• Journal of Life Science
• /
• v.6 no.2
• /
• pp.94-103
• /
• 1996

To understand effect of inoculum size, cell density, sucrose concentration and concentrations of MS basal on suspension culture and protoplast isolation of wild viola(Viola patrinii DC.) callus from petiole segments this experiment was conducted. In the lot of 30 mesh inoculum size, two observations were; One was that a considerable increase in the fresh and dry weight of callus was determined. Another was that the callus mass was relatively compact compared with others. A recommendable cell density was 0.4g for 20ml culture medium and the higher sucrose concentration, the higher fresh and dry weight were obtained. The dilution of MS basal salt was differently affected on fresh and dry weight; the highest fresh weight was found in 1x MS salt, while the higest dry weight was in 1/3x dilution.The addition of casein hydrolysate(3g/L) was more effective to increase of both fresh and dry weight. THe contents of protein was great in the inoculum lots with larger inoculum sizeand higher concentration of MS basal salts contenting 3g/L of casein hydrolysate and higher sucrose compared with others. The greatest protoplasts were isolated from the lot of 10 mesh size treated with 1%pectinase SE-150 and 2% cellulase YC. In general, for optimal protoplast isolation the followingconditions were recommended; 1) Use of smaller cell size cultured for 2-5 weeks, and 2) more than 5 hours incubation using the combined mixture of the enzymes with proper concentrations.

• Applied Biological Chemistry
• /
• v.48 no.4
• /
• pp.425-430
• /
• 2005

[${\beta}-sitosterol$] is a plant sterol that reduces cholesterol levels and inhibits the growth of human prostate and colon cancer cells. Optimal conditions for ${\beta}-sitosterol$ production were examined from cell suspension cultures of Chrysanthemum coronarium L. The callus induction was optimal in MS medium containing 1 mg/l NAA and 1 mg/l BAP. Cell suspension culture was also established from the callus. Optimal ${\beta}-sitosterol$ production was obtained when the cells were cultured at an initial density of 2 mg DCW/l in MS medium containing 1 X sucrose (30 mg/l), 1 X nitrogen (1900 mg/l $KNO_3$, 1650 mg/l $NH_4NO_3$), and 1 X phosphate source (170 mg/l). In cell suspension cultures of C. coronarium L. using shake flasks, the peak content of ${\beta}-sitosterol$ was $150{\mu}g/g$ DCW. In cell suspension cultures of C. coronarium L. using an air-lift bioreactor, the maximum ${\beta}-sitosterol$ content of $143.8{\mu}g/g$ DCW was obtained at an air-flow rate of 100 cc/min.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.2
• /
• pp.121-126
• /
• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Korean Journal of Plant Resources
• /
• v.21 no.1
• /
• pp.60-65
• /
• 2008

Kalopanax pictus was cultured in vitro to find out optimal condition for embryogenic cells proliferation in liquid media rapidly. Embryogenic cells were induced from leaves and petiols of Kalopanax pictus. Optimum culture medium appeared to be a 1/2MS medium supplemented with 2.0mg/L 2,4-D and 0.1mg/L BA. To find out optimal conditions, embryogenic cells were cultured some condition as different concentrations of 2,4-D, medium and sucrose. There was cultured on 1/2MS liquid medium containing different concentration of 2,4-D. When embryogenic cells were cultured on 1/2MS liquid medium supplemented with 1.0mg/L 2,4-D, cell propagation rate was higher than other concentration of 2,4-D. When embryogenic cells were cultured on different media that MS, Gambols B5, N6, White, SH medium, observed the highest multiplication rate among Gambols B5 and White medium. To find out of effect of sucrose to embryogenic cells propagation, we tested cells under different concentrations. Optimal concentration of sucrose appeared to be a basal medium added 3% sucrose. Above results suggest that optimal conditions for proliferation of embryogenic cells were established Gambols B5 and White medium added 1.0mg/L 2,4-D and 3% sucrose. There is every possibility achieving embryogenic cells proliferation via bioreactor culture system in Kalopanax pictus.

• KSBB Journal
• /
• v.30 no.6
• /
• pp.307-312
• /
• 2015

Transgenic rice cells using RAmy3D promoter can provide high productivity, and the production of recombinant protein is induced by sugar starvation. In this system, productivity was reduced during the scale-up processes. To ensure the influences of shear stress and oxygen transfer rate, working volume and mixing performances were investigated under various agitation speeds and working volumes. In addition, inoculation methods including suspended cells and filtered cells were compared. Working volumes and shaking speeds were 300, 450 mL and 80, 120 rpm, respectively. Hydrodynamic environment of each condition was measured numerically like mixing time and $k_La$. Good mixing performance and high shear stress were measured at high agitation speed and low volume. The highest level of hCTLA4Ig was 30.7 mg/L at 120 rpm, 300 mL. When conditioned medium was used for inoculation, increased cell growth was noticed during the day 0~4 and decreased slower than filtered cells. Compared with filtered cells, the maximum hCTLA4Ig level reached 37.8 mg/L at 120 rpm, 300 mL and lower protease activity level was observed. In conclusion mixing performance is critical factor for productivity and conditioned medium can have a positive effect on damaged cells caused by hydrodynamic shear stress.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.8
• /
• pp.1176-1184
• /
• 2013

Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.

