• Journal of Plant Biotechnology
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• v.48 no.4
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• pp.246-254
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• 2021

Environmental stresses are estimated to have reduced global crop yields of wheat by 5.5%. However, traditional approaches for the transfer of resistance to these stresses in wheat plants have yielded limited results. In this regard, genetic transformation has undoubtedly opened up new avenues to overcome crop losses due to various abiotic stresses. Particle bombardment has been successfully employed for obtaining transgenic wheat. However, most of these procedures employ immature embryos, which are not available throughout the year. Therefore, the present investigation utilized mature seeds as the starting material and used the calli raised from three Moroccan durum wheat varieties as the target tissue for genetic transformation by the biolistic approach. The pANIC-5E plasmid containing the SINA gene for drought and salinity tolerance was used for genetic transformation. To enhance the regeneration capacity and transformation efficiency of the tested genotypes, the study compared the effect of copper supplementation in the induction medium (up to 5 μM) with the standard MS medium. The results show that the genotypes displayed different sensitivities to CuSO₄, indicating that the transformation efficiency was highly genotype-dependent. The integration of transgenes in the T₀ transformants was demonstrated by polymerase chain reaction (PCR) analysis of the obtained resistant plantlets with primers specific to the SINA gene. Among the three genotypes studied, 'Isly' showed the highest efficiency of 9.75%, followed by 'Amria' with 1.25% and 'Chaoui' with 1%.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.317-322
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• 1995

As a basic research for breeding new varieties, reciprocal -intergeneric crosses between Citrus junos and P.trifoliata were made. F$_1$ hybrid production using in vitro ovule culture, gametogenesis, and fertilization phenomena were investigated. Frequency of fruit set resulting from crossing of Citrus junos and Poncirus Trifoliata was 16.6% while that of Poncirus Trifoliata and Citrus junos was 11.7%. Callus formation occurred well when ovules at the 6th week after pollination were cultured on MT (Murashige and Tucker) medium supplemented with zeatin 0.5 mg/L and NAA 1.0 or 3.0 mg/L. Immature ovules developed into mature embryos of the MT medium supplemented with 2,4-D 0.1 or 3.0 mg/L. Immature ovules developed into mature embryos of the MT medium supplemented with 2,4 D 0.1 or 0.5 mg/L. The invitro germination rates of 20-week-old ovules set C. junos $\times$ P. Trifoliata and P. Trifoliata $\times$ C. junos were 54.5% and 48.6%, respectively. The emergence ratios of trifoliate hybrids obtained by C. junos $\times$ P. Trifoliata and P. Trifoliata $\times$ C. junos were 56.7% and 100%, respectively. The chromosome number of C. junos and P. Trifoliata was n = 9 or 2n = 18, and the sizes of their pollen grain were 33.75 $\mu$ and 25.0 $\mu$. The length and width of embryo sac in C. junos and P. Trifoliata were 69.38~79.23 $\mu$ and 27.50~38.56 $\mu$, and those of egg cells were 17.50~41.50 $\mu$ and 6.25~8.12$\mu$. Fertilization of C. junos and P. trifoliata terminated 72 h after pollination.

• Development and Reproduction
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• v.4 no.1
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• pp.45-52
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• 2000

