Protoplasts were isolated from the primary leaves of lettuce (Lactuca sativa L.) seedlings 10 days after in vitro germination. The leaves were stripped and incubated in an enzyme mixture consisting of 1.2% Cellulase R-10 and 0.3% Macerozyme R-10 in cell and protoplast washing solution (CPW) overnight. The average protoplast yield was 8.25 x 106 protoplasts per g of fresh leaf tissue. When protoplasts were cultured at a density of 3.0 × 105 protoplasts/mL in agarose solid KM8P/KM8 medium, first and second divisions were observed in the protoplasts within a week. Protoplast-derived microcolonies formed after 4 weeks of culture, and visible colonies were present after 3 months of culture. Protoplast-derived microcalli were transferred to Murashige and Skoog medium supplemented with 2.0 mg/L kinetin and 0.1 mg/L NAA and incubated in the light for 3 weeks. They grew into callus, which then regenerated into plants after 7 weeks of culture. The regenerated plants grew as apparently normal flowering fertile plants.

This study aimed to develop an environmentally friendly horticultural substrate that promotes the growth of organic onion(Allium cepa L.) seedlings. Four substrates were prepared by mixing different ratios of peatmoss, cocopeat, perlite, vermiculite, and zeolite. Their pH and electrical conductivities ranged from 5.12 to 5.60 and from 0.07 to 0.08 dS/m, respectively. Beneficial microorganisms, molasses, sesame oil cake, and sulfur were added to one substrate combination, which was named “environmentally friendly horticultural substrate” (EFHS). The chemical properties of the EFHS were analyzed and compared with a commercial organic horticultural substrate (OHS) and a commercial general horticultural substrate (GHS). The organic matter and inorganic ion (nitrogen, potassium, calcium, zinc, and sulfur) contents in the EFHS were higher than those in the OHS and GHS. The germination rates of onion seeds in the EFHS were higher than in the OHS and GHS. The mean number of leaves, sheath diameter per seedling, and weight of 30-day-old seedlings grown on the EFHS were greater than those of seedlings grown on the OHS and GHS. The length of the seedlings grown on the EFHS was comparable to that of the seedlings grown on the OHS and greater than that of the seedlings grown on the GHS. Overall, the growth parameters of onion seedlings grown on the EFHS were better than those of seedlings grown on the OHS and GHS, suggesting that the EFHS may be used as an organic horticultural substrate for growing organic onion seedlings.

본 연구에서는 고구마(Ipomoea batatas L.) 수확 후 버려지는 잔재물의 활용가치를 위해 이들로부터 추출한 발효 추출물과 열수 추출물의 생리활성과 세포독성을 분석 하였다. 추출물의 pH는 모두 산성을 나타내었고, 유기물 함량은 발효 추출물과 열수 추출물에서 각각 0.98%와 0.97%로 비슷하게 나타내었다. 다량원소 중 인산, 칼륨, 칼슘, 마그네슘 성분의 함량은 열수 추출물에서 발효 추출물보다 모두 높게 나왔고, 질소 함량만 두 추출물에서 동일하게 나왔다. 미량원소 함량은 아연을 제외하고 발효 추출물보다 열수 추출물에서 높게 나타내었다. 총 폴리페놀 함량은 열수 추출물에서 60.5±2.7 mg/g로서 발효 추출물의 총 폴리페놀 함량의 22.7±4.2 mg/g 보다 37.8 mg/g 높은 함량을 나타내었다(p<0.05). 총 플라보노이드 함량은 열수 추출물에서 50.7±2.7 mg/g로서 발효 추출물의 함량인 14.0±2.1 mg/g 보다 36.7 mg/g 높은 함량을 나타내어 총 폴리페놀 함량과 마찬가지로 열수 추출물이 발효 추출물보다 높은 함량을 나타내었다(p<0.05). DPPH와 ABTS radical 소거능력은 모두 열수 추출물에서 발효 추출물 보다 높은 항산화력을 보였다(p<0.05). MTT assay를 이용한 추출물의 세포독성 실험에서는 두 추출물의 모든 농도에서 세포독성이 미약한 것으로 확인되었다. 따라서 고구마 수확 후 잔재물의 추출물이 향후 각종 바이오 소재로 이용 시에도 큰 문제가 없을 것이라 판단된다.

