Crop breeding schemes can be significantly accelerated by using(doubled)haploid plants.In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes,an...Crop breeding schemes can be significantly accelerated by using(doubled)haploid plants.In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes,and haploidization rates are generally very low.Therefore,methodological improvements to and new concepts for haploidization are required.Here,we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases(ECSs).We show that after successful sperm–egg cell fusion,ECSs play a critical role to ensure male and female nucleus fusion after fertilization.The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice.In summary,our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.展开更多
In flowering plants(angiosperms),fertilization of the egg cell by one sperm cell produces an embryo,whereas fusion of a second sperm cell with the central cell generates the endosperm.In most angiosperms like Arabidop...In flowering plants(angiosperms),fertilization of the egg cell by one sperm cell produces an embryo,whereas fusion of a second sperm cell with the central cell generates the endosperm.In most angiosperms like Arabidopsis,a pollen grain contains two isomorphic sperm cells required for this double fertilization process.A long-standing unsolved question is whether the two fertilization events have any preference.A tool to address this question is the usage of the cyclin-dependent kinase a1(cdka;1)mutant pollen,which produces a single sperm-like cell(SLC).Here,we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC.Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell,precursor of the two sperm cells.Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2%of the ovules,single fertilization of the egg cell occurred.Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery,which fertilized the central cell,resulting in 20.7%double-fertilized ovules.This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell.Taken together,our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.展开更多
Electrofusion between blastula cells and unfertilized eggs in loach were investigated usingdielectrophoretic field where, under alternating sinusoidal electric field, blastula cells formed beads-like chain in close co...Electrofusion between blastula cells and unfertilized eggs in loach were investigated usingdielectrophoretic field where, under alternating sinusoidal electric field, blastula cells formed beads-like chain in close contact with the unfertilized egg and cell fusion occurred between eggs and thecells in tight contact with them. The nuclei ofblastula cells were brought into the cytoplasm of therecipient eggs, where they promoted the development of the fused eggs just like the zygote nuclei.But the development of the fused eggs was different from that of zygotes. Several nuclei might enterone and the same egg simultaneously and all of them could undergo division, resulting in severalblastomere after the first cleavage of the recipient egg. Before blastula stage, the embryo developingfrom the fused egg showcd irregular shape, but it was soon regulated and developed to a normalblastula which often continued its development into a normal individual. Cell/egg electrofusion cameto its highest fosion rate (80%) 8nd hatching rate (20%), with cell density at 1×10~3 cells/ml, Ca^(++)concentration at 10 mM, mannitol at 0.2 M and when the blastula cells were digested with 100μg/ml pronase E for 6-10 min at 20℃. The mechanism underlying development of electrofused eggsis discussed. As the result indicates, electrofusion might prove to be a promising biotechnology justas nuclear transplantation.展开更多
Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nucl...Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.展开更多
A new cell line was established from 5-day-old embryonated eggs of Dendrolimus superans and has been designated NEAU-Ds-950821 .The cell line consists of mixture of cell types, including majority of spherical shaped c...A new cell line was established from 5-day-old embryonated eggs of Dendrolimus superans and has been designated NEAU-Ds-950821 .The cell line consists of mixture of cell types, including majority of spherical shaped cells and a few of spindle shaped cells. The cell line has a population of doubling time of 52.6 h. Chromosome analysis levealed typical lepidopteran chromosomes. lsozyme characterization of Esterase showed the patterns were different from other three cell lines (Ms-927311. Xc-920730, and SF21AE). Virus infectivity tests revealed the cell linc can support D. superans cytoplasmic polyhedrosis virus.展开更多
基金supported by the National Natural Science Foundation of China(grants 32130031 and 32000248)the Major Project of Hubei Hongshan Laboratory(2022hszd017)the China Postdoctoral Science Foundation(grants 2021M702525 and BX20200256).
文摘Crop breeding schemes can be significantly accelerated by using(doubled)haploid plants.In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes,and haploidization rates are generally very low.Therefore,methodological improvements to and new concepts for haploidization are required.Here,we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases(ECSs).We show that after successful sperm–egg cell fusion,ECSs play a critical role to ensure male and female nucleus fusion after fertilization.The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice.In summary,our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.
基金supported by the National Natural Science Foundation of China(Grant No.31991202,32122014,31830004,and 32070854)Young Elite Scientists Sponsorship Program by China Association of Science&Technology(Grant No.2019QNRC001)supported by the Peking-Tsinghua Joint Center for Life Sciences。
文摘In flowering plants(angiosperms),fertilization of the egg cell by one sperm cell produces an embryo,whereas fusion of a second sperm cell with the central cell generates the endosperm.In most angiosperms like Arabidopsis,a pollen grain contains two isomorphic sperm cells required for this double fertilization process.A long-standing unsolved question is whether the two fertilization events have any preference.A tool to address this question is the usage of the cyclin-dependent kinase a1(cdka;1)mutant pollen,which produces a single sperm-like cell(SLC).Here,we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC.Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell,precursor of the two sperm cells.Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2%of the ovules,single fertilization of the egg cell occurred.Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery,which fertilized the central cell,resulting in 20.7%double-fertilized ovules.This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell.Taken together,our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.
基金This work was supported by grants from Chinese National High-Tech.Project.
文摘Electrofusion between blastula cells and unfertilized eggs in loach were investigated usingdielectrophoretic field where, under alternating sinusoidal electric field, blastula cells formed beads-like chain in close contact with the unfertilized egg and cell fusion occurred between eggs and thecells in tight contact with them. The nuclei ofblastula cells were brought into the cytoplasm of therecipient eggs, where they promoted the development of the fused eggs just like the zygote nuclei.But the development of the fused eggs was different from that of zygotes. Several nuclei might enterone and the same egg simultaneously and all of them could undergo division, resulting in severalblastomere after the first cleavage of the recipient egg. Before blastula stage, the embryo developingfrom the fused egg showcd irregular shape, but it was soon regulated and developed to a normalblastula which often continued its development into a normal individual. Cell/egg electrofusion cameto its highest fosion rate (80%) 8nd hatching rate (20%), with cell density at 1×10~3 cells/ml, Ca^(++)concentration at 10 mM, mannitol at 0.2 M and when the blastula cells were digested with 100μg/ml pronase E for 6-10 min at 20℃. The mechanism underlying development of electrofused eggsis discussed. As the result indicates, electrofusion might prove to be a promising biotechnology justas nuclear transplantation.
文摘Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.
文摘A new cell line was established from 5-day-old embryonated eggs of Dendrolimus superans and has been designated NEAU-Ds-950821 .The cell line consists of mixture of cell types, including majority of spherical shaped cells and a few of spindle shaped cells. The cell line has a population of doubling time of 52.6 h. Chromosome analysis levealed typical lepidopteran chromosomes. lsozyme characterization of Esterase showed the patterns were different from other three cell lines (Ms-927311. Xc-920730, and SF21AE). Virus infectivity tests revealed the cell linc can support D. superans cytoplasmic polyhedrosis virus.
