• Fisheries and Aquatic Sciences
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• v.20 no.11
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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.

• Journal of Aquaculture
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• v.12 no.3
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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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• v.4 no.1
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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.

• Journal of Aquaculture
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• v.20 no.3
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• pp.184-189
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• 2007

Treatment conditions for the induced diploid gynogenesis, a maternal-exclusive form of artificial parthenogenetic reproduction, were optimized in bagrid catfish (Leiocassis ussuriensis, Siluriformes). Optimal amounts of ultraviolet (UV) irradiation for the genetic inactivation of spermatozoa in bagrid catfish and Pseudobagrus fulvidraco were proven to be ranged from 3,600 to 4,800 $ergs/mm^2$ based on the examination of viability of embryos and haploid incidence. Haploid embryos were restored to diploidy by preventing the extrusion of the second polar body using cold shock treatment. Thermal treatments (4 or $6^{\circ}C$ for 30, 40 or 50 min) were carried out 3, 5 or 7 min post insemination. Best scores for embryo viability (38.6% of total eggs taken) and incidence of normal diploidy (87.9% of hatched larvae) were observed at the embryo group treated at $4^{\circ}C$ for 40 min, 5 min after insemination. Restoration of gynogenetic diploidy was confirmed based on the absence of haploid syndrome, cell size and/or nucleolar organizing region (NOR) counts.

• Journal of Ecology and Environment
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• v.31 no.4
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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.

• Journal of Aquaculture
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• v.13 no.4
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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.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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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.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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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.

• Development and Reproduction
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• v.6 no.2
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• pp.71-76
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• 2002

Present study was aimed to summary the recent reports of chromosomal technology such like a polyploidv, sex differentiation, gynogenesis, transgenic fish and gene manipulation. Triploid cells for rainbow trout and channel catfish were induced through thermal shocks of varying temperature levels and produced as a industrial use. A monosex fish with homogametic females of 15 species of high valued fish were produced by exposing to irradiation. It seemed that different irradiation was suitable to inactivate the sperm and block the formation in producing the gynogenetic diploids. Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using about 40 fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in marine biotechnological applications.

• Korean Journal of Plant Tissue Culture
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• v.24 no.6
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• pp.319-322
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• 1997

The optimal conditions for callus formation and plant regeneration were determined by the manipulation of culture method in unpollinated ovary culture of rice. The effect of cold pretreatment on callus formation and plant regeneration varied with duration of pretreatment. The maximum frequency (38.7%) of plant regeneration was obtained from the unpollinated ovary pretreated for 10 days at $12^{\circ}C$. The ability of callus formation and plant regeneration was higher on the medium with picloram (1 mg/L) than that of 2, 4-D (1 mg/L). However, the high concentration of picloram increased markedly the frequency of albino plant from unpollinated ovary-derived callus. Floral parts inoculated as a unit play important roles in callus formation and plant regeneration. Best result was obtained when ovary with partial cut glume, pedicel and secondary branch as a unit was cultured.