• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.180-185
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• 2015

Leaf discs from 'Yeobong' and 'Maehyang' strawberry plants were used as explants for transformation. The Agrobacterium tumefaciens strain EHA105 harboring the monellin gene under the control of the CaMV 35S promoter was used in co-cultivation experiments. The frequencies of callus formation and plant regeneration from leaf explants after co-cultivation in 'Yeobong' were higher than those of 'Maehyang'. These transgenic plants showed normal growth patterns and flowering. PCR and Southern hybridization confirmed that 1 to 2 copies of the monellin gene were integrated into genome of the transgenic strawberry plants. Northern blot analysis confirm that the transcripts were expressed in transgenic strawberry plants. Although long-term subcultured transgenic strawberry plants showed a phenomenon to escape the transgene, the transformation system established in this study provides new opportunities for genetic improvement of strawberry plants.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.181-186
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• 2005

This work was conducted to evaluate the effects of rooting on tissue cultured M.9 (Malus domestica Bork. cv, tcM.9) after layering in field. We investigated an appearance period of first root in shoot, rooting ratio, contents of indole-3-acetic acid (IAA), abscisic acid (ABA), inorganic matters, sugars, and lignin in rooting areas of stems by layering. First root in shoot of tcM.9 and natural M.9 appeared 25 and 30 days after layering (DAL), respectively. Rooting ratio was much higher in tcM.9 than in natural M.9. The content of IAA was higher in tcM.9 than in natural M.9 before layering, but it was reversed at 20 and 30 DAL. In contrast, the content of ABA was much higher in natural M.9 than in tcM.9 in case of both before and 10 and 20 DAL. The contents of N, B, Mn, and Zn were significantly higher in tcM.9 than in natural M.9 both before and 10 and 20 DAL. The contents of sugars in tcM.9 had the similar pattern of the contents of inorganic materials. There were statistically significant differences in the contents of sucrose and glucose at 30 DAL as well as the content of maltose at 20 and 30 DAL. The content of lignin was significantly higher in tcM.9 than in natural M.9 before layering and 10 and 30 DAL while there was no difference 20 DAL. Therefore, improvement of rooting ability in the tissue cultured root stock M.9 might be due to the changes of inorganic matters or lignin rather than that of sugars and hormones.

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.135-138
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• 2002

Cotyledonary explants from immature zygotic embryos of each 85 recombinant inbred lines (RILs) were cultured on medium containing MS salts, B5 vitamins, 40 mg$^{-1}$ 2,4-dichlorophenoxyacetic acid and 30 g$^{-1}$ sucrose. Frequency of somatic embryo formation on cotyledonary explants showed in thirty-six lines(＜10%), in thirty-seven lines (11~49%), in nine lines (50~89%), and in three lines(>90%), respectively, The highest frequency (up to 90%) and number (6.36 per cotyledon) of somatic embryos were obtained from lines of KM1010, KM1032 and KM1064. Primary somatic embryos produced from three lines produced numerous secondary somatic embryos on the surfaces, which were subcultured for over one year. Upon transfer to maturation and conversion medium (Komatsuda, 1992), somatic embryos converted to plantlets at a frequency of approximately 25%.

• Horticultural Science & Technology
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• v.16 no.4
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• pp.520-524
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• 1998

This study was carried out to investigate the effect of thidiazuron(TDZ) on callus and shoot primordia formation, to determine the most optimum multiple shoot induction medium, and to obtain the plantlets on solid medium via shoot organogenesis. TDZ 0.01 mg/L in MS medium was most effective on callus formation, and BA 0.1 mg/L was most effective on shoot growth, while TDZ 0.01 mg/L was most effective on callus formation. TDZ 0.001 mg/L was most effective in shoot primordia formation. Shoot tips were cultured with TDZ 0.01 mg/L for 8 weeks and induced callus was transferred to regeneration medium containing TDZ 0.001 mg/L. After 4 weeks induced shoot primordia were resubcultured at growth regulator-free medium for 4 weeks. The induced multiple shoots rooted more efficiently at NAA 1.0, 5.0 mg/L, or IBA 5.0 mg/L.

