Torpedo Stage Research Articles

Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5′-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.

PRODUCTION OF HIGH QUALITY SEEDLINGS BY IMMATURE ZYGOTIC EMBRYO CULTURE OF WASABIA JAPONICA MATSUM.

Immature zygotic embryos of Wasabia japonica (var. Dalma) were isolated from immature seeds of field-grown plants and cultured on modified MS media supplemented with auxins and cytokinins. The stages of zygotic embryos were classified into torpedo, early cotyledonary, and late cotyledonary stages. The early cotyledonary zygotic embryos were more productive than the torpedo stage or late cotyledonary ones in somatic embryogenesis, and they also produced greater numbers of somatic embryos. At the early cotyledonary stage, the highest overall embryogenesis was achieved in basal media containing 2.0 mg.L -1 IAA. The generation of somatic embryos was best in media with 2.0 mg.L -1 of kinetin, and plantlets generated from somatic embryos had phenotypically normal leaves and roots. Shoots lacking roots were separated individually, and transferred to medium for multiple shoot and root formation. Rooting and root growth were best in media with 0.01 mg.L -1 IBA, and multiple shoots were produced in media containing BA. But, the combinations of BA and 0.01 mg.L -1 IBA showed little or no effect on rooting.

溶存酸素濃度制御によるニンジン不定はい培養の同調性改良

Low synchronization of somatic embryogenesis prevents the application of a large-scale production bioreactor to a transplant production system. We attempted to improve synchronization of carrot somatic embryogenesis for large-scale production by controlling the dissolved oxygen concentration. A suitably low dissolved oxygen concentration repressed excessive development of cotyledonary-stage embryos but did not affect the development of globular, heart, and torpedo-stage embryos. As a result the development of somatic embryos stopped at the torpedo stage under this condition. Moreover, the number of torpedo-stage embryos increased, and it was larger than the maximum number of torpedo-stage embryos in culture with ambient oxygen aeration. These torpedo-stage embryos regenerated normal plantlets after mild dehydration treatment. This method does not require changing the medium nor filtration of the somatic embryos during the culture period for synchronization of the culture. If this simple method is used for large-scale production of somatic embryos with a bioreactor, effective synchronization can be achieved by only controlling the dissolved oxygen concentration.

Somatic embryogenesis in Canary Island date palm

Zygotic embryo and shoot tip explants of Phoenix canariensis were cultured on MS (1962) basal medium supplemented with 100 μM Picloram and 9.5 μM kinetin or 10.8 μM or 45.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.8 μM N6-(2-isopentenyl) adenine (2iP). These explants after 12 weeks in darkness at 28 °C, produced embryogenic callus with very compact, pale yellow, nodular structures. Proliferation and maintenance of embryogenic callus was on MS basal medium with 2.26 μM 2,4-D, 0.83 μM kinetin and 2 μM abscisic acid (ABA), with a regular subculture every 3–4 weeks. Somatic embryo development was promoted by two months of culture on MS liquid medium enriched with 2 μM ABA, for torpedo stage development, then on liquid MS medium with 1 μM N6-benzyladenine (BA) and 0.46 μM kinetin, for shoot induction. Germinated embryos were transferred to basal media enriched with 0.45 μM BA and 0.06 μM naphthaleneacetic acid (NAA) for root and shoot induction and elongation. Viable plants were recovered on basal MS free of plant growth regulators (PGRs), but percentages of plant conversion have to be improved.

Induction of somatic embryogenesis from nucellus-derived callus of Anacardium occidentale L

Calli were induced from nucellar explants excised from 1-month-old developing fruits of Anacardium occidentale L. (cashew). The medium consisted of Murashige and Skoog's formulation containing 6.78 μM 2,4-D. Differentiation of somatic embryos from calli was noticed when they were transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on the ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical or filamentous pro-embryos. Subsequent divisions in the pro-embryo led to globular, heart and torpedo stages of somatic embryos. There was no further development of the torpedo stage in the liquid medium containing 2,4-D.

Direct somatic embryogenesis and plant regeneration of carnation (Dianthus caryophyllus L.)

Conditions for efficient direct somatic embryogenesis and plant regeneration of leaf explants from carnation cultivars Lena (SIM group) and Bulgarian spray cultivars Nasslada, Yanita, Regina and Line 84 were established. Murashige and Skoog (MS) liquid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-benzylaminopurine was used for direct induction of embryoids without an additional callus phase. The first globular structures were observed after 20 days of cultivation. Their further development to the torpedo stage was correlated with the presence of polyethylene glycol (PEG 6000). Somatic embryo maturation was promoted by casein hydrolysate (1000 mg/l) in MS liquid media. The percentage conversion of embryos and polyembryos to whole plants varied between 10 and 75% among studied cultivars. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in a greenhouse.

