Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and ...Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.展开更多
The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA r...The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.展开更多
CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and ...CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.展开更多
In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bomb...In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30～40mg/L Hyg or 150～200mg/L G418 or 10～20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ～24h before bombardment to 24～48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.展开更多
Nitrogen(N)fertilization is necessary for obtaining high rice yield.But excessive N fertilizer reduces rice plant N efficiency and causes negative effects such as environmental pollution.In this study,we assembled key...Nitrogen(N)fertilization is necessary for obtaining high rice yield.But excessive N fertilizer reduces rice plant N efficiency and causes negative effects such as environmental pollution.In this study,we assembled key genes involved in different nodes of N pathways to boost nitrate and ammonium uptake and assimilation,and to strengthen amino acid utilization to increase grain yield and nitrogen use efficiency(NUE)in rice.The combinations OsNPF8.9a×OsNR2,OsAMT1;2×OsGS1;2×OsAS1,and OsGS2×OsAS2×OsANT3 optimized nitrate assimilation,ammonium conversion,and N reutilization,respectively.In co-overexpressing rice lines obtained by co-transformation,the tiller number,biomass,and grain yield per plant of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of wild-type ZH11,the OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line,and the OsGS2×OsAS2×OsANT3-overexpressing line.The glutamine synthase activity,free amino acids,and nitrogen utilization efficiency(NUt E)of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of ZH11 and other lines that combined key genes.N influx efficiency was increased in the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line and OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line under a low ammonium and a low nitrate treatment,respectively.We propose that combining overexpression of OsAMT1;2,OsGS1;2,and OsAS1 is a promising breeding strategy for systematically increasing rice grain yield and NUE by focusing on key nodes in the N pathway.展开更多
基金supported by the National Major Project for Developing New GM Crops (2016ZX08001-003)
文摘Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.
文摘The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.
基金supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No.Z305650 and No.Y3080361)the Science and Technology Department of Zhejiang Province,China (Grant No.2004C12020)+1 种基金the National Key Technology Research & Development Program,China (Grant No.2006BAD01A01-5 and No.2008ZX08001-001)the 151 Foundation for the Talents of Zhejiang Province,China
文摘CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.
基金the Chinese 863 High-Technology Program(NO.863-101-01-01-01)and Chinese Post-Doctoral Science Foundation(2001-14)
文摘In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30～40mg/L Hyg or 150～200mg/L G418 or 10～20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ～24h before bombardment to 24～48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.
基金supported by the National Natural Science Foundation of China(32260498)the Guizhou Provincial Science and Technology Project(qiankehejichu-ZK(2022)Key 008)+2 种基金the Wuhan Science and Technology Project(2020020601012259)the Guizhou Provincial Science and Technology Support Plan(qiankehezhicheng(2022)Key 026)the Key Cultivation Project of Guizhou University(201903)。
文摘Nitrogen(N)fertilization is necessary for obtaining high rice yield.But excessive N fertilizer reduces rice plant N efficiency and causes negative effects such as environmental pollution.In this study,we assembled key genes involved in different nodes of N pathways to boost nitrate and ammonium uptake and assimilation,and to strengthen amino acid utilization to increase grain yield and nitrogen use efficiency(NUE)in rice.The combinations OsNPF8.9a×OsNR2,OsAMT1;2×OsGS1;2×OsAS1,and OsGS2×OsAS2×OsANT3 optimized nitrate assimilation,ammonium conversion,and N reutilization,respectively.In co-overexpressing rice lines obtained by co-transformation,the tiller number,biomass,and grain yield per plant of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of wild-type ZH11,the OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line,and the OsGS2×OsAS2×OsANT3-overexpressing line.The glutamine synthase activity,free amino acids,and nitrogen utilization efficiency(NUt E)of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of ZH11 and other lines that combined key genes.N influx efficiency was increased in the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line and OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line under a low ammonium and a low nitrate treatment,respectively.We propose that combining overexpression of OsAMT1;2,OsGS1;2,and OsAS1 is a promising breeding strategy for systematically increasing rice grain yield and NUE by focusing on key nodes in the N pathway.
