• Journal of Forest and Environmental Science
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• v.35 no.1
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• pp.54-60
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• 2019

Plant tissue culture techniques offer quick methods of regeneration of plants of medicinal importance but the survival chances of such plants are always questionable when shifted to the in vivo conditions. The present study enumerates the micromorphological developments in the leaves of in vitro regenerated and field transferred plantlets of Oldenlandia umbellata. The leaves developed in vitro after $4^{th}$ subcultures of multiplication phase and after 6 weeks of field transferred plants were used. Statistically significant differences in the number of stomata, veins, raphides, crystals and trichome density per square mm were observed. The improvements in stomatal apparatus and density (decreased from 41.85 to 32.20), developments in leaf architectural parameters and emergence of defense mechanism through increased numbers of raphides (8 to 15), crystals and trichomes (13.5 to 18.2) proved acclimation of tissue culture raised plantlets from in vitro to the in vivo environments lead to 100 % success in field establishment of the plantlets. The in vitro induced foliar abnormalities (changes in stomata, venation pattern, vein density, trichomes, crystals etc.) were repaired while hardening of plantlets in the greenhouse and finally in the field. The observed micromorphological response of leaves under altered environmental conditions could help in determination of proper stage of field transfer and prediction of survival percentage of in vitro regenerated O. umbellata plantlets.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.121-125
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• 1993

Early development of bovine oocytes fertilized in vitro in the medium with caffeine and heparin was examined in different culture systems. When the oocytes were transferred into culture medium 8 h after insemination, 12%(7/60) of penetrated oocytes cleaved to 4-cell stage 24 h after insemination. The proportions of oocytes cleaved to 80to 16-cell stage 48 h after insemination had also a to be higher in oocytes transferred into culture medium 8 h (29%) than 16 h(10%) or 24 h(4%) after insemination. 52% of the 4-cell embryos developed to morula and blastocyst stages when they were co-cultured with oviductal epithelia, whereas only 5% of embryos cultured without the epithelial cells(P<0.001). In another experiment, embryos were co-cultured with ampulla, isthmus or utero-tubal junction of oviducts. There are no significant differences in the proportions of embryos developed to morula and blastocyst stage.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.347-353
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• 1997

This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.241-248
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• 1996

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17$\beta$ and heat inactivated fetal calf serum at 39$^{\circ}C$ under 5% $CO_2$ in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.230-239
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• 2022

Holostemma annulare (Family Asclepiadaceae) is an invaluable vulnerable medicinal plant; the root tubers are used in Ayurveda medicine and by folk healers to treat various ailments. In this study, Schenk and Hildebrandt medium fortified with the cytokinins 6-benzyl adenine, kinetin, and auxins, including indole 3-butyric acid, indole 3-acetic acid, α-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, were checked for their efficiency on root tuber induction from different explants. Adventitious root tubers were more successfully induced from in vitro leaf segments and shoots when cultured in Schenk and Hildebrandt medium supplemented with 0.5 mg/l of α-naphthaleneacetic acid. In addition, preliminary phytochemical analysis of in vitro root tubers and identification of different secondary metabolites were conducted. Thin layer chromatography and high performance thin layer chromatography analysis of the crude methanolic extracts of the in vitro root tuber identified the presence of lupeol, a bioactive triterpene. Adventitious root tuber induction offers a novel method for the in vitro production of bioactive metabolites that can be scaled up by bioreactors, thus ensuring the conservation and sustainable utilization of H. annulare. The study warrants further scale-up production and pharmacological investigation that can be extended for pharmaceutical needs.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.131-135
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• 1995

