Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Co-culture ofFrankia alni subsp.pommerii (strain ACN1 AG ) with birch (Betula papyrifera) protoplasts

The present study was undertaken to set up an experimental system in which barriers to infection of a non-host plant related to the presence of the cell wall, at the level of recognition and/or the necessity of penetrating the cell wall, might be bypassed. Co-cultures betweenFrankia alni subsp.pommerii (strain ACN1 ^AG ) andBetula papyrifera protoplasts were established. Betula protoplasts remained viable after 2 weeks with no substantial cell wall regeneration. Suppression of the wall barrier was not sufficient to allowFrankia infection under the conditions tested. The non-infectivity ofFrankia on Betula protoplasts may also reflect difficulties inherent to thein vitro environment, which might not permit duplication of infection mechanisms.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Formation of cytoplasts from giant protoplasts in culture

Summary Protoplasts isolated from calli ofNicotiana plumbaginifolia can dramatically increase in volume without showing indications of cell wall synthesis. After reaching a critical size, the plasma-rich giant protoplasts show multiple formation of cytoplasts, which are released from the mother cells. The anucleate cytoplasts display the same increase in size as the nucleate protoplasts. Both cell types retain a spherical shape for several months, indicating that no major synthesis of cell wall occurred.     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.     

Synthesis of the inner cell wall layer of the chlamydomonad flagellate,Gloeomonas kupfferi

Summary An antibody to the inner wall layer ofGloeomonas kupfferi was isolated and used in a developmental analysis of cell wall processing, secretion and extracellular assembly. The focus of the processing of this matrix layer is the endomembrane system, in particular the Golgi apparatus (GA) and contractile vacuole (CV). During interphase, inner wall materials are processed in the GA, packaged in trans face vesicles and transported to the CV, the final internal depository of wall precursors until release to the cell surface. During cell division, significant changes occur in the inner wall layer processing. Early on in cytokinesis, the GA does not label with our antibody, suggesting that other wall layers are being processed. In later stages of cytokinesis, the GA changes in morphology and begins to produce inner wall layer materials. These wall precursors are shuttled to the CV where they are released around the daughter cell protoplasts. The first wall layer that is formed around daughter cells is the crystalline median wall layer. Once assembled, the inner wall layer condenses upon the crystalline layer and grows in size.     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Über die Abgrenzung derChlorosarcinales von denChlorococcales

It is proposed to use amongst other characters the type of cell division in order to delimit theChlorosarcinales from theChlorococcales. A definition of the two processes of division occuring in these orders is given. It differs from that of other authors. In theChlorosarcinales only those genera should be assembled in which vegetative daughter cells arise by bipartition followed by firm association of the wall between the daughter cells with that of the mother cell. In contrast, autospores, the vegetative daughter cells of a number ofChlorococcales, develop by multiple division, their cell walls are formed all around the protoplasts and are free from that of the mother cell. The chlorococcalean generaTrebouxia andDictyochloropsis incorporate species which multiply by zoo-, aplano- and autospores as well as others having no autospores. Autospores possibly have arisen more than once during evolution.

     

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

Candida albicans mycelial wall structure: supramolecular complexes released by Zymolyase,chitinase and β-mercaptoethanol

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118–1125]. Zymolyase, chitinase and -mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (>180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.     

Isolation and culture of protoplasts ofCapsicum annuum L. and their regeneration into plants floweringin vitro

Summary Axenic shoot cultures ofCapsicum annuum cv.California Wonder were used as the source for isolation of protoplasts from mesophyll cells. Protoplasts underwent sustained mitotic activity and proliferated to form callus masses on NT or DPD medium enriched with 2,4-D, NAA and BAP each at 1 mg/l level. The callus could be differentiated into whole plants on the differentiation media and plants floweredin vitro under long day conditions.     

Physiological properties and cytological features of protoplasts prepared fromChlorella saccharophila

Summary Protoplasts were prepared from cells ofChlorella saccharophila by treatment with a mixture of pectinase and cellulase. The yield of protoplasts is dependent upon the culture conditions prior to cell wall digestion. In thin section chemically-fixed protoplasts were without wall remnants at the surface of the plasma membrane. Of particular interest is the relationship between the Golgi apparatus and a nuclear envelope-endoplasmic reticulum continuum. Protoplasts have a photosynthetic capacity lying between 70 and 80% of that of normal cells, but show the same response towards CO₂ concentration and DCMU inhibition. Protoplasts also respond to changes in the osmolarity of the surrounding medium in accordance with the Boylevan't Hoff equation as if they are an osmometer. The nonosmotic volume (NOV) was calculated.Abbreviations GA Golgi apparatus - ER endoplasmic reticulum - NE nuclear envelope - PM plasma membrane - N nucleus - S starch - M mitochondria - V vacuole