• Journal of Plant Biotechnology
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• v.29 no.1
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• pp.45-49
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• 2002

The leaf and petiole segments of Farfugium japonica were cultured to investigate the influence of growth regulators on their callus induction and plant regeneration. The callus induction and growth showed a good response both leaf and petiole on MS media supplemented with 1∼2 mg/L 2,4-D and 1∼2 mg/L BA. Callus induction and growth were more effective in petiole segments than leaf one. The highest percentage of plant regeneration was obtained from 60-day-old calli on MS medium supplemented with 1 mg/L NAA and 2 mg/L BA. When subcultured to the same medium for about 60 days, multiple shoots were developed from regenerating callus. The shoots produced roots after transferring to rooting medium containing 0.5 mg/L IAA. The plantlets over 50 mm in height were successfully acclimatized in vermiculite, and the survival rate was over 95%.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.179-184
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• 2007

Culture method of somatic cells is one of the important factors in the production of transgenic pigs by somatic cell nuclear transfer. In this study, we established an efficient culture method of porcine fetal fibroblasts. Porcine fetal fibroblasts were isolated from 33-day-old fetuses. The proliferation of porcine fetal fibroblasts was analyzed by different serum types and culture media. The cultures in medium supplied 15% ES screened FBS showed faster increase in cell number than 15% FBS. Also, fetal fibroblasts have been propagated continuously for $7{\sim}8$ passages in ES modified DMEM and DMEM medium. We transfected $PGK-neo^r$ vector (pKJ2) into porcine fetal fibroblasts to estimate colony formation in this culture condition. The formation of colonies was confirmed in the medium containing $300\;{\mu}g/ml$ G418 at 12 day. These data show that this culture system can be used screening of porcine somatic cells transfected transgene.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.41-41
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• 2018

차꼬리고사리(Asplenium trichomanes L.)는 남방계식물로 제주도에 자생하는 것으로 알려져 있으며, 잎에 광택이 있고 총생하는 식물이다. 주로 실내 외 조경 및 분화소재로 이용되며, 한방에서는 철각봉미초라하여 뿌리를 포함한 전초를 이질, 임병, 만성질염, 월경불순 및 요통의 약재로 사용한다. 또한 식물구계학적 특정식물종 IV급, 적색자료목록 준위협 (NT)에 분류된 식물로 보호가 필요하다. 본 연구는 차꼬리고사리의 대량생산을 위한 기내전엽체 증식 및 기외포자체 형성조건을 구명하고자 수행되었다. 실험재료는 포자를 기내 발아시켜 전엽체를 획득한 다음 8주 간격으로 계대하면서 확보하였다. 전엽체의 증식과 생육에 적합한 배지를 비교하고자, 1/4, 1/2, 1, 2MS와 Knop배지를 조성하여 배양하였다. 배양은 전엽체 300mg을 메스로 균일하게 다지는 방법을 이용하였으며, 배양환경은 온도 $25{\pm}1.0^{\circ}C$, 광도 $30{\pm}1.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, 광주기 16/8h(light/dark)로 조절되었다. 실험결과, 1MS배지에 배양된 전엽체의 생체중이 4.3g으로 가장 많이 증가하였다. 형태형성발달도 하트형의 전엽체로 정상적으로 유도되었으며, 생식기관도 관찰되었다. 전엽체로부터 포자체의 형성을 유도하고자, 원예상토, 피트모스, 펄라이트 및 마사토의 비율을 5종류로 달리하여 혼합된 토양을 사각분($7.5{\times}7.5{\times}7.5cm$)에 충진하였다. 전엽체 1g과 증류수 25mL를 핸드블랜더로 10초간 분쇄하여 토양표면에 분주하는 실험방법을 사용하였다. 재배환경은 온도 $25{\pm}1.0^{\circ}C$, 광도 $43{\pm}2.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, 광주기 16/8h(light/dark)로 유지하면서 12주간 재배되었다. 실험결과, 원예상토 단용, 원예상토와 펄라이트가 2:1(v:v)로 혼합된 토양, 원예상토와 마사토가 2:1(v:v)로 혼합된 토양에서 각 31.7, 24.3, 19.3개의 포자체가 생산되었다. 한편 포자체의 생육은 원예상토 단용 토양에서 엽수, 엽장, 엽폭 등의 수치가 비교적 우수하였다. 따라서 차꼬리고사리의 전엽체는 MS배지에 배양하고 증식된 전엽체를 원예상토에 분주하여 포자체의 형성을 유도하는 것이 가장 효과적이었다.