Somatic and zygotic embryos of Daucus carota L. display different protein patterns until conversion to plants.

Total protein patterns of different developmental stages of carrot zygotic and somatic embryos revealed by one- and two-dimensional gel electrophoresis were compared using statistical dissimilarity index matrix, and some major polypeptides were partially sequenced. In spite of similar morphology, the protein patterns of somatic embryos at the torpedo stage were clearly different from those of zygotic embryos. In particular, none of the proteins specific of zygotic embryos required for maturation, previously identified, were accumulated in somatic embryos, namely the daucin (a globulin-type storage protein), the RAB25 protein (a late embryogenesis abundant protein) (Dodeman et al. 1998), as well as a novel globulin of M(r) 30,000, that we proposed to name apiacin. Somatic plantlets and seedlings also showed different patterns. This discrepancy likely reflects culture conditions, since somatic embryos recover a protein pattern close to that of seedlings after conversion to plant and growth on a carbon-free medium.

Temporal and transient expression of stearoyl-ACP carrier protein desaturase gene during olive fruit development

To study the regulation of gene expression during olive (Olea europaea) fruit development, a full length cDNA encoding the stearoyl-ACP desaturase (EC 1.14.99.6), which catalyses the conversion of saturated stearic acid to mono-unsaturated oleic acid was isolated. The primary structure of the protein as deduced from the nucleotide sequence of the cDNA includes a putative 27-amino acid transit peptide. Amino acid sequence comparison showed substantial similarity to Δ9-desat-urases isolated from other plant species. The two conserved motifs D/EEXXH, involved in the catalytic reaction are localized in similar positions to the other desaturases. Δ9-desaturase gene expression is developmentally regulated. Comparative Northern blot analysis exhibited distinct patterns of temporal gene regulation and mRNA accumulation in the three tissues of the developing olive fruit. In embryos and in the corresponding endosperms, Δ9-desaturase expression starts early during heart-stage, while in the mesocarp it starts later. Maximum expression and transcript accumulation in embryos and endosperms is observed at 13-14 WAF (early torpedo stage), whereas at 22 WAF (late torpedo stage), the Δ9-desaturase message was almost undetectable. In the mesocarp, gene expression and mRNA accumulation is active upto 28 WAF. This accumulation during fruit growth parallels the synthesis of oil in this oleogenic tissue. ABA modulates transiently Δ9-desaturase gene expression in immature olive embryos. Short exposures to ABA positively influenced gene expression whereas longer exposures decreased mRNA accumulation. Fluridone, an inhibitor of ABA synthesis and accumulation, decreased mRNA levels while ABA added to fluridone supplemented media restored Δ9-desaturase transcript accumulation. These results suggest that Δ9-desaturase gene expression in olives is temporally and developmentally regulated in olive fruit.

Comparative Morphological Study of Zygotic and Microspore-derived Embryos ofBrassica napusL. as Revealed by Scanning Electron Microscopy

A comparative morphological study of microspore-derived (MD) and zygotic embryos ofBrassica napusL. was conducted, illustrating substantial similarities in external morphology of these embryos throughout their development. Haploid embryos were produced from isolated microspores cultured on high molecular weight polyethylene glycol (PEG), replacing sucrose as an osmoticum. Morphological changes during the time-course of microspore embryo development induced on PEG (25%) and sucrose (13%) are described in detail as revealed by scanning electron microscopy (SEM) and compared to the corresponding stages of zygotic embryos developedin ovulo. At the heart, torpedo and early cotyledonary stages, microspore-derived (MD) embryos on PEG closely resemble their zygotic counterparts. In contrast, the external morphology of embryos induced on high sucrose medium differs from that of PEG and zygotic embryos indicating that a high concentration of sucrose in culture has a morphogenetic effect on MD embryo development inB. napus. Fragments of the original pollen wall are regularly observed at the root pole region and at the tips of suspensors in MD embryos throughout their development. This suggests that polarity in MD embryos might originate from structurally polarized late uninuclear microspores and early bicellular pollen.

Protoplast fusion between Lycopersicon esculentum and L. peruvianum -complex: somatic embryogenesis, plant regeneration and morphology

Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid.

