The cells of bacteria, fungi, and plants may be converted to protoplasts (or spheroplasts) by the enzymatic degradation of their cell walls. This opens up a range of new possibilities for the investigation of cells. Protoplasts may be fused with other protoplasts from strains of the same or different species or even with animal cells (1) or liposomes (2). The motivations for carrying out such protoplast fusions are diverse and include genetic investigations of species that are not amenable to normal genetic study, the improvement of strains of organisms of agronomic or biotechnological importance, the study of nucleo-cytoplasmic interactions and the introduction of foreign materials into cells (see Transfection).
The spontaneous fusion of protoplasts is usually quite rare, and the event must be induced in some way. The fusion technique itself involves two processes: 1) bringing the two protoplasts into close membrane contact; and 2) limited, localized disruption of adjacent membranes, permitting the formation of cytoplasmic continuities between the cells. The presence of residual cell wall material on the surface of spheroplasts may interfere with the formation of close contacts between the cytoplasmic membranes, but the fusion of spheroplasts is quite often possible although it occurs at a lower frequency than protoplast fusion (3, 4).
Two basic techniques have been employed for inducing fusion. The first, and currently most widely used, technique involves chemical induction. Polyethylene glycol (PEG) causes a non-specific aggregation of protoplasts and also causes protoplasts to shrink by withdrawing water. This serves to bring cytoplasmic membranes into close contact and, of itself, is adequate to bring about a small number of protoplast fusions. For efficient protoplast fusion, PEG treatment must be accompanied by calcium-ion treatment that results in local disturbances in the membrane, leading to fusion. The optimal molecular mass and concentration of PEG should be investigated as a preliminary step to any previously untried fusion. For plant and fungal protoplasts, it is often desirable to use re-crystallized PEG, as degradation products or other impurities may interfere with fusion yields (5, 6). Degradation products formed during the autoclaving of PEG are similarly toxic and, when good yields are critical, it is necessary to sterilize by filtration. Calcium chloride is the usual source of calcium ions, but improved fusion yields have been achieved for Candida albicans and Saccharomyces cereuisiae (7) when calcium acetate or propionate is used.
The alternative to the chemical induction of protoplast fusion is electrofusion. This was first achieved by Senda et al (8), who used a DC pulse to fuse pairs of plant protoplasts micro-manipulated into contact. The process has been refined and applied to bacterial (9), fungal (10), and plant protoplasts (11). Contact is established between protoplasts by exposing them to an alternating, weakly inhomogeneous field that leads to polarization of the cells and their migration to regions of higher field density in a process known as dielectrophoresis. Because they form dipoles, the protoplasts line up on meeting, parallel to the field lines, in what have been termed "pearl chains." The AC field also causes the lateral diffusion of membrane proteins and formation of protein-free regions at points of close contact (12). The fusion of adjacent cells is induced by a DC-field pulse of short duration and high intensity. This leads to electrical breakdown at the area of membrane contact causing pore formation and cytoplasmic continuity. The technique has a number of advantages over chemical fusion. It permits controlled observation of the fusion, limits the possibility for multiple cell fusion, and results in fusion products that have not been subjected to prolonged toxic effects of the fusogenic agent. The major disadvantage is that relatively small numbers of protoplasts may be processed compared with chemical fusion. It is possible to combine the PEG-induced aggregation of protoplasts with electrical-field-induced fusion.
Whichever technique is chosen for protoplast fusion, it is necessary to have some means of selecting the fused cells (fusants) and a suitable regimen for the regeneration of a cell wall and reversion to dividing cells. Complementary auxotrophic mutations in the parent protoplasts are most commonly used to select fusants that should grow on a minimal medium. Other selection techniques may be based on (1) the complementation of resistances to antibiotics or other inhibitors (2), in the case of yeasts, the restoration of respiratory competence in a respiration-deficient mutant and (3), in plants, the complementation of an albino mutation. The use of mutant parent strains may not always be desirable, particularly in fusions aimed at producing new strains with agronomically or biotechnologically improved characteristics, where the presence of such mutations, even in a complemented situation, may be deleterious. In such situations, it may be advantageous to fuse protoplasts that have been differentially labelled, eg, with contrasting fluorescence signals.
