• Journal of Plant Biotechnology
• /
• 제34권4호
• /
• pp.363-373
• /
• 2007

효율적이며 반복성 있는 고추의 소포자배양 시스템을 확립하기 위해 꽃봉오리로부터 소포자의 수확 시 나출 방법이 소포자 활력에 미치는 영향을 조사하는 동시에 모 식물 생육 시의 광도, 치상배지의 조성, 및 배양 광 조건이 배의 발생 및 발달에 미치는 영향을 조사하였다. 광도가 10,000 lux 와 5,500 lux로 각기 다른 곳에서 모식물이 생육된 경우 활력 있는 소포자의 비율은 큰 차이가 없었으며, 소포자 배의 발생이나 발달은 저광도인 5,500 lux에서 생육된 경우에 다소 높아 모 식물 생육 시의 광도가 소포자배의 발생에 미치는 영향이 크지 않은 것으로 나타났다. 그러나 5,500 lux에서 생육된 식물에서는 꽃봉오리의 수가 적어 배양에 필요한 소포자를 준비하기가 매우 어려웠다. 활력 있는 소포자의 비율은 체나 막자사발을 이용한 maceration 방법에 비해 blender를 이용하는 경우 13배 이상 높았다. 배의 발생 및 발달에 가장 좋았던 배지는 사용한 배지 중 MN배지였으며, 배양 광 조건은 암 상태에서 배양한 4주 후에 다시 광 상태로 옮겨 1주 배양하는 것이었다. 이상에서와 같은 결과들은 고추에서 형질전환이나 돌연변이 연구에 이용될 수 있을 정도로 많은 배를 생산할 수 있는 소포자 배양 시스템을 확립하는데 중요한 기초 자료가 될 것이다.

• 한국가축번식학회지
• /
• 제7권2호
• /
• pp.41-52
• /
• 1983

Animal in dustry in Korea urgently needs the domestic introduction and the industrial a, pp.ication of embryo transfer technique. Namely, this technique can be utilized effectively, as means of the improvement of livestocks, as means of the increase of meat production, as means of substitute for the livestock import, and dissemination of new breed. However, as this technique avaliable in our country is remaining initial stage, we can not make use of the technique industrially unless we make much improvement as follows; induction of superovulation, non-surgical recovery of embryos, synchronization between the estrus such cycles of donor and recipient, non-surgical transfer of embryos, etc. Simultaneously, the basic studies such as harvesting oocytes from ovary, in vitro culture of oocytes, in vitro capacitation of spermatozoa, cloning by culture of blastomeres and transfer of nuclei, sexing embryo, etc. should not be neglected in order to make the technique of embryo transfer more simple and convenient. For the success of these studies, universities, national and public institutes, large scale cattle farms, and small scale cattle farms should cooperate each other. For instance, universities undertake basic researches, and the national and public institutes a, pp.y the results of the researches to animal industry along with cooperation by large scale cattle farms. By the help of the cooperative organizations, the technique relevant to our environment and farm condition may be able to be finalized, and to be a, pp.ied to samll scale cattle farm. Consequently, being served to stimulate animal productivity, this technique can be contributed to the development of livestock industry in Korea.

• Clinical and Experimental Reproductive Medicine
• /
• 제14권2호
• /
• pp.93-100
• /
• 1987

To determine the effect of calcium on the preimplantational development of mouse two-cell embryo, the various concentrations of calcium were added into the culture media and the rate of blastocyst formation was observed. Also, to examine the effect of trifluoperazine, an inhibitor of calmodulin which is involved in the several intracellular calcium functions, embryos were cultured for 48 hours at the various concentrations of this inhibitor. An additional 24 hour culture was done to examine the effect of this drug on the transformation from morula to blastocyst. The results are as following ; 1. About 1.71mM of extracellular calcium is adequate for blastocyst formation and the higher concentrations of calcium (3.43mM and 8.55mM) do not affect on the blastocyst formation and the degenerating rate. 2. Trifluoperazine $100{\mu}M$ presents the inhibitory effect on the blastocyst formation while $1{\mu}M$ and $10{\mu}M$ do not so. 3. After an additional 24 hour culture, there is transformation of morula to blastocyst and the degenerating rate of embryo is increased all together.

