Morphological and histological changes during somatic embryo formation from coconut plumule explants

Summary Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing., At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.     

Somatic embryogenesis of mangosteen

Young red leaf segments from 1 to 3?year-old seedlings of mangosteen (Garcinia mangostana L.) were incubated on different media, consisting of either B5 or Murashige and Skoog (MS) basal media and supplemented with several plant growth regulator at different levels, including 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin or 2,4-D and 6-benzyladenine (BA) or thidiazuron (TDZ) and BA. The highest percentage of globular structures (36.0?%) was obtained using MS medium with a combination of TDZ and BA, each at a concentration of 0.7?mg?l^?1. Histological observation of sections of globular structures showed accumulation of dense meristematic cells, a common characteristic of globular formation during somatic embryogenesis. Molecular analysis detected the gene somatic embryogenesis receptor-like kinase 1 (SERK1). This gene sequence showed a high similarity (73.0?C87.0?%) to previously reported plant SERK1.     

Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.     

Morphological, histological and ultrastructural changes in the olive pistil during flowering

Sexual reproduction is essential for the propagation of higher plants. From an agronomical point of view, this is a particularly key process because fertilization guarantees fruit formation in most cultivated fruit species. In the olive, however, in spite of its agricultural importance, little attention has been paid to the study of sexual reproduction. In order to investigate the cellular and molecular mechanisms that regulate pollen-pistil interactions in the olive during the progamic phase, it is essential to first have a good knowledge of the reproductive structures involved in such interactions. This study characterizes the anatomical and ultrastructural changes in the olive pistil, beginning from the young pistil developing within the bud until the time of petal loss and visible stigma senescence. We have correlated changes in the pistil with a series of defined floral developmental stages and determined that olive pistil structures cannot be considered completely mature and ready to be pollinated and fertilized until the onset of anthesis. Our results clearly show histological and ultrastructural variation during the diverse flowering events. We discuss whether the changes observed might influence or result from pollen-pistil interactions during the progamic phase.     

Physiological and biochemical changes during organogenesis and somatic embryogenesis of HBsAg-transgenic cherry tomato mutant

Leaf explants of the HBsAg-transgenic cherry tomato (Solanum lycopersicum) mutant were cultured on Murashige and Skoog (MS) basal medium, supplemented with 1.0 mg/L 6-BA and 0.05 mg/L IAA for callus induction, to clarify the physiological and biochemical characteristics of morphogenesis development. Therefore, the physiological and biochemical changes during the development of organogenic shoots and somatic embryos in the mutant were studied. Superoxide dismutase (SOD) activities of the mutant had only one peak value on the 21st day. Peroxidase (POD) activities of the mutant declined less sharply since the explants were cultured. IAA oxidase activity of the mutant increased steadily until 42 days from culturing and then decreased sharply. Malondialdehyde (MDA) of the mutant showed a significant decreasing trend after 21 days from culturing. Growth rate of the mutant was at times lower than that of the control during its callus differentiation, and the soluble protein content of the mutant callus decreased from explant cultivation until the 28th day of culture. The mutant had greater values of chlorophyll a, carotenoid and Chlorophyll contents than the control after 14 days of culturing, and Chlorophyll b content of the mutant showed a declining trend. The electrical conductivity trend of the mutant was consistent with that in the control. It indicated that in terms of the organogenesis or somatic embryogenesis pattern, protein synthesis and catabolism were very active, and a number of antioxidant enzyme activities were consistent in the early development stages of the two regeneration systems. These findings were useful for the regeneration of the mutant.     

Morphological and histological changes in the swim bladder during maturation of the Japanese eel

Silver eels Anguilla japonica ( I _G 1·5–3·5) had more developed rete mirabile, gas gland and submucosa than yellow eels ( I _G 0·4–1·5) and the development of swim bladder components increased with sexual maturity only in the early maturation process ( I _G 3·5). These observations indicate that the Japanese eel develops its swim bladder in either the river or shallow sea water and leaves for the open sea after the swim bladder has become adapted to a deeper-sea environment.     

枣树体细胞胚发生和组织学研究

以临泽小枣子叶切块为外植体,在附加０．２mg/L IBA 1．0mg/L 6-BA的ＭＳ培养基上１周后切块边缘可诱导出白色胚愈伤组织,继续培养１个月后愈伤组织中产生体细胞胚。体细胞胚发生不同步,经历球形胚、心形胚、子叶胚等阶段,与合子胚发育途径相似。组织切片表面胚性愈伤组织细胞体积小,细胞核大、细胞质浓,细胞排列紧密;而非胚性愈伤组织细胞体积大、细胞核小、细胞质稀薄,子叶胚时期体细胞胚内部出现维管束,并观察到螺纹导管。     

Acquisition of embryogenic competence during somatic embryogenesis

This review focuses on investigation in acquisition of embryogenic competence during somatic embryogenesis in the last five decades. In tissue culture, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells during the induction phase. These embryogenic cells are important because they differentiate to form somatic embryos at a later time. Various molecular and structural markers for detecting embryogenic cells or enhancing embryogenic competence are summarized and implications of the findings are discussed.     

Meiosis-like reduction during somatic embryogenesis of Arabidopsis thaliana

Summary Somatic meiosis-like reduction was observed in some cells of the embryogenic callus of Arabidopsis thaliana. Two types were identified. One type was somatic chromosome reductional grouping, in wich the chromesomes in a cell were separated direetly at either prophase or metaphase. Chromosome reductional grouping happened more frequently in polyploid cells, and the morphology of the chromosomes did not show the role of the spindle fibers. The other type was somatic meiosis which was analogous to the process of gametogenesis, characterized by the pairing and synapsis of homologous chromosomes. The roles of somatic meiosis-like reduction in somatic embryogenesis and somaclonal variations are discussed     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

AtLTP1 luciferase expression during carrot somatic embryogenesis

The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo.     

珍稀濒危植物银鹊树体细胞胚时期同工酶分析

采用聚丙烯酰胺凝胶电泳技术对银鹊树体细胞胚胎发生过程中的酯酶(EST)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和淀粉酶(AMY)进行了同工酶分析.结果表明:球形胚时期的EST、POD、SOD、AMY同工酶活性最强;在体细胞胚胎形态建成过程中,SOD同工酶有新酶的合成,而POD同工酶则表现为活性表达增加并有新酶合...     

Synthesis of morphinane alkaloids during opium poppy somatic embryogenesis

Opium poppy (Papaver somniferum L.) tissue cultures were examines by thin-layer and high performance liquid chromatography (TLC; HPLC) for qualitative and quantitative changes in morphinane alkaloid content during somatic embryogenesis. Somatic embryos were examined at weekley intervals over a 7-week induction and maturation period. Thebaine was the only morphinane positively identified in tissue extracts and in spent growth media. Neither morphine nor codeine were produced in detectable quantities during somatic embryogenesis. Production of thebaine was developmentally regulated, gradually increasing following the removal of auxin from the culture medium. Accumulation of this alkaloid in the growth medium paralleled its appearance in somatic embryos. Alkaloid synthesis in somatic embryos appeared to require a minimum level of organization that could also be disrupted by the spontaneous loss of embryogenic potential that was observed in some culture lines.     

Expression of the BBM gene during somatic embryogenesis of Arabidopsis thaliana

The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture. BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis.     

DNA methylation during sexual embryogenesis and implications on the induction of somatic embryogenesis in Castanea sativa Miller

From anthesis to mature seed formation, burrs from cross-pollinated adult Castanea sativa Miller trees were characterized and seven developmental stages defined based on macro and micromorphological traits. In order to get an insight into the involvement of epigenetic mechanisms in sexual embryogenesis and to define somatic embryogenesis induction capability, global DNA methylation and the somatic embryogenic competence were quantified. On cross-pollinated trees once fertilization takes place, at least one ovule per ovary becomes dominant, and transient DNA demethylation occurs coinciding with the start of the sexual embryogenic programme. Unfertilized ovules from the same cluster, which maintain their prior size, increase their methylation level and undergo degeneration. These results were validated using non-cross-pollinated trees and the asynchrony of flower receptivity. When testing in vitro somatic embryogenesis response of isolated dominant ovules and axes from zygotic embryos under cross-pollinated conditions, the highest competence was found for reaching seed maturity. Thus, a “developmental window” of somatic embryogenesis in chestnut has been characterized. It includes from fertilization to embryo maturity, and a transient decrease in methylation is necessary after fertilization for the development of the somatic embryogenesis response.     

Analysis of the heat-shock protein pattern during somatic embryogenesis of carrot

Plant Molecular Biology - The pattern of proteins synthesized by carrot cells after heat-shock was analyzed by SDS-polyacrylamide gel electrophoresis. A study was made of the time of appearance and...     

Morphological and histological changes of dermal scales during the fish‐to‐tetrapod transition

Witzmann F. (2011). Morphological and histological changes of dermal scales during the fish‐to‐tetrapod transition. —Acta Zoologica (Stockholm) 92 : 281–302. The gastral scales of limbed tetrapodomorphs evolved from the ‘elpistostegid’‐type of scale by an enlargement and differentiation of the articulation facets and a shortening and broadening of the keel. These changes caused a tighter connection between gastral scales within a scale row and a greater overlap between the rows. Dorsal round scales of limbed tetrapodomorphs developed from a gastral scale‐type by an alteration of the ontogenetic pathway. The posterolateral direction of scale rows in ‘elpistostegids’ was retained in the gastral scalation of most limbed tetrapodomorphs, whereas the arrangement of round dorsal scales is modified to a transverse orientation. Both gastral and dorsal scales of limbed tetrapodomorphs consist solely of parallel‐fibred bone with circumferential growth marks. The proportionally larger overlap surfaces of gastral scales and their mode of articulation in the ventral midline indicate that the body of limbed tetrapodomorphs might have been more flexible than that of their finned relatives. The alteration of dermal scales was one of the most rapid morphological changes during the fish‐to‐tetrapod transition. Once established, gastral and dorsal scales were retained as a conservative character in different lineages of basal tetrapods, in both the amphibian and the amniote lineages.