigenomic studies indicate that an intricate epigenetic regulation takes place during the formation of male and female reproductive lineages. In this mini review, we assess the fast growing body of evidence for the epigenetic regulation of the developmental fate and function of plant gametes. We describe how small interfereing RNAs and DNA methylation machinery play a part in setting up unique epigenetic landscapes in different gametes, which may be responsible for their different fates and functions during fertilization. Collectively these studies will shed light on the dynamic epigenomic landscape of plant gametes or 'epigametes' and help to answer important unresolved questions on the sexual reproduction of flowering plants, especially those underpinning the formation of two products of fertilization, the embryo and the endosperm.     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

In vitro fertilization: analysis of early post-fertilization development using cytological and molecular techniques

Methods have been developed which enable us to obtain in vitro fusion of pairs of sperm and egg cells, and sperm and central cells of angiosperms. Cultured products of such cell fusions develop progressively into zygotes, embryos and fertile plants, and endosperm, respectively. In vitro fusion of isolated gametes allows precisely timed examination of the earliest developmental processes following fertilization. When cultured, in vitro produced zygotes and primary endosperm cells organize themselves independently, and without any requirement for supporting tissues. This technology thus constitutes a unique model system for studies of early stages of zygotic embryogenesis and endosperm development. Following the adaptation of molecular techniques for use with only a few cells, it has proved possible to investigate developmental processes in these systems. This review describes the successful combination of molecular techniques with in vitro fertilization methods, and highlights results obtained with small numbers of reproductive cells isolated by microdissection.     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.