The cells of most bacteria, plants and fungi are surrounded by a more or less rigid cell wall. This is a multifunctional structure separated from the protoplasmic contents of the cell by the plasma membrane. Cell walls provide protection for cells against mechanical damage and allow cells to survive in a medium of lower osmotic potential than that of its protoplasm. They may also provide a locus for enzymes, ligands, and receptors, and, in some cases, they represent a barrier to the penetration of exogenous materials. These cell walls may be removed from most bacterial, plant, and fungal cells, leaving protoplasm surrounded by a plasma or cytoplasmic membrane. Such cells are referred to as protoplasts.
Protoplasts are osmotically sensitive, as in a suspension medium hypotonic to that of its protoplasm, water enters the cells by osmosis. This inflow is prevented in walled cells by the rigidity of the cell wall. It has been calculated (1) that the hydrostatic pressure of the cytoplasm of bacterial cells suspended in a dilute aqueous medium may be as much as 30 atm. In intact cells, this is contained by the inward pressure of the wall. In contrast, protoplasts transferred to a hypotonic medium swell and burst. It is consequently necessary for them to be suspended in an iso-osmotic or hypertonic medium for the protoplasts to survive. In iso-osmotic conditions, protoplasts assume a spherical shape. It is often difficult to be certain that a cell wall has been totally removed because cells become osmotically sensitive and adopt a spherical shape before the cell wall is completely removed, so neither of these can be regarded as an adequate indicator of protoplast formation. Cells from which the cell wall has been incompletely removed are referred to as spheroplasts. When it is not certain whether protoplasts or spheroplasts are being used, these are often referred to as osmotically fragile cells.
1. Bacteria
Bacterial protoplasts or spheroplasts can be prepared by the treatment of cells with the enzyme lysozyme (murein hydrolyase) that digests the peptidoglycan murein component of their cell walls. This was first achieved using Gram-positive Bacillus cells (2, 3). In the case of Gram-negative bacteria (4), the situation is significantly more complicated, as the typical wall (or envelope) structure comprises a relatively mobile inner or cytoplasmic membrane, a thin layer of murein and an outer membrane rich in lipopolysaccharide and stabilized by divalent cations. Both lipopolysaccharide and murein contribute to the rigidity of the envelope. The outer membrane prevents access of lysozyme to the murein, so it is usual to include EDTA (ethylene diamine tetraacetic acid) in the isolation medium to remove divalent cations. This destabilizes the outer membrane, causing the loss of significant quantities of lipopolysaccharide, and permits access for lysozyme to the murein layer. This technique invariably leaves significant quantities of outer membrane in place, so the usual products are spheroplasts rather than protoplasts.
Regeneration of bacterial protoplasts or spheroplasts to normal cells is variable in its success. Removal of the induction regime (lysozyme/EDTA) may permit regeneration of the cell wall if a suitable medium is provided. Success may depend on the growth phase of the cells used initially or the temperature at which the cells were grown and the regeneration is attempted (5). The nature of the osmotic stabilizer used to prevent the lysis of bacterial protoplasts can be critical, especially if the regeneration and reversion of protoplasts are required. Those most commonly used are inorganic salts (KC1, NaCl, MgSO4) or nonmetabolizable sugars or sugar derivatives (sucrose, mannitol, sorbitol), or combinations of these.
Most, and possibly all, species of bacteria are capable of converting to L-forms. These, named after the Lister Institute where they were first discovered (6), resemble normal protoplasts or spheroplasts in being spherical in shape and osmotically sensitive but differ in their replication capabilities. Electron microscopic (EM) examination of L-form cells shows that they can be either devoid of any obvious cell wall material (protoplast-type L-forms), or they may contain some cell wall material (spheroplast-type L-forms). Large-scale conversion of Bacillus subtilis and other species of bacilli was first achieved by the removal of cell walls with lysozyme and plating the resultant protoplasts (spheroplasts) onto hypertonic soft agar medium (7). L-forms are most effectively induced by treatment with inhibitors of cell wall biosynthesis, especially penicillin and other b-lactam antibiotics. It is not always clear what heritable change leads to the L-form transition. Although there have been reports of changes in DNA sequence associated with the transition (8, 9), the ways in which L-forms are induced and the abilities of most L-forms to revert fairly readily suggest that, in most cases, the transformation is likely to be epigenetic in character. It has been suggested that the transition from an unstable to a stable L-form in Proteus mirabilis may be the result of a genetic change (4).
L-forms may be stable or unstable. The latter readily revert to the normal bacterial growth pattern and must be maintained in the presence of penicillin; the former are more difficult to revert, and, in some cases, reversion has not been possible. They grow by a budding process and shed envelope components as vesicular fragments. Murein is present in normal quantities and fully cross-linked but lacks its normal organization. Stable L-forms are usually devoid of an outer membrane and murein layer (10, 11). The cytoplasmic membrane of stable L-forms of Proteus mirabilis has been shown (4) to be modified from its normal composition, containing significant quantities of lipopolysaccharide and with a shift toward shorter-length fatty acids. L-form bacteria exhibit several properties that distinguish them from normal cells, such as resistance to bacteriophage in protoplast but not in spheroplast types, resistance to antibiotics, whose primary mechanism is to inhibit cell wall assembly, and increased sensitivity to antibiotics that act intracellularly. Early work suggested that L-forms of pathogenic bacteria were not pathogenic unless they showed a significant tendency to revert spontaneously. More recently (12), L-forms of some pathogens have been shown to retain pathogenicity. Some other pathogens show a tendency to adopt the L-form when patients are treated with penicillin.