Precociously germinating somatic embryos of Vitis vinifera have lower ABA and IAA levels than their germinating zygotic counterparts

Somatic embryos of Vitis vinifera (cv. Grenache noir) develop normally up to the torpedo stage, but they germinate precociously and form viable plantlets with very low frequency. Because a peak in abscisic acid (ABA) in mid‐embryogenesis could be one factor preventing precocious germination during normal seed development, we followed the development of ABA content concurrent with that of the somatic embryos. Additionally, we measured changes in indoleacetic acid (IAA) levels. We also compared the levels of both hormones during precocious germination of somatic embryos and during normal germination of their zygotic counterparts. Somatic embryos were able to accumulate ABA and IAA throughout their development but no peak in ABA concentration was detected during embryogenesis. This suggests that the switch from mid‐ to late‐embryogenesis is not triggered. Furthermore, during precocious germination, i.e. from the torpedo stage onwards, the concentrations of ABA and IAA in somatic embryos were much lower than during normal germination of zygotic embryos. Thus, it is likely that when precocious germination occurs, grape somatic embryos do not accumulate ABA and/or IAA in sufficient concentrations to support normal plantlet development. Therefore, for grape somatic embryos we propose that prevention of precocious germination, i.e. triggering late‐embryogenesis, is attainable by an ABA treatment followed by slow desiccation, as already shown for conifer somatic embryos. Our results also suggest that the role of ABA and IAA for improving normal germination after imposed quiescence should be investigated.

Comparison of starch, ADP-glucose pyrophosphorylase and starch synthase levels in non-embryogenic cells and developing embryos in Hevea brasiliensis

Summary Sucrose, glucose, fructose and starch levels, and ADP-glucose pyrophosphorylase (EC 2.7.7.27) and starch synthase (EC 2.4.1.21) activities were studied in an embryogenic strain of Hevea brasiliensis during the periods of multiplication of proembryogenic clusters and development of somatic embryos. For comparison, a non-embryogenic strain was also investigated. The development of somatic embryos was studied on a medium containing polyethylene glycol and abscisic acid and a medium devoid of both. First, a correlation between ADP-glucose pyrophosphorylase activity and starch accumulation was shown during the multiplication period of proembryogenic clusters. Second, starch accumulation in somatic embryos was observed at the end of the developmental period on the medium containing polyethylene glycol and abscisic acid but not on the medium devoid of these substances. In non-embryogenic cells, starch levels were similar whatever the culture conditions. However, this difference in starch content was not reflected in the starch biosynthetic capacity inferred from the ADP-glucose pyrophosphorylase and starch synthase activities, which were similar in the two culture conditions and in somatic embryos and non-embryogenic cells. Also, somatic embryos were more numerous, especially at the torpedo stage, on the medium containing polyethylene glycol and abscisic acid than on the medium devoid of these substances.

Pattern of DNA binding of nuclear proteins to the proximal Agrobacterium rhizogenesrolC promoter is altered during somatic embryogenesis of carrot

Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages. Since the proximal −255 bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation on the uidA gene in transgenic carrot cell culture, we investigated the interaction of nuclear proteins with the proximal −255 bp upstream sequences to characterize the mechanism of somatic embryogenesis-related activation. Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses), globular embryos, and heart/torpedo embryos. The binding activity associated with a fragment (−203 bp to −92 bp) of one protein (BI) was most abundant in globular embryos. Another DNA binding protein (AII) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and globular embryos. Such an altered pattern of DNA binding activities of nuclear proteins may contribute to somatic embryogenesis-related activation of the rolC promoter. Competition experiments with oligonucleotides revealed that the BI protein interacts with AT-rich sequences.

Molecular and biochemical characterization of a proteasome subunit from rice and carrot cells.

Proteasomes function mainly in the ATP-dependent degradation of proteins that have been conjugated with ubiquitin. We isolated a cDNA clone for a rice protein that exhibited high homology to subunit C2 of proteasomes. Southern blot analysis revealed that the corresponding gene was present as a single copy in the rice genome. After fractionation of a crude extract from cultured cells, a 35-kDa protein that cross-reacted with antibodies against the C2 subunit was recovered in the peak fraction of both 20 S and 26 S complexes. The same antibodies cross-reacted with two proteins in seedlings, one of which was the same as that detected in cultured cells. The level of the protein was reduced in roots under conditions of high salinity. The 35-kDa protein was not detected in the nuclei of rice of carrot cells. However, during somatic embryogenesis of carrot cells, the C2 subunit was found in the nucleus at the globular stage, and it gradually disappeared in the period from the heart to the torpedo stage. Cells at the globular stage are proliferating rapidly, thus, it is possible that proteasomes are associated with the proliferation of plant cells.

Fertilization-independent seed development in Arabidopsis thaliana.

We report mutants in Arabidopsis thaliana (fertilization-independent seed:fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, approximately 50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.

