Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0...Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.展开更多
[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF wa...[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.展开更多
Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticid...Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.展开更多
In this paper, populus xeuramericana cv. Guariento was transformed with bean chitinase by Agrobacterium tumefaciens-mediated leaf disc method. Firstly, the leaf explants were pre-cultured at 25℃ for 2d. Secondly, the...In this paper, populus xeuramericana cv. Guariento was transformed with bean chitinase by Agrobacterium tumefaciens-mediated leaf disc method. Firstly, the leaf explants were pre-cultured at 25℃ for 2d. Secondly, they were infected in Agrobacterium tumefaciens suspension （OD600=0.5） for 20 rain, then were co-cultured for 3d in the dark. Thirdly, the explants were transferred to the selection culture medium （containing Kanamycin 40 mg.L^-1 and Cefotaxime Sodium 800 mg-L1） and incubated at 25℃ until resistance buds formed. Chitinase activity was determined for the positive plants by PCR and PCR-Southern blot hybridization analysis. And, chitinase activity of positive plants was significantly higher than that of control plant, and the highest ratio of activity of NO.4 to that of control was 3.41. It showed that bean chitinase gene had been expressed in the plant genome.展开更多
Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of ...Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of studying infectivity in sweet potato. Unlike other begomoviruses, it has proven difficult to infect sweet potato plants with sweepoviruses using infectious clones. A novel sweepovirus, called Sweet potato leaf curl virus-Jiangsu(SPLCV-JS), was recently identified in China. In addition, the infectivity of the SPLCV-JS clone has been demonstrated in Nicotiana benthamiana. Here we describe the agroinfection of the sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone using vacuum infiltration. Yellowing symptoms were observed in newly emerged leaves. Molecular analysis confirmed successful inoculation by the detection of viral DNA. A synergistic effect of SPLCV-JS and the heterologous betasatellite DNA-β of Tomato yellow leaf curl China virus isolate Y10(TYLCCNV-Y10) on enhanced symptom severity and viral DNA accumulation was confirmed. The development of a routine agroinoculation system in sweet potato with SPLCV-JS using vacuum infiltration should facilitate the molecular study of sweepovirus in this host and permit the evaluation of virus resistance of sweet potato plants in breeding programs.展开更多
文摘Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.
基金Supported by Bioreactor Important Special Item of 863-Program inthe "Eleventh Five-Year" Plan (No. 2007AA100503)Science and Technology Development Key Plan of Jilin Province( No.20070922)+1 种基金Cultivation Fund of Scientific and Technical Innovation Project Major Program of Higher Education Institutions ( No.70S018)Science and Technology Plan of Changchun City (No.06GG150)~~
文摘[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.
文摘Immature embryos of rice varieties "Xiushui11" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.
基金This work was supported by Heilongjiang Key Technologies R＆D Programme （No. GB06B303-4） and Heilongjiang Natural Science Foundation（No. ZJN04-0101）.
文摘In this paper, populus xeuramericana cv. Guariento was transformed with bean chitinase by Agrobacterium tumefaciens-mediated leaf disc method. Firstly, the leaf explants were pre-cultured at 25℃ for 2d. Secondly, they were infected in Agrobacterium tumefaciens suspension （OD600=0.5） for 20 rain, then were co-cultured for 3d in the dark. Thirdly, the explants were transferred to the selection culture medium （containing Kanamycin 40 mg.L^-1 and Cefotaxime Sodium 800 mg-L1） and incubated at 25℃ until resistance buds formed. Chitinase activity was determined for the positive plants by PCR and PCR-Southern blot hybridization analysis. And, chitinase activity of positive plants was significantly higher than that of control plant, and the highest ratio of activity of NO.4 to that of control was 3.41. It showed that bean chitinase gene had been expressed in the plant genome.
基金supported by grants from the National High Technology Research and Development Program of China (2012AA101204)Shanghai Municipal Afforestation and City Appearance and Environmental Sanitation Administration (G102410, F132427)
文摘Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of studying infectivity in sweet potato. Unlike other begomoviruses, it has proven difficult to infect sweet potato plants with sweepoviruses using infectious clones. A novel sweepovirus, called Sweet potato leaf curl virus-Jiangsu(SPLCV-JS), was recently identified in China. In addition, the infectivity of the SPLCV-JS clone has been demonstrated in Nicotiana benthamiana. Here we describe the agroinfection of the sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone using vacuum infiltration. Yellowing symptoms were observed in newly emerged leaves. Molecular analysis confirmed successful inoculation by the detection of viral DNA. A synergistic effect of SPLCV-JS and the heterologous betasatellite DNA-β of Tomato yellow leaf curl China virus isolate Y10(TYLCCNV-Y10) on enhanced symptom severity and viral DNA accumulation was confirmed. The development of a routine agroinoculation system in sweet potato with SPLCV-JS using vacuum infiltration should facilitate the molecular study of sweepovirus in this host and permit the evaluation of virus resistance of sweet potato plants in breeding programs.