Fusant cells with hybrid fluorescence may be selected either visually or using a fluorescence-activated cell sorter (FACS) (see Flow Cytometry). It is not always necessary that partners in a fusion should be individually viable. Protoplasts from cells killed by chemical, irradiation, or heat treatments may be fused with viable protoplasts. In some situations, this may improve the chances of obtaining hybrids; in others, it can be used to bias the genetic input of the parents. The heat killing of one partner in a bacterial fusion may inactivate restriction systems that could interfere with the success of the fusion. Regeneration and reversion conditions for hybrids may need to be investigated, as it is not necessary that hybrid protoplasts will behave similarly to the parents. It is not always possible to regenerate hybrids under direct conditions of selection. When bacterial protoplasts are fused, a wider variety of hybrid types can be isolated if they are initially grown on rich medium. Plant hybrids may need to be grown alongside nurse cells that provide growth factors for the protoplasts. The general requirements for the regeneration of protoplasts are discussed in Protoplasts.
Natural genetic systems in bacteria have three characteristics (13): 1) Genetic transfer is unidirectional; 2) DNA alone, and no cytoplasm, is transferred; and 3) It is an unusual event for the full genome to be transferred. Protoplast fusion seemed to offer great potential for studying genetic recombination between whole genomes and interactions between the nucleus and cytoplasm. Much work has been done with the Bacillus species, as these are easy to convert to protoplasts. Attempts at genetic mapping by recombination analysis in protoplast fusion hybrids, however, have not always been very successful. When protoplasts of complementary auxotrophic strains of B. subtilis were fused and fusants selected on a minimal medium, complemented diploids were extremely hard to find (14). When fusants were regenerated on a complete medium and well-isolated colonies were examined, practically no prototrophs were found. A significant number of fusants that grew on minimal medium plus the nutritional requirements of either of the parents, but not on a minimal medium alone, were discovered. These were referred to as noncomplementary diploids (NCDs). The physical presence of chromosomes from each parent in NCDs was demonstrated, and it was postulated that only one of the two chromosomes was being expressed. The silence of the chromosome is not the result of methylation but rather of a total shutdown of transcription (15). The genomes of the NCDs can undergo recombination, and this is the probable origin of the small numbers of prototrophic fusants obtained. Gabor and Hotchkiss (16) examined the recombination patterns and found that recombination was frequent and multiple in some NCDs and reciprocal recombinants were frequently uncovered, often together within a single regenerated colony. Recombination occurred in all chromosomal regions, but the frequencies of recombination were considerably greater in the chromosomal replication origin and termination regions. The latter was attributed to structural characteristics at the origin and termination regions associated with membrane attachments. The genetic manipulation of organisms when the requisite genes are known and accessible is best performed by transformation. Protoplast fusion for genetic manipulation is most useful when target genes are not known or a polygenic trait is to be transferred (17). It is also of value for the manipulation of organellar genetic systems.
The use of protoplast fusion for genetic manipulation has perhaps its greatest success, and potential with plants and has involved intraspecific, interspecific, intergeneric, and intertribal fusions. Intraspecific fusions are generally performed to effect the pooling of desirable characteristics from varieties within a species. Attempts at interspecies (and higher-order) hybrid production have aimed to increase the gene pool available for the improvement of a crop or ornamental plant. Particular objectives have been the acquisition of disease and pest resistance, characteristics often found in wild species but lost from cultivated species or the breeding of new types of ornamental plants. With respect to crop plants, it is generally important that hybrids are fertile and they maintain the advantageous characteristics of the cultivated parent. Somatic hybrids often display low fertility, in particular for pollen, but this may be improved and desirable crop plant genes maintained by using the hybrid as the female parent in a back-cross to the cultivated species. The last few years have seen a rapid growth in the range of species for which protoplast formation, fusion, and regeneration are possible. Notably, techniques have been developed for woody species and members of the Poaceae (grasses), many of which are of economic importance and had previously proved recalcitrant, particularly in protoplast regeneration.
Whatever the outcome of a fusion process with respect to the contributing nuclei, cytoplasmic union is an inevitable outcome. When this occurs without nuclear fusion or the exchange of genetic information between the nuclei, the product is a cybrid (cytoplasmic hybrid). Mixing of the cytoplasms may be a slow process, but the time taken for the regeneration of protoplasts ensures fairly thorough cytoplasmic mixing before cell division occurs. In the yeasts Saccharomyces cerevisiae (18) and Kluyveromyces lactis (19), cybrid formation is the most common outcome of the fusion of two protoplasts. When nuclear fusion does not take place, loss of one of the two nuclei is likely to occur during the early divisions of the regenerating protoplasts. Fusion of a respiratory deficient, mitochondrial DNA mutant strain of Candida utilis with a respiratory competent S. cerevisiae gave rise to fusants having C. utilis nuclear and mitochondrial genomes, the latter transformed to respiratory competence (20). In addition, respiratory competence has been restored in a respiratory deficient strain of S. cerevisiae by fusion of protoplasts with mini-protoplast-containing functional mitochondria. This demonstrates the possibility of selectively introducing organelles into specific cells via protoplast fusion. In fusions involving protoplasts of respiratory competent strains, the mitochondrial genomes may not be retained equally in the hybrids. In a fusion of K. lactis and K. fragilis, it was demonstrated that the mitochondrial genome of the latter was always preferentially retained. Protoplast fusion may also be used in fungi to demonstrate the mitochondrial DNA location of a mutation (21). The transfer of dsRNA viruses between Aspergillus species has been achieved by protoplast fusion, although the strains involved were mating incompatible, demonstrating the potential of this technique to overcome mating barriers (22).
