一个高频率的活体-离体胚胎发生研究系统

通过对受精后烟草(Nicotiana tabacumL.)胚珠的体外培养,建立了一个简便、实用、高频率的胚胎发生研究系统。采用MS培养基附加6%蔗糖,然后换以2%蔗糖对烟草胚珠进行培养,结果受精后胚珠在体外可以完成正常的胚胎发生并可直接萌发成幼苗。追踪观察表明,合子体外胚胎发生过程中关键发育事件,如合子休眠期间的定向生长、一次不对称分裂的完成、胚柄的形成和解体、胚的分化等过程均可在体外重现。体外培养形成的胚珠及胚胎在形态上与自然状态下形成的胚珠及胚胎几乎没有差异。此体系操作简单,稳定性好,且关键发育事件的时空调控与自然胚胎发育进程相吻合。     

Zygote implantation to cultured ovules leads to direct embryogenesis and plant regeneration of wheat

Direct embryogenesis and plant regeneration were obtained by implantation of individual wheat ( Triticum aestivum L.) zygotes into cultured ovules of wheat or barley. The zygotes were isolated mechanically from emasculated spikes, 3–9 h after hand-pollination. In 13 independent experiments, a total of 186 zygotes were implanted into excised ovules obtained from emasculated spikes which had been treated previously with 2,4-dichlorophenoxyacetic acid to induce parthenocarpic, embryoless ovary development. On average, 17.2% of the implanted zygotes gave rise to dorsiventrally differentiated embryos. The embryos resembled those growing in planta with no obvious deviation from the zygotic embryogenesis pathway. In contrast to previously described regeneration systems from individual zygotes of higher plants, this is the first study in which direct embryo formation is reproducibly obtained without intermediate tissue dedifferentiation. Most embryos germinated when transferred to regeneration medium, and later formed phenotypically normal, fully fertile plants. Regenerants were confirmed to be derived from the implanted zygotes by means of AFLP and/or morphological analyses. Although zygote implantation has long been established as a useful method in sexual animal reproduction, an equivalent technique for plants is described here for the first time. Since the zygotes enter the embryogenic pathway directly, the genome is presumably as stable as during embryogenesis in planta . With this new approach, isolated wheat zygotes are accessible to micromanipulation without affecting their subsequent embryonic development.     

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.     

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.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

One hundred years of zygotic embryo culture investigations

Summary Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.     

Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes

Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryogenesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed. Received: 2 August 1996 / Accepted: 4 February 1997     

Differentiation of isolated wheat zygotes into embryos and normal plants

Efficient and reproducible embryo development has been obtained from fertilized wheat (Triticum aestivum L.) egg cells isolated 3–6 h after hand-pollination of emasculated spikes. It is possible to routinely isolate viable zygotes from about 75% of the excised ovaries from cultivars of both winter and spring types. Co-culture with barley microspores which had been stimulated to sporophytic development resulted in embryonic development of the cultivated wheat zygotes. Within 23 h of pollination; the zygotes underwent their first cell division. They proceeded to develop into club-shaped embryos, most of which turned subsequently to dorsiventral differentiation. The morphological patterns of in-vitro-grown embryos were in accordance with those of normal zygotic embryos growing in planta. The formation of twin or multiple embryos originating from a single zygote was dependent on genotype and exogeneously supplied auxin. Upon transfer onto a suitable solidified medium, zygote-derived embryos usually germinated and developed into plants. After optimizing the feeder system, the nutrient medium and the concentration of 2,4-dichloro phenoxyacetic acid (2,4-D), more than 80 and 90% of the zygotes eventually developed into plants in genotypes Florida and Veery #5, respectively. All regenerated plants were morphologically normal and fertile. The in-vitro development from isolated zygotes of a higher-plant species into typically patterned zygotic embryos is shown here for the first time. Since the entire process, including early zygotic development, is now freely accessible to observation and micromanipulation, the method presented opens up new approaches in fundamental as well as applied fields of reproductive biology. Received: 4 September 1997 / Accepted: 28 November 1997     

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Patterns of somatic embryo formation in Siberian larch: embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

Monitoring individual development of isolated wheat zygotes: a novel approach to study early embryogenesis

Summary A culture method has been established by which development of isolated wheat (Triticum aestivum L.) zygotes can be monitored individually until formation of multicellular structures. As was shown recently, these isolated zygotes have a high capacity to form differentiated embryos and normal plants, and thus constitute a suitable object to study early embryogenesis. After being isolated within 6 h after pollination (hap), zygotes were immobilized in an agarose droplet directly on a microscopic chamber slide, which allows for both subsequent development through co-culture with feeder aggregates, as well as detailed observation and photographic documentation of individiual behavior. Shortly after fertilization, the wheat zygote, like the unfertilized egg cell, is characterized by one conspicuous nucleolus. Typically, a second and a third nucleolus appeared between 5 and 8.5 hap. Between 7 and 15 hap, we observed nucleolar vacuolation indicating enhanced ribosomal activity. Continuous cell expansion with slight cell elongation was detected until around 15 hap, followed by a period of transitory reduction in cell volume which roughly corresponded with mitosis. Mitotic prophase of a zygote could easily be detected by the disappearance of all nucleoli within a few minutes. The division plane was generally established perpendicular to the formerly established cell elongation axis. At cytokinesis, which was completed by 19 hap in 90% of the individuals observed, 2 or 3 nucleoli were detected again per daughter cell. The first cell division, including the establishment of a cleavage furrow with intercellular spaces, was completed in all cases within 23 hap. Since this result is in accordance with what is known from earlier studies based upon fixed material, and since the zygotes subsequently continue embryogenesis, in vitro development is assumed to be analogous to that in planta. This experimental system constitutes a valuable experimental tool for further detailed research, both at the cellular and at the molecular level.Abbreviations hap hours after pollination - NOR nucleolus-organizing region     

