본 실험은 일정한 발달단계(發達段階)에 있는 생쥐배(胚)를 응집시킬때 세포응집소인 Phytohemagglutinin-P(PHA-P)를 첨가 라화배(裸化胚)의 응집율과 응집된 배(胚)를 in vitro에서 배양하였을때의 배양율 및 적당한 PHA-P의 첨가농도를 조사하여 Chimera배(胚)를 생산하는데 필요한 기초지식을 얻기 위하여 실시(實施)하였다. Albino BALB/C와 CBA계통 및 C57BL 계통의 생쥐에 pregnant mare Serum gonadotropin와 human chorionic gonadotropin를 투여하여 과배란 생쥐에 PMSG와 hCG를 투여하여 과배란을 유기, 회수된 생쥐의 4세포기, 8세포기 및 상실배기 수정란을 1.0% Protease 용액으로 투명대를 제거(除去)하고 PHA-P를 첨가한 배양액에서 미세한 초자봉(硝子棒)으로 계통(系統)이 다른 두 계통(系統)의 생쥐의 배(胚)를 응집시킨 다음 응집된 배를 , 5% , 95% Air의 배양기 조건하에서 13~50 시간 배양하면서 Chimera배(胚)의 발달상태를 조사하였다. 본(本) 실험에서 얻어진 결과를 요약하면 다음과 같다. 1. 1.0% Protease 또는 1.0% Protease 및 PHA-P가 첨가된 산성 Tyrode액에서 투명대를 제거한 라화배(裸化胚)를 배반포까지 배양했을때 유의차는 없었으나 4세포기배와 8세포기배 보다 상실기배가 더 잘 발달되었으며 또한 PHA-P를 첨가하였을때가 첨가하지 아니한 때 보다 다소 좋은 경향을 보였다. 2. PHA-P 첨가시 4세포기, 8세포기 및 상실기배의 응집율은40.0~82.0%, 첨가시에는 52.0~94.0%, 첨가시에는 48.0~96.0%로 배(胚)의 발달단계(發達段階)에 따라서는 4세포기, 8세포기가 상실배기 보다 유의적으로 높았다 (P<.05). PHA-P의 처리수준에 의한 응집율에 있어서는 5 또는 첨가구가 첨가구 보다 조금 높게 나타났으나 유의차는 없었다. 3. 응집배의 상실배까지의 배양율은 PHA-P의 각(各) 수준간 및 각(各) 세포기간에 유의차가 인정되지 않았다. 응집배의 배반포까지의 배양율은 PHA-P 수준사이에는 유의차가 없었으나 4세포기와 8세포기의 배(胚)는 상실기 배(胚)보다 유의적으로 높은 배양율을 보였다 (P<.05). 4. 응집된 배(胚)가 배반포까지 발달하는데 소요되는 평균시간은 4세포기배가 38.5~40시간, 8세포기배가 26~27시간, 상실기배가 19~20시간이었다. 5. 응집율은 34.0~94.0%의 범위로서 PHA-P를 첨가할때 응집율이 더 좋은 경향을 보였으나 유의성은 인정되지 않았다. 4세포기와 8세포기의 배(胚)가 상실기배 보다 유의적으로 높았다(P<.05). 6. 응집배의 상실배까지의 발달율은 4세포기배가 52.7~84.7%, 8세포기배가 73.8~872% 였으며 세포기 사이에는 유의차는 나타나지 않았다. 그러나 PHA-P를 첨가한 것이 발달이 더 좋았다. 7. 응집배의 배반포까지의 발달율은 4세포기의 배(胚)가 41.7~77.7%, 8세포기의 배(胚)는 78.7~83.0% 상실기배가 0~19.2%였으며 4세포기의 PHA-P 처리가 미처리(未處理) 보다 유의적으로 높았다(P<.05). PHA-P를 처리했을 때 4세포기와 8세포기가 상실기의 배(胚) 보다 더 높은 발달율을 보였다.

