On the occurrence of somatic meiosis in embryogenic carrot cell cultures

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

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.     

Developmental cell death in dictyostelium does not require paracaspase

Apoptotic cell death often requires caspases. Caspases are part of a family of related molecules including also paracaspases and metacaspases. Are molecules of this family generally involved in cell death? More specifically, do non-apoptotic caspase-independent types of cell death require paracaspases or metacaspases? Dictyostelium discoideum lends itself well to answering these questions because 1) it undergoes non-apoptotic developmental cell death of a vacuolar autophagic type and 2) it bears neither caspase nor metacaspase genes and apparently only one paracaspase gene. This only paracaspase gene can be inactivated by homologous recombination. Paracaspase-null clones were thus obtained in each of four distinct Dictyostelium strains. These clones were tested in two systems, developmental stalk cell death in vivo and vacuolar autophagic cell death in a monolayer system mimicking developmental cell death. Compared with parent cells, all of the paracaspase-null cells showed unaltered cell death in both test systems. In addition, paracaspase inactivation led to no alteration in development or interaction with a range of bacteria. Thus, in Dictyostelium, vacuolar programmed cell death in development and in a monolayer model in vitro would seem not to require paracaspase. To our knowledge, this is the first instance of developmental programmed cell death shown to be independent of any caspase, paracaspase or metacaspase. These results have implications as to the relationship in evolution between cell death and the caspase family.     

Tunicamycin affects somatic embryogenesis but not cell proliferation of carrot

The antibiotic tunicamycin which specifically blocks the first step in the lipid-linked oligosaccharide pathway is capable of arresting somatic embryogenesis in a reversible way. At the same drug concentration cell proliferation is not affected. The quantitative and qualitative changes induced by tunicamycin in glycolipids and glycoproteins are the same in embryogenic and non-embryogenic conditions and this might therefore indicate some proteins whose glycosylation is essential for development.     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.     

Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension

In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

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.     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.     

Purification and immunohistochemical detection of an embryogenic cell protein in carrot

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.     

Induction of secondary cytotoxic T-lymphocytes in vitro does not require cell proliferation.

Using a mouse in vitro allograft model, evidence has been obtained that, in contrast to the accepted view, the generation of cytotoxic effector function in T-lymphocytes does not necessarily require cell division.     

Chromosome replication does not trigger cell division in E. coli

An essential part of the chromosome replication origin of E. coli K-12 and B/r was replaced by the plasmid pOU71. The average initiation mass of replication for pOU71 decreases with increasing temperature. The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry. The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected. Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature. The increased DNA content could not be explained by an increase in the length of the C period. It is concluded that chromosome replication does not trigger cell division in E. coli, but that the chromosome replication and cell division cycles of E. coli run in parallel independently of each other.     

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.     

Arginine and ornithine decarboxylases in embryogenic and non-embryogenic carrot cell suspensions

The in vivo activities of arginine and ornithine decarboxylases, key enzymes in the biosynthesis of putrescine and thus polyamines, were measured in three different cell lines of carrot (Daucus carota) during growth and somatic embryogenesis. The activities of these two enzymes differed in the different cell lines in the presence of various levels of auxin (2,4 dichlorophenoxy acetic acid), but was highest during periods of active cell division. During somatic embryo development, the activities of both enzymes were highest during globular stage formation. Thus, both enzymes were found to be active during growth and somatic embryogenesis and could contribute to polyamine biosynthesis.     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

Optimization of culture medium extends response time of embryogenic carrot cells but does not restore initial high response of young cultures

The greatest number of embryos was obtained using young carrot callus cultured on Gamborg's B-5 medium containing 1 g l-1 casein hydrolysate and 10 -10 M 2,4-dichlorophenoxyacetic acid. The largest increase in embryogenesis for old cultures also was obtained using Gamborg's B-5 medium supplemented with 1 g l-1 casein hydrolysate and 10 -10 M 2,4-dichlorophenoxyacetic acid; however, no combination of factors for the older culture restored the initial vigorous morphogenetic response seen in young cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.