• 한국자원식물학회지
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• 제12권2호
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• pp.107-119
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• 1999

본 연구에서는 탱자의 미성숙배로부터 캘러스 유도와 재분화에 관하여 연구하였다. 탱자 배발생 캘러스들은 3% sucrose와 44.4 $\mu\textrm{m}$ BA로 처리된 $\frac{1}{2}$MS배지에서 광조사(2,000 1x, 16h)시에 미성숙종자의 배로부터 유도되었으며 식물체 재분화는 MS배지에 5.0 $\mu\textrm{m}$ BA를 첨가하였을 때 가장 양호하였다. 탱자는 배양2~3주경에 담황색의 캘러스가 유도되었으며 배양5~6주경부터는 딱딱한 녹색의 캘러스로 변하였다. 기내 재분화는 3% sucrose와 5.0 $\mu\textrm{m}$ BA가 첨가된 MS배지에서 배발생 캘러스로부터 직접 유도되었다. 또한 배양 12주 후에 Multishoot가 재분화되었으며, 배양 16주 후에 뿌리가 형성되었다. 또한 뿌리가 형성된 식물체들은 토양으로 옮겨져 완전한 식물체로 잘 자랐다. 광학 현미경 관찰시 탱자에서는 배양4주경에 하얀색의 조밀한 배양된 세포들이 나타났다. 배양6주경부터는 세포들이 신장되면서 배발생 돌기가 나타났으며 주변의 세포들은 액포화 되었다. 또한 배양12주경에는 미성숙 잎에서 발달된 유낭과 전형성층을 관찰하였다.

• Journal of Plant Biotechnology
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• 제7권1호
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• pp.75-80
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• 2005

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.

• 한국수정란이식학회지
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• 제15권3호
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• 한국수정란이식학회지
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• 제23권4호
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• 한국작물학회지
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• 제37권3호
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• pp.257-263
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• 1992

GA$_3$ 농도 및 저온처리 기간이 보리의 춘화처리효과에 미치는 영향을 조사하고자, 추파성 정도가 IV인 올보리를 공시하여 출수후 13 및 20일이 경과한 미숙배를 GA$_3$농도별로 B$_{5}$배지에 치상하여 저온처리한 후 온도 20/15$^{\circ}C$, 14시간 일장으로 온냉조절실에 이식재배하여 실험한 결과는 다음과 같다. 1. 미숙배의 생육은 출수후 13일배 보다 20일배가 양호하여 발아율 및 유묘장은 증가하였고, 평균발아일수는 감소하였는데, GA$_3$1 및 10ppm이 효과가 좋았다. 2. GA$_3$농도가 증가할수록 상엽전개 및 출수일수가 단축되었으며, 춘화처리에 효과가 높은 GA$_3$농도는 1,10ppm이었고 GA$_3$와 저온처리가 병행될 때 춘화처리효과가 높았다. 3. GA$_3$ 처리에 따른 상엽전개일수 단축 효과는 3주 저온처리의 GA$_3$무처리구보다 2주 저온처리의 GA$_3$1,10ppm에서 각각 3, 16일 단축되었고, 3주저온처리의 1ppm에서 18일, 10ppm에서 20일 단축되었다. 4. 간장, 수장 및 일수립수는 공히 3주 저농처리에 GA$_3$ 10ppm 처리가 최소치를 보였는데, 실제육종상 중요한 일수립수의 확보는 충분할 것으로 보였다. 5. GA$_3$농도와 주요 형질과의 상관은 1주 저온처리시 각 형질들과 모두 상관 관계가 인정되지 않았으나, 2주 저온처리에서는 고도의 부상관이 인정되었고, 3주 저온처리에서는 출수일수를 제외한 모든 형질이 부의 상관이 있었다. 6. 저온처리 기간과 주요 형질들과의 상관은 GA$_3$무처리시 간장만 유의적인 부의 상관이 있었고, 기지 상관이 없었으며 GA$_3$1, 10ppm 처리시 모든 형질이 고도의 부상관이 있었다.다.

• 한국자원식물학회지
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• 제37권3호
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• pp.263-269
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• 2024

Embryos of early-ripening peaches could not achieve physiological maturation or undergo abortion before harvest. Embryo rescue is an effective strategy to rescue embryos from early-ripening peaches. Thus, the current study was carried out to determine the appropriate developmental stage and optimal medium composition for embryo rescue in early-ripening peach. Development of open-pollinated 'Yumi' fruit was investigated from 20 to 90 days after full bloom (DAFB) to explore period occurring endocarp hardening. After endocarp hardening, embryo development was observed by light microscopes. Shoot and root meristems were observed at 65 DAFB and embryo size rapidly increased at 75 DAFB. Embryos collected at 75, 80, 85, and 90 DAFB were cultured on four media based on Driver and Kuniyuki (DKW) medium. Germination rate of embryos cultured on four media gradually increased from 75 to 90 DAFB and reached 100% at 90 DAFB. Notably, M3 medium (0.5 DKW supplemented with 6-benzylaminopurine (BAP) 1.0 ㎎/L) displayed the highest germination rate at 75 and 80 DAFB stages. Growth and development of shoot and root were pronounced in plantlet cultured at 90 DAFB stage. While delayed shoot growth was evident in plantlets cultured at 75, 80, and 85 DAFB stages, this retardation could be overcome through the application of growth regulators, particularly in M3 and M4 (0.5 DKW supplemented with BAP 1.0 ㎎/L and indole-3-butyric acid 0.5 ㎎/L) media. Remarkably, roots of plantlet grown in M4 medium exhibited limited elongation. In conclusion, germination rate of embryo and growth of embryo cultured plantlet can be enhanced by collecting seeds from early-ripening 'Yumi' at the 90 DAFB stage and conducting embryo culture using the M3 medium.

• 식물조직배양학회지
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• 제24권3호
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• pp.129-135
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• 1997

한국 야생차나무의 잎절편과 배배양에서 생장조절물질의 첨가에 따른 형태형성 과정의 변이를 조사하였다. 그 결과 접합자배는 싸이토키닌을 5-20$\mu\textrm{M}$을 첨가한 MS 배지에서 직접적인 체세포 배와 부정아 및 액아 발생률이 높았으며 옥신의 함량이 높아질수록 형태형성율이 급격히 저하되었다. 1/2 MS 및 1/4 SH배지에 $10\mu\textrm{M}$ IBA가 첨가된 배지에서 모든 줄기가 발근 되었다. MS배지에 2,4-D를 첨가한 배지에서 미성숙 접합자 배를 배양한 결과 체세포배성 캘러스가 유발되었다. 성숙된 접합자 배를 발아시킨 후 어린잎을 채취하여 고농도의 옥신 (IBA와 NAA) 또는 싸이토키닌 (BAP)이 함유된 MS배지에 배양한 결과 체세포배 형성 캘러스가 발생되었으며 또한 직접적인 체세포배가 발생하였다. 그러나 뿌리와 줄기 형성에는 각각 요구하는 옥신의 농도와 종류가 각각 달랐다.

• 식물조직배양학회지
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• 제21권1호
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• pp.7-14
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• 1994

국내 5가지 품종의 대두 미숙배로부터 발생되는 체세포배 형성빈도와 자엽변이에 대한 2,4-D 농도의 영향을 알아보였다. 여러가지 농도의 2,4-D가 첨가된 MS 기달배지에 각 품종의 미숙배를 6주동안 암소에서 배양한 결과 '울산콩', '금산콩', '뱀콩' 및 '반청두'의 경우 1-2 mg/L 2,4-D를 첨가한 배지에서, '아주까리'는 20 mg/L 2,4-D를 첨가한 배지에서 가장 높은 체세포배 형성빈도를 보였다. 또한 횡으로 반분한 체세포배를 0.1-1 mg/L 2,4-D가 첨가된 배지에서 배양하였을 때 최고 70%의 빈도로 이차배를 형성하였다. 미숙배(접합자배)에서 유도된 체세포배의 형태는 2,4-D 농도에 따라 다르게 나타났는데 즉, 0.2 mg/L 이하의 농도에서는 하나의 자엽을 갖는 배와 나팔형보다는 두개의 자엽을 갖는 정상적인 형태가, 0.5-4 mgh 농도에서는 정상적인 형태보다는 나팔형의 체세포배 형성빈도가 높게 나타났다. 또한 체세포배의 자엽형태에 따라 식물체 재생률을 조사하기 위하여 ME 기본배지와 0.1 mg/L IAA, 1 mg/L BA, 1mg/L Gh₃ 및 0.5% 활성탄을 각각 U 기본배지에 첨가하여 16시간 광주기하에서 4주동안 배양한 결과, 식물체 재생률은 일반적으로 두개의 자엽을 갖는 정상적인 배가 하나의 자엽을 갖는 배, 나팔형 및 구형기의 체세포배에 비하여 높았으며, 특히 1 mg/L GA3를 첨가한 배지에서 가장 높은 재생률(25%)을 보였다.

• Plant Resources
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• 제2권1호
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• pp.1-9
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• 1999

The callus from the hypocotyl region of immature embryo of Citrus junos Sieb. was efficiently induced in the $\frac{1}{2}$ MS medium containing 45uM BA after 8 weeks culture. The callus was developed into the two callus type, embryogenic callus and nonembryogenic callus, which can be distinguished by visual examination depending on color and appearence. In vitro regeneration of callus established efficiently in the hormone-free MS medium from the embryogenic callus. In order to investigate the physiological changes depending on the developmental stage of embryo, the embryo was formed in the MS medium. The embryogenic and nonembryogenic callus, and the various stages of the somatic embryo were examimed the changes of esterase activity, and their isozyme patterns as well. The protein content and esterase activities was gradually increased on the developmental stages of embryo. Total protein pattern were different by the SDS-PAGE and were appeared strong band of 23 KD in the torpedo stage. The pattern of the esterase isozyme was exhibited a difference between embryogenic callus and nonembryogenic callus. It was appered pI 6.0, 8.0, 8.2 in the embryogenic callus. Also the new band of pI 4.75 was appeared in the cotyledon. These results suggest that the changes of esterase activities and isozyme patterns are importent factor in the differentiation and development of citrus.

• 한국수정란이식학회지
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• 제32권3호
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.