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.     

The molecular biology of self-incompatibility systems in flowering plants

Self-incompatibility is a common mechanism by which flowering plants can exert some control over the process of fertilization. Typically, the self-incompatibility response involves the recognition and rejection of self-incompatible pollen which leads to a block in self-fertilization and, as a consequence, promotes outcrossing. In recent years, considerable progress has been made in the molecular understanding of several self-incompatibility systems. Interestingly, a common mechanism for self-incompatibility is not employed by all flowering plants, but in fact quite diverse mechanisms have been recruited for the rejection of self-incompatible pollen. In this review, the recent advances in the self-incompatibility systems of the Solanaceae, Papaveraceae, and Brassicaceae will be described as well as some of the molecular work that is emerging for the Poaceae and the heteromorphic self-incompatibility systems.     

A novel in vitro system for gamete fusion in maize

Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of plant gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. In this study, we developed a bovine serum albumin （BSA）-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell and sperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG system, and it will be especially useful for the in vitro study of the calcium dynamics during plant fertilization.     

In vitro double fertilization in Nicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy

In vitro double fertilization in tobacco was carried out with attention to fusion behavior and gamete interaction. Structural and cytological events indicating possible reaction to the fusion of sperm-egg and especially sperm-central cell were recorded by video-enhanced microscopy. Generative cells were fused with the egg cell or central cell as a control system to better understand gamete interaction. As early as adherence of the male cell, the female cell showed response by means of cytoplasm strand formation. After gamete fusion, cytoplasm activation in the egg cell was observed as long distance movement of organelles. In fertilized central cells, however, fusion did not result in notable cytological change within 30 min. Male nuclear movement recorded in the female cell illustrated two different patterns of movement which showed similarity to organelle movement. The dynamics of male and female nuclear fusion after in vitro fertilization was also recorded in the central cell. It revealed that the fusion process requires only a few seconds and is similar to that of gamete fusion in vitro. This may offer a new clue for understanding how female and male nuclei attract, adhere and finally fuse each other. Received: 13 October 1999 / Revision accepted: 6 December 1999     

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。     

From in vitro fertilization to early embryogenesis in maize

Summary The development of in vitro fertilization methods in plants, the characterization of developmental mutants, and the adaptation of molecular biology techniques to construct cDNA libraries from minute samples, all represent important recent technical break-throughs. They allow the study of fertilization and early embryogenesis at a molecular level and considerable improvement in the under-standing of higher plant reproduction can be predicted over the next few years. Important biological questions, such as polyspermy, gamete fusion physiology, asymmetrical cell division, embryo axis formation, can now be addressed experimentally in maize, which appears as a major study model in this area.Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Inositol 1,4,5-trisphosphate as fertilization signal in plants: testcase Chlamydomonas eugametos

Within the first 2 h of sexual reproduction, gametes of the green alga Chlamydomonas eugametos agglutinate, fuse via their mating structures, de-agglutinate and swim off as vis-à-vis pairs. During this period, increases in intracellular inositol 1,4,5-trisphosphate levels and changes in polyphosphoinositide synthesis were associated with cell fusion. The protein-kinase-C inhibitor staurosporine (0.1–0.2 M) inhibited the de-agglutination of pairs and therefore prevented them swimming away, while earlier stages of mating such as agglutination or cell fusion were unaffected. The results suggest that inositol 1,4,5-trisphosphate and diacylglycerol are fertilization signals in C. eugametos. The idea that they could also be fertilization signals in higher plants is discussed in relation to in vitro embryogenesis.Abbreviations DAG diacylglycerol - InsP₃ inositol 1,4,5-trisphosphate - mt⁺/mt^– mating-type plus or minus - PKC protein kinase C - PtdOH phosphatidic acid - PtdInsP phosphatidylinositol - 4-phosphate PtdInsP₂ phosphatidylinositol 4,5-bisphosphate     

Micromanipulation of male and female gametes ofNicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture

