Origin of somatic embryos from cultured shoot tips ofSorghum bicolor (L.) moench

Summary Histologic examination of shoot-tip explants, 1 wk after culture initiation on Murashige and Skoog medium with 2.5 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/liter kinetin, reveals active meristematic centers inside cultured tissue. Clusters of cells in these meristematic centers exhibit remarkable resemblance to the initial three divisions in the zygotic embryo. Several such meristematic groups of cells are observed in the cultured explant at this stage. Embryogenesis is obviously initiated very early in this tissue in the presence of 2,4-D. A well-defined, white globular embryogenic callus develops in culture in about 4 wk, and it consists of clusters of embryoids with large cells characterized by thick cell walls, numerous lipoidal vesicles, and localized areas of carbohydrate storage. These cells resemble the scutellar tissue of the embryo. However, there are cells within this tissue that themselves appear embryogenic. They undergo cell division giving rise to small clusters of cells. As long as 2,4-D is present in the medium, the cells apparently retain the capacity to proliferate and to produce more cells capable of embryogenesis. Embryogenesis seems to occur via two processes, initiation of somatic embryos early in culture and secondary embryogensis from the scutellar tissue that forms in vitro.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cytohistological Study on Somatic Embryogenesis in Asparagus officinalis L.

The single cell was the first cell of somatic embryoid in Asparagus officinalis L.. Electron microscopic observations revealed that early embryogenic cells and meristematic cells had the same characteristics in cell shape and structure. The embryogenic cell was small in size, with large nucleus, dense cytoplasm, thin wall, lots of small vacuoles, and rich in organelles. At later stage, the polarity of embryogenic cell appeared, i.e. nucleus situated at one end of the cell, while the other end was occupied by a large vacuole. The polar type of the embryc)genic cell was similar to that of a zygote. Light microscopic observations revealed :hat ihe embryoids were sequentially differentiated through 2-cells, 4-cells, 8-cells, multicellular proembryo, globularshaped, pear-shaped, rod-shaped, cotyledonary-differentiated and mature embryo stages. The heart-shaped and torpedo-shaped stages were observed during the early stage of embryogenesis. In addition, a typical embryoids were also found in vitro.     

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

Summary The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E₁ callus and those of the callus arising from it, according to the culture conditions.The peculiar characteristics of E₁ are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells.An embryogenic friable callus (E₂) is formed in time when E₁ is maintained on solid culture medium. The E₂ cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials.Shaking E₁ in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.Abbreviations c cell - cr crystal - cw cell wall - d dictyosome - er endoplasmic reticulum - m mitochondrion - mb microbody - n nucleus - p plastid - s starch - v vacuole     

The Origin and Development of Embryoids in Suspension Cultures of Carrot (Daucus carota)

In suspension cultures of carrot, embryoids are initiated ator near the surface of characteristic cellular aggregates (embryogenicclumps) and are released from these aggregates as free-floatingstructures capable of further development into plantlets. Embryoiddevelopment is promoted by transfer from a medium containing2,4-D to one from which it is omitted. The structure of theembryogenic clumps, in these two media, has been studied bythe thin section technique involving fixation in glutaraldehydeand embedding in glycol methacrylate. The superficial cellsof the clumps are clearly marked off from the central cellsand contain large starch grains, a large central nucleus, abundantcytoplasm and a number of vacuoles. It is from individual superficialcells that the embryoids are seen to arise in the 2,4-D omittedmedium but it has not been possible to distinguish, in advanceof embryogeny, such cells from within the superficial cell layers.The fragmentation of the proliferating clumps in 2,4-D containingmedium and the release of embryoids into the 2,4-D omitted mediumis promoted by some of the superficial cells becoming free ofstarch and undergoing pronounced enlargement. A sequence of segmentations leading from single superficialcells of the clumps to globular embryoids has been traced. Thisdiffers from that reported to occur during early embryogenyfrom the egg cell of carrot. The resemblance to zygote embryogenyis closer from the late globular stage of embryoid development;embryoids then differ from zygotic embryos in their shortersuspensors. Attention is drawn to the initiation of additionalembryoids from the epidermis of some of the embryoids developingin culture.     

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Pearl Millet (Pennisetum americanum)

Immature embryos of Pennisetum americanum (pearl millet), culturedin the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) produceda pale-yellow and compact callus tissue by proliferation ofthe scutellum. Teased pieces of the compact callus were placedin a liquid medium on a gyrotory shaker to establish suspensioncultures. The cultures were composed of large, elongated andhigly vacuolated cells, and a population of richly cytoplasmiccells. The latter, here termed embryogenic cells, containednumerous plastids with starch, and occurred in tight groupsof four or more cells, and occasionally as single cells. Structuresresembling various stages of embryogenic development were foundin the suspension cultures. When the cultures were plated ina 2,4-D-free agar medium containing abscisic acid, embryoidswith the typical organization of cereal embryos were produced.The embryoids ‘germinated’ in vitro to give riseto plantlets, which were successfully transferred to soil. Theregenerated plants showed the normal diploid chromosome numberof 14. Embryoids apparently arose from single embryogenic cells,either directly or after the formation of a proembryonal massof cells. embryogenesis, pearl millet, Pennisetum americanum, regeneration, suspension culture     

