• Journal of Mushroom
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• v.12 no.2
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• pp.96-106
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• 2014

The primary objective of the present study is the characterization of the somatic hybrids of dikaryon-monokaryon (di-mono) crosses in mushroom breeding. We employed this technique for developing superior strains from Pleurotus ostreatus strains with 48 intraspecific hybrids of 12 combinations between six P. ostreatus strains and one P. florida strain. The results on the experiments of hybridization rate, nuclear DNA patterns, and colors and morphology of fruit-bodies, are presented. In di-mono crosses, somatic hybrids among Pleurotus strains showed 100% of crossability as seen in those between P. ostreatus and P. florida strains indicating that the nuclei of a dikaryon is inferred to be migrated to a recipient. 87.5% of the somatic hybrids among Pleurotus strains were similar to the donor dikaryons, and 12.5% of the somatic hybrids presented DNA patterns of both parents. In 16.6% of di-mono crosses between P. ostreatus and P. florida, the nuclear DNA patterns of all hybrids showed the same or similar patterns compared to the donor dikaryons. 70.9% of the hybrids between P. ostreatus and P. ostreatus were similar to the donor dikaryons and 12.5% of them presented the DNA patterns of both parents. 79.2% of fruiting body morphology of the hybrids among Pleurotus strains were similar to the dikaryons and 20.8% of them were similar to both parents. Interestingly, the morphology of all dikaryons were dissimilar each other. All hybrid strains between dikaryon P. florida and monokaryon P. ostreatus showed the fruiting body of which colors were similar to those of the dikaryon, while the hybrids between dikaryon P. ostreatus and monokaryon P. florida were showed the combined colors of both parents. Therefore, the fruiting body color of P. florida tends to be generally dominant. In conclusion, the present study provides a way to find out and suggest superior hybrid strains using the nuclear DNA patterns of hybrids between Pleurotus strains as well as the characteristics of their fruiting bodies. The advantages of the di-mono crossing are needs to be fully utilized in mushroom breeding because it is an ideal way to develop the superior strains of Pleurotus.

• Journal of Microbiology
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• v.34 no.1
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• pp.37-37
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• 1996

The internal regions of nuclear small subunit rRNA from 6 plaeurotus species and 5 Pleurotus ostreatus strains were amplified by PCR and sequenced. The DNA sequences of 8 Pleurotus strains (P. ostreatus NFFA2, NFFA4501, NFFA4001, KFFA4001, KFCC11635, P florida, P. florida, P. sajor-cuju, P. pulmonarius, and P. spodoleucus) were idential, but P. cornucopiae differed from them in two bases out of 605 bases. However, p[hylogenetic analysis of the sequences by DNA-distance matrix and UPGMA methods showed that P. ostreatus NFFA2m1 and NFFA2m2, known as mutants of P. ostreatus NFFA2, belonged to anther group of Basidiomycotina, which is close to the genus Auricularia. The difference of the SSU rDNA sequences of P. cornucopiae from other Pleurotus species tested corresponds to the difference of mitochondrial plasmid type present in Pleurotus species as observed by Kim et al. (1993, Korean J. Microbiol. 31, 141-147).ishement of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with .alpha.-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de nono protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 /.mu.g/ml) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

• The Korean Journal of Mycology
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• v.17 no.1
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• pp.27-30
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• 1989

F. velutipes Leu 2 gene (${\beta}-isopropylmalate$ dehydrogenase gene) was used for transformation of P. florida leucine requiring auxotrophic mutant P101. Transformation frequency was very low but the transformed colony can grow on minimal medium very slowly. Transformation was identified by Southern hybridization and reverse transformation into E. coli using chromosome DNA isolated from transformed P. florida.

• The Korean Journal of Mycology
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• v.15 no.4
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• pp.250-253
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• 1987

The transfer of the isolated nuclei from P. florida into protoplasts of P. ostreatus was induced with polyethylene glycol and $CaCl_2$. Three types of transfer products of nuclei were obtained when transferred to MMM. Type 1 colonies were more vigrously growing mycelium and stable on MCM. One of the type 1 colonies, appeared segregation on MCM plus benomyl. The mycelium did not form clamp connection. These results suggest that type 1 colonies were nuclear hybrids or allodiploids. Type 2 was main products of nuclear transfer. The mycelium formed clamp connection and fertile on sawdust media. Type 3 was very slow growing or non-viable colonies after debris of nuclei or chromosomes transfer into recipient protoplasts. Isozyme pattern of esterase in type 1 produced a new band. Type 2 and type 3 could be characterized by parental bands.

• The Korean Journal of Mycology
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• v.20 no.2
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• pp.118-125
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• 1992

Interspecific fusion products were obtained by protoplast fusion induced by polyethylene glycol, from auxotrophic mutants, Pleurotus cornucopiae and P. florida. The fusants were classified into allodiploidy, stable heterokaryon, and spontaneously segregated heterokaryons. Fruiting body of the clamped or clampless fusants was produced by light-dark cycle on the sawdust medium in glass bottles. Most of these clampless fusants produced mature basidiocarps. The pilei showed various mixed colors resembling the parents. All fruit bodies presented clamp connections except two fusants. When small tissues of stipe from basidiocarps were cultured on a complete medium, mycelial colonies grew more vigorously than that of the original clampless fusants. Five fusants in three crosses were analysed with the distribution of progenies and segregation of genetic characters by random spore analyses. The genetic markers were shown to segregate and recombine in the first generation of monospores isolated from basidiocarps. The analysis indicated the heter-okar-yosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis. Genotypes of a large number of auxotiophic progenies were not detected. The aberration ratio of segregants indicated the gene interaction resulting from different genome structure between vegetatively incompatible species.

• The Korean Journal of Mycology
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• v.24 no.2 s.77
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• pp.111-126
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• 1996

Transfer of the isolated nuclei from Lentinus edodes into protoplasts of Pleurotus florida was induced with polyethlene glycol (PEG) and $CaCl_2$. The intergeneric transfer products were classified into nuclear hybrid, heterokaryon or synkaryon, and reconstituted cell. These progenies except nuclear hybrids formed mature fruiting bodies on sawdust rice bran medium. Formation of fruit bodies was influenced by several factors such as light, temperature, nutrition and physic state of the culture media. Most of fruiting body characters were similar to those of P. florida in synkaryon and L. edodes in reconstituted cell, respectively. All these basidiocarps had clamp connections though initial heterokaryon colonies were lacking. Isozyme patterns of intergeneric progenies were quite different from those of parents. DNA polymorphisms of transfer products were also compared by random amplified polymorphic DNAs (RAPD) analysis based on polymerase chain reaction. The RAPD patterns were different from those of donor and recipient. DNA fingerprints ranged in size from 0.25 to 4.0 Kb. On the basis of RAPD, the transfer products were classified into five groups. Two synkaryon were analysed with distribution of progenies and segregation of genetic markers by random spore analyses. The genetic markers were segregated into wild type and riboflavine requiring auxotrophs.

• The Korean Journal of Mycology
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• v.18 no.2
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• pp.77-83
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• 1990

Factors influencing the fusion frequency of protoplasts were investigated with auxotrophic mutants of Pleurotus florida and Pleurotus ostreatus. Immediately after the polyethylene glycol (PEG) solution was added, the protoplasts adhered firmly and shrank. During the subsequent dilution with 0.6 M sucrose, the protoplasts regained their normal size and larger bodies were observed. Interspecific heterokaryons were obtained by fusion of the nutritionally complementing protoplasts. Hyphae of the heterokaryotic fusants formed true clamp connections. The optimum conditions were a total of 1 to 15 million protoplasts per ml, 30% polyethylene glycol 8000 solution with adjustment to pH 8.0 and 0.6 M sucrose stabilized regeneration medium. Other parameters such as $CA^{++}$, glycine, exposure time and temperature influenced mainly the viability of the protoplasts.

• The Korean Journal of Mycology
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• v.14 no.1
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• pp.9-15
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• 1986

Interspecific hybrids of Pleurotus ostreatus and Pleurotus florida were formed by using protoplasts of complementing auxotrophs. The genetic markers were shown to segregate and recombine in the first generation of monosporus isolates from basidiocarp of seven fusion products. The analysis provides proof of heterokaryosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis. In all the crosses there was no evidence of linkage between the genetic markers. Clamp connections were formed in monosporus mycelia from basidiocarp of fusion products.

• The Korean Journal of Mycology
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• v.20 no.3
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• pp.222-228
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• 1992

Pleurotus florida was transformed by complementation of auxotrophic mutant using chimeric plasmid containing Flammulina velutipes leu 2 gene and pBR 322 replicon. Mycelial morphology of transformants was grown and compared on mushroom complete and minimal medium. Transformants were mated with monokaryon and their genetic recombination was investigated for the morphology of fruitbody and spore analysis. $Leu^+$ transformant showed same mating type of $A_1B_1$ as to the untransformed mutant. The transformant and the untransformed mutant were mated with monokaryon of which mating type is $A_2B_1$, respectively. Although fruitbody of the untransformed mutant was not produced, $leu^+$ transformant produced fruitbody. Spore analysis showed that leucine requiring spores from fruitbody of $leu^+$ transformant were diminished when compared with those of untransformed mutant.

• Applied Biological Chemistry
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• v.37 no.5
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• pp.326-333
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• 1994

Pleurotus florida with high cellulase activity as well as lignin degradability was selected out among strains for fermentation of the rice straw to improve the nutritive value. When the rice straw was fermented by P. florida, the contents of hemicellulose, cellulose and lignin were decreased to 22.5%, 11.4% and 28.1%, respectively, whereas the contents of rice straw fermented after pretreatment with $H_2O_2$ or alkaline hydrogen peroxide were decreased much in the lower concentration. The content of T-N (total-nitrogen) and crude fat was increased with the longer fermentation period. The amino acid content of rice straw fermented by P. florida in 30 days was increased to 28.9% and 35.1% as the rice straw was fermented after pretreatment without and with 4% $H_2O_2$, respectively. The crystalline intensity of rice straw was decreased by pretreatment with 4% $H_2O_2$ and fermentation by P. florida. However, the crystall intensity was increased by treatment with alkaline hydrogen peroxide and the more when the straw was washed after the treatment. When the rice straw was fermented by P. florida for 30 days, the in vitro organic matter digestibility was increased up to 6% of $H_2O_2$ pretreatment.