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Protoplast fusion of candida pseudotropicalis the conditions for protoplast formation regeneration and fusion



Protoplast fusion of candida pseudotropicalis the conditions for protoplast formation regeneration and fusion



Korean Journal of Microbiology 24(3): 243-250



Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 73%, respectively. When bovine serum albumin (4 mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol (0.5 mg/ml), 96-99% of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the additon of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and CaCl2 are 20% and 100 mM while optimal pH and exposure time are 6.0 and 30 min. The fusion frequencies between complementary mutants ranged from 1.5 .times. 10-3 to 8.8 .times. 10-3 and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

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Accession: 006216465

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