• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.343-356
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• 2010

Plant micropropagation techniques include bud cultures using apical or axillary buds, organogenesis through callus culture or adventitious bud induction, and somatic embryogenesis. In Korea Forest Research Institute (KFRI), the first tissue culture trial in woody plant was initiated from the bud culture of hybrid poplars (Populus alba x P. glandulosa) in 1978. Since then several mass propagation techniques have developed from conifer and hardwood species, resulting in allowing practical application to Poplars, Birches and some oak species. In addition, useful micropropagation and genetic resources conservation techniques were established in some rare and endangered tree species including Abeliophyllum distichum. Among various in vitro propagation techniques, somatic embryogenesis is known to be the most efficient plant regeneration system. Since the first somatic embryo induction was reported in Tilia amurensis by KFRI in 1986, various protocols for direct or indirect somatic embryogenesis systems have developed in conifer and hardwood species including Larix leptolepis, Pinus rigida x P. taeda F1, Kalopanax septemlobus and Liliodendron tulipifera, etc. However, most of these technologies have been developed using juvenile tissues, i.e. immature zygotic embryos or mature embryos. Therefore it has been difficult to directly application to tree breeding program due to their unproven genetic background. Recently remarkable progresses and new approaches have been achieved in mature tree somatic embryogenesis. In this article we reviewed several micropropagation techniques, which have been mainly developed by KFRI and recent international progresses.

• 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%.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Korean Journal of Medicinal Crop Science
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• v.17 no.4
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• pp.238-242
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• 2009

Somatic embryos on growth regulator-free medium can be produced directly from cotyledon explants of Panax ginseng C. A. Meyer. When the embryo developmental stage was torpedo and cotyledon, the germination rate of embryos was quite high on MS medium supplemented with gibberellic acid ($GA_3$). However, the percentage of plantlet formation at the cotyledon stage was higher than that at the torpedo stage. This result demonstrates that the embryo at the cotyledon stage was the most appropriate for increasing germination by $GA_3$. Embryos cultured on medium including four levels of $GA_3$ concentrations (3, 5, 10, or 20 mg/$\ell$) showed all quite high germination rates (87-91%). When the well-developed embryos were continuously cultured on media including high concentrations of $GA_3$ from 10 to 20 mg/$\ell$, the percentage of formation of normal plantlets was lower than that seen under low concentrations from 3 to 5 mg/$\ell$. This treatment of high concentrations resulted in shoots with abnormal shape. The optimal $GA_3$ treatment provides a basis for the efficient method obtaining healthy plantlets derived from ginseng somatic embryos.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.99-105
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• 2018

To determine the differences in the in-vitro ovum maturation process of bovine, we compared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte's maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

• Journal of Plant Biology
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• v.39 no.2
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• pp.127-135
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• 1996

An efficient system of somatic embryogenesis was established for the red pepper plant (Capsicum annuum L. cv. Nokkwang) usign immature zygotic embryos. The size of the immature zygotic embryos and the concentrations of 2, 4-D and sucrose were found to be critical. Somatic embryos were induced via callus or directly from explants and regenerated into plantlets successfully. When zygotic embryos 1~2 mm long were cultured on the modified Murashige-Skoong (MS) medium supplemented with 2 mg/L 2, 4-D for 3 weeks in the dark, somatic embryos were induced directly from the apical region of zygotic embryos with the highest frequency being approximately 90%. To mature the somatic embryos, ABA and an ethylene inhibitor AgNO3 were used. The highest frequency of shoot regeneration (25% in each) resulted at 2$\mu$M ABA or 20$\mu$M AgNO3 treatment at rates 3.7 and 1.6 times control, respectively. Shoots developed mainly from the cotyledonary node on CoCl2-containing medium, and from the upper side of cotyledon on medium containing AgNO3 while the embryos on the control medium produced shoots from both the cotyledonary node and the upper region of cotyledons both at frequencies of 50%. Indirect somatic embryogenesis via callus was induced at an efficiency of approximately 10% with zygotic embryos 3~4 mm long cultured on MS medium containing 5~10 mg/L, 2, 4-D for 5~7 weeks under a continuous light condition. The plants regenerated from the somatic embryos were morphologically normal. Using scanning electron microscopy, the direct and indirect somatic embryogeneses were observed to follow the globular, heart and torpedo stages, similar to zygotic embryogenesis. Also, suspensors appeared in the early globular and ovoid-shaped late globular embryos during indirect somatic embryogenesis.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.97-106
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• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.

• Applied Microscopy
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• v.26 no.3
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• pp.329-348
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• 1996

The prenatal development of lateral motor columns in the lumbar spinal cord was studied by electron microscopy in human embryos and fetuses ranging from 9 mm to 260 mm crown-rump length ($5{\sim}30$ weeks of gestational age). At 9 mm embryo, the lateral motor column were developed from ventro-lateral projection into the marginal layer and composed of primitive neuroblasts. At 20 mm embryo the primitive motor neurons were packed closely together and could readly be distinguished from primitive glioblasts by a presence of large nuclei. The primitive multipolar neurons were observed in lateral motor column at 40 mm fetus. At 80 mm fetus multipolar neurons were characterized by their many dendrites and axons in the vicinity of motor neuron perikarya. At 260 mm fetus, the motor neurons were large and contained all intracytoplasmic structures in the cytoplasm which were also found in mature motor neuron in lateral motor column. The first axo-dendritic synapses found at 40 mm fetus and increased in number throughout fetal development. Axo-somatic synapses with spherical vesicles were first observed at 80 mm fetus. A few axo-somatic synapses were found at next prenatal stages. Axo-dendritic and axo-somatic synapses contained mixed populations of spherical and flattened vesicles by 120 mm fetus. These findings indicate that axo-dendritic synapses develop prior to axo-somatic synapses in the spinal cord during neurogenesis.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.91-96
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• 2004

The use of liquid-medium-based procedure relative to the solid media led to a 4.5-fold increase in the number of cotyledon-stage embryos. The most efficient system for multiplication and regeneration of somatic embryos was CP6 procedure with the media MSD40/MSD20/MSM6AC/FNL0S3S3GM. However, the rate of regeneration was lower. About 71％ of the embryos with dicotyledon were continued to develop the roots after desiccation treatment and 92％ of the germinated embryos produced shoots in 10 days. Of the four morphologically different types of embryos, dicotyledonous ones showed a high frequency of conversion, while only a few with fused and horn type cotyledon developed shoots. Mature somatic embryos were desiccated in empty petri dishes for 12-72 h. Embryo survival rate was the highest after 12 h of desiccation, but maximal germination was observed at 24 h. After desiccation, they were placed on MS medium without growth regulators for germination. Germinating embryos were transferred to small pots with vermiculite for plant regeneration. The etiolating the plants during the growth was resolved to add 1％ activated charcoal on hormone-free MS medium.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.179-183
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• 1999

This study was performed to determine the significance of a baseline ovarian cyst on the response to controlled ovarian hyperstimulation and the outcome of IVF-ET. One hundred one patients who underwent IVF-ET were enrolled in this study. The outcome of 31 patients, who had an ovarian cyst of >10mm detected at ultrasound examination performed on day 3, was compared with that of 70 patients who underwent a similar protocol and did not have an ovarian cyst. E2 level on the day of hCG administration, the number of follicles, the number of oocytes retrieved, the number of embryo transferred and the pregnancy rate were evaulated. The E2 level on the day of hCG adminstration and the number of mature oocytes retrieved were lower in the group with a baseline cyst. The pregnancy rate also was significantly lower in the group with a cyst (21% versus 38%). Therefore a baseline ovarian cyst on cycle day 3 was associated with a poorer outcome after IVF-ET.