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。     

Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae. Received: 12 February 1999 / Revision accepted: 24 May 1999     

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.     

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。     

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.     

Generation and characterization of alpha-chymase-Cre transgenic mice

The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

Molecular mapping,phenotypic expression and geographical distribution of genes determining anthocyanin pigmentation of coleoptiles in wheat ( Triticum aestivumL.)

Three major gene loci determining the anthocyanin pigmentation of coleoptiles were mapped on the short arms of chromosomes 7A, 7B and 7D, respectively. All three genes map about 15 to 20 cM distal from the centromere and, therefore, it may be concluded that they are members of a homoeologous series and should be designated Rc-A1, Rc-B1 and Rc-D1, respectively. Further homoeologous loci exist in Triticum durum, Triticum tauschii, and most probably in Secale cereale and Hordeum vulgare. By analyzing a synthetic×cultivated wheat cross (ITMI mapping population) under different environmental conditions it was shown that the expression of the genes determining anthocyanin pigmentation of the coleoptiles varies. One additional locus was detected on chromosome 4BL. Beside the mapping data, results of a screening for red coleoptile color genes in 468 mainly European wheat varieties are presented. Received: 2 July 2001 / Accepted: 6 August 2001     

A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum

The investigation of plant cytochrome P450 genes and enzymes is a field of growing interest. Apparently, an even greater diversity of cytochrome P450 genes exists in plants in comparison to other eukaryotes. This may be due to their role in the biosynthesis of secondary metabolites that are present in plants in an enormous variety. Most cloning approaches are hampered by the large sequence diversity of plant cytochrome P450 genes. We present a method to clone divergent cytochrome P450 ESTs by a nested RT-PCR-strategy. These ESTs were used for the subsequent cloning of the corresponding full-size cDNAs of divergent families via cDNA-library screening. Sixteen cytochrome P450 genes belonging to different cytochrome P450-families have been identified in this way, proving the efficacy of the strategy. Received: 1 December 2000 / Accepted: 26 February 2001     

PCR-mediated recombination in amplification products derived from polyploid cotton

PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirement of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more divergent genomes (”homoeologues”) in a common nucleus, intergenic chimeras can form during the PCR amplification of any gene. Here we report a high frequency of PCR-induced recombination for four low-copy genes from allotetraploid cotton (Gossypium hirsutum). Amplification products from these genes (Myb3, Myb5, G1262 and CesA1) range in length from 860 to 4,050 bp. Intergenomic recombinants were formed frequently, accounting for 23 of the 74 (31.1%) amplicons evaluated, with the frequency of recombination in individual reactions ranging from 0% to approximately 89%. Inspection of the putative recombination zones failed to reveal sequence-specific attributes that promote recombination. The high levels of observed in vitro recombination indicate that the tacit assumption of exclusive amplification of target templates may often be violated, particularly from polyploid genomes. This conclusion has profound implications for population and evolutionary genetic studies, where unrecognized artifactually recombinant molecules may bias results or alter interpretations. Received: 28 February 2001 / Accepted: 8 June 2001     

Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world. Received: 22 March 1999 / Accepted: 6 December 1999     

Attraction and transport of male gametes for fertilization

 Two capabilities are critical in attracting and transporting male gametes for fertilization: (1) the pollen tube must locate, enter and discharge its contents at the correct site within the female gametophyte, and (2) once inside the embryo sac, the non-motile male gametes must be transported to the egg and the central cells for double fertilization. This review summarizes current information about evidence for communication between embryo sac and pollen tube and the means by which the non-motile male gametes are transported from the aperture of the pollen tube to the site of gamete fusion. Received: 6 June 1996 / Revision accepted: 9 July 1996     

Polymorphism for interspecific hybridisation within a population of wild radish (Raphanus raphanistrum) pollinated by oilseed rape (Brassica napus)

The within-population polymorphism of wild radish (Raphanus raphanistrum) for interspecific hybridisation with two cultivars of oilseed rape (Brassica napus) was investigated by hand crossing experiments and fluorescence microscopy. Wide variability among plants was observed in the ability of oilseed rape pollen to germinate on the wild radish stigma; the frequency of pistils showing pollen tubes ranged from 0 to 1, depending on the female plant. The ratio of fertilised ovules to the total number of ovules in ovaries where pollen tubes arrived ranged from 0.02 to 0.51. Overall, the results provide evidence for the presence of different phenotypes. In 40% of the plants, pistils had no or very few pollen tubes and few fertilised ovules. In 23%, the foreign pollen tubes grew through the style towards the ovary, but had low ovule fertilisation efficiency. The remaining 37% showed a large number of pollen tubes in the style and frequent ovule fertilisation, and two plants showed no difference between foreign and conspecific pollen. With regard to post-zygotic barriers, pollen germination and ovule fertilisation represent minor barriers to interspecific hybridisation between oilseed rape and wild radish. It is suggested that the effectiveness of these barriers could be improved through plant breeding; this could reduce the risk of gene flow from transgenic oilseed rape to populations of wild relatives. Received: 15 April 2001 / Accepted: 24 May 2001