본 연구는 우리나라에 자생하고 있는 비비추속 식물 6종(일월비비추, 주걱비비추, 다도해비비추, 좀비비추, 한라비비추, 흑산도비비추)의 정단을 이용하여 대량증식과 품종개발 등을 위한 기내증식체계를 확립하고자 하였다. 정단은 0.5, 1.0, 2.0, 4.0 ㎎/L BA와 0.1, 0.5, 1.0, 2.0 ㎎/L TDZ를 0.1 ㎎/L NAA와 각각 혼용한 조건과 PGRs을 무첨가한 조건(control)의 MS배지에 배양하였다. 배양 8주 후에 embryogenic callus, somatic embryo, crown bud, 그리고 신초와 뿌리의 분화 및 생육, 생체중 등에 대하여 조사하였다. 6종의 비비추 식물 모두에서 control에 비하여 PGRs 처리구의 embryogenic callus와 somatic embryo 형성율, 다신초 분화율이 높았다. 분화된 신초의 개수는 일월비비추는 2.0 ㎎/L TDZ에서 5.4개, 주걱비비추와 다도해비비추는 1.0 ㎎/L TDZ에서 각각 3.3개, 5.8개, 좀비비추는 0.5㎎/L BA에서 11.1개, 흑산도비비추는 0.5 ㎎/L TDZ에서 9.8개, 한라비비추는 1.0 ㎎/L BA, 0.1 ㎎/L TDZ에서 8.1개로 가장 많았다. Somatic embryo 형성에서는 다도해비비추와 흑산도비비추가 처리한 PGRs에 대해 효과적이었고, 일월비비추, 주걱비비추, 좀비비추, 한라비비추에서는 상대적으로 효과가 적었다. 4종의 자생 비비추(일월비비추, 주걱비비추, 다도해비비추, 흑산도비비추)에서 조사된 crown bud도 control에 비하여 PGRs 처리구에서 더 많이 형성되었다. 주걱비비추는 cytokinin의 종류 및 농도와 상관없이 callus와 신초 분화에 큰 효과가 나타나지 않았지만, crown bud의 형성에는 TDZ에서 약간 증가하였다.

Background : Ligusticum chuanxiong Hort is a perennial herb of the Umbelliferae family and an important traditional oriental medicinal plant. The compounds contained in L. chuanxiong can be divided into five kinds, essential oil, alkaloids, phenolic acids, phthalide lactones, and other constituents. These compounds have cardiovascular and cerebrovascular effects, antioxidants, neuroprotection, anti-fibrinolytic, antidotes, anti-inflammatory and antitumor activities. In this study, we anticipated to establish the in vitro propagation system of L. chuanxiong, which is a high economic value as medicinal herb, by plant tissue culture to solve the problem of root stocks contamination. Methods and Results : The whole study was carried out in the department of Herbal crop research, Eumseong, RDA. In this study, L. chuanxiong nodes was used as an explant and it was surface sterilized by 2% sodium hypochlorite for 1 minutes, then washed with ddH2O several times. Further the surface sterilized nodes were placed on MS basal media. Multiple shoots were induced on MS, SH, WPM media with 0.1 - 2 ㎎/ℓ auxin (NAA, IBA) and cytokine (BA). In this study we obtained 4.6 multi-shoots per an explant, and growth of the shoot was also favorable in the presence of 1.0 ㎎/ℓ BA. Subsequent transfer of these regenerated shoots on 1/2 MS media resulted in root formation. The rooted plantlets were able to grow in soil after 3 weeks of acclimatization. Conclusion : The optimal conditions for in vitro propagation of L. chuanxiong were established through this experiment.

This study was performed to develop the mass propagation system using tissue culture technique to supply the seeds of Elephant garlic (Allium ampeloprasum L.) which has difficulty in propagation. Immature spathe of Elephant garlic was cultured on Murashige & Skoog (MS) medium supplemented with two plant growth regulators, naphthaleneacetic acid (NAA) and kinetin. After 6 weeks of culture, the highest number of shoot (14.9/explant) was obtained when the immature spathe with 10 ㎝ length was cultured right after harvesting. In MS medium supplemented with 2 ㎎/L kinetin and 0.5 ㎎/L NAA, the most vigorous growth characteristics was observed, the shoot number was 14.9/explant, its length was 11.3 ㎝, and its fresh weight was 2.5 g. When the bulblets were cultured in MS medium with 2 ㎎/L kinetin and 0.5 ㎎/L NAA, the addition of 30 ㎎/L adenine improved their proliferation and growth significantly, the highest bulblet formation rate (48%) was obtained. The addition of 7% sucrose also increased the bulblet formation rate at the highest frequency of 98.2%. The shoots were shown be more vigorously proliferated at the secondary subculture stage rather than primary culture stage, their propagation rate was 80% after subculture.