2. Filamentous Fungi and Yeasts
Protoplasts may be formed from both filamentous fungi and yeasts. The usual method for making fungal protoplasts is to treat a culture with an enzyme cocktail that digests the major polysaccharide components of the cell wall: b-glucan, mannan, and chitin. Digestion of the cell wall was first achieved for Saccharomyces cerevisiae by Giaja (13) using the gastric juices of the snail Helix pomatia as the digesting enzyme mixture, although no protoplasts were formed in the absence of an osmotic stabilizer. These were obtained by Eddy and Williamson (14) also using snail gut juice (Suc d’Helixpomatia). Emerson and Emerson (15) produced the first protoplasts in filamentous fungi. There are now several commercially available sources of snail gut juice (b-glucuronidase, glusulase, helicase), and these are known to be complex mixes of glucanases, mannanases, lipases, and proteinases. Subsequently, other enzyme preparations have become available—some from bacteria, principally Bacillus and Streptomyces spp., others from fungi. The most widely used fungal enzyme preparations are Novozyme 234 (from Trichoderma harzianum) and Funcelase (from Trichoderma viride), which contain chitinase as well as the other enzymes mentioned.
Enzymic release of protoplasts from yeasts follows one of two pathways. Either the cell wall is gradually eroded until a protoplast (or spheroplast) remains or, when a suitably sized portion of cell membrane is exposed, the protoplast may be extruded through what is left of the cell wall. In yeasts this often occurs through a bud scar. In the case of filamentous fungi, protoplasts are often smaller than individual fungal cells, particularly in the case of coenocytic fungi, and are variable in size and nuclear content. The growing tips of hyphae are generally more susceptible to enzymic digestion, so protoplasts tend to be released from these regions. In some species, however, the release of protoplasts may occur at any position on the hyphal surface. Because of the method of release, the osmotically fragile products of filamentous fungi are usually protoplasts rather than spheroplasts. The rate at which fungal protoplasts are formed may be limited by the accessibility of digesting enzymes to the inner layers of the cell walls. In particular, the access of glucanases to the inner glucan component may be restricted by the outer mannoprotein layer. The inclusion of disulfide-reducing agents (e.g., b-mercaptoethanol, dithiothreitol) may open up the mannoprotein matrix and enhance the rate of protoplast release. This is most important for stationary phase cells, from which it is often quite difficult to release protoplasts. For some species, however, the release of protoplasts is not enhanced by the presence of reducing compounds, and their presence may be deleterious insofar as they may interfere with the reversion of the protoplasts.
Sorbitol or mannitol are mostly preferred as osmotic stabilizers for yeasts and inorganic salts for filamentous fungi. Optimal stabilizers and concentrations of these differ from species to species, from strain to strain, and with growth conditions. For any new species or strain, it is usually necessary to establish optimal conditions for protoplast formation and maintenance. The minimum condition is that the osmolality of the suspending medium be at least isotonic with that of the cytoplasm. Hypotonicity results in lysis; hypertonicity may be tolerated up to a point but can result in a reduction of the rate of protoplast release (16, 17). There is a cell wall-less mutant strain of Neurospora crassa (18) that grows on the surface of osmotically stabilized agar as a cluster of multinucleate protoplasts. This mutant, to an extent similar to bacterial L-forms, has been used as a source of protoplasts whose cell membranes have not been exposed to lytic enzymes.
Protoplasts from some fungal species (eg, Schizosaccharomycespombe, Nadsonia elongata) can regenerate a cell wall in liquid suspension (19). In many species, however, regeneration requires embedding protoplasts in a solid or semisolid medium (20, 21). This is necessary to prevent the diffusion and loss of cell wall materials secreted from the protoplast. In some species, it has proved difficult to regenerate dividing cells from true protoplasts but relatively easy to regenerate from spheroplasts (22). The first polymer to appear on the surface of a regenerating protoplast of the yeast Candida albicans appears to be chitin, which is then followed by b (1, 3) and b (1, 6) glucan. Once these polymers have been deposited on the protoplast surface, mannans and mannoproteins are secreted and, if restrained by the presence of a physical barrier, aggregate to form a nascent cell wall. In the case of yeasts, the initial regenerated cells are spherical because the cell wall is assembled on the surface of a spherical protoplast. The typical shape of the yeast cell is reinstated only in cells arising during subsequent budding divisions. Protoplasts of filamentous fungi may also regenerate a spherical cell that subsequently produces either a germ tube or pseudohyphal structure before the establishment of true hyphae (23).
