Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr.

Protoplasts of the ectomycorrhizal ascomycete Cenococcum geophilum were isolated from mycelium grown in liquid medium. The method was optimized with regard to culture conditions, preincubation, lytic enzyme system, pH value of the incubation medium, osmotic buffer and incubation temperature for C. geophilum strains SIV and 1448. The yields were 1-3·10⁸ and 7·10⁶ protoplasts per gram fresh weight for C. geophilum SIV and C. geophilum 1448, respectively. Protoplasts from C. geophilum SIV exhibited plasma membrane integrity close to 100% (fluorescein diacetate staining). At least 50% of the protoplasts contained a nucleus (staining with acridine orange). The regeneration of protoplasts from C. geophilum is described for the first time. The regeneration frequency was up to 13%, and, dependent on the conditions of culture (liquid medium, agarose, agar), four types of regeneration patterns could be distinguished Regenerated protoplasts of C. geophilum were capable of forming mycorrhizas with spruce (Picea abies) seedlings.     

Flow sorting and culture of protoplasts: Conditions for high-frequency recovery,growth and morphogenesis from sorted protoplasts of suspension cultures of nicotiana

Protoplasts were prepared from suspension cultures of Nicotiana tabacum cv Wisconsin 38 that had been prelabeled with FITC. The protoplasts were subjected to flow sorting based on fluorescence content using a Coulter EPICS V Flow Cytometer — Cell Sorter. Conditions were established that allowed the recovery after sorting of approximately 30% of the initial protoplasts in a viable state. These were subsequently regenerated into calli that underwent shoot morphogenesis.Abbreviations FITC Fluorescein isothiocyanate - FDA fluorescein diacetate     

Optical compartmentation of vegetating algae species as a basis for their growth-specific characterization

BACKGROUND: The number of microalgal strains known to date is enormous and continuously growing, and their characterization accordingly requires quick and reliable methodologies. METHODS: Asynchronously growing logarithmic (3- and 6-day cultures) and stationary (9-day cultures) phase cell populations of two algae species that are difficult to distinguish microscopically (one Chlorella sp., C. vulgaris [c-27], and another that might belong to the same genus, SA-3 algae exsymbiotic from Paramecium bursaria) were characterized by means of flow cytometry (FCM). Forward light scatter (FSC) of algae was monitored in association with their 90 degrees side light scatter (SSC) and fluorescence of endogenous chlorophyll (FL3-height). RESULTS: Two-parameter FSC versus SSC and FSC versus FL3-height plots distinctly showed growth-specific compartmentation of algae into discrete cell subpopulations staying at a particular stage of the life cycle, and numbers of cells constituting these subpopulations could be quantitated. The growth pattern of C. vulgaris (c-27) differed substantially from that of SA-3 algae, particularly in the late-logarithmic (6-day) cultures. At this phase of growth, C. vulgaris (c-27) cells compartmentalized into three subpopulations, whereas SA-3 cells compartmentalized into two subpopulations. Different compartmentations of optical signals from late-logarithmic phase SA-3 algae and C. vulgaris (c-27) likely were caused by the differences in timing of the life cycle stages of these types of cells. CONCLUSIONS: Growth-specific compartmentation of vegetating microalgae by FCM provides a good basis for characterization of morphologically similar algae species. Because algae are also present in symbiotic relationships with other organisms, this tool might be of potential interest for the study of symbiosis mechanisms.     

Comparison of physiological responses of guard cell protoplasts of Nicotiana glauca isolated from leaves collected before dawn or at midday

We observed that guard cell protoplasts isolated from leaves collected at midday from Nicotiana glauca Graham (tree tobacco) did not give the same physiological responses to light as those isolated from leaves collected in early morning. Based on that observation, we attempted to determine whether there were significant differences between the physiological responses of guard cell protoplasts isolated from leaves collected before dawn (with closed stomata) and those isolated from leaves collected at midday (with open stomata). We isolated guard cell protoplasts from leaves collected before dawn and at midday and compared (1) rates of red and blue light-induced pH changes in weakly buffered media caused by changes in their metabolism, (2) their rates of oxygen consumption in darkness and oxygen evolution in light and (3) relative rates of decay of variable chlorophyll a fluorescence in their chloroplasts. Studies with the vital stain fluorescein diacetate failed to reveal any significant differences in the viabilities of protoplast preparations from leaves collected before dawn and at midday. Furthermore, protoplasts from leaves collected at these times swelled to similar extents in an osmotic medium containing 10 µM fusicoccin and 5 mM KCI. Nevertheless, rates of light-induced pH changes, rates of oxygen consumption and evolution and rates of decay of variable chlorophyll a fluorescence were all lower in preparations of guard cell protoplasts from leaves collected at midday than in preparations from leaves collected before dawn. Initial volumes of guard cell protoplasts isolated from leaves collected at midday were 150% of those of guard cell protoplasts isolated from leaves collected before dawn. We suggest that the differences in responses of guard cell protoplasts isolated from leaves collected before dawn and at midday may be caused by (1) nonoptimal isolation conditions for guard cell protoplasts prepared from leaves collected at midday, (2) the lower surface-to-volume ratio of guard cell protoplasts isolated from leaves collected at midday or (3) diurnal and/or circadian regulation of guard cell metabolism over the course of a day.     

ABA synthesis in protoplasts of different origin in response to osmotic stress

All the protoplasts analysed in this study whatever the original plant material were able to accumulate ABA under osmotic stress. The time course of ABA accumulation strongly differed according to the plant material. In both rose petal or Amaranthus leaf protoplasts, the increase in ABA level was significant but transient. Protoplasts prepared from Amaranthus cell suspensions behaved differently, showing a late and durable accumulation of ABA. Similar patterns of changes in ABA accumulation were observed in the original plant material under osmotic stress. A pretreatment of plant material by fluridone induced a strong inhibition of ABA accumulation whatever the origin of protoplasts was. This result suggests that ABA could be synthesised via the carotenoid pathway in the absence of the cell wall.     

