• Journal of Korean Society of Forest Science
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• v.81 no.2
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• pp.183-190
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• 1992

The influence of various levels of major medium components such as sucrose, nitrate, phosphate, plant growth regulators, and light intensity for cell growth and the production of anthocyanin content in cell cultures of Populus alba ${\times}$ P. glandulosa were investigated. Best results for anthocyanin yield were obtained using Murashige and Skoog(MS) medium containing 5% sucrose, 12.5% nitrate, 200% phosphate, 1.0mg/l indole-3-acetic acid(IAA), 1.0mg/l benaylaminopurine(BAP), and continuous illumination of 7,000 lux. On the other hand, maximum cell growth was achieved with 5% sucrose, 50% nitrate above 400% phosphate compare with that of MS basal mediumi, and 0.5mg/l 2, 4-dichlorophenoxyacetic acid(2, 4-D). Anthocyanin accumulation in a suspension cultured cells of given genotype was stimulated by subculturing onto the medium lacking 2, 4-D. Pigmented cell clusters were extracted with methanol containing 1% hydrochloric acid (HCl) and then anthocyanin was identified by thin layer chromatography (TLC) and U. V. spectrophotometer.

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.157-163
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• 2010

A protocol for in vitro propagation from pseudobulb sections of Lycaste armomatica (Graham ex Hook) Lindl., an ornamental and fragrant orchid, was developed. The effect of four cytokinins: kinetin (K), metatopolin (mT), $N^6$-benzyladenine (BA), and thidiazuron (TDZ), in equimolar concentrations, was investigated. Shoot formation from apical and basal pseudobulb sections was obtained in all treatments. A few medial sections cultured in media supplemented with BA formed protocorm-like bodies. Shoot formation was greater from the basal section than the apical, and mainly occurred in explants cultured in media containing TDZ. The highest average numbers of shoots per explant were achieved from basal sections cultured in media supplemented with TDZ at 4.4, 8.87 and 2.2 ${\mu}M$, forming on average 9.9, 8.6 and 7.3 shoots per explant, respectively. Since the medial pseudobulb section was the worst explant for propagation of L. aromatica, we recommend that pseudobulbs be divided into two sections; the basal half should be cultured in MS medium supplemented with TDZ at 4.4 ${\mu}M$ and the apical half with TDZ at 2.2 ${\mu}M$. Subculturing individual shoots in MS medium without plant growth regulators allows further development and rooting. A survival rate of more than 90% under greenhouse conditions was achieved. This research represents a direct contribution to the conservation and sustainable use of this valuable natural resource.

• Ocean and Polar Research
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• v.33 no.3
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• pp.303-308
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• 2011

Polar psychrophiles which thrive under extreme conditions such as cold temperature, high salinity, and high dose ultraviolet light, emerge as novel targets for biotechnology. To prevent genetic drift and the possibility of contamination by subculturing, cryopreservation was employed for two psychrophilic microalgae, Porosira sp. (KOPRI AnM0008) and Pyramimonas sp. (KOPRI AnM0046), which have anti-freeze activities. Five cryoprotectants (dimethyl sulphoxide, ethylene glycol, glycerol, methanol and propylene glycol) showed toxicity at 20-30% (v/v). The optimal cryoprotectant concentration and equilibration time were less than 20% and 10 min, respectively. Cryopreservation was carried out in the presence of cryoprotectants either by direct freezing in liquid nitrogen ($LN_2$) or controlled freezing using a controlled rate freezer followed by storage in the $LN_2$ tank. As a result, Pyramimonas sp. (KOPRI AnM0046), a psychrophilic chlorophyta was revived. Cryopreserved Porosira sp. was not revived from either freezing protocols probably due to the silicic cell wall and its relatively large cell size. In the case of Pyramimonas sp. (KOPRI AnM0046), the controlled freezing method showed higher revival yield than the direct freezing method.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.342-347
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• 2003

Clumping of cells is one of the major obstacles to culture wild myxobacterial strains in liquid media. In an effort to solve this problem, we tried a method isolating spontaneous mutants that grow dispersed in liquid media from a wild myxobacterial strain. Myxococcus stipitatus KYC1001, a newly isolated strain from Gyearyongsan National Park in Korea, clumps and sticks to the surface of culture vessels as other wild myxobacteria behave in liquid media. Taking an advantage of the characteristics that dispersed mutant cells would grow dispersed while most other wild type cells would clump and stick to the surface of culture vessels, spontaneous dispersed mutants were enriched by repeated subculturing of culture supernatant. A resultant mutant, KYC2001, did not form any clumps nor stick to the surface of culture flasks, but grew completely dispersed in liquid. Meanwhile, three other spontaneous mutants, KYC2002, KYC2003, and KYC2004, shelved partially dispersed phenotype. A major portion of the cells grew dispersed in liquid but they still formed some clumps.

