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.     

Morphological and ultrastructural studies on protoplast production and reversion of the higher basidiomyceteAgaricus bisporus

Protoplasts have been isolated from young vegetative mycelia ofAgaricus bisporus by an enzyme mixture of novozym and chitinase. Protoplasts were released through ruptures in the wall, initially at the apices, but also later from older parts of the hyphae, indicating that they may lack the cell wall. The process of regeneration of these protoplasts has been investigated in liquid medium in which the protoplasts produced short chains of convoluted cells that finally produced a hypha. Electron microscopy has shown that at the start of regeneration two different kinds of fibrils were produced at the external surface of the protoplasts. Later, the thickness of the cell wall increased, and there was a deposit of amorphous material giving rise to a complete new wall.     

Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 m sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 m sorbitol layer. Regeneration occurred over a period of 10–24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater. Received: 26 September 1997 / Revision received: 17 January 1998 / Accepted: 7 February 1998     

ELECTRON MICROSCOPIC OBSERVATIONS OF CULTURED CELLS AND PROTOPLASTS OF AMMI VISNAGA

Protoplasts were prepared from cultured cells of Ammi visnaga (Umbelliferae) by enzymatic digestion of the cell walls and examined microscopically. Staining of fresh protoplasts with Calcofluor and silver hexamine demonstrated the apparent absence of wall material. Protoplasts contained more cell organelles than the whole cells, particularly endoplasmic reticulum and associated polysomes. The plasmalemma of most protoplasts appeared smooth; some protoplasts were connected by structures resembling plasmodesmata. Multinucleates resulting from fusion were frequently observed.     

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.     

Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Selectivity to K+ and Na+ of protoplast fractions isolated from different regions of Aspergillus nidulans hyphae

The selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1.5 mM-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.     

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).     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

Protoplast Cultures from Cell Suspensions of Daucus carota

Protoplasts, enzymatically isolated from cell suspension cultures of Daucus carota, have been grown in small Petri-dishes. After enzyme treatment and washing the protoplasts were plated in agar media. Growth and divisions were viewed through the bottom of the Petri-dishes with a light microscope. Different osmotic stabilizers were tested with respect to their ability to promote wall formation and growth of the protoplasts. Combinations of sucrose, sorbitol and “Modopeg” gave the best results. Electron micrographs of cultured protoplasts revealed normal as well as abnormal nuclear conditions.     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

Direct somatic embryogenesis from protoplasts of Foeniculum vulgare

Protoplasts prepared from an embryogenic cell suspension culture of fennel gave rise to somatic embryoids directly through unequal cell divisions of enlarged, ellipsoidal cells, when embedded in hormone-free LS agarose medium. On the other hand, protoplasts embedded in LS agarose medium containing 2,4-D and kinetin proliferated through unpolarized cell divisions to form calli, which gave somatic embryoids on the surface upon transfer onto the same medium. In either case, somatic embryoids germinated to develop into normal plantlets when cultured on hormone-free LS agar medium under illumination.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Simulated weightlessness and hyper-g results in opposite effects on the regeneration of the cortical microtubule array in protoplasts from Brassica napus hypocotyls

Enzymatic digestion of the cell wall of Brassica napus hypocotyls gave a heterogeneous suspension of protoplasts with the cortical microtubules (CMTs) randomly organised or CMTs organised in parallel. The effect of variable g- influences has been tested on CMT organisation. In contrast to the 1 g- protoplasts, which reorganised the CMTs into parallel arrays during the 96 h test period, the frequency of randomly-oriented CMTs in the protoplasts exposed to simulated weightlessness (0 g ) on a 2-D clinostat increased significantly during the same period. The opposite effect was obtained when the protoplasts were exposed to hyper -g (7 or 10 g ), where the reorganisation of the CMTs into parallel arrays was accelerated compared to the 1 and 0 g- protoplasts. These results indicate that a unidirectional gravity force is a necessity for the reorganisation of CMTs in protoplasts to parallel arrays and that CMTs act as responding elements that are able to sense different levels of gravity. Besides the inability of the protoplasts to reorganise the CMTs into parallel arrays, the quantity of CMTs in the individual protoplast decreased during 4 days of simulated weightlessness, both compared to the CMTs quantity in the protoplasts immediately after isolation and compared to the 1 g- and hyper -g- protoplasts after 24 and 48 h of g- exposure. The size of the protoplasts was also affected by the g- exposure. Protoplasts exposed to simulated 0 g increased significantly after 24 and 48 h, whereas the 1 g- and 10 g- protoplasts maintained the same size during the 48 h test period.     

The Elicitation of Ethylene Biosynthesis by a Trichoderma Xylanase Is Not Related to the Cell Wall Degradation Activity of the Enzyme

A [beta]-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.     

Ascospore Formation in the Yeast Saccharomyces cerevisiae

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Isolation of protoplasts from zygotes of Fucus distichus (L.) powell (Phaeophyta)

Two-step procedures were developed for the isolation of protoplasts from 6-hour-old zygotes of Fucus distichus L., using commercial cellulases and alginate-lyases from marine molluscs. Protoplasts were obtained either as a suspension or attached to a substratum. Protoplasts yields were greater than 95% of the cell population and over 80% regenerated a wall, germinated and divided into a polar, multicellular embryo. Zygote development from regenerated protoplasts was also quite synchronous. The same procedures were less effective in removing the cell wall from zygotes older than 8 h, and did not yield significant numbers of protoplasts from two-celled embryos.     

Protoplasts in organelle research: Transfer and transformation of plastids

Protoplasts, because they lack the wall of a typical higher plant cell, offer unique opportunities for experimental manipulation of their organellar constituents. Here, we report on modification of the organellar content of Nicotiana tabacum protoplasts by microfusion-induced transfer of defined numbers of chloroplasts into albino recipient cells. A single chloroplast is found to be sufficient for establishing a new plastid population in the progeny of the recipient cell. The frequency of green or variegated regenerants is shown to be genotype dependent. It can be drastically increased by using selection pressure for the transferred organelle. We also report on transient expression of plastid specific reporter gene constructs in plastids after PEG-mediated direct gene transfer into Nicotiana plumbaginifolia protoplasts. The expression is shown to be localized in the plastids by determining gene expression in isolated chloroplasts under conditins which completely remove cytoplasmic enzyme activity derived from a nuclear reporter gene construct. These data demonstrate for the first time that functional DNA, introduced into the cytoplasm by direct gene transfer, enters the organellar compartment and is expressed.     

Preparation of protoplast-like structures from conidia ofFusarium culmorum

Protoplast-like structures have been formed by digestion of the cell walls ofFusarium culmorum conidia by lytic enzyme preparations ofMicromonospora AS. Under the test conditions extrusion of the protoplasts was not observed. It seems that digestion of the cell wall occurs in different stages. Digestion of the septa preceded the formation of protoplasts of the individual cells of the multicellularF. culmorum conidia. A few protoplasts survived the lytic enzyme treatment. “Protoplasts” obtained from conidia are much more stable than those obtained from young hyphae and were able to germinate with the formation of normal mycelium. Lysis of some of the protoplast bodies led to the formation of a membranous structure. The protoplasts derived from each of the constituent cells of the conidia could be isolated with the micromanipulator. No differences were found in the ability of the isolated cells to germinate.