• FLOWER RESEARCH JOURNAL
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• v.18 no.2
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• pp.83-86
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• 2010

This study was conducted to develop in vitro propagation techniques of new cultivar, 'Greenstar', bred by Highland Agriculture Research Center. The multiple shoot induction and plant growth of in vitro plant were analyzed by MS media concentration (1/2 MS, 1 MS and 2 MS medium), plant growth regulators and its proper concentration; CPPU [Forchlorfenuron(N-(2-chloro-4-pridyl)-3-phenylurea) (0, 0.5, 1.0 and $2.0mg{\cdot}L^{-1}$), thidiazuron [(TDZ), (0.01, 0.1, 0.5 and $1.0mg{\cdot}L^{-1}$), zeatin (0, 0.5, 1.0 and $2.0mg{\cdot}L^{-1}$), and BA [6-benzylaminiopurine(BA), (0, 0.5, 1.0 and $2.0mg{\cdot}L^{-1}$)] in MS (3% sugar and 0.8% agar with pH 5.7) media. The highest number of induced shoots, leaves and roots were shown in 1/2 MS medium concentration. On the 1/2 MS medium, shoot numbers, shoot length, leaf numbers and root numbers were 11.0, 1.9 cm, 24.7, and 8.0, respectively. On the absence of CPPU in the 1/2 MS medium, shoot length and root numbers was greater than CPPU treatment, but the highest number of shoots was induced by the $2.0mg{\cdot}L^{-1}$ of CPPU concentration in 1/2MS medium. TDZ, zeatin, and BA treatments were not effective on the induction of multiple shoot in vitro culture. As a result, in vitro culture of new Saxifraga fortunei, 'Greenstar' with $2.0mg{\cdot}L^{-1}$ of CPPU in 1/2 MS medium was most effective for the rapid multiplication.

• Development and Reproduction
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• v.12 no.3
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• pp.221-229
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• 2008

For the clinical application, it is needed to keep characteristics of stem cells after storage for a long time. In the present study, we examined stem cell properties of human cord-derived stem cells (HUC) after cryopreservation. Cells were isolated from human umbilical cord and cultured in vitro. At passage 2 or 3, HUC were suspended at a concentration of $1.0{\times}10^6/m{\ell}$ in cryomedium consisting of DMSO and FBS. After freezing at $-80^{\circ}C$ overnight, HUC were cryopreserved at $-196^{\circ}C$ nitrogen gas. After 6 months, HUC were thawed and cultured in vitro. Assessment for the stem cell properties was made upon the morphology, population doubling time, and expression profiles of genes and various proteins. Cryopreserved HUC showed more than 70% viability and maintained fibroblast-like morphology similar to HUC before cryopreservation. Throughout the culture, they underwent average 42.8 doublings and produced $6.75{\times}{10^{18}}$ cells. RT-PCR analyses showed that cryopreserved HUC expressed Oct-4, nanog, SCF, NCAM, nestin, GATA-4, BMP4, and HLA-1 genes. They did not express Brachyury and HLA-DR genes. Immunocytochemical studies showed that cryopreserved HUC reacted with antibodies against SSEA-3, -4, Thy-1, vimentin, fibronectin, HCAM, ICAM, HLA-1 proteins. They did not react with antibody against HLA-DR protein. Theses genes and proteins expression patterns of cryopresserved HUC were similar to those of HUC before cryopreservation. These results suggest that cryopreserved HUC could retain proliferative potential and they expressed various genes and proteins similar to HUC before cryopreservation. Thus, cryopreservation might be useful for HUC for future research and clinical application.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.