In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture

Ovules of the wheat breeding line Veery #5 were excised and transferred to culture within 24 h after pollination. When ovules were cultured on Phytagel-solidified medium, and the pericarp removed exclusively at the micropylar tip and the abaxial side, zygotes from up to 79.2% of the ovules underwent embryogenesis with the same developmental pattern as found in planta. Embryos from more than 50% of the cultured ovules germinated when transferred to regeneration medium. More than 100 plantlets were randomly chosen for transfer to soil, all of which developed to phenotypically normal and fertile plants. With this system, the entire process of zygotic embryogenesis can be studied using living material. Furthermore, the method could be used as an embryo rescue technique for plant breeding purposes. Received: 17 June 1996 / Revision received: 22 October 1996 / Accepted: 15 December 1996     

Regeneration of fertile wheat (Triticum aestivum) plants from isolated zygotes using wheat microspore culture as nurse cells

A new method for growing isolated wheat zygotes by co-cultivation with microspores isolated from the same species has been developed. Although the mortality of isolated zygotes within 6 h after the transfer into the nurse culture was relatively high (70%) in this method, the majority of the structures which survived developed into fertile plants (61%). Zygotic structures grown in these cultures were morphologically not as regular as embryos grown in planta, but they resembled them closely in cytological properties and developmental pattern.     

Comparative ultrastructural analysis of the in vitro microspore embryoids and in vivo zygotic embryos of wheat as a basis for understanding of cytophysiological aspects of their development

Ultrastructures of in vitro microspore embryoids and in vivo zygotic embryos of spring wheat have been analyzed and compared. Along with the similarity of ultrastructural characteristics of embryoid and embryo cells at the corresponding developmental stages, some differences have been revealed. Unlike embryos, embryoid cells are characterized by lipid inclusions and numerous mitochondria with well-developed internal membranes. According to our hypothesis, lipids represent an alternative energy source required for active cell divisions in the forming embryoids. Unlike embryos, since the earliest developmental stages, embryoid cells accumulate a significant amount of starch and then utilize it during the organogenesis and germination. A conclusion has been made that embryoid cells create their own reserve of carbohydrates, which is then mobilized during their development. The concept of T.B. Batygina (1987, 1997, 2014) about the universal character of the plant morphogenesis in vivo, in situ, and in vitro has been confirmed. The prospects for the use of microspore embryoidogenesis in vitro as a model to study cytophysiological aspects of zygotic embryogenesis in vivo are discussed.     

Specific features of the development of Siberian stone pine megagametophytes and embryos in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitro cultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

Auxin distribution and transport during embryonic pattern formation in wheat

Inhibitors of auxin polar transport disrupt normal embryogenesis and thus specific spatial auxin distribution due to auxin movement may be important in establishing embryonic pattern formation in plants. In the present study, the distribution of the photoaffinity labeling agent tritiated 5-azidoindole-3-acetic acid ([3H],5-N3IAA), an analog of indole-3-acetic acid (IAA), was visualized in zygotic wheat (Triticum aestivum L.) embryos grown in vitro and in planta, and used to deduce auxin transport pathways in these embryos. This study provides the first direct evidence that the distribution of auxin, here [3H],5-N3IAA, is heterogeneous and changes during embryo development. In particular, the shift from radial to bilateral symmetry was correlated with a redistribution of [3H],5-N3IAA in the embryo. Furthermore, in bilaterally symmetrical embryos, that is, embryos in the late transition stage or older, the localization of [3H],5-N3IAA was altered by N-1-naphthylphthalamic acid, a specific inhibitor of auxin polar transport. No significant effect was observed in radially symmetrical embryos, that is, globular embryos, or very early transition embryos. Thus, the shift from radial to bilateral symmetry is associated with the onset of active, directed auxin transport involved in auxin redistribution. A change in the distribution of [3H],5-N3IAA was also observed in morphologically abnormal embryos induced on media supplemented with auxin or auxin polar transport inhibitors. By means of a microscale technique, free IAA concentration was measured in in vitro- and in planta-grown embryos and was found to increase during development. Therefore, IAA may be synthesized or released from conjugates in bilaterally symmetrical embryos, although import from surrounding tissues cannot be excluded.     

Comparative study of reserve lipid accumulation during somatic and zygotic <Emphasis Type="Italic">Acca sellowiana</Emphasis> (O. Berg.) Burret embryogenesis

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.     

In vitro development of globular zygotic wheat embryos

Summary We have established in vitro culture conditions for globular zygotic wheat embryos (Triticum aestivum L.). Their nutritional requirements have been systematically investigated. The initial sucrose concentration, as well as the sucrose concentration during the culture, a 6-benzylaminopurine supplement, the use of nitrates and ammonium as nitrogen source have a major influence on the embryo development. Proline has an inhibitory effect on the germination. A double layer system with different media was used to give a continuous variation of the medium composition with time. These culture conditions allowed normal direct embryogenesis in up to 47% of the globular embryos.Abbreviations BAP 6-benzylaminopurine - MES 2-N-morpholinoethane-sulfonic acid - MS Murashige and Skoog (1962)