Fungal protoplast fusion - A revisit

Biotechnology Centre, I.I.T. Kharagpur 721302, West Bengal, India
Bioprocess and Biosystems Engineering (Impact Factor: 2). 04/2000; 22(5):429-431. DOI: 10.1007/s004490050755


Protoplast fusion is a non-specific recombination technique used for transfer of cytosolic organelles including genetic material. The process involves cell wall breakdown, regeneration of protoplasts, chemofusion and electrofusion. This review article discusses all the stages involved in fusion of protoplasts and some of the applications of protoplast fusion technique in fungal systems.

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    • "Protoplast fusion technique has a great potential for genetic analysis and for strain improvement. It is particularly useful for industrially useful micro-organisms (Murlidhar and Panda 2000). These techniques have been widely used for enhanced yield in conversion of cellulose to ethanol (Knowles et al. 1987), strain improvement for alcohol fermentation (Lima et al. 1995), citric acid-producing strains of Aspergillus niger (Kirimura et al. 1986). "
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    ABSTRACT: AimsProtoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste.Methods and ResultsThe filamentous chitinolytic fungal strains Aspergillus oryzae NCIM 1272 and Trichoderma harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg mL−1 lysing enzymes from T. harzianum, 0.06 mg mL−1β-glucuronidase from H. pomatia and 1 mg mL−1 purified P. ochrochloron chitinase in 0.8 M sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers viz., colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE).Conclusions The present study revealed protoplast fusion between Aspergillus oryzae and Trichoderma harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of present study which can be emerged as a fundamental step in fusant identification.Significance and Impact of the StudyNow it is need to provide attention over effective chitin degradation in order to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 11/2014; 118(2). DOI:10.1111/jam.12711 · 2.48 Impact Factor
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    • "Afr. J. Biotechnol. fungal breeding (Gokhale, 1992; Muralidhar and Panda, 2000). Up to date, no report on the breeding of high taxol-production strain by inactivated protoplast fusion is available. "
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    ABSTRACT: Inactivated protoplast fusion by UV irradiation and UV+LiCl mutation was conducted using Nodulisporium sylviforme strain UV 40-19 and UL 50-6 to breed a high taxol-producing fungus. Qualitative and quantitative analysis of taxol production was confirmed using thin-layer chromatography, high performance liquid chromatography and mass spectrometry. The protoplasts of UV 40-19 and UL 50-6 were fully inactivated by heating at 54°C for 5 min and by UV irradiation (30 w UV light and vertical distance 30 cm) for 85 s. The highest fusion rate (14.31 ± 1.13%) between UV 40-19 and UL 50-6 was obtained under the conditions of 35% PEG, 90 s fusion time and the addition of 0.01 mol/l CaCl 2 . One high taxol production strain HDF-68 was obtained. The taxol production was up to 468.62 ± 37.49 g/l, which was increased by 24.51 and 19.35% compared with the parental strain UV 40-19 and UL 50-6 , respectively. This study provided a good basis for the application of this technique to the breeding of the strains with high taxol output.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 05/2011; 10(20). · 0.57 Impact Factor
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    ABSTRACT: An attempt was made to improve the stability toward centrifugation of protoplast fusion between Shewanella sp. and Escherichia coli. Stability of the cell membrane is an important factor in protoplast fusion. In order to change the fatty acid composition of the cell membrane phospholipids, eight fatty acids [caprylic acid, capric acid, palmitic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid] were added to each nutrient medium of Shewanella sp. and E. coli. The protoplasts were treated with lysozyme, and fusion occurred in the presence of a polyethylene glycol solution. The stability of the protoplast of Shewanella sp. decreased after EPA was added to the culture medium, and the stability of the protoplast of E. coli increased after the addition of linoleic acid or linolenic acid. Some fusant colonies that developed on the regenerated medium selected for E. coli with antibiotic tolerance. The efficiency of this fusion was higher than that of initial condition using protoplasts from Shewanella sp. and E. coli incubated without fatty acids. Protoplasts improved the fatty acid composition of the phospholipids. Cell membrane stability can change in order for the weak cells to be taken in by strong cells. These results suggested that the fatty acid composition of cell membrane phospholipids affected the fusant yield of the fusion of these bacteria.
    Fisheries Science 12/2008; 74(6):1290 - 1296. DOI:10.1111/j.1444-2906.2008.01654.x · 0.88 Impact Factor
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