• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.23-34
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• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Journal of the korean academy of Pediatric Dentistry
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• v.45 no.4
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• pp.492-498
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• 2018

This study was conducted to investigate the characteristics of subculture times in the early, middle, and late passages by measuring the time under subculture until it was judged that the supernumerary tooth-derived pulp stem cells (sDPSCS) were no longer proliferating. Three supernumerary teeth from two healthy six-years old boys were extracted and stem cells were obtained from the pulp tissue. This was called SNT1 (supernumerary tooth 1), SNT2, and the supernumerary tooth from another child was named SNT3. SNT1 and 2 were subcultured at the same time and SNT3 was subcultured a little faster. The mean time of complete subculture was $3.6{\pm}1.1$ days. Total passages were cultured up to $23.3{\pm}0.6$ and took 83 days. These were divided into three groups based on the passage. The increase rate of time taken in subculture between group I and group II was 11.9%, but the rate between group II and group III was 28.6%, which was 2.4 times increased. The time taken between passages during long-term subculture up to 22 passages shows a regressive pattern y = 0.1169x + 2.25 and y = 0.1169x + 2.0. In conclusion, the passage time of SPSCs increased in late passages, and it shows a similar pattern.

• Journal of Veterinary Clinics
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• v.23 no.4
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• pp.411-415
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• 2006

Ascorbic acid has been used widely as a medium supplement to stimulate cell proliferation, but its effects on cell proliferation have not yet been elucidated, and no reports have analyzed effects on subcultured chondrocytes. Subcultured canine chondrocytes of passage one, two and four were cultured in monolayer and alginate beads with and without ascorbic acid. Cell proliferation was examined by 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) (XTT) colormetric assay. Ascorbic acid stimulated cell proliferation significantly in both culture methods (p<0.05). The increased cell numbers by stimulation with ascorbic acid were significantly high in passage one cells compared to that of other passages. Differences in cell proliferative capacity by subculturing were not determined. These results suggest that ascorbic acid stimulated the proliferation of subcultured canine chondrocytes and enhanced it more in low-passage cells than in the other cells tested.

• Current Research on Agriculture and Life Sciences
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• v.9
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• pp.21-28
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• 1991

The effect of subculture intervals and passages on plant regeneration from seed-derived callus was determined. Regeneration capacity of callus varied with rice cultivars and subculture intervars tested. The callus subcultured every 2 weeks produced more plants than that of 4 weeks. The calli from a Tongil-type rice cultivar, Milyang 23, lost easily their regeneration ability when the calli were subcultured every 2 weeks and 4 weeks. The callus induced from a japonica cultivar, "Yeongdeogbyeo", showed to maintain high frequency(>70%) of plant regeneration when it was subcultured every 2-week intervals. Casein hydrolysate supplemented in callus induction medium enhanced callus growth and its regeneration. High frequency of plant regeneration was obtained from the calli transferred on $N_6$ medium supplemented with kinetin(2mg/1) and NAA(1mg/1). The subcultured calli in the medium supplemented with casein hydrolysate(2 g/1), myo-inositol(200mg/1) and thiamine-HCl(2mg/1) increased the frequency of embryogenic callus formation and plant regeneration.

• The Korean Journal of Mycology
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• v.30 no.1
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• pp.23-30
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• 2002

The genetic variations and the rate of mycelial growth in the dikaryon and the monokaryon stages of Pleurotus spp. were surveyed during their subcultures. The highest growth rate was observed on both the 3rd and the 4th subcultures. The remarkably rapid growth rate was detected in P. ostreatus dikaryon. Genetic similarities in the dikaryon and the monokaryon of P. ostreatus were more than 57.5% and 85.7%, respectively, and those of P. eryngii were more than 85.2% and 84.8%, respectively. The genetic similarities of monokaryotic P. ostreatus were higher than those of dikaryotic. The topology of phylogenetic trees showed that the divergence and the clustering patterns of branch did not correlated with the number of subcultures. This suggests that genetic variations occur very randomly on mycelial cultures. These results suggest that the monokaryotic mycelia is genetically more stable than the dikaryotic in subcultures, and that it is very useful to stock monokaryotic mycelia for making spawns and breeding of Pleurotus spp.

• Korean Journal of Plant Tissue Culture
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• v.26 no.1
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• pp.53-58
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• 1999

The gamma radiation-induced changes of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in callus cultures of cassava (Manihot esculenta Crantz) selected as a high yield of cell line for SOD were investigated. In normal cultures, the cell growth reached a maximum at 30 days after subculture (DAS), followed by a rapid decrease with further cultures. The SOD and POD specific activities (units/mg protein) showed the highest at the immediately after subculture and subsequently decreased to 20 DAS, and then increased to 30 DAS, whereas the CAT activity showed the lowest at just after subculture, and it continuously increased from 15 DAS to 30 DAS, showing a good correlation with the cell growth. Irradiation of gamma-ray of 50 and 70 Gy on 7 DAS inhibited significantly the cell growth by 50% and 80% at 14 days after treatment (DAT), respectively. In the cells irradiated with 70 Gy, SOD and POD specific activities increased by 4 and 2.5 folds at 14 DAT, respectively, whereas CAT activity was not affected. The results indicate that SOD and POD may be involved in the antioxidative mechanism in relation to oxidative stresses induced by subcultures and by gamma radiation in callus cultures of cassava.

