• Fisheries and Aquatic Sciences
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• 제20권11호
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• pp.29.1-29.7
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• 2017

The objective of this study was to determine a simple and reliable ploidy identification protocol for the rainbow trout (RT), Oncorhynchus mykiss, in the field condition. To evaluate the ploidy level and compare different detection protocols, triploid RT and gynogenesis were induced by UV irradiation and/or heat shock. The hatching rate at day 30 was 85.2% and the survival rate at day 90 was 69.4% (fingerling). The sex ratio of female RT was 93.75% in the gynogenesis group, illustrating that the UV irradiation inactivated the sperm DNA. The hatching rate and survival rate were 82.0 and 74.7%, respectively, in the triploid-induced group. The triploid induction rate by heat shock procedure was 73.9%. Cytogenetic protocols for ploidy identification such as chromosome counting, erythrocyte nuclear size comparison, and analysis of nucleolar organizing regions (NORs) by silver staining were compared. Silver nitrate staining showed the greatest success rate (22/23 and 32/32 for the triploid-induced group and gynogenesis group, respectively), followed by erythrocyte nuclear size comparison (16/23 and 19/32 for the triploid-induced group and gynogenesis group, respectively) and, lastly, chromosome preparation (2/23 and 6/32 for the triploid-induced group and gynogenesis group, respectively) with the lowest success rate. Based on our findings, silver staining for RT ploidy identification is speculated to be highly applicable in a wide range of research conditions, due to its cost-effectiveness and simplicity compared to other numerous ploidy detection protocols.

• 한국양식학회지
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• 제12권3호
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• pp.167-173
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• 1999

Blocking the 1st mitotic cleavage was performed in mud loach (Misgurmus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and ternal shocks Optimum UV range for inactivation of cyprinid loach sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to 4,050 ergs/m$m^2$. Heat shock treatment ($41^{\circ}C$ for 3mins) with various treatment initiation times ranged from 22 to 50 min post insemination resulted wide range of success for induced gynogenesis. Best result was obtained when haploid egges were shocked at 28 min after insemination (corresponding to metaphase division of the 1st cleavage); 26% of total eggs inseminated were viable diploid gynogens. The hatching success and early survival of the both meiotic and mitotic gynogenetic groups were significantly lower than those of control crosses (P<0.05). Maternal origin of induced gynogenetic mud loach was verified by multi-locus DNA fingerprinting.

• Fisheries and Aquatic Sciences
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• 제4권1호
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• pp.39-46
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• 2001

To examine the effect of copy number-dependent transgenic genotype on the expression of foreign gene, stable hemizygous and homozygous transgenic breeding line was established using artificial parthenogenesis. For this purpose, induced diploid gynogenetic transgenesis was optimized in mud loach (Misgurnus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to $4,050\;ergs/mm^2$ The UV-irradiated sperm were inseminated into eggs from recessive color strain (yellow) or heterozygous transgenic mud loach containing CAT gene. Cold shock at $2^{\circ}C$ for 60 min, 5 min post fertilization successfully restored the diploidy of eggs inseminated with UV-irradiated sperm. Restoration to diploidy was confirmed by flow cytometry and gynogenetic status was verified by examining maternal exclusive inheritance of multi-locus DNA fingerprints, body color and transgenic marker. Putative isogenic transgenic fish clearly showed homozygous status at trans gene locus based on Southern blot hybridization and progeny testing. Further, such homozygous gynogenetic diploids revealed the increased levels of transgene expression, when compared to those of heterozygous (hemizygous) transgenic fish.

• 한국양식학회지
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• 제20권3호
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• pp.184-189
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• 2007

대농갱이, Leiocassis ussuriensis의 유전육종학적 연구의 일환으로 자성발생성 이배체 유도 기법을 최적화하였다. 대농갱이 반수체 유도를 위해 다양한 농도의 자외선 조사를 통해 대농갱이 및 동자개 정자의 유전학적 불활성화 실시하였으며 최적 자외선 처리 조건은 4,800 $ergs/mm^2$로 나타났고, 이 때 Hertwig effect와 함께 97% 이상의 반수체 유도가 가능하였다. 반수체 대농갱이는 전형적인 반수체 증후군(haploid syndrome)을 보였으며, 반수체 대농갱이를 이배체로 복원시키기 위해 $4^{\circ}C$ 또는 $6^{\circ}C$에서 인공 수정 후 3, 5 및 7분 후에 30, 40 및 50분 간의 처리를 수행한 결과, 가장 높은 생존율(38.6%)과 이배체 복원률(87.9%)은 수정 5분 후 $4^{\circ}C$에서 40분간 처리 조건에서 관찰되었다.

• Journal of Ecology and Environment
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• 제31권4호
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• pp.255-260
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• 2008

A commonly held view in studies of character displacement is that character states of both species are shifted in areas of sympatry. This view has been confirmed in an overwhelming number of cases for ecological character displacement. Excluding species pairs in which one of the two interacting species is found only within the distribution of the other species and species displaying gynogenesis, the pattern of reproductive character displacement is asymmetrical in that the shift in character states between areas of symaptry and allopatry occurs in only one of the two interacting species. Hypotheses for the reasons behind this asymmetry in reproductive character displacement include (1) homogenization by gene flow, (2) other mechanisms of reproductive isolation, and (3) sufficient reproductive isolation being provided by one of the interacting species exhibiting a pattern of reproductive character displacement. Because reproductive isolation can be achieved by divergence at any point in a sequence of premating reproductive behaviors and postmating developments, it is necessary to understand the mechanisms of reproductive isolation of two interacting taxa in areas of sympatry and allopatry and to analyze the relative contributions of potential factors to reproductive isolation to disentangle hypotheses for the patterns of asymmetry.