This research is part of an attempt to establish an in vitro fertilization system in tobacco to aid in understanding mechanisms of fertilization. Fusions of isolated male and female gametes were induced in a polyethylene glycol solution. Fusion appears similar to that in maize. One nuclear division of both an unfertilized egg cell and a synergid was induced in KM8p medium with 1 mg/l 2,4-dichlorophenoxyacetic acid in a microchamber culture; one cellular division of the egg cell was also induced in the same medium in solid-drop culture. The osmolality of suspension culture feeder cells was critical for the development of these cells. These results indicate that in vitro fertilization is possible in tobacco, which would be the first such system in dicots.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - PEG Polyethylene glycol     

Identification and characterization of segregation distortion loci along chromosome 5B in tetraploid wheat

Segregation distortion genes are widespread in plants and animals and function by their effect on competition among gametes for preferential fertilization. In this study, we evaluated the segregation distortion of molecular markers in multiple reciprocal backcross populations derived from unique cytogenetic stocks involving the durum cultivar Langdon (LDN) and wild emmer accessions that allowed us to study the effects of chromosome 5B in isolation. No segregation distortion of female gametes was observed, but three populations developed to analyze segregation of male gametes had genomic regions containing markers with skewed segregation ratios. One region of distortion was due to preferential transmission of LDN alleles over wild emmer alleles through male gametes. Another region required the presence of LDN 5B chromosomes in the female for preferential fertilization by male gametes harboring LDN alleles indicating that the corresponding genes in the female gametes can govern genes affecting segregation distortion of male gametes. A third region of distortion was the result of preferential transmission of wild emmer alleles over LDN alleles through male gametes. These results indicate the existence of different distorter/meiotic drive elements among different genotypes and show that distortion factors along wheat chromosome 5B differ in chromosomal location as well as underlying mechanisms.     

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.     

Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization

The aim of this study was to determine the influence of hamster oviductal glycoprotein (Oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5-diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus-free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona-free oocytes, indicating that sperm-zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin sperm-zona interaction was reduced when cumulus-enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin-sperm complex and maintaining the fertilizing ability. © Wiley-Liss, Inc.     

Gametophytic self-incompatibility: contrasting mechanisms for Nicotiana and Papaver

Since Darwin first noted that not all plants produce self-seed, several mechanisms that regulate the acceptance or rejection of pollen during fertilization have been recognized, of which self-incompatibility (SI) is the most widespread. Over the past few years much progress has been made in understanding the molecular and cellular processes involved in SI. Here we review recent studies of the SI systems of Nicotiana alata and Papaver rhoeas. The SI systems are both determined by a single, multi-allelic gametophytically controlled S-gene, but involve quite different mechanisms.     

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。     

Cell-cell membrane fusion during mammalian fertilization

The mechanism of sperm-egg fusion in mammals is a research area that has greatly benefited from the use of gene deletion technology. Because fertilization is internal in mammals and the gametes (particularly the eggs) are sparse in number, in vitro studies have considerable limitations. Using gene deletions, a few cell surface proteins in both gametes have been identified as essential for gamete fusion. Ongoing studies are directed at analysis of the function of these proteins and the search for additional proteins that may be involved in this process. So far, no mammalian proteins have been found that also function in sperm-egg fusion of non-mammalian species or in other types of cell-cell fusion.     

Boron translocation in coffee trees

Boron deficiency in coffee trees (Coffea arabica) is widespread, however, responses to B fertilizer have been erratic, depending on the year, method, and time of application. A better understanding of B uptake, distribution, and remobilization within the plant is important in developing a rational fertilization program. Field and greenhouse experiments were conducted to study B distribution and remobilization in coffee trees. Boron was provided either in the nutrient solution or sprayed on the leaves of trees grown under adequate or transient B deficiency. There was clear evidence for B translocation via symplast (remobilization) to coffee grains, even in well-nourished plants. When ¹⁰B was present in the nutrient solution during most part of fruit filling, from 33 to 40% of the B found in coffee fruits was absorbed during this period, depending on the timing and duration of the B deficiency treatment. In the field, when B was sprayed once on the leaves, around 4% of the fruit B was derived from the foliar fertilizer. Boron remobilization within coffee trees is limited in well nourished plants, but it can be significant during periods of temporary B deficiency in plants otherwise well nourished with B. The implications of these findings for B fertilization practice, are discussed.