Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusualin that oblique cell divisions result in the formation of severalsuspensor cells prior to the development of the embryo proper.Characteristic changes in microtubular distribution can be foundwithin the zygote and the proembryo during their development.After fertilization, the ellipsoid-shaped zygote has randomlydistributed microtubules within its cytoplasm. As the zygotetakes on a more rounded appearance, microtubules organize intoa dense meshwork. Furthermore, microtubule bundles appear atthe chalazal region of the cell prior to the first mitotic divisionof the zygote. At the preprophase stage of mitosis, a preprophaseband of microtubules appears in the cytoplasm of the zygote.The zygote divides obliquely and unequally and gives rise toan apical cell and a slightly larger basal cell. Many randomly-alignedmicrotubules can be found in the cortex of the basal cell. Theincrease in the abundance of microtubules coincides with theisotropic expansion of the basal cell. The early division ofthe basal cell and subsequent division of the apical cell resultsin the formation of a four-celled embryo, of which three cellsnear the micropylar pole develop as suspensor cells. In thesuspensor cells, the microtubules tend to orient in the samedirection as the long axis of the cell. In addition, prominentmicrotubules can also be found near the adjoining cell wallsof the four-celled embryo. The terminal cell is highly cytoplasmicwith abundant microtubules within the cell. Subsequent divisionsof the terminal cell give rise to additional suspensor cellsand the embryo proper. In the mature embryo, five suspensorcells are usually present; one eventually grows through themicropyle of the inner integument and four grow towards thechalazal pole. The cortical microtubules of suspensor cellsredistribute from a longitudinal to a transverse direction asthey grow towards their respective poles.Copyright 1998 Annalsof Botany Company Embryogenesis, endosperm, microtubules, preprophase band, suspensor cells,Cymbidium sinense(Andr.) Willd.     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

Ultrastructural Changes of the Egg Apparatus Associated with Fertilization and Proembryo Development of Soybean, Glycine max (Fabaceae)

As part of a study involving pod retention in soybean, Glycinemax (L.) Merr., we investigated changes occurring in the eggapparatus of non-abscised flowers from the time immediatelypreceding fertilization through early embryogeny. Prior to theentry of the pollen tube into the embryo sac, one of the synergidsbegins to degenerate as evidenced by increased electron densityand a loss of volume. This cell serves as the site of entryfor the pollen tube. The cytoplasm of the second, or persistentsynergid, remains unaltered until after fertilization. Bothsynergids contain, in addition to a filiform apparatus, a singleunidentified inclusion of flocculent material located in thechalazal portion of each cell. The zygote can be distinguishedfrom the egg by its consistently narrow wall; and it dividesto form a proembryo, a mass of cells not yet differentiatedinto embryo proper and suspensor. The basal cells of the proembryoare more vacuolate than the apical ones, characteristic of thebasal vacuolation of both egg and zygote. Cells of the proembryoare connected to one another via plasmodesmata, and with theexception of the basal-most cell, are isolated symplasticallyfrom the surrounding endosperm. Wall ingrowths frequently occurin certain cells of the proembryo, notably those cells in contactwith the degenerate synergid and embryo sac wall. At a laterstage of ontogeny, by which time the globular embryo properhas become distinct from the suspensor, the wall ingrowths areconcentrated in the suspensor. Glycine max, soybean, embryogeny, synergids     

Alisma embryogenesis: The development and ultrastructure of the suspensor

Summary The development of the suspensor (consisting of a basal cell and a few chalazal cells) inAlisma plantagoaquatica andA. lanceolatum was investigated using cytochemical methods, light and electron microscopy. The basal cell becomes differentiated during the first three days of embryo development. As a result of endopolyploidization the volume of the nucleus rapidly increases, as does the quantity of chromatin it contains and the size of the nucleolus. As basal cell grows, its cytoplasm increases in volume and the number of organelles increase, and wall ingrowths begin to form on the walls at the micropylar pole of the cell. The full development and functioning of the suspensor occurs during the next three days. The enormous basal cell then attains its maximum degree of differentiation: its nucleus reaches a ploidy of 256n or 512n, the micropylar transfer wall is fully developed, as is the cytoplasm, rich in proteins, ribonucleic acids (RNA) and organelles, particularly dictyosomes and long cisternae of the rough endoplasmic reticulum. The chalazal suspensor cells joining the embryo proper to the basal cell also become differentiated. In the seven-day embryo the suspensor begins to degenerate which coincides with the cellularization of the endosperm at the micropylar pole of the embryo sac. The senescence of the suspensor involves the degradation of the nucleus, increasing cytoplasmic vacuolization, and a distinct decrease in protein and RNA content, first in the basal cell, then in the chalazal suspensor cells. Analysis of the development and ultrastructure of the basal suspensor cell suggests that it plays the role of an active metabolic transfer cell, translocating nutrients from the maternal tissues via the chalazal suspensor cells to the growing embryo proper.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