본 연구는 사람의 다양한 세포주를 이용하여 활성산소종(과산화수소수)이 세포의 노화에 미치는 영향을 비교 조사하였다. 여러 농도의 과산화수소수에 세포주를 일주일 동안 배양하여 MTT 방법으로 과산화수소수에 대한 세포 성장의 반억제농도를 구하였다. 그 결과, 50대에서 유래하는 피부 섬유아세포와 10대의 노화 유도 피부 섬유아세포와 비교하여 10대에서 유래하는 피부 섬유아세포에서 과산화수소수에 대한 반억제농도의 값이 유의적으로 더 높았고, 10대의 피부 섬유아세포보다는 10대의 여러 조직 기원하는 성체줄기세포에서 반억제농도의 값이 유의적으로 더 높게 관찰되었다. 또한, 50 ppm 과산화수소수를 1주일 동안 처리한 후, 50대의 피부 섬유아세포에서 다른 세포주에 비해 세포 성장이 현저히 억제되었고, 노화 관련 베타-갈락토시다아제의 활성이 증가되는 것을 관찰하였다. 또한, 활성산소의 세포 독성을 중화시키는 두 유전자, 글루타티온 과산화효소(GPX)와 카탈라아제(CAT)의 발현을 각 세포주에서 조사하였을 때, CAT의 발현은 모든 세포주에서 대체로 낮았지만, GPX 유전자의 발현이 50 대의 피부 섬유아세포보다 10대의 피부 섬유아세포와 성체줄기세포에서 현저히 높게 발현되는 것을 관찰하였다. 이상의 결과에서 활성산소는 세포 노화를 유도하고, GPX의 발현이 높은 10대의 피부 섬유아세포와 줄기세포보다는 50대의 피부 섬유아세포와 노화된 피부 섬유아세포에서 활성산소종에 대해 더 큰 민감성을 가지고 있는 것을 알 수 있었다.

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p< 0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

Although the function and utility of RNA transcripts derived from matured spermatozoa remains unclear, they might play important roles in the establishment of a paternal genome and subsequently embryo development. Herein, we investigated the expression of X-chromosome linked RNA transcripts in matured bovine spermatozoa. The total RNA was extracted from the matured spermatozoa, and then converted to cDNA. Autosomal genes (ACT-β and H-2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) were analyzed for the characterization of X-chromosome linked RNA transcripts and compared to female fibroblasts by RT-PCR. The transcripts of autosomal genes (ACT-β and H2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X and ZFX) were not detected in spermatozoa. However, XIAP (X-linked inhibitor of apoptosis protein) and XIST (X-chromosome inactive-specific transcript, a kind of paternal imprinted gene) transcripts were detected in spermatozoa, and relative levels of XIAP and XIST transcripts were similar and 0.5-fold lower when compared to female fibroblasts, respectively. Based on the findings, it is summarized that the presence of RNA transcripts of XIAP and XIST in the isolated spermatozoa may imply their role in inhibition of apoptosis and induction of X-chromosome inactivation in embryo development.

