• Journal of Mushroom
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• v.2 no.3
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• pp.115-126
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• 2004

Somatic hybrids of inter-compatible and inter-incompatible strains were obtained by protoplast fusion. The fusion products between compatible strains, Pleurotus ostreatus and P. florida, formed heterokaryons, while fusants between incompatible strains such as P. cornucopiae + P. florida, P. ostreatus + Ganoderma applanatum, P. florida + Ganoderma lucidum, and P. ostreatus + Flammulina velutipes formed synkaryons that retained genes from both parents. The heterokaryons showed the same level of basidioma development. In contrast, the synkaryons showed unique characteristics including clamp connection formation at mitosis, either partner basidioma development, and abnormal segregation and recombination compared with inter-compatible strains. Synkaryons can be classified into homokaryoyic and heterokaryotic type. A comparison of somatic hybrids with compatible and incompatible strains was made using random amplified polymorphic DNA (RAPD) analysis. The heterokaryons between compatible species showed the same level of variability and contained both parental RAPD bands. In contrast, most of the synkaryons between incompatible species showed similarity to those of either parental bands and non-parental RAPD bands. Synkaryons can be classified into microgenome insertion type and macrogenome insertion type. A tetrapolar mating system was found among monospore isolates in somatic hybrids and wild type P. ostreatus. Homokaryons from each somatic hybrid combination were paired with tester homokaryons of the initial wild type of P. ostreatus. The changed mating types were identified in progenies. The pattern of mating type switching in somatic hybrids depends on compatibility of fusion partner. There are several factors related to the mechanism of clamp connection formation and fruiting body development of synkaryons. Of these,the major factor may be associated with self-fertility and mating type switching such as homokaryotic fruiting of wild type P. ostreatus. This review will discuss these aspects.

• Plant Biotechnology Reports
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• v.2 no.3
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• pp.219-226
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• 2008

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient ${\beta}-glucuronidase$ (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and $100{\mu}F$ in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1-2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.294-301
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• 2001

The effects of including glycine and isonicotinic acid hydrazide (INH) in the growth medium (Luria broth, LBG) on the subsequent lysozyme-imduced protoplast formation and transformation efficiency of Corynebacterium glutamicum were studied. The transformation efficiency of C. glutamicum AS019 increased up to 100-fold as the ocncentrationof glycine in the media increased from 0% to 5% (w/v), relative to cells grown in the absence of glycine. The presence of 5 mg/ml INH in the growth medium led to a further 10-fold increase in transformation efficiency. In addition, this transformation protocol was successfully applied to other strains of C. glutamicum. Both chemicals affected the mycolic acid attachment to the cell surface of C. glutamicum, when INH, the relative percentage of fatty acids of AS019 to the total lipids (mycolic acid plus fatty acids) decreased from 76.9% (in LBG) to 72.9% (in LBG-2% glycine) and 66.4% (in LBG-8 mg InG/ml), thereby suggeting that these chemicals also inhibit fatty acid synthesis.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1528-1531
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• 2015

Sclerotia of Wolfiporia cocos are of medicinal and culinary value. The genes and molecular mechanisms involved in W. cocos sclerotial formation are poorly investigated because of the lack of a suitable and reproducible transformation system for W. cocos. In this study, a PEG/CaCl₂-mediated genetic transformation system for W. cocos was developed. The promoter P_gpd from Ganoderma lucidum effectively drove expression of the hygromycin B phosphotransferase gene in W. cocos, and approximately 30 transformants were obtained per 10 μg DNA when the protoplast suspension density was 10⁶ protoplasts/ml. However, no transformants were obtained under the regulation of the PtrpC promoter from Aspergillus nidulans.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.182-186
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• 1988

The possibility of strain improvement of cellulolytic fungus, Penicillium verruculosum via protoplast fusion was investigated. The cellulolytic activities of the six fusants, finally selected for their hyper-cellulolytics were 2 times of those of wild type and 1.2 to 4.4 times of those parental auxotrophs. It was confirmed that the nuclear fusion occurred in fusants by their DNA contents and nuclear staining with Giemsa. It was also found that the fusants were aneuploids, and their genetic stability was demonstrated from the subculture for four months.

• Journal of Life Science
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• v.7 no.4
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• pp.282-288
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• 1997

To obtain a basic information on the development of Genus Viola, ultrastructure and electrofusion process between the two protoplasts from wild Viola callus cells and pansy mesophyll cells were observed with a scanning electron microscopy(SEM) and transmission electron microscopy(TEM). In the ultrastructural observation of wild viola callus protoplasts and pansy mesophyll protoplasts using SEM, their cell walls were removed completely. A knob-like formation was observed on the enlarge surface of viola callus protoplasts. On the surface of pansy mesophyll protoplasts net-like chloroplasts were observed. In SEM observation of pansy mesophyll protoplasts, chloroplasts devoid of membrane were observed on the surface the protoplasts. Pearl chain was formed by applying AC field of 200 V/cm at 1.0 MHz for 43 sec. The lysis of plasma membranes and fusion process occurred by applying a 1,600 V/cm DC pulse twice for 1 sec. After 1-2 hours of a DC pulse application, it was observed that the two protoplasts were fused completely into one cell. In TEM observation of the fused cell, many small vacuoles were located in the fusion area of the two protoplasts. Indeed, two distinct regions were observed during fusing process; in one region, a nucleus was found, while in the other region, both nucleus and nucleous were found.

• 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.

• 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.

• Korean Journal of Food Science and Technology
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• v.21 no.1
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• pp.154-163
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• 1989

This studies were designed to improve the productivity of L-lysine by protoplast fusion and immobilized system of fusants using strains of Brevibacterium flavum ATCC 21528, Brevibacterium lactofermentum ATCC 21086 and Corynebacterium glutamicum 820. Mutants were isolated with concentration method of $300{\mu}g/ml$ penicillin-G after treatment of $250{\mu}g/ml$ N-methyl-N-nitro-N-nitrosoguanidine. B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;AEC^r)$ and C. glutamicum 57-5$(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ were isolated from mutants. Protoplasts were induced by being incubated with $500{\mu}g/ml$ lysozyme of lysis solution for 6 hr and the ratio of protoplast formation and regeneration were ranging from 97-99% and 33-37%, respectively. Fusion frequencies of fusants of BBFL 21, BCFG 37 and BCLG 59 were shown in the range from $1.25{\times}10^{-6}\;to\;5.83{\times}10^{-7}$ under the optimum conditions. The fusant BBFL 21 showed the highest productivity of $411.1\;ng/ml{\cdot}hr$ L-lysine in the lysine productivity broth at $30^{\circ}C$ for 72hr. In the immobilization systems, fusant BBFL 21 was employed in various polymer matrices such as sodium alginate, polyacrylamide, agar and ${\alpha}-carrageena$. The immobilization of sodium alginate showed the highest productivity of $413\;ng/ml{\cdot}hr$ L-lysine in the batch system. Continuous fermentation of immobilization system by using tube fermentor was produced the highest productivity $416.7\;ng/ml{\cdot}hr $ L-lysine under optimum condition.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.143-151
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• 1997

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.