Isolation and Cell Wall Regeneration of Protoplasts from Botrytis cinerea Pers.

Abstract A procedure for efficient isolation and cell wall regeneration of protoplasts from Botrytis cinerea is described. Protoplasts were obtained from mycelia using a lytic enzyme mixture containing β-Glucuronidase, Cellulase R10 and Driselase with mannitol for osmotic support. The digestion of cell walls was checked by fluorescence microscopy. Protoplasts were purified from cell debris and lytic enzymes. Regeneration and reversion were performed by incubation on agar plates.     

Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phureja

Protoplasts from diploid S. tuberosum and diploid S. phureja were electrofused followed by selection of the heterokaryons with a micromanipulator. Visual identification of the heterokaryons was facilitated by fluorescein diacetate staining of the protoplasts from one of the parents, which was grown on herbicide containing medium to induce bleaching of the chlorophyll. In total, 840 heterokaryons showing red (chlorophyll) and yellow-green (fluorescein) fluorescence were selected and cultured at relatively low densities using various feeder systems. Finally, 18 putative hybrid plantlets were obtained and grown to maturity. DNA histograms indicated that the plants were hexaploid, octoploid or mixoploid. With Giemsa C-band pattern analysis of the chromosomes the hybrid character and the combinations of the chromosome sets of all plants investigated could be established.     

Monoclonal antibody against non-histone chromosomal protein high mobility group 1 Co-migrates with high mobility group 1 into the nucleus

Non-histone chromosomal protein high mobility group 1 (HMG-1) rapidly migrates into the nucleus when injected into the cytoplasm of bovine fibroblasts and HeLa cells by red cell-mediated microinjection (Rechsteiner, M., and Kuehl, L. (1979) Cell 16, 901-908). We isolated hybridomas secreting monoclonal antibodies against HMG-1. One of these monoclonal antibodies, FR-1, inhibited in vitro binding of 125I-HMG-1 to chromatin isolated from FL cells. When 125I-HMG-1 was co-introduced with antibody FR-1 by red cell-mediated microinjection, antibody FR-1 did not prevent the accumulation of 125I-HMG-1 in the nucleus. When 125I-antibody FR-1 or fluorescein isothiocyanate antibody FR-1 was introduced into the cytoplasm of FL cells, most of the antibody did not accumulate in the nucleus. But when 125I- or fluorescein isothiocyanate antibody FR-1 was co-introduced with HMG-1 into the cytoplasm of FL cells, it did migrate into the nucleus.     

Sugar effects on membrane damage during desiccation of pea embryo protoplasts

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.     

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.     

Abscisic acid synthesis and metabolism in barley leaves and protoplasts

Protoplasts isolated from barley (Hordeum vulgare L. cv. Clipper) leaves contained abscisic acid (ABA). The ABA content of these protoplasts did not change when they were incubated for up to 6 h in media of decreasing osmotic potential. There was a substantial, but transient, increase in ABA in barley leaf segments during protoplast isolation. The magnitude of this increase was inversely dependent on the osmotic potential of the isolation medium. Maximum ABA content was recorded after 2 h of exposure to the sorbitol-containing medium. The subsequent decline was due to conversion of ABA to phaseic acid (PA) and to other metabolites.Barley mesophyll protoplasts were not able to metabolise ABA, PA or any of the other metabolites formed from ABA by intact leaf tissue.     

Studies on Isolated Protoplasts and Vacuoles: I. GENERAL PROPERTIES

Protoplasts and vacuoles have been isolated in large quantitiesfrom the parenchymatous placental tissue of immature tomatofruit. Their mechanical stability and osmotic stability havebeen investigated. A small proportion of initially discretebut adjacent vacuoles were seen to coalesce; the possible significanceof this phenomenon in the growth of plant cells is discussed.The overall chemical composition of isolated protoplasts hasbeen determined, and their lipids have been analysed by thin-layerchromatography.     

Protoplast production in Chondrus crispus gametophytes (gigartinales,rhodophyta)

Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×10⁸ protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.     

Embryogenic callus formation from maize protoplasts

Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA fluorescein diacetate - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Secondary phenolic products in isolated guard cell,epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination

Summary Guard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.Abbreviations uE adaxial (upper) epidermis - IE abaxial (lower) epidermis - GCP guard cell protoplasts - ECP epidermal cell protoplasts - MCP mesophyll cell protoplasts - PP protoplasts - HPLC high performance liquid chromatography - TLC thin layer chromatography - CC column chromatography - HOAc acetic acid     

钙在6-BA诱导黄化绿豆幼苗下胚轴原生质体体积膨大中的作用

对照和仅用6-BA(无Ca~(2 ))处理的原生质体的体积均无变化。含钙培养液中的原生质体经6-BA处理后10min~(45)Ca~(2 )积累明显增多,15min后开始膨大;处理30min时~(45)Ca~(2 )积累最多,此时原生质体的膨大效应最好;随后~(45)Ca~(2 )积累和膨大效应逐渐下降。两者的时间进程十分相似。K~ 、Zn~(2 )、Ba~(2 )、Mg~(2 )等可在不同程度上代替Ca~(2 )的作用。EGTA、verapamil和LaCl_3处理均可使原生质体~(45)Ca~(2 )累积降到对照的水平,膨大效应完全消失。表明Ca~(2 )可能在6-BA诱导原生质体膨大的过程中起着重要作用。