Complanate Scytosiphon lomentaria (Lyngbye) J. Agardh (Scytosiphonales: Phaeophyta) from Southern Australia: The effects of season,temperature, and daylength on the life history

Cultures of the complanate form of Scytosiphon lomentaria (Lyngbye) J. Agardh were initiated at different times of year, maintained in several temperature daylength regimes, and grown through at least three generations. The composition of the F₁ generation varied on a seasonal basis, with a greater proportion of complanate individuals in winter and a predominance of ralfsioid and and cylindrical individuals in spring-summer.In culture, complanate thalli were only initiated from zooids derived from thalli (complanate or ralfsioid) grown in short days; ralfsioid progeny developed in long days. Some kinds of ralfsioid thalli and the cylindrical thalli developed and reproduced in long and short day conditions. The effects of temperatures between 11° and 20°C were subtle, and principally related to the rate of growth of cultured plants and not to the determination of particular stages in the life history.The relationship between the seasonal occurrence of complanate S. lomentaria, its seasonal pattern of reproduction, and the effects of daylength on the life history are discussed.     

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.     

The Lichen Connections of Black Fungi

Many black meristematic fungi persist on rock surfaces—hostile and exposed habitats where high doses of radiation and periods of desiccation alternate with rain and temperature extremes. To cope with these extremes, rock-inhabiting black fungi show phenotypic plasticity and produce melanin as cell wall pigments. The rather slow growth rate seems to be an additional prerequisite to oligotrophic conditions. At least some of these fungi can undergo facultative, lichen-like associations with photoautotrophs. Certain genera presenting different lifestyles are phylogenetic related among the superclass Dothideomyceta. In this paper, we focus on the genus Lichenothelia, which includes border-line lichens, that is, associations of melanised fungi with algae without forming proper lichen thalli. We provide a first phylogenetic hypothesis to show that Lichenothelia belongs to the superclass Dothideomyceta. Further, culture experiments revealed the presence of co-occurring fungi in Lichenothelia thalli. These fungi are related to plant pathogenic fungi (Mycosphaerellaceae) and to other rock-inhabiting lineages (Teratosphaeriaceae). The Lichenothelia thallus-forming fungi represent therefore consortia of different black fungal strains. Our results suggest a common link between rock-inhabiting meristematic and lichen-forming lifestyles of ascomycetous fungi.     

A cold shock-syringe method for the preparation of an axenic culture of the giant coenocytic alga Valonia ventricosa

We developed a new method for the preparation of an axenic culture of the giant coenocytic green alga Valonia ventricosa. Aplanospore formation was induced by exposing the organism to cold shock (15°C) for one day. The aplanospore was then aseptically isolated with a syringe. The aplanospore grown on ESS₁ agar medium developed into axenic thalli (2 mm in diameter) after 2 months.     

Production of berberine in cultured cells of Coptis japonica

A culture of small fragments of tissues (rootlets) of Coptis japonica on a solid medium followed by successive liquid culture produced friable cell lines with high growth rates and a high berberine content. Light inhibited growth and berberine production in these cell lines whereas high aeration stimulated both.     

AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU-1) cultured aseptically in defined synthetic media . Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.     

Synergistic effects of metabolically related amino acids on the growth of a multicellular plant

The effects of several amino acids related to the metabolism of aspartic acid on the growth and development of gemmalings of the liverwort Marchantia polymorpha were investigated under axenic conditions. Lysine and theonine synergistically inhibit the growth of these plants and cause a loss of normal pigmentation at concentrations as low as 1 μm. These effects are highly specific for this pair of amino acids, are partially reversible upon removal of the effectors, and can be prevented by low concentrations of methionine or its metabolic precursor, homoserine. Alterations in the growth and development of gemmalings in the presence of natural amino acids are discussed in relation to metabolic regulatory mechanisms which have been well established in microorganisms.     