Membrane type matrix metalloproteinases(MT-MMPs) have been suggested to play an important role during structural remodeling of various tissue. Expression patterns of MT1-MMP and MT2-MMP mRNAs were investigated in oocytes, embryos, ovary and oviduct of mouse during their differentiation or periovulatory period using RT-PCR technique. Both cDNA products of MT1- and MT2-MMP of immature oocytes were barely discernable with a minimum amount but the expressions were distinct in mature oocytes regardless that they were matured in vivo or in vitro. MT2-MMP was not expressed by 2-cell embryos but was expressed by 4-cell stage embryos. From the morula stage untill hatched blastocyst stage, embyos showed intesnse expression of MT2-MMP with a sudden increase at blastocyst stage. While mouse ovarian tissues showed both expression of MT1- and MT2-MMP, there was no stage-specific difference throughout the estrous cycle. Mouse oviducts also exhibit constant amount of both MT1- and MT2-MMP expressions throughout periovulatory period, i.e., before or after ovulation. These observations lead to suggest that the differential expressions of maternal MT1- and MT2-MMP during meiotic resumption of mouse oocytes and embryonic expression of MT2-MMP particularly at blastocyst stage might play a role in the differentiation of mouse oocytes and／or embryos. The precise function of MT1- and MT2-MMP with regards to their participation in the remodeling of ovarian and oviductal tissues remains in a question.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.142-147
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• 2011

Objective: This study was performed to evaluate testicular sperm chromatin condensation using aniline blue-eosin (AB-E) staining and its effects on IVF-ET. Methods: Chromatin condensation was analyzed using AB-E staining in 27 cases of testicular sperm extraction. There were 19 cases of obstructive azoospermia (OA) and 8 cases of non-obstructive azoospermia (NOA) in IVF-ET. Mature sperm heads were stained red-pink whereas immature sperm heads were stained dark blue. The percentage of sperm chromatin condensation was calculated from the ratio of the number of red-pink sperm to the total number of sperm analyzed. Results: The overall percentages of chromatin condensation in OA and NOA were $31.1{\pm}11.2%$ and $26.3{\pm}14.4%$, respectively. The fertilization rate was significant higher in OA than NOA ($p$ <0.05); however, the rates of good embryos and clinical pregnancy did not show statistical differences. In OA and NOA, statistical differences were not observed in the rate of chromatin condensation, fertilization, good embryos, and clinical pregnancy between the pregnant group and non-pregnant group. Conclusion: Chromatin condensation is less stable than OA and showed a low fertilization rate in NOA. While there were no significant differences in chromatin condensation results between NOA and OA, we propose that a pattern of decreased chromatin condensation in NOA is one of the factors of low fertilization results requiring further study.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.2
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• pp.119-125
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• 2016

Objective: The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods: Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results: A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion: Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.

• Korean Journal of Medicinal Crop Science
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• v.22 no.6
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• pp.423-428
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• 2014

This study was performed to identify optimal harvesting time of ginseng seeds and to examine the effect of $GA_3$ treatment for improvement of seed stratification rate. Ginseng seeds harvested from Land race, Chunpoong and Yunpoong cultivar in July 20 were tested for stratification rate. It was shown that stratification rates of land race, Yunpoong and Chunpoong cultivar were 94.1%, 93.1%, and 82.6%, respectively. Seeds of Chunpoong cultivar harvested 10-15 days later showed a comparable stratification rate to that of Land race, indicating that late harvest of Chunpoong seeds is beneficial for the increase of stratification rate. The higher stratification rate was found in mature seeds (92.3%) than immature seeds (37.8%), both of which were harvested in July 20. Stratification rate of mature seeds harvested in July 15 was 87.5%, demonstrating optimal harvesting time of ginseng seeds with higher stratification rate is after mid-July. An exponential growth of endosperms of ginseng seeds was observed from early June to mid-June and then slow growth was observed. There was no obvious growth of embryos from fertilization to mid-August. After the this time, embryos quickly grew until late October. Thus, appropriate stratification control is essential during the period (from early September to late October) in order to optimize embryo growth and development. While no increase of stratification rate was observed in seeds treated with 50 ppm of $GA_3$, significant increases were observed in seeds treated with 100 ppm of $GA_3$. At this concentration of $GA_3$, the stratification rate of Land race, Chunpoong and Yunpoong cultivar was 95.0%, 95.3%, and 96.5%, respectively.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.85-92
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• 1993

In the previous study, we observed that Purine has a time dependent effect in maintaining the oocytes in meiotic arrest, and human fetal cord serum(HFCS) and human mature follicular fluid(HMFF) reverse the GVBD suppressed by purines. And it was reported that purine has a harmful effect on the development of oocytes or embryos, when they were cultured for a long time, in vitro. Therefore this study was performed to investigate the effects of purine on extrusion rates of 1st pb and viability of oocytes cultured for a long time, in vitro. Immature oocytes(GV stage) were collected from ovaries of 25~28 day old ICR mice at 48 hrs after PMSG injection. Cumulus-enclosed and denuded oocytes collected were assigned randomly to one of several culture conditions. Some of the oocytes were cultured in 4mM hypoxanthine for 24hr, and the extrusion rates of 1st pb and viability of the oocytes were assessed at every 12 hrs. In the viability, the oocytes showed granulation, pigmentation of cytoplasm or lysis of 1st pb extruded were regarded as degenerating oocytes. Also some of the oocytes were cultured in hypoxanthine for 12 hrs then the resulting oocytes were transferred to hypoxanthine-free medium and cultured for 12 hrs to determine whether the inhibitory effect of hypoxanthine on the 1st pb extrusion was reversible. The rest of the oocytes were cultured in medium containing hypoxanthine and adenosine for 18 hrs to compare the 1st pb extrusion be attendant upon hte concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominently. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine from the cultrue medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominetly. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine fromthe culture medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.61-65
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• 2002

This study was carried out to study the viability of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified at 10, 14 and 20 hrs after the onset of maturation, respectively). The oocytes remained vitrified for 24 hrs, and then were thawed in 3$0^{\circ}C$. Survival and cleavage rates were investigated by results of in vitro culture and aceto-orcein staining or FDA test. No difference in the incidence of diploid oocytes was observed among the control, non-vitrified group(3.6%) and oocytes vitrified at 14 hrs(6.7%) or 20 hrs(1.7%). However, more diploid oocytes were detected after vitrification at 0 hr.(26.7%) and 10 hrs(21.7%) post maturation. The survival rate of all vitrified immature oocytes(12.0~38.0%) was low, 48.0% of unvitrified oocytes and oocytes vitrified before maturation or 0~ 10 hrs after the onset of maturation were higher than that of other groups. The overall fertilization and cleavage rates of vitrified immature oocytes (32.3 ~ 64.6% and 4.6 ~ 32.3%) were low, and 55.0% of unvitrified oocytes and the rate of immature oocytes were very higher than that of mature oocytes.

• Korean Journal of Environment and Ecology
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• v.35 no.3
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• pp.285-293
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• 2021

This study was conducted to elucidate spawning period characteristics and early life development of eight barbel loach, Lefua costata (Balitoridae) at the Jusucheon stream, Okgye-myeon, Gangneung-si, Gangwon-do, Korea between January and December in 2018. The spawning period was estimated to be from May to August, considering the change in the gonad-somatic index, the appearance of young fry, and frequency distribution in egg diameter. It was a multi-spawning type. The gender ratio was 1:0.79 with 1,117 females and 879 males collected. The egg size was 0.24-0.93 mm, with mature and immature eggs found during the spawning period. The size of mature eggs was 0.71±0.02 mm, and the average number of fecundity was 1,786±818 (n=31). Observation of the egg development showed that the fertilized eggs were the sticky, gray, segregated, and demersal type with 0.76±0.03 mm in diameter. The hatching of the embryos began at about 34 hours (hatching rate 50%) after fertilization underwater temperature of 25℃. The average length of the newly hatched pre-larvae was 2.7±0.11 mm. The average length of pre-larvae at 4 days after hatching was 4.5±0.16 mm, and the yolk sac was completely absorbed and entered the post-larvae stage. At 20 days after hatching, the average length of post-larvae was 11.5±0.67 mm, and their fin rays were formed before they transitioned to the juvenile stage. At 100 days after hatching, the average length reached 49.8±2.60 mm, and the appearance and the lateral sideband patterns were similar to those of the adult fish.