Particle bombardment transformation using minimal gene cassette （containing the promoter, open reading frame and terminator） is the novel trend in plant genetic transformation, and its use helps to alleviate the und...Particle bombardment transformation using minimal gene cassette （containing the promoter, open reading frame and terminator） is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T1 to T3 generations of the transgenic rice （Oryza sativa L.） lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of ‘false homozygotes’, with abnormal segregation ratios ranging from 35：1 to 144：1 （Basta-resistant： sensitive plants） in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T2 progeny. The homozygotes were obtained in three transgenic lines in T1 generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expres- sion interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.展开更多
Bar gene driven under the control of CaMV35s promotor was delivered into the immatureembroys of a japonica rice cultivar Jingying 119through biolistic approach. Two putativetransgenic plants were produced, which ex-pr...Bar gene driven under the control of CaMV35s promotor was delivered into the immatureembroys of a japonica rice cultivar Jingying 119through biolistic approach. Two putativetransgenic plants were produced, which ex-pressed Basta-resistance. One of the Basta-re-sistance transgenic rices was completely sterile(JY 119-3), the other was self-fertile (JY119-4).展开更多
The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. ...The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. The germination and growth of rice pollen grains on barnyard grass stigmas at 30 min, and 1-4 h after crossing by hand were observed with an optical microscope. The results were compared with the germination and growth of barnyard grass pollen grains at the corresponding time after self-pollination. The results showed that germination and growth of the pollen grains of the two varieties were similar on barnyard grass stigmas, but differed significantly from self-pollination of barnyard grass. Pollen grains germinated and pollen tubes penetrated stigmas normally, and the number of pollen grains being condensing or releasing their inclusions or having released them increased with the time after self-pollination. Pollen grains of transgenic rice on the stigmas of barnyard grass couldn't germinate or grow normally after crossing, neither could they penetrate the stigmas of barnyard grass. Therefore, it could be concluded that the sexual incompatibility between transgenic rice with bar gene and barnyard grass is due to the rice pollen being unable to penetrate the stigma of barnyard grass. Further proof of incompatibility lies in the fact that the emasculated barnyard grass pollinated with the rice pollen grains could not seed.展开更多
The pollen tube pathway method of transformation has been reported to be successful in most crops, but less successfu in maizc. DNA can be transferred by cutting the stigma following pollination and applying the DNA s...The pollen tube pathway method of transformation has been reported to be successful in most crops, but less successfu in maizc. DNA can be transferred by cutting the stigma following pollination and applying the DNA solution in a suitable period DNA presumably reaches the ovary by flowing down the pollen tube and then integrates into the just fertilized but undivided zygotic cells. To provide the molecular evidence for this procedure, the plasmids pGBIRC carrying a CaMV35S promoter-PPT acetyle transferase （bar） gene-nos terminator genc fusion construct were used. Total 3 276 seeds were produced from the ears trcated with DNA. It was found that 35 scedlings were GUS assay positive, but less intense than that of the positive controls, of which 17 were PCR amplification positive. But, only 13 of the seeds from the plants treated with DNA containing the bar gene were found to be resistant compared with the negative control. Less than 1.07% of progeny seedlings tested cxpressed a herbicide positive reaction and polymerase chain reaction （PCR） with seedling DNA did detect the bar genc. Morphological variation was observed in six plants. We succeed in obtain PPT-resistant maize inbred lines via pollen tube pathway展开更多
基金This work was supported by the National Natural Science Foundation of China (No. 30300221 and No. 30370132).
文摘Particle bombardment transformation using minimal gene cassette （containing the promoter, open reading frame and terminator） is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T1 to T3 generations of the transgenic rice （Oryza sativa L.） lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of ‘false homozygotes’, with abnormal segregation ratios ranging from 35：1 to 144：1 （Basta-resistant： sensitive plants） in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T2 progeny. The homozygotes were obtained in three transgenic lines in T1 generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expres- sion interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.
文摘Bar gene driven under the control of CaMV35s promotor was delivered into the immatureembroys of a japonica rice cultivar Jingying 119through biolistic approach. Two putativetransgenic plants were produced, which ex-pressed Basta-resistance. One of the Basta-re-sistance transgenic rices was completely sterile(JY 119-3), the other was self-fertile (JY119-4).
基金the Ministry ofScience and Technology,P.R.ChinaEvaluation of Safety of Transgenic Crops(E200102).
文摘The possibility of gene flow between two varieties of transgenic rice with bar gene (Y0003 and 99-t) (male) and barnyard grass(Echinochloa crusgalli var. mitis ) (female) was studied by means of reproductive biology. The germination and growth of rice pollen grains on barnyard grass stigmas at 30 min, and 1-4 h after crossing by hand were observed with an optical microscope. The results were compared with the germination and growth of barnyard grass pollen grains at the corresponding time after self-pollination. The results showed that germination and growth of the pollen grains of the two varieties were similar on barnyard grass stigmas, but differed significantly from self-pollination of barnyard grass. Pollen grains germinated and pollen tubes penetrated stigmas normally, and the number of pollen grains being condensing or releasing their inclusions or having released them increased with the time after self-pollination. Pollen grains of transgenic rice on the stigmas of barnyard grass couldn't germinate or grow normally after crossing, neither could they penetrate the stigmas of barnyard grass. Therefore, it could be concluded that the sexual incompatibility between transgenic rice with bar gene and barnyard grass is due to the rice pollen being unable to penetrate the stigma of barnyard grass. Further proof of incompatibility lies in the fact that the emasculated barnyard grass pollinated with the rice pollen grains could not seed.
文摘The pollen tube pathway method of transformation has been reported to be successful in most crops, but less successfu in maizc. DNA can be transferred by cutting the stigma following pollination and applying the DNA solution in a suitable period DNA presumably reaches the ovary by flowing down the pollen tube and then integrates into the just fertilized but undivided zygotic cells. To provide the molecular evidence for this procedure, the plasmids pGBIRC carrying a CaMV35S promoter-PPT acetyle transferase （bar） gene-nos terminator genc fusion construct were used. Total 3 276 seeds were produced from the ears trcated with DNA. It was found that 35 scedlings were GUS assay positive, but less intense than that of the positive controls, of which 17 were PCR amplification positive. But, only 13 of the seeds from the plants treated with DNA containing the bar gene were found to be resistant compared with the negative control. Less than 1.07% of progeny seedlings tested cxpressed a herbicide positive reaction and polymerase chain reaction （PCR） with seedling DNA did detect the bar genc. Morphological variation was observed in six plants. We succeed in obtain PPT-resistant maize inbred lines via pollen tube pathway
