• The Korean Journal of Mycology
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• v.17 no.3
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• pp.119-123
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• 1989

Interorder heterokaryons were obtained by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Pleurotus ostreatus in agaricales and Ganoderma applanaturm in aphyllophorales. When transferred to MMM plates, all fusion colonies exhibited an extremely growth rate. During three times subcultivation on MCM the growth rate of fusants showed faster little by little. Seventy-five % fusion products of potoplasts showed mixed morphologies between those of P. ostreatus and G. applannatum in the first subcultivation on MCM and MGM. The phenotype of these fusants changed similar those of P. osteatus type after three times subcultivation on MCM. However, phenotype of 25% stable strains did not change on subcultivation. Hyphae of all fusion products did not form true clamp connection. All these types did not produce primordia. A comparrison of interorder somatic hybrids was made using isozyme analysis of esterase, malate dehydrogenase and peroxidase. In most cases the enzyme patterns of G. applanatum were not distinct, however, fusant showed non-parental bands.

• Journal of Mushroom
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• v.13 no.3
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• pp.270-273
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• 2015

Hypsizigus marmoreus is commercially the most important edible mushroom in Japan. This mushroom is usually cultivated for a longer period (about 85~120 days) than other mushroom. In order to develop a new cultivar that has a shortened cultivation period, the genome analysis of this strain has been considered. This study aims to obtain parental monokaryotic strains reproducing 'Haemi' cultivar in Hypsizigus marmoreus for reference genome sequencing. The mycelia were cultured in MCM and MYG media for various incubation periods. Homogenized mycelia were treated with commercial cell wall degrading enzymes to maximize protoplasts production yield from Hypsizigus marmoreus. The greatest number of protoplasts was obtained from mycelia cultured in MCM media for 3 days using Novozyme enzyme. The isolated protoplasts were grown in regeneration agar media after two weeks. Regenerated colonies were picked and moved on separated dishes for microscopic observation. Neohaplonts regenerated from dikayotic strains were identified by the absence of clamp connections. We confirmed that one of monokaryotic strains is a parental strain by crossing with an original compatible strain of 'Haemi' cultivar. This parental strain will be used for reference genome sequence analysis.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.81-86
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• 2008

This study describes conditions for plant regeneration from protoplasts-derived from embryogenic callus of satsuma mandarin. Plants were generated via somatic embryogenesis. Protoplasts isolated directly from nucellar callus induced from immature ovule of satsuma mandarin cv. Okitsu (Citrus unshiu Marc.) were cultured in 0.6M $BH_3$ medium. Cell division and plating efficiency were affected by protoplast culture method. The liquid over solid method was the most effective for formation of microcalli. Most of microcalli grew rapidly and transferred onto embryoid formation medium. Optimum embryoid formation medium was MT medium containing 1.5 g/L malt extract, 0.146 M sucrose and the medium for plantlet regeneration was MS medium containing 0.09M sucrose, 1.0 mg/L $GA_3$. No differences were noticed in growth habits and leaf characters such as shape, thickness, and colour between protoplast-derived plants and nucellar seedlings. This plant regeneration system from protoplasts-derived from embryogenic callus provides an alternative way for producing new scion and rootstock cultivar from citrus species which can not be crossed.

• The Korean Journal of Mycology
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• v.16 no.2
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• pp.79-86
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• 1988

Interspecific fusion products were obtained from fusion of Ganoderma applanatum and Ganoderma luridum. Frequency of fusion was 0.77-1.38%. Fusion products were selected by the comparision of morphology and color of colony. Fusion Products had chracteristics of two parental strains and generally grew faster than the parents. Some fusants were segregated on GCM. Fusion products were confirmed by mycelial morphology and electrophoretics pattern of esterase isozyme from mycelium. Most of fusion products were lack in clamp connection but those fusion products, that were segregated produced mycelium with and without clamp connection. Some of the fusion products produced fruit body on sawdust medium.