Background : Sea buckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values has wide spread distribution from Eurasia to South east Asia. In recent times the medicinal benefits of vitamin tree are inclining, hence, efforts were taken to propagate them in vitro to exploit their medicinal property. Methods and Results : The tissue culture potential of them was investigated for the ability to induce shoot organogenesis in leaf explant, and induction of direct somatic embryogenesis from leaf tissue. Moreover, we also determined the effective induction medium for callus and somatic embryo production from H. rhamnoides. To induce the callus form leaf tissue, several phytohormone combinations such as α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA), 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA), and Kinetin (K) were tried with the Murashige and Skoog (MS) as well as woody plant medium (WPM). In MS basal medium, the combination of 2,4-D and K showed the best callus induction rate of 71%, whereas in WPM basal medium the combination of NAA and BA showed the best callus induction rate of 91%. The adventitious root induction form callus was also attempted by using MS and B5 medium with the phytohormone combinations of IBA 1 – 5 g/ℓ. In MS medium, root was induced only at 4 g/ℓ of IBA and 64%, 51% and 55% root induction results were obtained at 3 g/ℓ, 4 g/ℓ and 5 g/ℓ in B5 basal medium, respectively. The somatic embryos were induced only in half strength MS with the triple phytohormone ratio of 2:1:2 of NAA, BA, and K. Conclusion : The in vitro propagation of sea buckthorn was successfully employed by generating callus, adventitious roots as well as the induction of somatic embryos form the leaf tissues derived callus. Our results provided a valuable addition to the utilization of H. rhamnoides thus enabling their propagation.

Background : Cnidium officinale Makino is a perennial herb of the family Umbelliferae, and an important traditional oriental medicinal plant in China, Japan and Korea. Cnidii Rhizoma, the dried rhizome of C. officinale have been used as traditional oriental medicine in Korea. It has been shown that the cnidii rhizomes are used in the treatment of pain, inflammation, menstrual disturbance, and anti-vitamin deficiency disease, and also act as a blood pressure depressant. In this study, we anticipated to establish the mass propagation system of C. officinale, which is a high economic value as medicinal herb, by plant tissue culture to solve the problem of root stocks contamination. Methods and Results : The whole study was carried out in the department of Herbal crop research, Eumseong, RDA. In this study, C. officinale root bud was used as a explant and it was surface sterilized by 1% sodium hypochlorite for 3 minutes, then several times washed with ddH2O. Multiple shoots were induction them on MS, B5, SH media with 0.1 - 2 ㎎/ℓ auxin (NAA, IBA) and cytokine (BA, Zeatin). In this study we obtained, 7.4 multi-shoots per an explant, and the shoot growth was also favorable in the presence of 0.2 ㎎/ℓ Zeatin. Subsequent transfer of these regenerated shoots on 1/2 SH media resulted in root formation. Rooted plantlets were able to grow in soil after a short period of acclimatization. Conclusion : This experiment was comducted to identify the optimal in vitro propagation condition of C. officinale.