Biphasic expression of a Vicia faba legumin B gene in developing seeds of transgenic tobacco

Summary We analysed the spatial and temporal expression of the Vicia faba legumin gene LeB4 in developing seeds of transgenic tobacco plants by means of in situ hybridization and immunohistostaining. The results indicate that the expression of the LeB4 gene is regulated in embryo and endosperm in a biphasic manner. Legumin accumulation starts during early seed development at low amounts in single cells of the integument. Accumulation proceeds successively in the proembryo, suspensor and endosperm reaching maximum levels in all cells of these organs around 10 to 12 DAP. With the beginning of the heart stage (around 13 DAP) legumin reserves disappear completely from all parts of the seed. The described early phase of legumin accumulation occurs in embryo and endosperm still before the onset of the cell expansion phase in a period of continuous mitotic activity. A second expression phase starts in embryo and endosperm around 18 DAP, as the embryo enters late torpedo stage. Within the embryo the accumulation is restricted to the parenchyma cells of cotyledons and hypocotyl, while the embryonic root and provascular tissue do not accumulate LeB4 protein. In addition, a low percentage ( V. faba for the legumin and vicilin genes during early embryogenesis (Panitz et al., 1995), but differs from that of tobacco 12S globulin genes, which are expressed in the embryo not before the late heart stage.

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by β-cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10-11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4-5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones "b" and "d" of esterase and zone "b" of phosphogluconate dehydrogenase coincided with vascular bundle formation.

An Ultrastructural Study of The Embryo/Endosperm Interface in the Developing Seeds of Solanum nigrumL. Zygote to Mid Torpedo Stage

The early developmental sequences in the formation of the Zone of Separation and Secretion in a hexaploid species of Solanum nigrumL. are described. Ultrastructural changes which occurred during the development of the embryo/endosperm interface could be related to the different stages in the embryo's development. The first step was the completion of the cell wall around the chalazal end of the zygote; a thin wall was formed along the endosperm cell(s) abutting the zygote. From the mature zygote stage to the quadrant stage, minute plasmalemma invaginations occurred along the endosperm wall facing the zygote. These invaginations enlarged, and from the mid-globular stage onwards became filled with a fine fibrillar material; this material accumulated between the endosperm cell wall and the plasmalemma before being released into the developing periembryonic and intercellular spaces to become the extracellular matrix. Cell wall development in the endosperm cells abutting the embryo followed an unusual path. During the quadrant stage, whilst the outer embryo wall increased in thickness due to vesicle fusion, the endosperm cell wall facing the embryo showed a loosening of the wall fibrils as well as partial separation of these same endosperm cells from each other. From the early-globular stage, the endosperm cell walls opposite the embryo became electron-translucent, disappearing into the extracellular matrix. Enzymic secretions by the embryo may account for the alteration in the abutting endosperm cell walls. Enzymic activity may also explain the development of a homogenous electron-opaque layer over the outer embryo wall as well as the differences in the width of the fibrillar layer which accumulated around the cotyledons as the embryo grew through the Zone of Separation and Secretion. The potential roles of the extracellular matrix are briefly discussed.

Histological Comparisons of Shoot-forming and Nonshoot-forming Somatic Embryo Variants of Sweetpotato (Ipomoea batatas (L) Lam)

A comparison of external morphology captured via a computer vision system and a study of internal anatomy of sweetpotato somatic embryos identified five different major morphological variants among torpedo and cotyledonary stage embryos. These included 1) Perfect Type, 2) Near Perfect Type, 3) Limited/No Meristematic Activity Type, 4) Disrupted Internal Anatomy Type, 5) Proliferating Type. Perfect and Near Perfect types of somatic embryos were categorized as competent, while Limited/No Meristematic activity, Disrupted Internal Anatomy, and Proliferating types were categorized as noncompetent with respect to their conversion ability. Lack of organized shoot development in somatic embryos of sweetpotato was attributed to the following abnormalities: 1) lack of an organized apical meristem, 2) sparsity of dividing cells in the apical region, 3) flattened apical meristem, 4) multiple meristemoids and/or diffuse meristematic activity throughout the embryo. A morphological fate map of most of the torpedo and cotyledonary embryo variants was identified, which will be beneficial in synthetic seeding and transgenic research and development of sweetpotato.

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine ( Vitis viniferaL.): Embryonic Organogenesis

Somatic embryos of Vitis viniferaL. ‘Grenache noir’ develop abnormally and form viable plantlets at very low frequencies. They grow continuously and, after the torpedo stage, they form giant structures which do not undergo further organogenesis. Morphological, histological and cytochemical data were used to study development from the globular to the giant-embryo stage. Histological organization of somatic embryos until the torpedo stage was similar to that of zygotic embryos. Somatic embryos formed bipolar axes, which differentiated precociously and simultaneously a root and a shoot meristem. However, they differed from their zygotic homologues by forming a cotyledonary crown or multiple cotyledons and by their rapid cellular differentiation. At the end of the torpedo stage and up to the giant-embryo stage, somatic embryos underwent some characteristic events of germination: the radical grew, tannins accumulated, and protodermal cells suberized. However the shoot apex was rapidly disorganized and disappeared. This peculiar behaviour is discussed in comparison with the phenomenon of precocious germination often observed for immature zygotic embryos in in vitroculture.