• The Korean Journal of Mycology
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• v.15 no.3
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• pp.135-141
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• 1987

Interspecific heterokaryons were obtained by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of P. ostreatus+P. cornucopiae, P. ostreatus+P. florida, P. ostreatus+P. sajor-caju, P. florida+P. cornucopiae, P. florida+P. sajor-caju and P. sajor-caju+P. cornucopiae protoplasts. The Fusion products of protoplasts were induced by MCM+benomyl, but segregation of sectors were not identified. Interspecific fusion products of protoplasts between incompatible strains did not form true clamp connections and did not produce fruit bodies. For induction of fertility, interspecific heterokaryons crossed with their parents by hyphal anastomosis.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.22-30
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• 1985

Protoplasts of Pisum sativum L. were isolated and cultured from leaf mesophyll tissue. The successful yield of protoplast was obtained in an enzyme solution of 2% 'Onozuka R-10' and 2 % 'Macerozyme R-10' contained 6mM $CaCl_2{\cdot}2H_2O$ within 4 hours. They were divided in B5 culture medium supplemented with 2mg/l kinetin, 1mg/l 2, 4-D and 0.2% Difcobacto agar. Divisions of the protoplasts were continued and led to colony formation for 1 months. The colony from protoplasts of pea mesophyll tissue was formed to callus after subculture in a medium contained macronutrients and amino acids of BII medium and micronutrients and vitamins of B5 medium, and also supplemented with 2mg/l kinetin 2mg/l NAA.

• 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.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.297-302
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• 1988

The protoplasts of Saccharomyces cerevisiae D-71, a thermophilic strain and Zygosaccharomyces rouxii SR-S, an osmotolerant strain were fused, and a fusant (FS-RN 1) was selected, then was characterized for its genetic stability, DNA content, cell capacity, growth rate, tolerance to salts and chemicals, $\beta$-fructofuranosidase level and ethanol fermenting activity. After 6 months of preservation, 5.8% of the fusant clones were segregated to parental types. The DNA content and cell capacity of the fusant were greater than those of the parental strains. Lag period of growth for the fusant was longer than those for the parents. The fusant colonies showed pink-color reaction to triphenyltetrazolium chloride(TTC) test. The fusant appeared to have resistances to NaCl at moderate levels between both parental strains, and resistances to KCI, sodium propionate and cycloheximide similar to either one of the parents. $\beta$-Fructofuranosidase activity of the fusant was 24.2$\times$10$^{-3}$/IU/g(dry wt) for 3 days culture. Ethanol yields ofter 4 days of fermentation by the fusant at 3$0^{\circ}C$ were : 6.0%(v/v) from 40% of glucose and 8.8%(v/v) from 20% of sucrose.

• The Korean Journal of Mycology
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• v.51 no.4
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• pp.389-396
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• 2023

In this study, dikaryotic strains of Lentinula edodes were generated by mono-mono hybridization using mating type analysis and their fruiting body characteristics were investigated. Approximately 100 monokaryotic strains were isolated from the basidiospores of Sanbaekhyhang, and homokaryotic strains were isolated from Chungheung 1ho using protoplast isolation and regeneration. Their mating types were evaluated and a total of 60 dikaryotic strains were hybridized. Using these strains, fruiting bodies were produced and their characteristics were examined after cultivation on sawdust media. The results indicated that the rate of hybridization was 100% and that 55 of 60 strains formed fruiting bodies. These showed normal pileus and gill structures; however,10 strains also produced fruiting bodies with abnormal pileus and gill structures. The weight and size of the fruiting bodies differed depending on the strains. Overall, further studies are needed for predicting the characteristics of hybridized strains based on their parental strains.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.144.1-144
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• 2003

Attenuated viruses can protect their hosts against challenge to their related viruses. Increasing evidence shows that mutations of the tobamoviral 126/183 kDa protein play a major role in the viral attenuation and contribute to the cross protection mechanism. In this study, four mutants of Pepper mild mottle virus (PMMoV) have been constructed by mutagenesis; two mutants, pTPpoly348 and pTPpoly762, were substituted in the middle of replicase gene, and the others, pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219, were deletion mutants made by deleting some parts of pseudoknot structures of the 3' noncoding region (NCR) of the virus. Progeny viruses generated from the four mutants were infectious on N. benthamiana plants with symptomless or mild mosaic symptom. Replication efficiency and viral product accumulations of four mutants were assessed by Northern and Western blot analyses on BY-2 protoplast cells. Accumulation of CP for the pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219 were lower than that of other mutants and wild type virus. These data suggest that the 3'-NCR mutations contribute to the viral gene expression in host tissues, while mutants of replicase gene rather govern the symptom expression.