Axenizing and Culturing Endomigratory Plant-Parasitic Nematodes using Pluronic F127, Including its Effects,on Population Dynamics of Pratylenchus penetrans

A non-chemical technique for surface sterilizing plant-parasitic nematodes for aseptic cultures is described. The method is most applicable to nematodes with active migratory infective stages and requires only a few starting specimens. Rate of achieving a primary aseptic culture with the technique ranged from 60%-100% depending on the conditions of the specimens collected for culturing. Aseptic cultures of species of Meloidogyne, Rotylenchuluz, Pratylenchus, and Radopholus initiated with the method remained contamination-free after 12 months of maintenance in tomato root explant or alfalfa callus cultures. Further studies of Pluronic F127, a polyol gel medium employed in the technique to confine the spread of contaminating bacteria or fungi associated with the nematodes, showed that the polyol gel was a suitable support medium for culturing corn root explant, alfalfa callus tissues, and consequently Pratylenchus species including P. agilis, P. brachyurus, P. scribneri, and P. penetrans. During the course of 10 months, P. penetrans reared in polyol-base medium followed a standard biological growth curve, multiplied to a higher population density, maintained a similar female-to-male ratio, and possessed a similar tendency to reside inside or outside host tissues as did P. penetrans reared in agar-base medium. The percentages of P. penetrans juveniles in the sub-populations residing outside or inside the host tissues reared in polyol-base medium also were similar to and fluctuated temporally in like manner as those reared in agar-base medium. Members of these sub-populations from the polyol- or agar-base were equally infective and reproductive after 9 months of culturing.     

Life histories of Colpomenia sinuosa and Hydroclathrus clathratus (Scytosiphonaceae, Phaeophyceae) in culture

Life-history studies in culture were carried out on Colpomenia sinuosa (Mertens ex Roth) Derbès et Solier and Hydroclathrus clathratus (C. Agardh) Howe (Scytosiphonales, Phaeophyceae) from Japan. These species showed a heteromorphic life-history pattern with an alternation between erect thalli bearing plurilocular zoidangia and prostrate thalli bearing ectocarpoid plurilocular and unilocular zoidangia. Plurizoids released from erect and prostrate thalli developed into prostrate thalli. Unizoids, however, developed into erect thalli. Prostrate thalli produced plurilocular zoidangia in long-day conditions and unilocular zoidangia in short-day conditions at 10-20°C. Prostrate thalli of C. sinuosa formed ascocysts. Germlings of both species did not grow at 5°C.     

Effects of deep seawater medium on growth and amino acid profile of a sterile Ulva pertusa Kjellman (Ulvaceae, Chlorophyta)

The potential use of deep seawater (DSW) for the cultivation of macroalgae was evaluated by analysis of the growth rate of thalli (by surface area), photosynthetic pigment content, and total free amino acid content of sterile Ulva pertusa cultured in the control medium (one-fifth strength Provasoli’s enriched seawater (PES) medium), surface seawater (SSW) medium and also seawater medium collected from 400 m depth (400-DSW) and 1,200 m depth (1,200-DSW). The growth rate of the sterile U. pertusa grown in 400-DSW and 1,200-DSW was similar to that of thalli cultured in the one-fifth strength PES medium that had previously been shown to be the optimum concentration for the cultivation of Ulva. Photosynthetic pigment content in the control medium, 400-DSW- and 1,200-DSW-cultured thalli was 1.7–2.0 times higher than that of the SSW-cultured thalli. The total free amino acids content of the control medium and SSW-cultivated thalli was 264?±?64 and 120?±?39 mg per 100 g dry weight, whereas the 400-DSW- and 1,200-DSW-cultivated thalli had significantly more amino acids (342?±?69–327?±?64 mg per 100 g dry weight). The results of this study indicate that DSW has a high potential for cultivation of edible macroalgae without any supply of extra nutrient until it grows to harvest size.     

New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata

Isolation of meristematic tissue of the red alga Palmaria palmata by a freezing-thawing method and further maintenance of the tissue in culture showed the existence of groups of meristematic cells in superficial cortical layers of thallus forming wart-like outgrowths. For the first time, proliferations (plantlets) were obtained from meristematic tissue of sporophytic and male gametophytic fronds and tetraspores from submeristematic tissue of sporophytic fronds within a short period (6 weeks). Tissue fragments (1 × 1 mm²) from upper margins of fresh thalli and cell aggregates (10−100,000 cells) from marginal meristem and meristematic warts of fresh thalli and thalli after the freezing-thawing procedure were cultured for getting plantlets. Tissue fragments (TF) and cell aggregates (CA) from submeristematic tissue of fresh thalli were cultured for obtaining tetraspores. For mass getting proliferations (plantlets) and tetraspores we recommend to use CA from marginal tissue of fresh fronds because of fast growth, high numbers of proliferations and simple techniques of the method. The freezing-thawing method allows also to identify meristematic tissue and to obtain plantlets of red algae with apical meristem (e.g., Gelidium spp.).     

