• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.168-172
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• 1999

The antifungal mechanism of the antifungal peptide against Aspergillus fumigatus, $K^{18,19}$-CA(l-8)-ME(l-12), derived from cecropin A(l-8)-melittin(l-12) was investigated by confocal laser scanning microscopy, cell wall regeneration, ATPase activity inhibition, and released potassium ion. By confocal laser scanning microscopy, $K^{18,19}$-CA(l-8)-ME(l-12) was detected on the surface of A. fumigatus, while cecropin A used as a negative control peptide was not detected. The protoplast of A. fumigatus treated with$K^{18,19}$-CA(1-8)-ME(1-12) failed to regenerate the fungal cell walls. Compared with cecropin A, the amount of potassium ion released by $K^{18,19}$-CA(l-8)-ME(l-12) was increased. Furthermore, $K^{18,19}$-CA(l-8)-ME(l-12) inhibited the ATPase activity on the plasma membrane. These results suggested that $K^{18,19}$-CA(l-8)-ME(1-12) acts on the plasma membrane of A. fumigatus and its antifungal action is due to the ion channel or pore formation on the plasma membrane.

• Korean Journal of Plant Tissue Culture
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• v.27 no.2
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• pp.125-131
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• 2000

Protoplasts of Arabidopsis thaliana were easily isolated from the shoot-forming (SF) suspension-cultured cell clusters with 4 hours-shaking condition (40 rpm) on CPD enzyme solution containing 1% cellulase R-10, 0.25% pectolyase Y-23 and 0.5% driselase. Protoplasts were cultured on liquid KAO medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L kinetin, 200 mg/L spermidine and 68 g/L glucose. Also, protoplasts were cultured on 0.2 $\mu$M membrane filter placed onto CP solid medium containing the suspension cells as feeder cells in the dark at $25^{\circ}C$ for 4 weeks. Protoplast-derived-SF calli were cultured on MS medium containing 0.05 mg/L IAA, 7 mg/L 2 ip and 30 g/L sucrose under the continuous illumination for four weeks. The frequency of shoot formation was about 60%. The regenerants were transferred into potting soil to grow mature plants. The regenerants formed the silques with seeds after 8 weeks of cultures.

• Korean Journal of Agricultural Science
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• v.15 no.1
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• pp.15-22
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• 1988

Factors affecting the regeneration, reversion of protoplasts from mycelium of F. velutipes were investigated and the selection of auxotrophic mutants from protoplasts of F. velutipes was performed. PDP medium stabilized with 0.6M sucrose was suitable for the regeneration of protoplasts, and regeneration frequency was 0.47-1.32. The regeneration frequency of protoplasts was increased when nutrients were added to the regeneration medium. Especially, yeast extract was the most effective to regeneration of protoplasts. Regeneration pattern of protoplasts was formation of germ tubes from bud-like cells. 13-18% of monokaryotic strains was appeared from reverted protoplasts. Five of auxotrophic mutants were isolated from strains showed survival frequency of 1.9-16.

• Journal of Korean Society of Forest Science
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• v.77 no.2
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• pp.208-215
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• 1988

The leaf mesophyll protoplasts of Populus alba ${\times}$ glandulosa were isolated from leaf of plantlet in vitro and cultured for plant regeneration. The MS medium (minus $NH_4NO_3$) with 0.5 mg/l BAP and 2.0 mg/l 2, 4-D showed the moderate frequency of dividing protoplasts cultured by the liquid plating method during the first week of culture. The percentage of colony formation was revealed the highest frequency by the gauze contained semi-solid agar plating method after 5 weeks cultured. Ridding out the gauze, the micro-callus was formed on the same semi-solid medium in 8 weeks after protoplasts culture. For proliferation of callus, mini-callus was transferred on the MS solid medium with 0.5 mg/l 2, 4-D and 0.1 mg/l BAP 12 weeks after culture. Shoot regeneration occurred when the calli derived from protoplasts were cultured on MS medium with 1.0 mg/l zeatin and such shoots could be readily rooted on the one half strengthen MS medium with non-phytohormone. Rooting shoots were planted in green-house 22 weeks after protoplast culture.

• 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.

• KSBB Journal
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• v.25 no.2
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• pp.155-166
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• 2010

Studies on the production of cell-wall bound innerpolysaccharides (IPS) (soluble ${\beta}$-D-glucan) have been performed by use of suspended myelial cultures of Inonotus obliquus. This product has promising potentials as an effective antidiabetic as well as an immunostimulating agents. As a first step to enhanced production of IPS, Intensive strain improvement programs were carried out by obtaining a large amounts of protoplasts for the isolation of single cell colonies. Rapid and large screening of high-yielding producers was possible because about fivefold higher amount of protoplasts ($2.3{\times}10^6$ protoplasts/mL) could be recovered with relatively high regeneration rates of $10^{-2}{\sim}10^{-3}$ by applying a modified filtration method, as compared to the previously used trapping method. A basic protocol necessary for UV-mutation of the protoplasts was also developed, resulting in several overproducing variants with good fermentation properties. Since the amount of IPS extracted from the mycelial cell walls of I. obliquus turned out to be almost constant per g DCW, increase in cell mass was considered the most important factor for the enhancement in IPS production. Therefore, attempts were made to screen mutant cells showing rapid mycelial growth rate in the final suspended cultures. Notably, the mutant strains showing an active cellgrowth in the preceding solid growth cultures were observed to produce higher amount of IPS in the suspended fermentations as well. A striking mutant, OBLQ756-15-5 strain, obtained from the survivors of a harsh UV-treated condition (97% death rate) was found to stably produce as high cell mass as 22 g DCW/L in the final fermentations. Currently, this strain is being tested for development of a scaled-up fermentation process for mass production of IPS.