Background : Platycodon grandiflorum has been used as food material and a traditional medicine in Korea. In order to develop an efficient in vitro micropropagation technique for a rare plant species and conservation for inbred line of plant breeding. Methods and Results : Plant regeneration via organogenesis and somatic embryogenesis was investigated in Platycodon grandiflorum. Leaf, stem, root tissues of 7-day-old seedlings were cultured on 1/2MS medium containing various concentration (0, 0.5, 1 and 2 ㎎/L) of IBA, BA and NAA. The results showed that 1/2MS medium supplemented with BA+NAA 2.0 ㎎/L yielded the highest callus formation ratio of 73.5%. When various tissues (leaf, stem, root) were tested on 1/2MS medium containing BA 2.0 ㎎/L+ NAA 2.0 ㎎/L for somatic embryogenesis, the optimum tissue for embryogenic shoot induction was stem tissue. Callus were cultured on MS medium containing various concentration (0, 0.5 and 1 ㎎/L) of BA and NAA. The best rooting rate was achieved by three different medium (B5, 1/2MS and MS) and 1/2MS medium cultured the highest rooting ratio (82%). Conclusion : This study suggested that above micropropagation techniques can be used for rapid multiplication as well as in vitro or in vivo conservation of the Platycodon species.

Plant regeneration via organogenesis and somatic embryogenesis was investigated in Korean soybean cultivars including Cheongja 3, Jinpumkong 2, Taekwangkong and Uram. Cotyledon, cotyledon+hypocotyl and hypocotyl segments of 7-day-old seedlings were cultured on MS medium containing various concentration (0, 1, 2 and 4 ㎎/L) of BA and TDZ. The results showed that MS medium supplemented with BA 2.0 ㎎/L yielded the highest shoot formation ratio of 83.3%. In 4 cultivars, Taekwangkong showed the highest ratio of shoot formation. When various sizes of immature cotyledons (S: 1∼ 2 ㎜, M: 3∼5 ㎜, L: 6∼8 ㎜) were tested on MS medium containing 2,4-D 40 ㎎/L for somatic embryogenesis, the optimum size for embryogenic callus induction was 3∼5 ㎜ in length of immature cotyledons. In 4 cultivars, Taekwangkong showed the highest percentage of embryogenic callus induction. The results indicate that Taekwangkong is the best soybean cultivar for plant regeneration via organogenesis and embryogenic callus induction among the 4 cultivars.

Background : A series of studies were conducted to optimize adventitious root induction in vitro from explants of Astragalus membranaceus using various nutrient media supplemented with plant hormones. Methods and Results : Levels of active components were analyzed from adventitious roots induced under different media conditions. Among the different media conditions, Murashige and Skoog medium supplemented with 1.0㎎• ℓ −1 indole-3-butyric acid resulted in the greatest adventitious root induction rate. The amount of the major active component of the adventitious roots of Ama1, calycosin-7-O-β-D-glucopyranoside was higher than that of other adventitious root samples. Conclusions : These results suggest that the adventitious roots of A. membranaceus could be used for the commercial production of medicines.

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/–) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/– (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/– (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture. Key words : Mouse embryonic stem cell, Feeder cell, Pluripotency marker, MEF feeder cell

Spermatogonial stem cells (SSCs) possess the unique capacity of self-renewal and differentiation and thereby can transmit genetic information to the next generation. Combination with techniques such as isolation, culture, preservation, and transplantation of the SSC has facilitated the efficient method for production of transgenic animals, and preservation of livestock and endangered species. The purpose of this study was to genetically modify enriched populations of pre-pubertal germ cells using lentiviral transduction and to develop an efficient in vitro culture system and cryopreservation technique for bovine SSCs. To maximize the efficiency of genetic modification of bovine SSCs, effective enrichment techniques need to be developed. Selection of bovine SSCs using a combination of laminin and gelatin was resulted in a 8-fold enrichment. Selected cells were then transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Next, to enhance the efficiency of proliferation for in vitro culture, the effects of various culture conditions and growth factors on bovine cell proliferation were evaluated. Based on the results, we developed the optimal culture conditions [2× rat sertum free medium (rSFM) containing 0.1% FBS together with GDNF, GFRα1, bFGF, EGF, LIF, and CSF-1] for maintaining bovine SSCs over 3 months without any alteration of stem cell characteristics and functions. Also, to develop an effective cryopreservation technique for bovine SSCs, the effects of different freezing methods and various cryoprotective agents were tested. The recovery rate, and proliferation capacity of bovine SSCs were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, and proliferation capacity of germ cells compared to control. As a results, cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Collectively, these findings can serve as a model for comprehensively understanding the biology of SSCs and the factors that regulate male fertility. Furthermore, results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage.