The ultrastructure of zygotic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of zygotic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Embryogenesis was initially characterized by the presence of a vacuolated egg cell and zygote. The increased presence of Golgi bodies in the zygote suggested it was metabolically more active than the egg cell. The first zygotic division resulted in a densely cytoplasmic apical cell and a highly vacuolated basal cell. The club-shaped proembryo displayed a large amount of endoplasmic reticulum (ER) and ribosomes, very few lipids, and a continuous gradient of vacuoles from the highly vacuolated basal suspensor cells to the densely cytoplasmic apical cells. The embryo had well-defined parts by 8 days after pollination, including shoot and root meristems, coleoptile, scutellum, provascular system, and the first leaf primordium. Large increases in ER, lipids, starch, and vacuoles occurred in the scutellum during the maturation of the embryo, except in the provascular cells. Throughout zygotic embryogenesis, embryo cells were connected by plasmodesmata except where intercellular spaces occurred. Ultrastructural, morphological, and histological observations of zygotic embryogenesis in pearl millet are in agreement with previous reports for other grass species.     

Effect of anti-microtubular drug amiprophos-methyl on somatic embryogenesis and DNA ploidy levels in alfalfa and carrot cell suspension cultures

A short treatment with the anti-microtubular drug amiprophos-methyl (APM) blocked somatic embryogenesis in alfalfa(Medicago sativa L.) and carrot (Daucus carota L.). The interruption was temporary and restoration of somatic embryogenesis was observed in long-term cultures. In addition to the effect on somatic embryogenesis, APM treatment induced polyploidization the extent of which was concentration dependent. In long-term alfalfa cultures, APM-induced loss of somatic embryogenesis led to ploidy instability and to a shift to DNA aneuploidy. Critical stages of somatic embryogenesis sensitive to disruption of microtubule-mediated processes were determined in carrot cell cultures. Complete embryogenic arrest occurred when APM was added within the first 5 d of embryogenesis from single cells. The role of the cytoskeleton in the first events of somatic embryogenesis and the relation between totipotency and ploidy stabilityin vitro is discussed.     

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.     

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in <Emphasis Type="Italic">Acca sellowiana</Emphasis> (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.     

Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems

Abstract

Whatever the in vitro regeneration pathway, it would be of interest to be able to distinguish regeneration-competent from non-regenerating cells and tissues, and as early in culture as possible, as this would allow a dramatic improvement of biotechnology breeding, particularly for the so-called recalcitrant species. With this aim, we examined a range of genotypes of pea (Pisum sativum) and grass pea (Lathyrus sativus), and of the two model species Medicago truncatula, another legume, and Arabidopsis thaliana. This was done by comparing cell suspension cultures of different ages (young [<6 transfers-old]vs. mature [>6 transfers-old]), densities (dense [>10⁷ cells/ml] or sparse [<10⁶ cells/ml]) and regeneration abilities (non-embryogenic vs. embryogenic), in order to identify early indicators of competence for somatic embryogenesis. All such cell suspensions were subcultured every 14 days and several parameters were assessed every 3–4 days during each 14-day cycle. These included the time course pH and osmolarity of the culture medium, the internal osmolarity of cells, the cell surface and the cell wall thickness (by examining cellulose accumulation in Calcofluor White-stained cells under UV light). As cells underwent embryogenesis they enlarged. Cellulose accumulated in the walls of non-embryogenic cells, but walls became thinner with the onset of embryogenesis, and diminished further as embryos matured. Although medium osmolarity decreased at the onset of embryogenesis, this was never observed for non-embryogenic cell suspensions. Conversely, there was a concomitant increase in intracellular osmolarity for embryogenic cells. Medium pH (analysed with the model species only) was not significantly correlated with regeneration competence of cells. For all genotypes and species, the kinetics of cell wall thickness and cell surface, and that of medium and cell osmolarity were reliable early indicators of the competence of cells to undergo somatic embryogenesis. The implication of these results for biotechnological breeding of grain legumes and for plant regeneration competence in general are discussed.     

Acetylsalicylic acid and ammonium-induced somatic embryogenesis and enhanced plumbagin production in suspension cultures of <Emphasis Type="Italic">Plumbago rosea</Emphasis> L.

Summary Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog’s medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM). but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.     

Cold-enhanced somatic embryogenesis in cell suspension cultures of Astragalus adsurgens Pall.: relationship with exogenous calcium during cold pretreatment

The inter-relationship between exogenous calcium (Ca²⁺) during cold pretreatment and cold-enhanced somatic embryogenesis was investigated using cell suspension cultures of Astragalus adsurgens Pall. Cell suspension was obtained from embryogenic callus and could be induced to form somatic embryos in the differentiation medium. Suspension cells, after cold-treatment at 8 °C for 2 to 3 wk, displayed an enhanced capacity for somatic embryogenesis as compared to those without cold pretreatment. Longer cold pretreatment (> 4 wk) resulted in the inhibition of somatic embryogenesis. The enhanced embryogenic response of cells to cold pretreatment was dependent on the Ca²⁺ level in the pretreatment medium. Ca²⁺ levels below 1 mM suppressed the cold-enhanced response. Addition of lanthanum into the pretreatment medium completely abolished the cold induced enhancement of somatic embryogenesis. These results suggest that embryogenic cells require a minimal concentration of Ca²⁺ during pretreatment for the expression of this cold-enhanced capacity for somatic embryogenesis in A.adsurgens and the influx of exogenous Ca²⁺ during pretreatment might also be involved.     

Autopolyploid induction via somatic embryogenesis in Lilium distichum Nakai and Lilium cernuum Komar

New ornamental varieties of high quality can be created via artificial polyploid induction. In the present study, the first system of polyploid induction with somatic embryogenesis of Lilium distichum Nakai and Lilium cernuum Komar. was developed. Somatic embryos were cultured on MS with 0.41 μmol L^?1 picloram and 1.07 μmol L^?1 NAA by scales (5 mm²). After 40 days, somatic embryos were transferred to MS with 2.21 μmol L^?1 BA for somatic embryogenesis. As determined from observations of paraffin sections, embryonic cells of L. distichum originated from outer cells at first, and somatic embryogenesis occurred through an indirect pathway. In L. cernuum, embryonic cells originated from inner cells at first, and somatic embryogenesis occurred through a direct pathway. Polyploids were successfully formed from somatic embryos and scales by the soaking and mixed culture methods with different colchicine concentrations (0.01%, 0.05%, and 0.1%; v/v) and durations (24, 48, and 72 h). The polyploid induction rate reached 57.14% and 46.15% with 0.05% colchicine treatment in L. distichum (48 h) and L. cernuum (24 h), respectively. Tetraploids (28.57% and 23.08%) and aneuploids without chimeras among the obtained polyploid plantlets were identified by chromosome counts of root-tip tissue squashes in L. distichum and L. cernuum. Tetraploid plantlets of L. distichum exhibited broader leaves, longer guard cells, larger stomata and higher stomatal conductance than diploid plantlets. Tetraploid plantlets of L. cernuum showed 1.76?×?higher chlorophyll content, significantly more leaves, longer guard cells, larger stomata and lower stomatal conductance than diploid plantlets.

     

Cytological features of developing embryogenic callus and somatic embryos of Iris pumila L. and Iris setosa Pall

Abstract

Cytological examination during somatic embryogenesis in Iris pumila L. and Iris setosa Pall, were performed using light and electron microscopy. The first sign of the cellular differentiation in the initial embryogenic callus (EC; stage 1) of both Iris species was the formation of short and elongated cell types. After the onset of embryogenesis, short cells divided producing a mass of densely packed meristematic cells, closely connected with numerous plasmodesmata. Further differentiation into globular embryos (GE) led to a loss of plasmodesmata and cell separation. In vacuolated elongated cells, cytoplasm was located near the wall and around the nucleus. In both cell types amyloplasts and small mitochondria with poorly developed crystae were abundant.

Cell of GE (stage 2) contained an increased number of mitochondria and plastids comparing to those from stage 1, indicating further differentiation. Thylakoids and starch grains were observed within the plastids, while the number of cristae within the mitochondria was increasing.

In cells of embryos with coleoptile (ECl) (stage 3), plastids differentiated into chloroplasts with thylakoids. In all stages of cell differentiation, short and long cisternae of endoplasmic reticulum with ribosomes were seen. Activity of dictyosomes was increased in stages 1 and 2, then reduced in stage 3.

Ultrastructure of EC cells was identical to that of proembryogenic cells, i.e. of early GE. Ultrastructural appearance of GE cells was identical in both Iris species, but evident, and increasing, differences in mitochondria and plastids were observed between GE and ECl embryos.The presence of bi-, three- and eight- cell proembryos demonstrates that they originate from a single cell in both Iris species.     

Expression of the gene <Emphasis Type="Italic">MyoD</Emphasis> and <Emphasis Type="Italic">m</Emphasis>-cadherin in the myogenic precursor cell culture isolated from muscles of rats at different stages of ontogenesis

Comparative analysis of expression of the MyoD gene and m-cadherin in the myogenic precursor cells isolated from rats’ muscular tissue on the 20th–21st day of embryogenesis, on the third to fifth day of postnatal development, and from adult animals was carried out. No significant differences in expression of the MyoD gene were observed. Nevertheless, we found that in contrast to myogenic cells isolated from postnatal muscular tissues, in the subpopulation of myoblasts isolated from embryonic tissues on the 20th–21st day of embryogenesis cells that were more progressive with respect to differentiation prevailed. These cells demonstrate little proliferative activity alongside with expression of m-cadherin, the protein responsible for cytoadherence, and, as a consequence, a higher rate of differentiation.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

Ontogeny of odorant receptor gene expression in zebrafish,Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26°C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ. © 1996 John Wiley & Sons, Inc.     

Effects of glycerol on somatic embryogenesis in Cichorium leaves

 Direct somatic embryogenesis was induced in leaf cells of a Cichorium hybrid (Cichorium intybus L var. sativum×Cichorium endivia L. var. latifolia) through a two-step procedure. Leaf tissue explants were cultured for 5 days in M17 liquid medium supplemented with 30 mM sucrose and 330 mM glycerol (M17S30Gly330 medium). Synchronised divisions of embryogenic cells occurred after transfer for 7 days onto glycerol free-medium (M17S30). By doubling the sucrose concentration (60 mM) in the presence of glycerol (M17S60Gly330) during the induction step, embryogenesis increased and the length of the induction step was reduced from 5 to 4 days. Compared to sucrose, glycerol as carbon source during the induction and the expression steps had an inhibitory effect on the embryogenic response. During culture, glycerol was not detected in M17S60 medium and was at a low level in leaf fragments incubated in this medium. Initially supplied as an osmoticum, glycerol disappeared from M17S60Gly330 medium during the 4-day induction period and penetrated into the tissues where most of was metabolised. Furthermore, glycerol modified it carbohydrate metabolism, particularly during the induction period of embryogenesis. Sucrose hydrolysis was affected in the medium and sucrose and hexose contents in tissues were higher than in glycerol-free medium. The effects of glycerol as osmoticum and as a molecule itself are discussed. Received: 30 January 1998 / Accepted after revision 25 February 1999