A CYTOCHEMICAL STUDY OF EMBRYO DEVELOPMENT IN STELLARIA MEDIA

The caryophyllad type of embryogenesis in Slellaria media was investigated using topological cytochemical methods for the demonstration of nucleic acids, proteins and polysaccharides. The primary suspensor cell initially contained high levels of cytoplasmic RNA, but these declined rapidly after the first few cell divisions. Cytoplasmic protein levels were high throughout the existence of the primary suspensor cell, becoming concentrated into proteinoplasts at the time of cotyledon initiation in the embryo. These plastids were RNA- and DNA-negative, and only slightly positive with methods for polysaccharides and lipids. Cytoplasmic and nucleolar RNA and protein levels were high in the embryo throughout its development. The cotyledons and endosperm also showed high RNA and protein levels. Cytoplasmic DNA was present in the embryonic apical meristematic cells in the form of Feulgen-positive, deoxyribonuclease-removable granules. Cytoplasmic histones were present in the embryo and endosperm. Polysaccharides were demonstrable in the cotyledons, cortex, endosperm, and nucellus.     

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vignaaconitifolia, V. mungo and V. radiata on MS medium supplementedwith NAA, picloram or 2, 4-D. On transfer to L-6 liquid mediumsupplemented with low concentrations of picloram, GA₃ and cytokinins,large number of somatic embryos differentiated from the callus.The cells destined to become somatic embryos divided to formspherical or filamentous proembryos. From the filamentous proembryo,the embryo proper developed either at single or multiple sites.Development of somatic embryos from multiple sites resultedin several embryos connected by a common suspensor at the radicleend. Continued divisions of the proembryos led to globular,heart shaped and dicotyledonary stages of somatic embryogenesis.The somatic embryos of V. mungo and V. aconitifolia differentiatedinto tiny plantlets at low frequency (1%) in liquid suspensioncultures supplemented with zeatin, picloram and GA₃. Vigna aconitifolia Jacq, Marechal, mothbean, Vigna mungo L. Hepper, urdbean, Vigna radiata L. Wilczk, mungbean, somatic embryo     

A light and electron microscope study ofPicea glauca (White spruce) somatic embryos

Summary Somatic embryos in embryogenic callus cultures derived from Immature zygotic embryos ofPicea glauca (White spruce) were examined by light and electron microscopy. Somatic embryos consist of an embryonic region of small densely cytoplasmic cells subtended by a suspensor consisting of long highly vacuolated cells. Mitotic figures are frequent in the embryonic cells but are not observed in the suspensor. Cell divisions in the embryonic region apparently produce rows of cells which elongate to form the suspensor. The presence of abundant polysomes, coated membranes and dictyosomes in the cytoplasm of embryonic and upper suspensor cells suggests rapid growth of the embryo. In contrast the basipetal suspensor cells appear to be senescing. While only a few scattered microfilaments are present in the meristematic cells, the upper suspensor cells contain numerous bundles of longitudinally oriented microfilaments. These bundles correspond to actin cables observed in light microscope preparations stained with rhodamine labelled phalloidin and are oriented parallel to the direction of active streaming in these cells.     

Embryo development in the lady's slipper orchid, Paphiopedilum delenatii, with emphasis on the ultrastructure of the suspensor

BACKGROUND AND AIMS: Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species. METHODS: Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy. KEY RESULTS: The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation. CONCLUSIONS: The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.     

The osmotic property and fluorescent tracer movement of developing orchid embryos of Phaius tankervilliae (Aiton) Bl

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.     