The present study compared the developmental potential, telomerase activity and transcript levels of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos derived from different age and cell cycle of female donor nucleus. In experiment 1, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was slightly increased in embryos cloned with fetal fibroblasts compared to those with adult fibroblasts, but there was no significantly (p<0.05) differences. Telomerase activity was also similar in blastocysts cloned with fetal and adult fibroblasts. Up-regulated RPS4X and down-regulated MeCP2, XIAP, and XIST transcript level were observed in blastocysts cloned with adult fibroblasts, compared to those with fetal fibroblasts. In experiment 2, the fusion rate, cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cells was significantly (p<0.05) increased in embryos cloned with fetal fibroblasts at early G1 phase of the cell cycle, compared to those of fetal fibroblasts at late G1 phase. DNMT1 transcript was observed to significantly (p<0.05) increased in the fetal fibroblasts at 3 hrs after trypsin treatment of confluent culture. Further, level of telomerase activity and transcribed X-linked genes was also significantly (p<0.05) higher in the early G1 SCNT blastocysts than those of late G1. The results imply that fetal fibroblasts at early G1 phase induces the enhanced developmental potential and up-regulated telomerase activity and X-linked gene, but aberrant transcript pattern of X-linked genes may be displayed in the SCNT embryos.

The present study investigated the nuclear remodeling, development potential with telomerase activity and transcription level of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos using two different fusion and activation methods. Female adult fibroblasts were injected into perivitelline space of in vitro matured oocytes. The oocyte-nucleus complexes were fused and followed by immediately either activated (Group 1), or activated at 1 h post-fusion (hpf) (Group 2), respectively. The incidence of normal premature chromosome condensation (PCC) at 1 hpf was slightly increased in the Group 2, compared to those of Group 1, but there was no significant (p<0.05) difference. The incidence of normal pronucleus (PN) and chromosome spread at 5 and 18 hpf were significantly (p<0.05) higher in the Group 2 than those of Group 1. The cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cell numbers were significantly (p<0.05) higher in the Group 2, compared to those of Group 1. Level of telomerase activity was significantly (p<0.05) higher in the SCNT blastocysts of Group 2, compared to those of Group 1. Transcript levels of HPRT, MeCP2 and XIST were not significantly (p<0.05) different between blastocysts of Group 1 and 2. However, transcript level of ANT3, RPS4X, XIAP and ZFX were significantly (p<0.05) up-regulated in the SCNT blastocysts of Group 2, compared to those of Group 1. Taken together, it is concluded that oocyte activation at 1 hpf induces the enhanced developmental potential by efficient nuclear remodeling and subsequent facilitation of the nuclear reprogramming of bovine SCNT embryos.

개 난자의 체외성숙율을 높이기 위하여 다양한 방법들이 시도되고 있지만 여전히 그 효율성은 낮다. 본 연구는 개 난자의 체외성숙 시, 성선 자극 호르몬인 황체형성호르몬(LH)과 난포자극호르몬(FSH), 상피세포성장인자(Epidermal growth factor, EGF) 그리고 시스테인(cysteine)을 각각 첨가하여 72시간 동안 체외성숙시킨 후 핵성숙율(GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphase I, MII: metaphase II, UK: unknown stage)을 확인하였다. LH와 FSH를 첨가하였을 때 첨가하지 않은 군과 GV, MI 및 MII율에는 유의적인 차이는 없었다. 하지만 GVBD율은 첨가군이 유의적으로(p<0.05) 높았다. 성선 자극 호르몬을 첨가한 배지에 10 ng/ml의 EGF를 첨가하였을 때 MII율이 첨가하지 않은 군보다 유의적으로(p<0.05) 높았다(4.54% vs. 7.06%). cysteine을 첨가하였을 경우, 핵성숙율에 유의적인 차이는 보이지 않았지만 전반적으로 핵성숙율이 향상된 것을 확인할 수 있었다. 따라서 개 난자의 체외성숙 시, 10 μg/ml의 LH와 FSH, 10 ng/ml의 EGF 그리고 0.57 mM의 cysteine을 첨가하는 것이 핵성숙율을 향상시키는 것으로 사료된다.

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ～45 day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, 30 μsec) and cultured with 7.5 μg/ml cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at 39℃, 5% CO2, 5% O2 in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT (79.3±8.5 and 25.5±6.1, and 85.0±6.4 and 38.6±5.5, respectively) than NT counterparts (65.1±5.2 and 15.6±3.0, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT (34.7±5.8 and 38.1±4.1) than NT embryos (24.8±3.2). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.