Megachromosomes in tissue culture ofAllium

Chromosome studies have been conducted on the long-term callus cultures of threeAllium species:A. porrum (2n=32),A. tuberosum (2n=32) andA. fistulosum (2n=16). In cultures ofA. fistulosum several interesting cytological abnormalities were observed. They included direct elimination of chromatin from nuclei, multiple chromosome fusions and formation of polycentric and megachromosomes. The rate of abnormalities increased with the time of culture. Allium fistulosum may provide an excellent model system to analyse cytogenetic and molecular aspects of callus-induced genomic changes and, thus, somaclonal variation.     

Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol

Calcium is an important regulator of a variety of neuronal activities including gene expression. However, it is not clear how Ca2+ influx affects intracellular Ca2+ concentration [( Ca2+]i) in the nucleus. We have taken advantage of laser photometry, the Ca2(+)-sensitive dye Indo-1 that allows ratio imaging, and confocal microscopy to eliminate the influences of unequal cell geometry and dye distribution. We show that Ca2+ influx into sympathetic neurons causes a significantly greater and faster increase in [Ca2+]i in the nucleus than in the cytosol. The differential increase in nuclear [Ca2+]i was apparent when Ca2+ entered from the extracellular medium during K+ depolarization, ionomycin or acetylcholine treatment, and brief periods of electrical stimulation. When intracellular Ca2+ was mobilized by caffeine the rise in nuclear [Ca2+]i was again greater than in any other region of the neuron. The increased nuclear Ca2+ levels were uniform throughout the nucleus and not associated with the nuclear envelope. The differential rise in nuclear Ca2+ was eliminated by acridine orange binding to nucleic acids. Nonexcitable cells (astrocytes, oligodendrocytes, and fibroblasts) did not show differential distribution of Ca2+ after ionomycin treatment. These results support the idea that activity-dependent gene regulation in sympathetic neurons may be mediated by changes in Ca2+ concentration at the level of the chromatin material.     

Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape.

Undifferentiated embryonic mesenchymal cells are round/cuboidal in shape. During development, visceral myogenesis is shortly preceded by mesenchymal cell elongation. To determine the role of the cell's shape on smooth muscle development, undifferentiated embryonic mesenchymal cells from intestine (abundant visceral muscle), lung (some visceral muscle) or kidney (no visceral muscle) were plated under conditions that maintained cell rounding or promoted elongation. Regardless of their fate in vivo, all the cells differentiated into smooth muscle upon elongation as indicated by the expression of smooth muscle-specific proteins and the development of membrane potentials of -60 mV and voltage-dependent Ca2+ currents, characteristic of excitable cells. Smooth muscle differentiation occurred within 24 hours and was independent of cell proliferation. Regardless of their fate in vivo, all the round cells remained negative for smooth muscle markers, had membrane potentials of -30 mV and showed no voltage-activated current. These cells, however, differentiated into smooth muscle upon elongation. The role of the cell's shape in controlling smooth muscle differentiation was not overcome by treatment with retinoic acid, TGF-beta1, PDGF BB or epithelial-conditioned medium (all modulators of smooth muscle differentiation). These studies suggest that the mesenchymal cell shape plays a main role in visceral myogenesis.     

Exogenous Carbohydrate Utilisation by Explants of Brassica napus Cultured in vitro

In 5; non-coding regions of genes for phytochrome A from horseradish (ArPHYAs) were fused with the 35S promoter of cauliflower mosaic virus in the antisense direction (CaMV35SantiArPHYAs) and introduced into horseradish hairy roots. Phytochrome levels of proximal areas in many hairy roots that had been transformed with CaMV35SantiArPHYAs decreased to levels of about one-half to one-quarter those of control hairy roots. The extent of the light-induced formation of adventitious shoots from hairy roots with less than half of the control level of phytochrome was lower than in the controls and not different between the three ArPHYAs. In contrast, the efficiency of phytochrome on the extent of shoot formation differed in hairy roots transformed with CaMV35SantiArPHYAs when phytochrome levels were more than 0.02 (;(;A) g^–1). The efficiency of ArPHYA3 to initiate shoot formation was greatest and that of ArPHYA2 was smallest. Furthermore, previous reports on hairy roots overexpressing ArPHYAs showed a similar efficiency of phytochrome on shoot formation. These results indicate that the ArphyA1 and/or ArphyA3 play major roles in the light-induced formation of adventitious shoots and that ArphyA2 has a minor role.     

Endosperm response to pollen irradiation in kiwifruit

 Cytological details of endosperm development after pollination with irradiated pollen were studied in Actinidia deliciosa (kiwifruit) cultivar Hayward. Pollinations were carried out involving five different sources of pollen (Matua, Tomuri, Burt, Berryman, and fruiting male) irradiated with gamma rays at doses of 700 and 900 Gy. Non-irradiated crosses were used as controls. Irradiated pollen induced development of approximately 25–30% of the ovules. Two types of ovules were observed: (1) with both embryo and endosperm and (2) with endosperm only. No mitotic abnormalities were found in control or irradiated endosperms. Mitotic divisions were regular and nuclei spherical and evenly spaced. However, the cells of irradiated endosperm usually contained low amounts of storage products. Ploidy level of the endosperms was evaluated by nuclear size (volume) with the use of image analyzis. Mean nuclear size in control and irradiated endosperms was 1598.3 and 750.9 μm³, respectively. It is concluded that endosperm produced after pollination with irradiated pollen is autonomous and represents the 2n level. Received: 14 October 1998 / Revision accepted: 10 March 1999     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

The reticuloplasmin calreticulun is released into the medium by carrot cell cultures

We report here the presence of a 58-kDa protein in the cells of Daucus carota L. cultivated in vitro. Two lines of carrot cells are used: wild-type line (wt) and mutant line (ts11). We describe here also presence of this protein in the media of cultured cells. Strong reaction of this intracellular and extracellular protein with an anti-calreticulin antiserum indicates that it is a major high capacity, low affinity Ca²⁺-binding reticuloplasmin–calreticulin. No differences in biochemical characterization is found between calreticulin purified from the wild-type line and the mutant line. Moreover molecular mass, type of glycosylation and the ability of extracellular protein to bind calcium is found to be indistinguishable from those of the purified intracellular calreticulin. Calreticulin release is attributed to some stress imposed on cultured cells by growth conditions. It is shown that this process can be also induced in CR-non-releasing systems such as carrot somatic embryos by applying a high-cell-density stress.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.