After fusion, the nuclei exist in a common cytoplasm for some time before karyogamy, or the loss of one nucleus occurs. Stable heterokaryons are characteristic of a wide range of filamentous fungi but not of yeasts or plant cells. However, heterokaryosis can be maintained by the continued application of selection in fusants when karyogamy does not take place. In Saccharomyces cerevisiae, the incidence of karyogamy can be greatly increased if the nuclei of the parents are synchronized by treatment with either a- or a-mating pheromones before protoplast isolation (18, 23).
The fusion of haploid strains of Saccharomyces cerevisiae usually produces stable diploids. Kluyveromyces lactis, however, gives stable diploids only if haploids of similar mating type are fused; ala hybrids produced by protoplast fusion sporulate spontaneously. In some cases with filamentous fungi, protoplast fusion produces stable, vigorously growing heterokaryons. In a similar manner to a fungal parasexual cycle, karyogamy may occur, producing heterozygous diploids and recombinants on segregation. This is the case for Aspergillus nidulans (24), Penicillium chrysogenum (25), Aspergillus oryzae (26), and Aspergillus niger (27). In the case of Paecilomyces fumosoroseus (28) or Aspergillus chrysogenum (29), heterokaryons grow very slowly, and heterozygous diploids are rarely found. Recombinant haploids can usually be obtained, probably resulting from karyogamy followed by early haploidization. In a number of instances, heterokaryons stabilized by the apparent random loss of chromosomes from one parent and gave rise to a genome containing the chromosomes from one parent together with a few chromosomes from the other.
The products of interspecific fungal protoplast fusion are less predictable. As a general rule, interspecific heterokaryons formed from filamentous fungi grow badly. Sporulation gives spores characteristic of either parent, often with a predominance of one of them (30). When the selection of yeast protoplast fusants is based on auxotrophic complementation, a common outcome is that these contain the full genome of one of the parents plus a small number (often one) of the chromosomes of the other parent (31-33). Aneuploids and diploids have been formed as a result of interspecific fusions of yeast protoplasts, but the fusants display the characteristics associated with the dominant contributor of DNA. It is not clear whether the retention of a single chromosome of one partner in a hybrid results from: 1) karyogamy followed by chromosome loss, or 2) from chromosome transfer between nuclei in a transitory heterokaryon.
As for plant protoplasts, the most significant potential for protoplast fusion is for the transfer of polygenic traits between species. When a characteristic to be transferred is determined by a small number of known genes, a direct molecular genetic approach to transfer is the most useful. When protoplast fusion is used, the fusant progeny are likely to have acquired an uncontrolled number of unwanted genes, as well as those being sought. Nevertheless, there have been many attempts to produce fusion hybrids with biotechnological value. Many of these have sought to construct strains in which the ability to metabolize novel carbon sources has been transferred from a poorly fermenting species to the efficient ethanol producer Saccharomyces cerevisiae. These include the fermentation of lactose, from Kluyveromyces lactis (34) and Kluyveromyces fragilis (35); of xylose, from Pachysolen tannophilus (36); and of cellulose hydrolysates, from Zygosaccharomyces fermentati (37). These studies have met with mixed success. Typically, fermentation levels in the hybrids are better than those of the parent strain that metabolized the substrate but still somewhat inferior to S. cerevisiae in terms of general fermentation efficiency. The stability of the hybrids is often an additional problem. Hydrids produced as a result of the fusion of protoplasts of the yeast Pichia stipitis stabilized during culturing as a result of ploidy reduction. Attempts have been made to improve the fermentation characteristics of yeasts by raising the ploidy level using protoplast fusion. In the case of xylose fermentation, raising ploidy levels increased rates of ethanol formation with P. tannophilus (38) and Candida shehetae (39) but not with Pichia stipitis (40). For filamentous fungi, improvements in citric acid synthesis by Aspergillus niger (27) and cephalosporin production by Aspergillus chrysogenum (29) have been achieved by the fusion of different strains. In many other recorded cases, however, protoplast fusion did not give rise to improved performance (30, 41). There is clearly some potential for the improvement of biotechnological performance using protoplast fusion with fungi, but the approach is so empirical that more rational strategies are preferable wherever possible.