• Korean Journal of Environment and Ecology
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• v.14 no.3
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• pp.205-211
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• 2000

본연구는 조경소재로 이용가능성이 크지만 현재 자생지가 파괴되어 복원이 요구되고 있는 야생자란의 대량번식을 위해 무균배양시 배지 내 담함량의 변화와 펩톤, 트립톤 등의 첨가가 종자발아와 계대배양 후 유식물의 생육에 미치는 영향을 알아보고자 실시하였다. 배지 내 펩톤과 트립톤의 첨가는 발아에 영향을 주지는 않았지만, 당의 함량은 그 농도가 10g/L까지 증가함에 따라 발아율을 높였다. 또한 발아 후 유식물의 생육시 당의 첨가는 뿌리의 생육을 두드러지게 향상시켰으며, 생체중도 거의 2~3배정도 많았다. 하이포넥스 배지(대조구)에서는 높은 발아율을 보였지만 유식물의 생육은 트립톤 첨가배지(2g/L)에서 많았는데 엽수, 뿌리수, 엽장 근장, 생체중 등이 모두 다른 처리구의 2~3배에 이르는 초기생육을 보였다. 계대배양 이후의 생육상은 펩톤 첨가배지에서 가장 많은 생육량을 보였는데 특히 엽장과 엽폭 그리고 근장이 다른 처리구보다 월등히 높은 경향을 나타냈다. 생체중도 한 개체당 0.18g으로 가장 높게 나타나 펩톤의 첨가가 계대배양 이후의 생육을 크게 촉지시키는 것으로 나타났다. 결론적으로 하이포넥스 배지에 트립톤 2g/L를 첨가하였을 때 발아율과 유식물의 생육이 다른 배지에 대해 매우 양호한 것으로 나타나 자생 자란의 종자발아용 배지로 가장 적당한 것으로 사료된다. 그리고 이후 계대배양시에는 펩톤의 첨가 (3g/L)가 유식물의 생육을 가장 촉진시키는 것으로 나타났다.

• The Korean Journal of Mycology
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• v.42 no.4
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• pp.322-327
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• 2014

Subculture is the most common method for preservation fungi, but has a disadvantage of accumulation of spontaneous mutations during the repeated subculture. To reduce the subculture frequency, the effect of preservation period at $4^{\circ}C$ in a slant culture was examined on the mycelia growth and lignocellulolytic enzyme activities of various basidiomycetes. Mushrooms, including Stereum ostrea, Coprinellus micaeus, Trametes versicolor, Hypholoma fasciculare, Wolfiporia extensa, Pleurotus pulmonarius, Piptoporus betulinus and Ganoderma applanatum were not affected by the preservation period more than two years, indicating that they can be maintained by subculture every two years. Some other tested fungal strains showed a significant decrease in both viability and enzyme activity when they were maintained for two years, suggesting that they should be subcultured at least once in a year. A little correlation was found between the recovery of mycelial growth and extracellular enzyme activity. In conclusion, mycelial activity and enzyme activity according to storage period is expected to be a way of deciding on subculture times for fungal preservation.

• Korean journal of applied entomology
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• v.18 no.2 s.39
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• pp.73-76
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• 1979

Two different isolates of Xanthomonas oryzae, KB 7785 of pathotype I and JN 7721 of pathotype III, that had been the most virulent isolates in the previous inoculation test, were reisolated from cultivar 'Milyang 23' and serially transferred to 10 times. They were inoculated to the 3 cultivars; 'Milyang 23' in Kinmaze group, 'Yushin' in Kogyoku group and 'Tongil' in Rantai-emas group cultivars. It was observed that the virulence of the isolate JN 7721 was more attenuated by the serial transfer on the Wakimoto's agar than the isolate KB 7785. The attenuation of virulence of the isolate JN 7721 was more significant at the cultivar 'Milyang23' than at the other cultivars. This suggests that the host-pathogen interactions and differences of the pathogenicity-maintenance ability among the pathogenic strains may be involved.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.187-192
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• 2022

The repeated monthly or weekly subculture of plant callus is labor intensive and increases the risk of somaclonal variation from the parental callus line. The most effective method for preserving plant callus is cryopreservation, which involves storage in liquid nitrogen. However, this method cannot be applied to the callus of different plant species in the same manner, so it is difficult to develop a standardized cryopreservation method. In addition, the survival rate of the frozen callus after thawing and the regeneration rate after survival are uncertain. Therefore, it is necessary to develop a method to extend the subculture interval of plant callus in an active state. In this study, active plant calli of various species without freezing was incubated at 15℃ for 4 to 12 weeks without subculture. After 12 weeks, 8 lines of plant callus grew less than 2-fold when cultured at 25℃, but at least 2 times as much when cultured at 15℃. Moreover, total antioxidant activity did not differ significantly between plant callus recovered at 25℃ after culturing at 15℃ or at 25℃. These results show that the subculture interval can be extended at a temperature of 15℃ without need for modified medium composition or additional processes. In addition, positive results in all calli of several plant species are expected to reduce labor as well as somaclonal variation by increasing the subculture.