• 한국양식학회지
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• 제13권4호
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• pp.359-362
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• 2000

Mitotic gynogenesis was induced in the far eastern catfish, Silurus asotus using UV-irradiated heterospecific sperm and cold shock treatment. Eggs were activated with the sperm of mud loach, Misgurnus mizolepis which has been irradiated with UV at dose of 9,000 ergs/$mm^2$. To determine the optimum duration required to prevent the first cleavage, a cold shock at 4$^{\circ}C$ with duration of 20, 30 or 40 min was applied to the eggs 50 min after activation. To induce diploidization of mitogenesis, the most effective protocol was to apply cold shock to 50-min old (after activation) eggs at 4$^{\Circ}C$ for 30min.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2003년도 추계학술발표대회 논문요약집
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• pp.38-38
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• 2003

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5$^{\circ}C$ for 1, 2 or 3 min followed by cold shock at $1.5^{\circ}C$ for 30, 45 or 60 min. When applied during the first cleavage metaphase of mud loach (Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7% and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2003년도 추계학술발표대회 논문요약집
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• pp.30-30
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• 2003

자성발생(gynogenesis)은 대상 어류의 성 결정기작이 암컷 동형접합성(female homogamety)인 경우 전 암컷 단성집단을 생산할 수 있으며, 단기간 내에 우수한 품종의 순계 획득과 유전적으로 동일한 clone 집단을 만들 수 있다(Thorgaard, 1986; 정 등 1996). 이에 제 1 난할 억제에 의한 메기 S. asotus에서의 체세포분열 억제성(mitotic) 자성발생 2배체가 유도된 바 있다(임 등, 2002), 염색체공학 기법 중 웅성발생 2배체(androgentic diploid)는 난자 핵을 불활성 시킨 후 정자 핵에 의해 제 1 난할억제에 의한 유전자 배가로 유도 될 수 있다. 본 연구는 염색체공학에 의한 메기 4배체 생산, 메기체세포분열 억제성 자성발생성 2배체의 효과적인 유도 조건 검정 및 메기 웅성발생성 2배체 생산을 위한 연구의 일환으로, 메기에서의 핵분열(korykinesis)에 관하여 조사하였다. 메기에서의 핵분열 수온 25$\pm$0.5$^{\circ}C$ 조건에서 수정 후 31분에 가장 현저하였다. 본 연구 결과를 Park and Im (2001)의 수온 24$^{\circ}C$에서의 제 1 난할 후기가 50분이며 mitotic interval이 18.5$\pm$1.2분인 결과와 비교시, 본 연구 결과의 수온 24$\pm$0.5$^{\circ}C$ 조건에서 핵분열이 수정 후 31분인 것은 핵분열과 세포질분열(cytokinesis)이라는 관점에서 정확히 일치한다. 염색체 수의 배가시, 핵분열 억제는 세포질 분열 억제에 비해 더욱 효과적으로서(Cherfas et al., 1993; Nam et al., 1999), 본 연구 결과의 핵분열 시간(수온 24$^{\circ}C$ 조건에서의 수정후 31분)을 기반으로 하여 차후, 4배체 메기와 체세포분열 억제성 자성발생 2배체 메기 및 웅성발생성 2배체 메기 유도 및 그의 생산성 향상에 관한 연구가 기대된다.

• 한국발생생물학회지:발생과생식
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• 제6권2호
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• pp.71-76
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• 2002

최첨단 연구로 성장하고 있는 해양생물공학 연구 중 염색체공학 연구의 배수체 조작, 성분화, 자성 발생, 형질전환 어류와 유전자 조작에 관한 최근의 연구 동향을 분석하고 향후 발전 방향에 관하여 살펴보았다. 배수체 연구 중 3배체 어류 생산 연구는 20여종의 어류들 중 무지개 송어와 메기가 물리적 충격을 이용한 실험법으로 산업적으로 이용 가능한 수준에 있다. 성분화를 포함한 약 15종의 고급 어종이 방사선 투사법에 의해 자성 발생 2배체를 생산 유도하였다. 형질전환 어류는 1985년 이래 약 40여종의 어류가 외래 유전자를 미세 조작이나 전기충격법에 의해 형질전환되고 있다. 최근에 역바이러스성 벡터들에 의한 외래 유전자를 이용한 형질전환 어류가 생산되었으며 이러한 형질전환 어류는 기초과학 육성은 물론 해양생물공학 연구의 발전이 기대되고 있다.

• 식물조직배양학회지
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• 제24권6호
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• pp.319-322
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• 1997

벼의 미수분자방배양에서도 저온처리의 효과가 인정되어 $12^{\circ}C$에 10일동안 전처리된 자방배양에서 캘러스형성률과 식물체 재분화율이 각각 10.7%와 38.7%로 가장 높게 나타났다. 액체배지에서 보다 6 g/L의 gelrite를 첨가한 고체배지에 자방을 배양하였을 때 식물체 재분화율이 높게 나타났다. 미수분 자방을 1 mg/L의 picloram이 첨가된 배지에 배양하였을 때 녹색체 분화율이 가장 높았고, picloram의 농도가 그 이상 증가함에 따라 백색체의 출현빈도가 높아지는 경향이었다. 저온처리된 이삭으로부터 자방만 분리하여 배양하였을때는 캘러스가 형성되지 않았으나 내외영의 일부와 자방 및 소지경이 2차지경에 부착된 상태로 배양하였을때 캘러스 형성률과 식물체 재분화율이 각각 9.4%와 39.3%로 가장 높게 나타났다.