Liquid Culture of the Entomogenous Nematode Steinernema feltiae with Its Bacterial Symbiont

The insect-parasitic nematode, Steinernema feltiae Filipjev strain 42, was reared in liquid culture along with its bacterial symbiont, Xenorhabdus nematophilus Thomas &Poinar. First-stage juveniles developed into reproducing adults in a maintenance salts medium containing resuspended Xenorhabdus cells and the yeast Kluyveromyces marxianus (Hansen) van der Walt or cholesterol. Cultures with media depths greater than 4 mm required aeration. Nematode populations increased as bacterial density increased. An optimal culture system was obtained when the bacteria and nematodes developed in a semidefined medium containing tryptic soy, yeast extract, and cholesterol and were incubated on a rotary shaker at 25 ± 1 C. Under these conditions, up to 86% of the final population were infective juveniles.     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

Effects of UV-B Radiation and Periodic Desiccation on the Morphogenesis of the Edible Terrestrial Cyanobacterium Nostoc flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.     

Human feeder layers for human embryonic stem cells

Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1). the ability to grow these cells under serum-free conditions, 2). maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3). the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.     

Changes in morphological plasticity of Ulva prolifera under different environmental conditions: A laboratory experiment

The large-scale green tides, consisting mainly of Ulva prolifera, have invaded the coastal zones of western Yellow Sea each year since 2008, resulting in tremendous impacts on the local environment and economy. A large number of studies have been conducted to investigate the physiological traits of U. prolifera to explain its dominance in the green tides. However, little has been reported regarding the response of U. prolifera to changing environmental factors via morphological variation. In our experiments, we found remarkable morphological acclimation of U. prolifera to various temperature (20 and 25 °C) and salinity (10, 20, and 30) conditions. U. prolifera had more, but shorter branches when they were cultured at lower temperature and salinity conditions. To investigate the significance of these morphological variations in its acclimation to changes of environmental factors, physiological and biochemical traits of U. prolifera grown under different conditions were measured. Higher temperature increased the relative growth rate while salinity did not affect it. On the other hand, higher temperature did not enhance the net photosynthetic rate whilst lower salinity did. The increased net photosynthetic rate at lower salinity conditions could be attributed to more photosynthetic pigments—chlorophyll a, chlorophyll b, and carotenoids—in thalli due to there being more branches at lower salinity conditions. Increased numbers of branches and thus an increased intensity of thalli may be helpful to protect thalli from increased osmotic pressure caused by lower salinity, but it led to more shading. In order to capture enough light when being shaded, thalli of U. prolifera synthesized more photosynthetic pigments at lower salinity levels. In addition, higher temperature increased nitrate reductase activity and soluble protein content but variations in salinity did not impose any effect on them. Our results demonstrate conclusively that U. prolifera can acclimatize in the laboratory to the changes of environmental factors (salinity and temperature) by morphology-driven physiological and biochemical variation. We suggest that the morphological plasticity of U. prolifera may be an important factor for it to outcompete other algal species in a changing ocean.     

The life history of Porphyra endiviifolium from the South Shetland Islands, Antarctica

This paper describes the reproduction and life history of an intertidal species, Porphyra endiviifolium, from Antarctica. Field specimens were examined microscopically, prepared for electron microscopy and used to establish cultures. Wild populations comprised two kinds of leafy thalli, morphologically similar but distinguished by their mode of reproduction, either sexual or asexual. Carpospores from monoecious leafy gametophytes developed into conchocelis filaments in culture, and under “winter-spring” conditions these formed conchospores that germinated to produce leafy thalli. Monospores from asexual leafy thalli developed directly into two different forms of leafy thalli. Only one of the cultured morphotypes became fertile, reproducing asexually by monospores. We conclude that the phases of the life history of P. endiviifolium show different ecological strategies, the conchocelis phase reproducing in response to short days unlike the leafy thalli in which growth and reproduction respond primarily to irradiance. Received: 2 May 1997 / Accepted: 11 October 1997     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

Stem cell maintenance by manipulating signaling pathways: past,current and future

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676]