Observations on Morphology and Cytology of Embryoids Derived from Callus of Maize

Observations on the callus sections showed that most embryoids were produced from surface layer cells of the callus and a few embryoids from inside layers of the callus. The initial cell of the embryoids possessed denser protoplasm and larger nucleus than the others cells did. The developmental sequence of the initial cell was similar to that of the zygotic embryos. First division gave rise to two daughter cells, the basal cell and terminal cell. The basal cell was able to divide or not divide again and changed into the suspensor, The terminal cell first divided longitudinally and then transversely into four cells. As the four cells further divided the embryo was formed properly. The embryoids possessed monody or polycotyledons. The production of the embryoids from a callus was not synchronous. So embryoids of different development stages could be found on the same callus.     

Morphohistological analysis of the origin and development of somatic embryos from leaves of mature Quercus robur

Summary In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.     

香雪兰的直接与间接体细胞胚胎发生

香雪兰的体细胞胚胎发生可通过两种途径进行,即直接发生与间接发生。在直接发生方式中,体细胞胚直接来源于尚未完全分化的外植体表皮细胞;体细胞胚与母体组织以一种类似胚柄的结构相联系。间接发生方式中,体细胞胚的形成要经过一个愈伤组织阶段。以是否能形成体细胞胚分类,可将愈伤组织分为胚性和非胚性愈伤组织。以间接方式形成的体细胞胚是由胚性愈伤组织中的一种决定细胞发育来的。这种体细胞胚不具有类似胚柄的结构,而与母体组织共同形成一个复合体。体细胞胚具有自己独立的维管束系统,在脱离母体组织后能够独立发育成株。     

Direct and indirect somatic embryogenesis in Freesia refracta]

Somatic embryogenesis can be induced in tissue cultures of Freesia refracta either directly from the epidermal cells of explant, or indirectly via intervening callus. In direct pathway, somatic embryos were in contact with maternal tissue in a suspensor-like structure. In indirect pathway, the explants first proliferacted to give rise to calluses before embryoids were induced. The two sorts of calluses were defined to embryogenic callus and non-embryogenic callus according to producing of somatic embryos. An indirect somatic embryo is developed from a pre-embryogenically determined cell. This kind of somatic embryo has no suspensor structure instead of a complex with maternal tissue. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.     

FORMATION OF HAPLOID EMBRYOIDS OF LARIX DECIDUA: EARLY EMBRYOGENESIS

Callus initiation of sections of megagametophytes of Larix decidua occurs just below the cut surface and is followed by the formation of one or more long protruding cells. The long cells then divide transversely at their tips to yield small cytoplasmically-dense terminal cells. The latter divide, forming loose aggregates of dense cells, microcalli, which develop long cells that radiate in all directions and divide terminally to produce aggregates of small cells. This long/short cell alternation is repeated a few times. Eventually the aggregates divide in a polarized manner producing files of long cells predominantly in one direction. These loosely bundled long cells form a suspensor-like structure. The meristematic small cells continue dividing forming a mass of embryonal cells. This early embryoid eventually turns green and produces both a root and shoot. Haploid embryoids are also derived from long cells in which karyokinesis but not cytokinesis occurs, resulting in a four-nucleate coenocyte. The nuclei migrate to one pole and become surrounded by cell walls. The cells thus formed are the originators of a new embryoid.     

The Diversity of Morphogenesis in Suspension Cultures of Atropa belladonna L.

Root formation from the cell aggregates formed in suspensioncultures of Atropa belladonna L. occurs either from peripheralmeristems or from endogenous meristematic nodules. The rootswhen released from the aggregates either continue to developas cultured roots or quickly develop a callus at their basalends and within this develops a shoot bud. When this shoot budis initiated both root and shoot may continue to develop togive a plantlet with either two equal first leaves or, lesscommonly, with several first leaves or with a single first leaf.Where the roots are of endogenous origin (and lack the ‘collar’characteristic of those derived from a peripheral meristem)and where some retardation of root growth is associated withbud formation, structures arise which have previously been termed‘embryo-like structures’ (Thomas and Street, 1970,Plate 10, F). Alternatively, root development may be suppressedand only the bud develops. It has been possible to trace the development also in thesecultures of embryoids whose development involves recognizablestages of normal embryology. These embryoids may arise fromsingle cells of the initial cell aggregate or from cells ofthe massive suspensors of these embryoids (these latter embryoidscan be described as adventive in origin). The embryoids mayshow two equal cotyledons or only one cotyledon.     