• KSBB Journal
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• v.13 no.1
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• pp.83-89
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• 1998

Using a cellulose macroporous microcarrier, HeLa cells were cultivated in 100mL spinner flask(Bellco Co., USA) and confluent cell laden microcarriers were subcultured by bead-to-bead cell transfer method. In macroporous mcirocarrier-HeLa system viable suspended cells played an important role in bead-to-bead cell transfer and that could be increased by use of RPMI-1640, a calcium-ion-reduced-media and high speed agitation. Successful bead-to-bead cell transfers were performed continuously three time in spinner flask. We applied this technique to produce recombinant Vaccinia virus which express $\beta$-galactosidase. Recombinant protein yield of bead-to-bead transferred culture was comparable to conventional microcarrier cultures that were inoculated by cells detached from T-flask. Although trypsinization is a useful method for subculturing microcarriers in some cases, that process adds quality control problem and handling steps to large scale cell production. There fore, bead-to-bead cell transfer technique offers another convenient and efficient scale-up method for continuous microcarrier cultures.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.479-484
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• 2000

This study was conducted to examine the effects of explant sources and plant growth regulators on mass propagation of Cyclamen persicum. Tuber, cotyledon, and petiole tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins and cytokinins. Shoots were not induced from calli on cotyledon and petiole explants cultured on MS medium containing various concentrations of 2,4-D or NAA. However, multiple shoots were formed directly from tuber explants cultured on the medium containing 0.5 and 1.0 mg/L 2,4-D or NAA. In MS medium with cytokinin alone, TDZ was more effective in shoot formation than BA or kinetin in all explants. The combinations of NAA and BA was found to be most effective in shoot formation from tuber, cotyledon and petiole explants. Especially, shoots were formed from all the tuber explants on the medium containing 0.5 mg/L of NAA and BA. Hormonal effects on root formation were examined by subculturing single shoots on MS medium containing NAA or IBA. The medium with 0.5 mg/L IBA was most effective in root induction and subsequent plantlet regeneration.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.289-291
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• 1999

Hypocotyl explants from 7 days old seedlings of one $F_1$ hybrid cultivar and two pure lines of cucumber formed embryogenic calli at frequencies of up to 8% when cultured on Murashige and Skoog medium (MS) supplemented with 1 mg/L 2,4-D for 3 weeks. Embryogenic calli gave rise to somatic embryos. When slices of somatic embryos were cultured on the same medium for 4 weeks, they formed embryogenic calli. Embryogenic cell suspension cultures were established with embryogenic calli in MS liquid medium with 1 mg/L 2,4-D. Embryogenic potential of cell suspension cultures was maintained by subculturing every seven days. When the level of 2,4-D in the medium was lowered to 0.2 mg/L by diluting with liquid MS basal medium, embryogenic cell suspension cultures underwent development into numerous somatic embryos. When plated onto MS basal medium, over 95% of somatic embryos developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity.

• Restorative Dentistry and Endodontics
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• v.22 no.1
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• pp.413-427
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• 1997

Porphyromonas endodontalis and Prevotella intermedia are black-pigmented anaerobic gram negative rods which have been isolated from infected root canals and submucous abscesses of endodontic origin. And they are associated with clinical symptoms such as pain, percussion, and foul odor. It has been reported that there are 3 serotypes according to capsule membrane in P. endodontalis and 2 DNA homology groups and 3 serotypes in P. intermedia, but there is no data available regarding genetic diversity for the species P. endodontalis and P. intermedia. The purpose of this study is to investigate genetic diversities between individual strains of P. endodontalis and P. intermedia which are indistinguishable by serotyping and biotyping using bacterial DNA restriction endonuclease analysis. 45 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted to the apex of the canal, leave there for 15 seconds, and finally it was placed into PRAS Ringer's solution and PBS solution. P. endodontalis and P. intermedia were identified by biochemical test and IIF after subculturing black and brown colonies which were produced after 7 days of incubation on BAP in anaerobic chamber. P. endodontalis and P. intermedia strains were grown in BHI broth and whole genomic DNA was extracted by phenol-chloroform extraction technique and digested by restriction endonuclease, Eco RI and Pst I. The resulting DNA fragments were separated by agarose gel electrophoresis, stained with EtBr and photographed under UV light. The results were as follows : 1. In both P. endodontalis and P. intermedia, different serotypes could be found within a root canal of same patient. 2. There were obvious genetic heterogeneity within a patient and within a serotype in both P. endodontalis and P. intermedia. 3. P. endodontalis serotype c, isolated from different patients, exhibited limited genotypic diversity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.155-161
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• 2005

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinifera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of $2.73\;\pm\;0.15,\;1.45\;\pm\;0.04,\;0.7\;\pm\;0.024\;and\;0.27\;\pm\;0.04$CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and $84\%$ for V. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be $9.7\%$, ranging from 4 to $17\%$. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.183-187
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• 1998

Chlorophyll mutants were produced by treating the rhizome of Cymbidium kanran with mutagen, EMS(ethyl methan sulfonate). The germination ratio of Cymbidium kanran seeds was 5.5 times higher when the seeds were treated with ultrasonic treatment for 20 minutes than untreated control. Fifty to sixty percent of the rhizomes became dark brown when they were cultured in a liquid growth medium containing 0.2% EMS for three weeks. When the dark-brown rhizomes were cultured in a solified MS medium, new rhizomes were developed from a part of the old ones. Chlorophyll mutant rhizomes were obtained from a meristem tissue by a subculturing the cuts of these new rhizomes for a year. Of the chlorophyll mutants, a zigzag-striped type of rhizome was dominant and light-yellow and albino ones were also produced. While the zigzag-striped type rhizomes were differentiated into green and striped plant, the light yellow and the white rhizomes produced yellow-striped and albino plants repectively.These results indicate that the EMS treatment on the rhizome is an effective means to induce a chlorophyll mutant. We believe that this method may be useful to produce variegated plants chlorophyll mutants from other orchids.