Once the cell wall has assembled around the protoplast, the process of reversion to the normal cellular state can take place. While in the protoplast state, nuclear division (karyokinesis) can proceed, but cytoplasmic division (cytokinesis) is impossible. Consequently, many protoplasts may be multi-nucleate by the time the cell wall has regenerated. During the first few generations, the "regenerated" cell reverts to the "normal" state, that is, the number of nuclei per cell is eventually reduced to one, the cell wall changes from one having a high amount of chitin to one with a relatively low amount and cellular metabolism re-commences.
3. Plants
Plant protoplasts have been prepared from plant types ranging from algae to flowering plants and from a variety of tissues. Many protoplasts are totipotent and, given suitable conditions, will regenerate cell walls, initiate and sustain cell division, and regenerate complete plants. The source of plant material for protoplast production is very important, particularly if good regeneration is required. Suspension cultures have been widely used, early exponential phase cultures giving the best yields (24). Sequential subculturing, however, leads to diminished protoplast yields, as well as to the accumulation of mutations and loss of totipotency (25). If regeneration is required, protoplasts isolated from leaf mesophyll cells of living plants grown under strictly controlled environmental conditions are usually most consistent. Shoot culture under axenic conditions is finding increasing usage as a source of mesophyll and shoot apical protoplasts (26, 27). This is particularly useful for woody plants. Other sources of protoplasts include roots, petals, cotyledons, somatic embryos, and pollen.
The first-ever protoplast isolation was completed by Klercker (28), who cut onion scale epidermis using a sharp knife under 1 M sucrose. A small number of protoplasts were released from cells whose cell walls had been cut without damaging the protoplasts. Enzymic isolation of plant protoplasts in quantity was later achieved by Cocking (29). In intact tissues, plant cell walls are surrounded by a layer of pectin that must be removed before a tissue may be dis-aggregated. The major component of plant cell walls is cellulose; a minor component, particularly in aging cells, is hemicellulose. Various enzyme products are available for the removal of plant cell walls. When purified enzymes are used, a mixture of pectinase, cellulase, and hemicellulase may be required, but cruder preparations often contain sufficient amounts of all the enzymes necessary for cell wall removal. An empirical approach may be necessary in any new investigation, as preparations that give good protoplast yields in one system may be damaging to the protoplasts in another. Fowke and Cutler (30) give detailed protocols for the isolation of protoplasts from soybean suspension cultures, pea leaves, somatic embryos, and the filamentous green alga Ulothrix. Protoplast yields may be enhanced by adding, in addition to the necessary enzymes and osmotic stabilizer (usually a sugar alcohol): 1) Tween 80 to encourage protoplast release (31), 2) potassium dextran sulfate (32) or bovine serum albumin (33) to improve protoplast stability, or 3) antibiotics to prevent the growth of bacteria present from the source material (34). Protoplasts may be purified from residual plant material by flotation on either sucrose or Ficoll gradients (30). Healthy protoplasts appear spherical, and cytoplasmic streaming is apparent. Viable protoplasts, on treatment with fluorescein diacetate, produce fluorescence inside the plasma membrane.
The regeneration of plant protoplasts was first reported by Takebe et al. (35) for tobacco and has now been described for well over 300 species from at least 50 families. The process is very complex but can be considered to take place in three stages: 1) preparation for cell division involves the synthesis of a cell wall and the onset of DNA replication and organelle assembly; 2) cell division results in the establishment of a callus (undifferentiated cell mass); and 3) morphogenesis comprises differentiation to produce a somatic embryo. Establishment of cell division of protoplasts has been achieved by embedding in agar medium or overlaying a liquid protoplast suspension onto an agar surface. The accumulation of toxic compounds in the region of the regenerating cells may interfere with their recovery and, for this reason, it may be useful to include filter paper at the liquid agar interface or activated charcoal in the agar (34). Other problems stem from the presence of toxic materials in some agars or the temperature shock experienced by protoplasts on suspension in molten agar. Success has been achieved in many cases by the use of agarose or alginate as low-temperature gelling agents.
The nutritional requirements for protoplast culture are generally similar to those required for plant cell suspension cultures and are complex. Plant protoplasts are remarkably variable in their nutritional demands, and slight variations in the quality of the protoplast source or of medium constituents have given rise to difficulties in reproducing results between laboratories. The nature of the nitrogen source and the levels of growth regulators provided are particularly critical.
4. Applications
Probably the most important use of protoplasts, and the one that gave the impetus to the rapid developments in protoplast technology during the last 20 years, is genetic manipulation of species using protoplast fusion. However, protoplasts have also found many other uses. One of the most important has been the isolation of cell organelles undamaged by the mechanical fragmentation required for walled cells. This approach has been used for the preparation of intact nuclei, mitochondria, chloroplasts, and vacuoles, as well as for cytoplasmic and tonoplast membranes. Protoplasts have also been employed for the study of cytoplasmic membrane permeability and transport properties, for the investigation of the biochemistry of cell wall synthesis, and to assist in the introduction of macromolecules into cells.
