the medium used in protoplast fusion experiments is

Rowlands (1992) suggested that they may be used in mutagenesis of non-sporulating fungi. Protoplasts are also useful in the filamentous fungi for manipulations other than cell fusion. Thus, the desirable characteristics of each strain were combined in the recombinant. A total of 238 colonies judged to be recombinants were screened for penicillin V production and the culture with the best combination of properties is shown in the table. To avoid any adverse effects no selective genetic markers were used and the regenerated colonies were screened on the basis of colony morphology and spore colour. Table 3.7 illustrates the properties of the two strains used in a protoplast fusion and one of the recombinants selected. This programme included random and directed selection as well as protoplast fusion. Lein (1986) reported the penicillin strain improvement programme adopted by Panlabs, Inc. Out of 100 stable colonies which were scored, two possessed the desirable morphology, high penicillin V and low OH-V penicillin productivities. Protoplasts from two strains, differing in colony morphology and the abilities to produce penicillin V (the desired product)Īnd OH-V penicillin (an undesirable product), were fused, followed by plating on a non-selective medium. (1982) utilized protoplast fusion to combine the desirable qualities of two strains of Pénicillium chrysogenum. a strain which demonstrated good sporulation, a high growth rate and produced 40% more antibiotic than the higher-yielding parent. A cross between an asporulating, slow-growing strain with a sporulating fast-growing strain which only produced one-third of the cephalosporin level of the first strain eventually resulted in the isolation of a recombinant which combined the desirable properties of both strains, i.e. Recombinants were obtained in both sister and divergent crosses. Electron microscopic examination of fused protoplasts indicated that up to 1% underwent immediate nuclear fusion. The results from conventional techniques suggested that nuclear fusion was difficult to achieve. acremonium with the protoplast fusion technique. These workers compared the effectiveness of conventional techniques of obtaining nuclear fusion between strains of C. still demonstrated that protoplast fusion could be successful where conventional techniques had failed.Ī demonstration of the use of protoplast fusion for an industrial fungus is provided by the work of Hamlyn and Ball (1979) on the cephalosporin producer, C. cyaneofulvum are not different species of Pénicillium (Samson et al., 1977), Peberdy et al. cyaneoful-vum and demonstrated the formation of diploids which gave rise to recombinants after treatment with /j-fluo-rophenylalanine or benomyl. (1977) who succeeded in obtaining heterokaryons between P. This situation is illustrated by the work of Peberdy et al.

Thus, this approach has allowed the use of the parasexual cycle for breeding purposes in situations where it had not been previously possible. (Fodor and Alfoldi, 1976), corynebacteria (Karasawa et al., 1986), filamentous fungi (Ferenczy et al., 1974) and yeasts (Sipiczki and Ferenczy, 1977).įusion of fungal protoplasts appears to be an excellent technique to obtain heterokaryons between strains where conventional techniques have failed, or, indeed, as the method of choice. Protoplast fusion has been demonstrated in a large number of industrially important organisms including Streptomyces spp. Thus, protoplasts may be used to overcome some recombination barriers. Cell fusion, followed by nuclear fusion, may occur between protoplasts of strains which would otherwise not fuse and the resulting fused protoplast may regenerate a cell wall and grow as a normal cell. Protoplasts may regenerate their cell walls and are then capable of growth as normal cells. Protoplasts are cells devoid of their cell walls and may be prepared by subjecting cells to the action of wall degrading enzymes in isotonic solutions.

The medium used in protoplast fusion experiments is