Capsella embryogenesis: The suspensor and the basal cell

Summary The suspensor and basal cell ofCapsella were examined with the electron microscope and analyzed by histochemical procedures. The suspensor cells are more vacuolate and contain more ER and dictyosomes, but fewer ribosomes and stain less intensely for protein and nucleic acids than the cells of the embryo. The end walls of the suspensor cells contain numerous plasmodesmata but there are no plasmodesmata in the walls separating the suspensor from the embryo sac. The lower suspensor cells fuse with the embryo sac wall and the lateral walls of the lower and middle suspensor cells produce finger-like projections into the endosperm. At the heart stage the suspensor cells begin to degenerate and gradually lose their ability to stain for protein and nucleic acids.The basal cell is highly vacuolate and enlarges to a size of 150 X 70. An extensive network of wall projections develops on the micropylar end wall and adjacent lateral wall. The nucleus becomes deeply lobed and suspended in a strand of cytoplasm traversing the large vacuole. The cytoplasmic matrix darkens at the late globular stage and histochemical staining for protein becomes very intense. The basal cell remains active after the suspensor cytoplasm has degenerated. It is proposed that the suspensor and basal cell function as an embryonic root in the absorption and translocation of nutriments from the integuments to the developing embryo.Research supported by NSF grant GB 3460 and NIH grant 5-RO 1-CA-03656-09.     

西瓜胚和胚乳的发育

应用显微技术对西瓜胚和胚乳的发育过程进行了观察并分析了西瓜胚珠败育的原因。西瓜胚发育属紫菀型。合子第一次分裂为不均等分裂 ,形成的基细胞体积明显较顶细胞大 ,两细胞均含有多个液泡。原胚发育过程中没有明显的胚柄。最外层的原胚细胞 ,与胚乳细胞相邻的壁上被胼胝质物质包围 ,且无外连丝存在 ;与胚囊壁相接的壁上无壁内突结构。胚的子叶体积增长的同时 ,子叶细胞内积累蛋白质和脂类物质 ,多糖物质的含量下降。胚乳发育属核型 ,在球形胚期开始自珠孔端向合点端细胞化 ,胚子叶分化出后开始自珠孔端向合点端退化。胚乳合点端在球形胚早期形成发达的胚乳吸器 ,开始呈游离核状态 ,后细胞化 ,在心型胚期之后退化。     

Features of the embryonic development of <Emphasis Type="Italic">Dienia ophrydis</Emphasis> (Orchidaceae)

A new Dienia type of the embryogenesis of orchid plants differing from the Liparis type, earlier observed for the tribe Malaxideae, has been described in Dienia ophrydis (J. Köenig) Seidenf. (Orchidaceae). The Dienia-type embryogenesis is characterized by the following features: (1) development of a single-celled suspensor formed by a cb-derivative, (2) linear arrangement of embryo cells at the tetrad stage, (3) atypical origin of some tiers, and (4) no divisions of the ci and cb cells. A hypothesis about the convergent similarity between the Dienia and Caryophyllaceae types of embryogenesis has been proposed. A number of embryo sac and embryo structures typical for D. ophrydis, including “petassum,” “fitting,” and “suspensor mantle,” have been first described. A “petassum” represents the remains of cell walls of the pollen tube and probably the filamentous apparatus of synergids sealing the micropyle side of a fertilized embryo sac. The sole suspensor cell has a special appendix (“fitting”), which connects it to the embryo. The suspensor and the fitting are surrounded by a special envelope (“suspensor mantle”), which does not cover the basal cell of the embryo (ci).     

ABNORMAL DEVELOPMENT OF THE SUSPENSOR IN AN EMBRYO-LETHAL MUTANT OF ARABIDOPSIS THALIANA

Developmental arrest of the embryo proper in aborted seeds from mutant 50B, a recessive embryo-lethal mutant of Arabidopsis thaliana, was shown to be followed by abnormal growth of the suspensor. Each of the 12 aborted seeds examined in sectioned material contained an abnormally large suspensor and an embryo proper arrested at a preglobular stage of development. Analysis of serial sections revealed that mutant suspensors contained 15–150 cells whereas wild-type suspensors were composed of only six to eight cells. Development of the mutant endosperm continued to a late nuclear or early cellular stage even in the absence of further development of the embryo proper. These results suggest that the missing gene product in mutant 50B is required for development of the embryo proper but not for continued growth of the suspensor or endosperm tissue. The pattern of abnormal development observed in this mutant provides further evidence that continued growth of the suspensor during normal development is inhibited by the developing embryo proper and that the full developmental potential of cells in the suspensor is expressed only when this inhibitory effect is removed through a mutation or experimental treatment that is lethal only to cells of the embryo proper.     