Physiologically modified stem nodes derived from ex vitro and in vivo explants of hybrid aspen (Populus alba L.X P.grandidentata Michx. 'Crandon') were tested for their multiple shoot regeneration capacity using a broad spectrum dosage of cytokinins. Ex vitro derived stem nodes with excised axillary buds at the time of culture produced 11 to 13 multiple shoots on 20 to 30 $\mu$M zeatin containing Woody Plant Medium (WPM) after 6 weeks. Excision of axillary bud sprouts after 2 weeks of culture and culture of the remaining stem nodes on WPM with 1.0 to 2.0 $\mu$M BA or 10 to 30 $\mu$M zeatin produced 13 to 15 and 7 to 8 shoots per explant, respectively, Multiple tiny shoots were produced when in vivo derived stem nodes (on which all leaves were removed) were cultured on WPM with 30 to 50 $\mu$M 2iP or 20 to 50 $\mu$M zeatin. The greatest number of multiple tiny shoot proliferation (32 to 50 shoots per explant) were obtained when the explants were cultured on media containing 20 $\mu$M zeatin. Successful transplanting of these multiple shoots into the greenhouse and/or nursery was achieved.

• Parasites, Hosts and Diseases
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• v.37 no.1
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• pp.13-19
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• 1999

The effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae ($L_3$) to rats were investigated. A significantly higher body length was observed in $L_3$ from filter paper culture ($597.3{\;}{\pm}{\;}32.2{\;}{\mu\textrm{m}}$) than those in fecal (($509.9{\;}{\pm}{\;}35.0{\;}{\mu\textrm{m}}$) and nutrient broth culture (503.3{\;}{\pm}{\;}31.0{\;}{\mu\textrm{m}}) (P<0.05). Larval infectivity was assessed by exposing rats to 1,000 $L_3$ from each culture and worms were recovered from the lungs and small intestines. Recovery rate of these worms did not show any significant difference. A significantly greater body length of adults was recorded in those corresponding to the $L_3$ harvested from filter paper (2,777.5{\;}{\pm}{\;}204.4{\;}{\mu\textrm{m}}$) and nutrient broth culture (($2.732.5{\;}{\pm}{\;}169.8{\;}{\mu\textrm{m}}$) than those corresponding to the $L_3$ obtained from fecal culture (($2.600.5{\;}{\pm}{\;}172.4{\;}{\mu\textrm{m}}$) (P<0.05). Although worm fecundity and EPG counts differed among culture methods but worm burdens and course of infection did not. These findings suggest that the methods of cultures have a significant effect on the morphological development of the larvae to the $L_3$ stage, but do not influence the infectivity to rats.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.70-76
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• 2023

Background: Despite considerable technological advancements, polyspermy remains a significant challenge in in vitro fertilization (IVF) procedures in pigs, disrupting normal embryonic development. Here, we aimed to determine whether optimal fertilization conditions reduce the polyspermy incidence in pigs. Methods: In vitro-matured oocytes were co-incubated with sperm according to a modified two-step culture system. Results: In the first experiment, oocytes were briefly co-incubated with sperm, washed in IVF medium, and then moved to fresh IVF medium for 5 or 6 h. Although the 6 h sperm-free cultured group had a higher penetration rate than the 5 h cultured group, the polyspermy rate significantly increased in the 6 h sperm-free cultured group. The gamete co-incubation period was either 20 or 40 min. The 40 min cultured group had a higher rate of blastocyst formation and number of total cells in blastocysts than the 20 min cultured group. In experiment 2, oocytes were inseminated with sperm separated by Pecroll treatment. Percoll treatment increased the rate of oocyte penetration and blastocyst formation compared to the control. In experiment 3, fertilized oocytes were cultured in 25 µL microdroplets (10 gametes/drop) or 500 µL (100 gametes/well) of culture medium in 4-well plates. The large volume of medium significantly reduced the number of dead oocytes and increased the rate of blastocyst formation compared to the small volume. Conclusions: Collectively, these results demonstrate that various fertilization conditions, including modified co-culture period, active sperm separation, and culture medium volume, enhance fertilization efficiency and subsequent embryonic development by decreasing polyspermy occurrence.

• The Korean Journal of Pain
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• v.35 no.2
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• pp.160-172
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• 2022

Background: The authors established an in vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons. Methods: SCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination. Results: The cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media. Conclusions: Insulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy.