In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method o...In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.展开更多
[ Objective] The cold shock method was used to induce diploid gynogenesis of Silurus astus. [ Mthod ] The Silurus astus sperms were irradiated by ultraviolet, then conducted for fertilization. The different cold shock...[ Objective] The cold shock method was used to induce diploid gynogenesis of Silurus astus. [ Mthod ] The Silurus astus sperms were irradiated by ultraviolet, then conducted for fertilization. The different cold shock starting time and duration were set to observe diploid gynogenesis of Silurus astus. [ Result] Under such inducing condition that ultraviolet irradiated 15 min,cold shock started 5 rain after fertilization, and cold shock duartion was 40 rain,the survival rate of diploid Silurus astus reached the highest（8.5% ）. [ Conclusion] The experiment laid foundation for culturing new excellent Silurus astus variety and accumulated original files tor further study of gynogenesis development mechanism.展开更多
A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on scre...A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.展开更多
Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynoge...Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.展开更多
The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to reg...The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to regenerate the vitro plantlets tolerant. This study aims in the first step in the regeneration of plantlets tolerant to salinity from mature embryos culture derived from two Tunisian durum wheat varieties: improved (Razzek) and landrace (Jenah Khotifa (JK)) varieties. The tolerance evaluation to salt stress was applied in vitro (100 mmol·l-1 NaCl) and was based on various parameters. Our results showed that JK variety was distinguished by a stable response for all parameters tested: average weight of callus (368.1 mg for control and 307 mg under salt stress), callus regenerated percentage (36.6% for control and 35.7% under salt stress) and green shoots number/callus (17 for control and 17 under salt stress). This stability of response translates the adaptability of this variety to salinity. In order to fix regenerated JK plantlets in single generation and obtain HDs homozygous stable lines, in vitro gynogenesis technical is tested for this genotype. The Evaluation of gynogenetic capacity focused on about 1200 unfertilized ovaries of JK and was based on its ability to induction, differentiation, development of green shoots, and haploid plantlets regeneration. JK showed good tolerance to salinity and a relatively good response to gynogenesis.展开更多
基金supported by the National Natural Science Foundation of China(31171963)the Major Project of Chinese National Programs for Fundamental Research and Development(2011CB100600)
文摘In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.
基金the Key Technology Research (0324030026)Development Program of Henan Province and Key Programs of Zoology of Henan Province~~
文摘[ Objective] The cold shock method was used to induce diploid gynogenesis of Silurus astus. [ Mthod ] The Silurus astus sperms were irradiated by ultraviolet, then conducted for fertilization. The different cold shock starting time and duration were set to observe diploid gynogenesis of Silurus astus. [ Result] Under such inducing condition that ultraviolet irradiated 15 min,cold shock started 5 rain after fertilization, and cold shock duartion was 40 rain,the survival rate of diploid Silurus astus reached the highest（8.5% ）. [ Conclusion] The experiment laid foundation for culturing new excellent Silurus astus variety and accumulated original files tor further study of gynogenesis development mechanism.
基金This work was supported by the National Nat-ural Science Foundation of China(Grant No.30130240),the Chinese Academy of Sciences(GrantNo.KSCX2-SW-303).
文摘A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.
基金supported by the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (No. 2015A SKJ02)the National Natural Science Foundation of China (No. 31540063)
文摘Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.
文摘The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to regenerate the vitro plantlets tolerant. This study aims in the first step in the regeneration of plantlets tolerant to salinity from mature embryos culture derived from two Tunisian durum wheat varieties: improved (Razzek) and landrace (Jenah Khotifa (JK)) varieties. The tolerance evaluation to salt stress was applied in vitro (100 mmol·l-1 NaCl) and was based on various parameters. Our results showed that JK variety was distinguished by a stable response for all parameters tested: average weight of callus (368.1 mg for control and 307 mg under salt stress), callus regenerated percentage (36.6% for control and 35.7% under salt stress) and green shoots number/callus (17 for control and 17 under salt stress). This stability of response translates the adaptability of this variety to salinity. In order to fix regenerated JK plantlets in single generation and obtain HDs homozygous stable lines, in vitro gynogenesis technical is tested for this genotype. The Evaluation of gynogenetic capacity focused on about 1200 unfertilized ovaries of JK and was based on its ability to induction, differentiation, development of green shoots, and haploid plantlets regeneration. JK showed good tolerance to salinity and a relatively good response to gynogenesis.