• Korean Journal of Veterinary Research
• /
• v.38 no.2
• /
• pp.359-365
• /
• 1998

This study was conducted to isolate the protozoan parasite Babesia gibsoni by intraerythrocytic culture method of micoraerophilous stationary phase(MASP) and evaluate the possibility of application for the detection of B gibsoni in canine babesiosis. Also, indirect fluorescent antibody test(IFAT) and thick blood smear(giemsa stain), direct light microscopy (DLM), as control diagnostic tests, were conducted to compare diagnostic effects between MASP, IFAT and DLM. The results obtained from this study were summarized as follows. The protozoan parasite B gibsoni multiplied in 24-well polystyrene plate containing 1.2ml of canine red blood cell suspension in RPMI 1640 medium(pH 7.0) which is contained 20~40% normal canine serum(NCS) under the MASP condition of 5% $CO_2$ and 95% air at $37^{\circ}C$ incubator. Under the above MASP culturing system the percentage of parasitized erythrocytes(PPE) after incubation for 9 days reached the peak. The levels of PPE in MASP culture were shown more higher by exchanging the medium at 24 hour intervals. The parasite were purely isolated from MASP culture of canine red blood cells collected from dogs(pit bullterrier) infected with B gibsoni naturally. Among the total of 83 heads of pit bullterrier blood samples the positive rate was 32 heads(38.5%) in DLM, 45 heads(54.2%) in IFAT and 42 heads(50.6) in MASP culture. In negative cases of IFAT and DLM the isolation rates of B gibsoni by MASP culture were 16 heads(42.1%) of 38 heads and 16 heads(28.6%)% of 56 heads, respectively. From this study it was suggested that MASP culture method by RPMI 1640 medium was a reliable and useful diagnostic test for the diagnosis of B gibsoni infections in canine babesiosis.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.10
• /
• pp.1360-1366
• /
• 2001

The aims of this study were first to evaluate the effects of different levels (20, 40 and 100%) and sources (follicular size: large, >7 mm; medium, >5-7 mm; small, 3-5 mm) of porcine follicular fluid (pFF) on the in vitro maturation (IVM) of porcine oocytes, and the effects of fertilization treatments and different culture conditions on development of fertilized oocytes were also investigated. No differences in the maturation (63.6-76.6%) and cleavage (24.8-34.3%) rates were observed among the 20,40 and 100% pFF groups (p>0.05). The cleavage rates of oocytes cultured and fertilized in 40% and 100% pFF maturation media were significantly higher than those fertilized in m199-NBCS (51.0-61.2% vs. 12.8-31.8%. p<0.05), regardless of sources of the pFF. When oocytes were fertilized in m199-NBCS followed by culture in rabbit oviducts for 4 days, the cleavage rate in 40% pFF group was better than that in 100% pFF group (46.9% vs. 32.5%, p<0.05). Two oocytes recovered from the oviducts in the 40% pFF group developed to blastocysts after IVC. However, none developed to blastocysts when fertilized in the IVM medium after being transferred to rabbit oviducts. In conclusion, addition of pFF accompanied with gonadotropins (FSH, LH) in IVM medium enhanced maturation and cleavage rates of porcine oocytes. Direct addition of sperm suspension to IVM medium may be an alternative to simplify the fertilization procedures and to reduce the mechanical lesion during manipulation. Furthermore, rabbit oviducts provide a better environment for the in vitro fertilized oocyte developing to the morula and blastocyst stages.

• Korean Journal Plant Pathology
• /
• v.14 no.5
• /
• pp.445-451
• /
• 1998

A severe Phytophthora rot of strawberry caused by a species of Phytophthora has been widely occurred at major cultivation areas of Kimhae on August in 1997. Incidence of the disease was obtained in the range of 69.2~83.6% in surveyed 4 fields and showed an average of 75.2%. A species of Phytophthora was mostly isolated from the crown of infected strawberry plants and all the isolates were identified as P. nicotianae var. nicotianae (=P. parasitica). The fungus showed strong pathogenicity on strawberry by inoculation test. As a result of the leaf inoculation using mycelial disks of the fungus, both leaves and petioles were darkly browned, and were finally blighted. As a result of the root inoculation of zoospore suspension, both roots and crowns were rotten with dark brown. Although the fungus produced sporangia either on V-8 juice agar medium or liquid medium, the sporangia observed on the liquid medium appeared to be broadly turbinate and noncaducous. Moreover the fungus cultured on the liquid medium often produced sporangia having two papilla. The number of zoospores in sporangia was found to be ranged from 3 or 4 to as many as 20 or 25. In addition, the released zoospore from the sporangium became the cystospore during the prolonged culture of the fungus. The sporangia were measured as av. 49$\times$35 ${\mu}{\textrm}{m}$ with l/b ratio of 1.43. All isolates from crowns were heterothallic and A1 mating type since oospores were abundantly formed on clarified V-8 juice agar by dual culture with P. capsici A2 mating type. Aplerotic oospores were sized 24-26 ${\mu}{\textrm}{m}$. Antheridia were always amphigynous and recoreded an average of 12$\times$10 ${\mu}{\textrm}{m}$. Hyphal swlling were easily observed, and terminal or intercalary chlamydospores were abundantly formed on V-8 juice agar as well as in C/Z solution and sized av. 28.2 ${\mu}{\textrm}{m}$. This is the first report of Phytophthora rot of strawberry in Korea.

• Horticultural Science & Technology
• /
• v.16 no.4
• /
• pp.525-527
• /
• 1998

This study was conducted to determine the optimum cultural condition and method for in vitro mass production of Hibiscus syriacus L. 'Honghwarang'. When callus induced in MS solid medium supplemented with 0.01 mg/L TDZ was cultured in liquid medium containing 0.01mg/L TDZ, callus growth and shoot primordia formation was most effective. Formed shoot primordia were regenerated into shoot in MS or 1/2 MS medium of growth regulator-free condition. Effects of mesh size, shaking speed on callus and shoot primordia formation were examined after 5 weeks. Callus and shoot primordia formation was formed most effectively at 10 mesh and 80 rpm shaking speed in liquid medium.