• The Korean Journal of Mycology
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• v.21 no.4
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• Journal of Microbiology and Biotechnology
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• v.7 no.3
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• pp.161-166
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• 1997

Nuclei were isolated from the protoplasts of Trichoderma harzianum T95 and treated with colchicine, a polyploid inducer. The nuclei were transferred into the protoplast of multi-auxotrophic Gliocladium virens G88 which cannot grow in minimal medium. The protoplast of G. virens G88 carrying the transferred nuclei were regenerated in a regeneration minimal medium containing $17{\mu}g/ml$ of chloroneb as a haploid inducer. Six intergeneric hybrids between G. virens and T. harzianum were isolated from the regeneration minimal medium. The hybrids could be classified into three types according to morphology, those with an isozyme pattern, those with an protein band and those with an randomly amplified polymorphic DNA(RAPD) pattern produced by random primers and repetitive sequences. The first group was identified to be a haploid recombinant, the second group a heterokaryon, and the third appeared to be petite.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.23-30
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• 1994

Intergeneric hybrids formed between Aspergillus oryzae var oryzae and Penicillium verruculosum F-3 were obtained by nuclear transfer technique. Several auxotrophic mutants isolated fromconidio-spores of the two strains mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were 3$\times $10$^{-5}$ $~$1$\times $10$^{-5}$. From observation of genetic stability, conidial size, DNA content, nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrid posses the 1.3$~$2.2 fold higher amylase activities than those of parental strains. It was also revealed that some hybids had different isozyme patterns compared to those of parental strains by amylase activity assays.

• Archives of Pharmacal Research
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• v.14 no.3
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• pp.282-283
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• 1991

Stable intergeneric hybrids involving allodiploid were obtained through protoplast fusion and nuclear transfer between the auxotrophic mutans of two basidiomycetes. Ganoderma lucidum and Corolus versicolor.

• Journal of Plant Biology
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• v.5 no.3
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• pp.21-24
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• 1962

Intergeneric crossings between the inbred line of Brassica pekinensis var. Chongbangkokun and inbred line of Raphanus sativus var. Oolsanjaelae were made using Brassica as female plant, and obtained two individuals of intergeneric hybrids. Morphological characters of the two F1 hybrids are mostly intermediate of the two parental species, and their somatic chromosome number is 19. Meiotic behaviors of the parental plants were normal, while those of F1 plants were extremely irregular. The mean pairing frequencies per cell of hybrids are 1.09II＋16.811(examined in May), and 1.76II＋15.47i(examined in June). Majority of the pollen grain of parent species are normal, whereas those of F1's are invariably abortive. No seed setting was obtained in the hybrid plants left in the open field.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.31-37
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• 1997

Intergeneric hybrids between Aspergillus niger and Perricillium ch~y.sop~um(Tyr ), hyperlipolytic enzyne-producing fungi, were obtained by nuclear transfer technique:. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts were obtainrd by 1% Novozym 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KC]. Frequencies of hybrid formation by nuclear transfer were $1.3{\times}$10^{-3}$$ $-3.8{\times}$10^{-3}$$. From the chervation of genetic stability, conidial size, DNA content, ;md nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed 1.4-2.2 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.

• The Korean Journal of Mycology
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• v.22 no.3
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• pp.276-280
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• 1994

To obtain hybrids producing antagonisms and plant growth promoting effects by intergeneric nuclei transfer, the nuclei were isolated from the protoplasts of Trichoderma harzianum T95 and treated with colchicine. The nuclei were tranferred into protoplast of multi-auxotrophic Gliocladium virens G88 which cannot grow in minimal medium. The nuclei tranferred into protoplasts of G. virens G88 were selected on the regeneration minimal medium containing chloroneb as a haploid inducer. Low transfer frequency of 0.08% was observed with three chemical treatment, however no segregants were found in the intergeneric nuclei transfer. The various types of hybrids with different morphology were detected when different concentration of chloroneb were treated. These morphologies were classified as parental, recombinant and petite type.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.528.1-528
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• 1986

Cellulose utilising hybrids between Cellulomonas fimi and Bacillus pumilus were isolated after PEG mediated protoplast fusion. 33% (w/v) PEG #6, 000 and 50mM $Ca^{++}$were optimum concentration. The intergeneric fusion frequency was 3.2$\times$10$^{-7}$ xtracellular CMCase and $\beta$-glucosidase activities were detected from one hydrid unlike only CMCase was detected from Cellulomonas fimi.

• Plant Biotechnology Reports
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• v.2 no.2
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• pp.145-152
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• 2008

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.

• Journal of Plant Biology
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• v.30 no.1
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• pp.1-9
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• 1987

Leaf mesophyll protoplasts of Nicotiana tabacum (nitrate reductase deficient mutant) were fused with cell suspension protoplasts of albino Petunia inflata in an electric field. Hybrid cell colonies were selected for nitrate reductase proficiency and chlorophyll synthesis. Five hybrid plant lines, regenerated from the selected calli lines, were analysed by electrophoresis, number of chromosomes and morphological characters. Somtic hybrid plants showed both parent patterns in the isozymesof isoleucine aminopeptidase and esterase. The hybrids had the expected chromosome number of 62 and exhibited an intermediate floral morphology when compared with the parents, but plant height and leaf arrangement were similar to N. tabacum.