• 한국가축번식학회지
• /
• 제19권2호
• /
• pp.89-93
• /
• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• 한국작물학회지
• /
• 제39권2호
• /
• pp.193-197
• /
• 1994

Hordeum bulbosum을 이용한 보리 반수체육종법의 실용화를 체계화하기 위해 일련의 실험을 실시하였으며, 유배의 적취시기, 유배의 크기에 따른 배배양 반응, 배지 및 환경조건 등을 조사하였다. 1. 유배의 적취시기는 배발육과 식물체 발생률을 고려할 때 수분 후 17일이 적기였다. 2. 식물체 발생에 유리한 유배의 크기는 0.9mm내외였다. 3. 식물체 발생에 효과적인 배지 및 배양조건은 MS배지의 암상태에서 발생시킨 후 명상태로 하는 것이 정상 식물체 발생률(3%)이 높았으나, 작업상의 편리성을 고려한다면 B5배지에서 24시간의 일장처리조건이 좋았다. 4. 반수체는 정상 2배체에 비하여 배 및 식물체의 발생이 현저히 부진하였다.

• 한국수정란이식학회지
• /
• 제11권3호
• /
• pp.201-210
• /
• 1996

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

• Journal of Ginseng Research
• /
• 제31권1호
• /
• pp.14-22
• /
• 2007

As the zygotic embryo of North American ginseng (Panax quinquefolius L.) matured during stratification over 203 days it grew from 0.75 to 5.2 mm. Embryo excision and culturing on media containing different concentrations of two growth regulators, gibberellic acid ($GA_3$, 1 to 10 ${\mu}M$) and benzyladenine (BA, 1 to 5 ${\mu}M$), during stratification, showed that shoot and root number and the shoot, root and cotyledon length increased with increased stratification time. Gibberellic acid was the more effective growth regulator for increasing shoot and root number and shoot, root and cotyledon lengths. Immature embryos (stratified for up to 63 days) needed growth regulators for further development. Cultures on $GA_3$ at the last culture date (stratified for 203 days) when embryos were mature, produced multiple shoots but there was no effect of $GA_3$ concentration. Benzyladenine inhibited shoot and root growth regardless of embryo stratification. Growth regulators had little effect on cotyledon length of mature embryos. Embryos cultured on $GA_3$ combined with BA were green on all culture dates whereas greening in the control and BA treatments increased with culture date. The BA treatments induced 100% swelling of the embryos on the final culture date while in the control and $GA_3$ treatments there was no swelling. There was little or no curling in the control and BA treatments and a linear decrease in curling with culture date in the $GA_3$ and $GA_3$ + BA treatments.

• 한국수정란이식학회지
• /
• 제29권3호
• /
• pp.229-234
• /
• 2014

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P<0.0001 and P<0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P<0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