Direct somatic embryogenesis from protoplasts of Foeniculum vulgare

Protoplasts prepared from an embryogenic cell suspension culture of fennel gave rise to somatic embryoids directly through unequal cell divisions of enlarged, ellipsoidal cells, when embedded in hormone-free LS agarose medium. On the other hand, protoplasts embedded in LS agarose medium containing 2,4-D and kinetin proliferated through unpolarized cell divisions to form calli, which gave somatic embryoids on the surface upon transfer onto the same medium. In either case, somatic embryoids germinated to develop into normal plantlets when cultured on hormone-free LS agar medium under illumination.     

Ultrastructure of sunflower protoplast derived ealluses differing in their regenerative potential

The ultrastructural properties of microcalluses derived from mesophyll protoplasts of commercial sunflower (Helianthus annuus L.) cultivars were investigated by light and electron microscopy. Two culture regimes were chosen: Regime A giving rise to callus formation but of little embryogenic potential and regime B resulting in higher embryogenicity. Bipolar colonies that developed during early stages of regime A were found to be composed of mostly degenerated structures. No differentiation or embryonal organization as suggested by the compactness and shape of the microcalluses could be observed. Amorphous calluses obtained at later stages of the same regime consisted of small groups of meristematic as well as vacuolated cells. Incomplete cellular divisions occurred in almost all colonies grown under the regime A, causing most probably the lack of further callus organization. In contrast calluses of irregular shape, cultivated under regime B, mostly lacked incomplete cell partitioning but showed the formation of organized regions. These structural investigations can give us a tool to identify and characterize the quality of embryogenic calluses.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - V-KM medium of Binding & Nehls (1978)     

Plant donor tissue and isolation procedure effect on early formation of embryoids from protoplasts of Helianthus annuus L.

The effects of factors influencing sunflower protoplast isolation yield, plating efficiency (PE) and the early differentiation into embryoids (embryogenic capacity, EC) have been studied. Only hypocotyl-derived protoplasts divided. The variations of PE and EC in the various treatments did not seem to be linked to the protoplast yields. From statistical analysis of the data, we concluded that, the sunflower genotype, the age and height of seedlings, the part of hypocotyl used, the incubation time (from 6 to 16 hours) in enzymes of explants or of protoplasts alone, influenced PE but large variations were detected for EC. A comparison of the factors effecting EC suggested an origin, inside the hypocotyl, of cells able to give rise, after induction, to embryogenic protoplasts.     

Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

Embryogenesis and ribosomal DNA in carrot cell suspensions cultured in vitro

The contents of nucleic acids and rDNA were estimated during the development of carrot cell suspensions cultured under two different conditions. The cells transferred from stock culture to the medium without 2,4-dichlorophenoxyacetic acid (2,4-D) induced the embryogenesis (embryogenic culture), while the cells inoculated to the medium with 0.2 mg/l 2,4-D did not form any embryos (non-embryogenic culture). The ratio of RNA to DNA of both cultures increased in the early stage of the culture. The rise of the ratio in embryogenic culture was much higher than that in non-embryogenic culture, which showed that embryogenic culture accumulated RNA prior to the formation of embryos. The rDNA amount of non-embryogenic culture remained constant throughout the culture period. Although embryogenic culture showed a slight change in rDNA amount, the differences were at most 12% and the quantitative stability of the rDNA was demonstrated during the development of carrot cell suspension cultures.     

Identification of a transitional cell state in the developmental pathway to carrot somatic embryogenesis

We have located a novel carbohydrate epitope in the cell walls of certain single cells in embryogenic, but not in non-embryogenic, suspension cultures of carrot. Expression of this epitope, recognized by the mAb JIM8, is regulated during initiation, proliferation, and prolonged growth of suspension cultures such that changes in the abundance of JIM8-reactive cells always precede equivalent changes in embryogenic potential. Therefore, a direct correlation exists between the presence of the JIM8-reactive cell wall epitope and somatic embryo formation. The JIM8-reactive cell wall epitope is expressed in the cell walls of three types of single cells and one type of cell cluster. One of the single cell types seems able to follow one of two phytohormone-controlled developmental pathways, either a cell elongation pathway that eventually leads to cell death, or a cell division pathway that gives rise to proembryogenic masses. We demonstrate that all JIM8-reactive cell types in embryogenic carrot suspension cultures are developmentally related, and that the switch by one of them to somatic embryogenesis is accompanied by the immediate dissipation of the JIM8-reactive cell wall epitope. The cell wall carbohydrate epitope recognized by JIM8 therefore represents a cell wall marker for a very early transitional cell state in the developmental pathway to carrot somatic embryogenesis.     

A new method for the purification of the different stages of carrot embryoids

An easy method is presented for the purification of the different stages of carrot embryoids. This is based on a synchronization of the regenerating culture and on a filtration through filters of various pore sizes. A differential sedimentation was used for removing undifferentiated cells. At the end of the process, the different stages: globular, heart- and torpedo-shaped were obtained with a degree of purity that always exceeded 90%. This method can be used for the separation of relatively large numbers of embryoids (from thousands to a million) of haploid and diploid carrot lines and is very gentle on embryoids in that it does not affect their viability or further development.Abbreviations 6BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - pol polymerase     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy