• Journal of Plant Biotechnology
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• 제2권3호
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• pp.123-128
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• 2000

To enhance in vitro plantlet regeneration efficiency of soybean through embryogenesis, the culture conditions such as material part and size of immature seed, 2,4-D, pH and solidifying agents for somatic embryogenesis were investigated. Somatic embryogenesis was induced from the immature embryo, immature cotyledon and embryonic axis explants of the immature seed on MS medium supplemented with 2.0 mg/L 2,4-D. The highest rate (up to 22.9%) of somatic embryogenesis was obtained from the immature cotyledon, following embryonic axis and the immature embryo. The rate varied with the developmental stages of seed. The maximum rate (25.4%) of embryogenesis was obtained from 3-4 mm length of the seed (after 25 days of flowering). The optimum concentration of 2,4-D for embryogenesis was 10 mg/L. The optimum pH was at 5.8 and solidifying agent for medium was better with 0.4% gelrite than with agar. For rapid multiplication of shoot tips from the germinating somatic embryos, they were cultured on MS medium containing 2 mg/L indole-3-butyyic acid (IBA) and 1 mg/L 6-benzyladenine (BA). After then somatic embryos with one and three cotyledons were transferred to the growth regulator free medium. The medium exhibited the higher rate (ca. 50%) of development than the multiplication medium.

• 한국자원식물학회지
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• 제8권2호
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• pp.153-157
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• 1995

초피(椒皮)나무(Zanthoxylum piperitum DC.)의 embryogenic callus로부터 부정배발생(不定胚發生)에 있어 $NH_4NO_3,\;KNO_3$와 casein hydrolysate의 첨가(添加) 효과(效果)를 알아 보기위하여 본(本) 실험(實驗)을 실시(實施)하였던 바 그 결과(結果)는 다음과 같다. 1. 2,4-D 0.1mg/1가 첨가(添加)된 MT배지(培地)에 $NH_4NO_3$를 3300mg/1 첨가(添加)시킨 배지(培地)에서 부정배발생(不定胚發生)이 가장 양호(良好)하였다. 2. $KNO_3$를 3800mg/1 첨가(添加) 시킨 경우(境遇)에는 MT 기본배지(基本培地)와 2,4-D 0.1mg/1를 첨가(添加)시킨 배지(培地)에서 비교적(比較的) 양호(良好)한 부정배(不定胚)를 발생(發生)시키었다. 3. 2,4-D 0.1mg/1가 첨가(添加)된 MT 배지(培地)에 casein hydrolysate를 700mg/1 첨가(添加)시킨 경우(境遇)에 부정배(不定培) 발생(發生)이 양호(良好)하였으며 2,4-D가 첨가(添加)되지 않는 배지(培地)에서는 부정배(不定培) 발생(發生)이 저조(低調)하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제27권2호
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• pp.266-277
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• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

• 식물조직배양학회지
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• 제24권2호
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• pp.107-112
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• 1997

본 연구는 미나리의 배발생 캘러스를 배양하여 정상적이고 균일한 형태의 체세포 배를 대량 생산할 수 있는 조건을 구명하고, 생산된 체세포배를 종묘로 이용하기 위한 기초연구로 실시하였다. 배발생 캘러스를 생장조절제, 당, 질소원등을 농도별로 첨가한 MS배지에 현탁배양한 결과는 아래와 같다. 배발생 캘러스를 생장조절제가 첨가되지 않은 액체배지에 현탁배양하면 배발생 세포괴 및 체세포 배가 왕성하게 증식되었다. 구형단계의 배를 IBA와 NAA를 첨가한 배지에 현탁배양하면 배발생 세포괴의 증식과 함께 배의 발달도 진행됐으나, 2,4-D가 첨가된 배지에서 배양하면 배발생 세포괴 및 세포만 증식하고 배의 발달은 진행되지 많았다. 질소원의 종류에 따른 배발생 세포괴의 생장 발육상태에서 $\textrm{KNO}_3$와 $\textrm{NH}_4\textrm{NO}_3$가 각각 20mM 첨가되었을 때 가장 좋았다. 유아 발달에는 $\textrm{NH}_4\textrm{NO}_3$가, 유근발달에는 $\textrm{KNO}_3$가 효과적이었다. 구형단계의 배를 sucrose가 3-6 % 첨가된 배지에 배양하면 발육이 좋았다. 구형단계의 배를 ABA가 첨가되지 않은 배지에 배양하면 하배축의 이상신장 및 다배현상이 일어나지만 ABA가 $10\mu\textrm{M}$ 첨가된 배지에 배양하면 하배축의 길이가 단축되고 이차배 발생도 현저히 감소하였다. 고체배지에 배발생캘러스를 계속 배양하여 다양의 구형배를 얻을 수 있고 이들을 생장조절제가 첨가되지 않은 배지에서 배양하면 종자 유내 식물체와 유사한 유식물을 얻을 수 있었다.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.1-6
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• 2004

본 연구는 체세포의 종류, 세포 제공 개체, 계대배양 수 및 세포의 trypsin 처리시간이 소 체세포 핵이식란의 체외발육에 미치는 영향을 검토하였다. 세 종류의 체세포(피부, 근육 및 난구세포) 와 암소 3 개체를 실험에 공시하였으며, 한 개체 유래의 피부세포는 5∼30회 계대배양하였고, 핵이식 전에 1∼3분간 trypsin처리하여 핵이식에 사용하였다. 핵이식과정은 상법에 따라 전기융합법을 이용하였다. 핵이식란의 배반포 발육율은 세포의 종류(16.5∼23.9%)나 개체 간(16.4∼19.5%)에 차이가 없으나, 30회 계대 배양한 세포를 사용한 경우(5.8%)에는 5회(25.3%) 또는 15회(23.5%) 계대 배양한 세포를 사용한 경우에 비해 유의적으로 낮았다(P＜0.05). 또한, 1분간 trypsinization 한 세포를 사용한 경우(30.7%)는 3분간 trypsinization 한 경우에 비해 배반포 발육율이 유의적으로 높게 나타났다(P＜0.05). 본 연구의 결과는 소 체세포 핵이식란의 발육이 체세포의 종류 및 세포 제공 개체에 따라서는 영향을 받지 않으나, passage 수 및 체세포의 trypsinization 시간에 따라서는 영향을 받을 수 있음을 시사한다.

• Journal of Plant Biotechnology
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• 제34권3호
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• pp.181-187
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• 2007

Orostachys japonicus A. Berger is a Perennial herbaceous plant which has been traditionally used as an anti-inflammatory agent to treat hepatitis and as an anticancer agent. The objective of this study was 1) to establish and proliferate in vitro plant of O. japonicus 2) to induce indirect somatic embryogenesis from O. japonicus. General calli and embryogenic calli in all ranges of 2,4-D and BA combination, were induced and were best at 22% (embryogenic cell) in 5.0 mg/L 2,4-D and 0.5 mg/L BA combination. Embryogenic cell line was maintained by subculture at 2 week intervals and transferred to solid and liquid medium for embryo formation. In solid medium culture, globular and heart shaped embryos were observed in MS medium containing 5.0 mg/L 2,4-D and 0.5 mg/L BA combination. The number of embryos was 6.5 per 0.5 g cell, and then the immature embryos transferred to MS basal medium for embryo development. In a suspension culture of embryogenic cells, globular and heart shaped embryos were emerged in MS medium supplemented with 3.0 mg/L 2,4-D and 0.3 mg/L BA combination after 10 days of incubation. The embryo formation rate was about 33% by suspension culture. The ratio of embryo germination was 60.9%, on the other side, the root formation rate was 74.3% in 1/2 MS continuously.

• 한국수정란이식학회지
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• 제25권2호
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• pp.117-125
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• 2010

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

• Journal of Plant Biotechnology
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• 제3권1호
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• pp.19-25
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• 2001

Induction of somatic embryogenesis from Brazilian eggplant variety F-100 was studied in response to four auxin types. NAA, at the optimal concentration of 54 $\mu\textrm{m}$, was the only one that resulted in the induction of somatic embryos in either leaf and cotyledon explant and, at murk lower intensity and frequency, in hypocotyl and epicotyl explants. The optimal temperatures for embryo induction were 28 and 35$^{\circ}C$ for cotyledon and leaf explants. Incubation at 22$^{\circ}C$ caused a significant reduction both in the frequency and intensity of induction. This system was used to study the effects of position and orientation of the tissue on the culture medium as well as of antibiotics and explant co-cultivation with Agrobacterium on the efficiency of somatic embryo induction. The intensity of embryo induction was greater in the midsections of cotyledons relative to apical and basal regions, when the abaxial surface was in contact with the culture medium. The presence of antibiotics resulted in approximately 40-60% reduction of embryo induction relative to control explants, which originated 335$\pm$26.6 embryos. Co-cultivation with Agrobacterium before treatment with antibiotics caused a more drastic reduction (80-99%). Ampicilin treatment after cocultivalion with Agrobacterium caused the least inhibitory effect, allowing the production of 60 embryos/explant.

• 식물조직배양학회지
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• 제21권5호
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• pp.303-308
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• 1994

가나 재래종 카사바 식물체의 경단 분열조직을 2 mg/L 2,4-D가 함유된 MS 배지에서 배양하였을 때 32%가 체세포배를 형성하였다. 절단한 체세포배를 1 mg/L 2,4-D가 함유된 배지에 배양하였을 때는 최고 93%가 2차배를 형성하였다. 체세포배는 식물체로 전환하지 못하였으므로 종단으로 자른 체세포배를 0.1-5mg/L BA가 함유된 배지에서 배양하여 부정아를 유도하였다. 8주 경과하였을 때 최고 100%가 부정아를 형성하였으며 이들은 발근된 후에 토양으로 이식하였다

• 한국수정란이식학회지
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• 제30권4호
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• pp.345-350
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• 2015

In the last 10 years, porcine somatic cell nuclear transfer to generate transgenic pig has been performed tremendous development with introduction and knockout of many genes. However, efficiency of porcine somatic cell nuclear transfer is still low and embryo transfer (ET) is one of important step for production efficiency. In porcine ET for production of transgenic cloned pig, we can consider many of points to increase production rates. In respect of seasonality and weather, porcine ET usually is not performed in summer and winter. Cloned transgenic embryos must be transferred into reproductive tracts of recipients where embryos are located after natural fertilization with similar estrous cycle. If cloned embryos with 2~4 cell stage are transferred, they must be transferred into oviducts in periovulatory stage. Number and deposition sites of transferred cloned embryos are important. And we must compare the methods of ET between surgical and non-surgical ones in respect of production efficiency. Sow recipients after natural estrus is most preferred recipients however its cost is must be considered. Here we will review many of current studies about porcine embryo transfer to increase production efficiency of transgenic pigs and strategies for further studies.