Histochemical Localization of Several Enzymes in Early Young Embryo of Pinus tabulaeformis

Histochemical localization of adenosine triphosphatase, acid phosphatase and peroxidase in young embryo of Pinus tabulaeformis has been studied. All cells of the embryo proper showed very intense lead phosphate disposite with ATP in the Wachs- tein Meisel medium and with B-glyceropbosphate in Gomori medium. In the suspensor 1–3 suspensor cells closed to embryo proper appear blackening with both the ATP and the B-glycerophosphate, and other cells of the suspensor showed less adenosine triphosphatase and B-glyeerophosphatase. In the young embryo the peroxidase distribution may vary with the developmental stages of the young embryo, for instance cone-shaped embryo proper showed dark blue, and which peroxidase occupies only 1–2 suspensor cells closed to embryo proper. When the young embryo developed further into columnus-shape, the peroxidase was mainly distributed in regions of both the posterior part of embryo proper and just derived suspensor cells. However the anterior part of embryo proper showed slight peroxidase. The experimental results indicate that all of these enzymes mentioned above are mainly present in both the embryo proper and the connecting end of the suspensor, and that these regions show great matabolic activity. From the above mentioned observation, the possible relationship between the young embryo and near surrounding tissue during the development of embryo is discussed.     

THE DEVELOPMENT OF THE SEED IN ANDROGRAPHIS SERPYLLIFOLIA

Mohan Ram , H. Y. (U. Delhi, India.) The development of the seed in Andrographis serpyllifolia. Amer. Jour. Bot. 47(3) : 215—219. Illus. 1960.–Andrographis serpyllifolia, a member of the Acanthaceae, has an embryo sac with a bifurcated chalazal part. At the time of fertilization both synergids and antipodal cells disintegrate. Early in its development the endosperm is composed of 3 distinct parts: (1) a binucleate densely cytoplasmic chalazal haustorium; (2) a large binucleate micropylar haustorium; and (3) a central chamber which develops into the endosperm proper. The divisions in the central endosperm chamber are ab initio cellular. A few of the endosperm cells elongate enormously, ramify into the integument and destroy the surrounding cells. These cells have been termed secondary haustoria. Due to the unequal destruction of the integument, the endosperm assumes a ruminate condition. The mature seed is nearly naked because the seed coat is almost completely digested. The embryo has a long suspensor. The micropylar cells of the suspensor are hypertrophied and multinucleate. Contrary to Mauritzon's (1934) belief, the course of endosperm development is markedly different from that observed in Thunbergia. So far, albuminous seeds have been reported only in the subfamily Nelsonioideae. The present investigation provides a case of its occurrence in the Acanthoideae also.     

The Developments of Embryo and Endosperm of Zizyphus jujuba Mill

The development of the embryo of Zizyphus jujuba Mill. conforms to the Asterad type. The transverse division of the zygote results in the formation of the terminal and basal cells. The basal cell of the two-celled proembryo divides transversely first and contributes to the suspensor and a minor part of the embryo proper in further development. The terminal cell first divides vertically and the divisions in various ways contribute to almost all parts of the embryo proper. The endosperm development is of the nuclear type. Three stages may be divided as follows: the free nuclear stage, the wall formation stage and the stage in which the disorganization of the endosperm cells takes place rapidly. The developments of the embryo and endosperm are well correlated. Thus the morphological development of the embryo may be used as an indicator to tell the development stage of the endosperm. The adequate stage for endosperm culture is in the wall formation stage.     

Frequenza di cellule con « DNA puffs » nel sospensore di Phaseolus coccineus in momenti diversi dello sviluppo embrionale

Abstract

Morphological aspects of Phaseolus coccineus suspensor cells at different periods of embryo development. — Embryo suspensor cells of Phaseolus coccineus have been analysed at different periods of seed and embryo development in field-grown plants and the frequency of cells involved in « DNA puffs » formation has been determined. The collected data show that: a) the relation between seed development and embryo development is not constant at different times in the season (from July to November); b) the frequency of cells showing « DNA puffs » is influenced by the above ratio. This fact seems to indicate that embryos of comparable developmental stages (as estimated by cotyledon length) show different metabolic patterns in relation to seed development and enviromental factors.