• Journal of Forest and Environmental Science
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• 제35권1호
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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.

• 한국가축번식학회지
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• 제17권2호
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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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• 제24권3호
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• pp.347-353
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• 1997

본 실험은 초자화 동결된 생쥐 배반포기배의 융해 후 배양조건 및 이식방법이 난자의 생존에 미치는 효과를 조사하고자 실시하였다. 체외수정후, M16배양액에서 4일동안 배양하여 얻어진 생쥐 배반포기배는 EFS40 (40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 함유된 PBS)으로 초자화동결하였다. 실험 I에서는 융해 후 배양조건에 따른 난자들의 체외/체내 생존율을 조사하였다. 융해된 난자가 M16과 4 mg/ml 소혈청알부민과 20 가지 아미노산이 함유된 m-CR1 (2% BME 아미노산 용액, 1% MEM 아미노산 용액) 및 단층배양이 유도된 난구세포 (10% FBS가 함유된 m-CR1배양액)에서 각각 배양되었을 때, 융해 후 24시간째 체외 생존율은 배양조건에 따라 차이가 없었다(75.6, 83.1, 82.4%). 그러나 체내 발달율에 있어서 임신 15일째 생존 산자율은 39.0, 49.0, 38.1%로서 유사한 성적을 나타냈으나, 전체 착상율에 있어서는 m-CR1 (80.4%)에 배양되었을 때, M16 (51.2%), 난구세포와 공배양시 (57.1%)보다 유의하게 높은 생존율을 보였다(p<0.05). 실험 II에서는 수정란 이식 방법에 따른 체내 발달율을 조사하였다. 배반포기배를 융해 후 체외배양없이 곧바로 가임신 2, 3일째 대리모에 이식을 실시하였을 때, 가임신 2일째 대리모에서는 임신징후를 얻지 못하였고, 가임신 3일째 대리모에서는 50.0%의 착상율과 15.4%의 정상산자율을 얻었다. 그러나, 이러한 결과는 융해 후, 16시간 배양하여 가임신 3일째 대리모에 이식 (73.5, 57.1%)하는 경우보다 유의하게 낮은 결과였다(p<0.05). 실험 III에서는 초자화 동결된 배반포기배의 융해 후 배양시 발달이 늦어진 수정란의 이용효율을 극대화시키기 위해 융해한 4일째 초기, 5일째 초기, 5일째 팽창 배반포기배의 체외/체내 생존율을 조사하였다. 가장 높은 체외 생존율은 5일째 팽창 배반포기배 (78.3%)에서 얻었으나, 체내 발달율 (산자율, 착상율)에 있어서는 4일째 초기 배반포기배 (33.3, 66.7%)의 경우가, 5일째 팽창 배반포기배(29.0, 38.7%)의 경우보다 높았다(p<0.05). 따라서 본 연구의 결과는 배양조건과 수정란 이식방법에 따라 초자화 동결된 배아의 체외/체내 발달율을 높일 수 있으며, 발달이 늦은 배반포기배의 체내 발달율은 체외 배양시간이 길어질수록 낮아짐으로, 5일째 팽창 배반포기배보다 4일째 초기 배반포기배를 동결하는 것이 더 유용하다는 것을 알 수 있었다.

• 한국수정란이식학회지
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• 제11권3호
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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%).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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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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• 제49권3호
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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.

• 식물조직배양학회지
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• 제22권3호
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• pp.131-135
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• 1995

잡종 사기나무의 기내 대량증식을 목적으로 줄기절간 유래의 기내 및 기외시료를 이용하여 다경줄기 유도에 관여하는 식물생장 조절물질의 효과에 대하여 조사하였다. 기외시료의 측아를 제거한 다음 20에서 30$\mu$M 의 zeatin이 함유된 WPM 배지에 6주간 배양하였을때 각각 11개와 13개의 다경줄기가 유도되었다. 그러나 기외시료의 측아가 붙어있는 상태에서 기내배양하여 2주후에 측아를 제거하고 동일 배지에 배양하였을 경우에는 BA가 1.0에서 2.0 $\mu$M 함유된 배지에서도 13개와 15개의 다경줄기를 유도할 수 있었으며, 특히 이때 생산된 줄기의 상태는 아주 건전한 것으로 나타났다. 기내줄기를 사용하였을때는 20 $\mu$M의 zeatin을 처리하였을때 가장 많은 줄기를 생산할 수 있었으며, 생산된 조직배양묘는 일정기간 순화를 시킨 후 온실이나 포지에 식재가 가능하였다.

• Parasites, Hosts and Diseases
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• 제37권1호
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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.

• 한국동물생명공학회지
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• 제38권2호
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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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• 제35권2호
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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.