• Journal of Plant Biotechnology
• /
• 제32권1호
• /
• pp.37-44
• /
• 2005

고추의 나출 소포자로부터 배를 유기하는데 최적인 조건을 확립하기 위하여 micro-blender를 사용하여 소포자들을 나출한 후 NLN 배지에 배양하였으며 $32^{\circ}C$의 고온처리 기간, 전처리 배지내 2-hydroxynicotinic acid의 첨가, 그리고 약과 소포자의 공동전처리가 소포자배의 발생에 미치는 영향을 조사하였다. 나출 소포자들을 기아배지에서 고온처리 한 후 NLN 배지에 배양하였을 때 다수의 배가 발생하였다. 배양 3주 후부터 구형 및 어뢰형배가 관찰되었는데 이때 배의 발생은 비동조적으로 일어났다. 배양4주가 되면 구형과 어뢰형배 이외에 자엽배들이 발생하였다. 발생한 배들 중자엽배늘은 $2\%$ sucrose가 첨가되고 생장조절물질은 첨가되지 않은 B5 고체배지로 옮겼을 때 정상인 유식물들로 발달하였다. 고온처리 기간이 $1\~2$일로 짧은 경우 소포자배의 발생은 높았으나 대부분이 발달초기의 구형 또는 심장형배이었으며 자엽배의 발생은 매우 드물었다. 고온처리 3일 이상에서는 배의 발생은 크게 감소하였으나 자엽배가 다수 발생하였다. Inducer chemical로 알려진 2-hydroxynicotinic acid를 약전처리배지에 첨가하였을 때 배의 발생은 다소 높았으나 발달은 오히려 억제되어 대부분이 구형 또는 심장형이었다. 소포자 전처리시 약을 첨가한 경우 배의 발생이나 발달 모두 억제되었다. 본 연구결과 고추의 나출 소포자로부터 다수의 배를 획득하였고 식물체를 재분화 시키는데 처음으로 성공하였다. 이와 같은 소포자 배양시스템은 앞으로 더 많은 배를 생산할 수 있는 배양조건이 확립되어야 하지만 homozygous한 배가 반수체의 생산 뿐만아니라 형질 전환과 열성 또는 우성의 돌연변이체 선발에 매우 유용하게 이용될 수 있을 것이다. 비해 $ 0.27\~0.79\;\cal{mg/g}$ F.W.가 높았으며 환기횟수 $0.1 h^{-1}$ 처리구는 광도의 증가에 따른 효과가 나타나지 않았으나 환기횟수 $0.1 h^{-1}$ 처리구는 높은 광도에서 감소하였다. 환기횟수 $4.9 h^{-1}$는 $0.1 h^{-1}$에 비해 잎의 공변세포가 크고 주변 부세포가 잘 발달되어 있었다. 특히 PPF $99\;{\mu}mol\;m^{-2}s^{-1}$에서 환기횟수 $0.1 h^{-1}$는 부세포의 발달이 미흡하고 기공이 많이 열려 있는 상태인 반면 환기횟수 $4.9 h^{-1}$는 부세포가 잘 발달된 잎을 지니고 있었다.:PR30, KB50:PR50, PR100:KB0 처리구의 $1m^2$ 당 개체 수는 각각 16,600개, 6,700개, 4,900개, 3,300개, 12,400개였으며 총직립경 수는 각각 33,200개, 22,800개, 18,000개, 15,000개, 62,000개였다. 개체 수는 KB100:PR0처리구가 가장 높았고, 다음은 PR100:KB0처리구였으며 혼합처리구의 경우는 Kentucky bluegrass 혼합비 율이 높을수록 높았다. 총직립경 수는 PR100:KB0 처리구가 KB100:PR0 처리구보다 오히려 높았으며 혼합 처리구의 경우는 개체수와 비슷한 경향을 보였다. 혼합처리구의 경우 초종별 개체수의 비율은 KB80:PR20는 87:13, KB70:PR30는 78:22, KB50:PR50은 48:52의 비율로 나타났다. 조성시기의 기상과 피복율과의 상관관계 2001년 가을과 2002년 봄의 일일평균기온을 비교하여 보면 가을(9월, 10월, 11월) 3개월간의 일일평균기온은

• 한국수정란이식학회지
• /
• 제23권3호
• /
• pp.217-222
• /
• 2008

In previous studies, we reported that sow which was transferred OPS-freezing embryos not able to deliver a piglet (Kim et al, 2004). This study was conducted to investigate a possibility of gilt as recipients which produce piglets after transfer of OPS-freezing embryos. All transferred embryos were prepared by in vitro production (IVP) system. In vitro culture (IVC) medium used glucose-free NCSU23 supplemented with 5mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$. From day 3 of IVC, 10% fetal bovine serum albumin was added to the culture medium. In preparing of freezing embryos, embryos were treated with 7.5 $\mu g/ml$ cytochalasin-B for 30 min and centrifuged at $13,000{\times}g$ for 13 min. And then, embryos were exposed sequentially to an ethylene glycol (EG) solution, aspirated into open pulled straw (OPS), and plunged or thawed into the liquid nitrogen. In embryo transfer (ET), we used two kinds of type (surgical method vs. non-surgical method). In surgical method of embryo transfer, $55\sim65$ embryo were transferred in both uterine horn of two recipient gilts by plastic straw. Non-surgical method which is like artificial insemination was performed on three gilts. Each 140 frozen embryos were transferred to two gilts and 40 fresh embryos to one gilt. Pregnancy establishment was shown one recipient at 45 days after ET. However, the one recipient was also aborted at 58 days after ET. These results suggest that gilts can be considered as a candidate of recipients for OPS-freezing embryo transfer.