Glycinebetaine(GB),an osmotic substance,could improve some stress tolerance in plants.CodA gene,originating from bacteria,could translate choline oxidase which stimulates the synthesis of GB in plants.To create lily l...Glycinebetaine(GB),an osmotic substance,could improve some stress tolerance in plants.CodA gene,originating from bacteria,could translate choline oxidase which stimulates the synthesis of GB in plants.To create lily lines resistant to heat,Belladonna lily and Yelloween lily had been transferred CodA gene through Agrobacterium tumefaciens.The bacteria harbored a binary vector carrying the hygromycin phosphotransferase,choline oxidase(CodA)and intron-containingβ-glucuronidase(Gus)genes were co-cultivated with lily bulb scales slides.The result showed that most the bulb scales had developed into bulblets in a regulator-free growth medium,while some expressed the hygromycin-resistance,heat tolerance and Gus gene expression.Among them,one line demonstrated primarily the transcription level expression through reverse transcription polymerase chain reaction(RT-PCR).Moreover,they were tested with the accumulation of GB which was evident that the transferred line had four times of GB volumes higher than that of wild type.The original evidence could open a right approach to enhance stress tolerance in lily 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
The green-fluorescent protein （gfp） gene was evaluated as a screening marker during cotton （Gossypium hirsutum L.） transforming and plant regeneration. High expression of GFP （green-fluorescent protein） was obse...The green-fluorescent protein （gfp） gene was evaluated as a screening marker during cotton （Gossypium hirsutum L.） transforming and plant regeneration. High expression of GFP （green-fluorescent protein） was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators （PGRs） was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T~ progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T~ progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.展开更多
文摘Glycinebetaine(GB),an osmotic substance,could improve some stress tolerance in plants.CodA gene,originating from bacteria,could translate choline oxidase which stimulates the synthesis of GB in plants.To create lily lines resistant to heat,Belladonna lily and Yelloween lily had been transferred CodA gene through Agrobacterium tumefaciens.The bacteria harbored a binary vector carrying the hygromycin phosphotransferase,choline oxidase(CodA)and intron-containingβ-glucuronidase(Gus)genes were co-cultivated with lily bulb scales slides.The result showed that most the bulb scales had developed into bulblets in a regulator-free growth medium,while some expressed the hygromycin-resistance,heat tolerance and Gus gene expression.Among them,one line demonstrated primarily the transcription level expression through reverse transcription polymerase chain reaction(RT-PCR).Moreover,they were tested with the accumulation of GB which was evident that the transferred line had four times of GB volumes higher than that of wild type.The original evidence could open a right approach to enhance stress tolerance in lily 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supported by the Key Project for International Cooperation and Exchanges,National Natural Science Foundation of China (30810103911)the National Natural Science Foundation of China(30771368)the Doctoral Fund of Ministry of Education for Young Scholar,China (20070504087)
文摘The green-fluorescent protein （gfp） gene was evaluated as a screening marker during cotton （Gossypium hirsutum L.） transforming and plant regeneration. High expression of GFP （green-fluorescent protein） was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators （PGRs） was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T~ progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T~ progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.
