| The purpose of this study is to obtain winter transgenic rapeseed suitable for China with bar gene as a target gene, which taking atvantage of the most widely used agrobacterium-mediated plant transformation methods. In addition, analysis the gene transferability, spontaneous outcrossing and survival fitness between transgenic rapeseed and the various subspecies of Brassica rapa to evaluate gene flow within transgenic rapeseed and B. rapa.Fourty six transgenic plants were obtained from 3 groups explants about 500 cotyledons and 600 hypocotyls with a double-low disease-resistant winter varieties rapeseed "zhongshuang 7". There were 35 positive plants wthin the transformats, and the positive rate was 76%, and the transformation efficiency was 3.2%. Different lines of transgenic plants generations were analysised on copy number, expression level of target gene and foreign gene integration location. For bar gene expression analysis, leaves, stem, flowers, roots and seed such 5 tissues from 6 lines with different copy number were choose. The result showed that the copy number has no effect of expression, and bar gene expressed higher in leaves and stems, low in seeds and roots. On the other hand, a comprehensive study on growth potential, growth duration, yield-related traits, stress analysis, seed quality analysis of transgenic rapeseed lines was carried out. After studying T4-generation of transgenic family, we could conclude that there was no significantly change in agronomic traits, quality and viability, growth period and resistance between transgenic lines and control. And Z7B10, Z7B16 and Z7B22 were final choosen for environment release experimental after comparison of all these aspect.In order to obtain release detection method of these environment release experimental GM rapeseed lines, the franking sequence of Z7B10, Z7B16 and Z7B22 were analysied by genomic walking PCR method, and strain-specific detection methods were also established. 677bp, 1100bp and 964bp left border franking sequence were separated from these three lines, respectively, and a 718bp right border flanking sequence was isolated form Z7B22. Sequence analysis showed that the above sequence retrieval included vector sequence and genomic sequence, indicating that flanking sequence is true insertion sequence between transformtion. According to the three strains of isolated left and right borders of the flanking sequence, qualitative detection method of these three lines were established, the size of amplified fragments were 314bp,259bp and 384bp, respectivly. The specificity of the established methods were tested good after testing with other transgenic rapeseed, and detection sensitivity of them were five copies (0.013%),50 copies (0.13%) and five copies(0.013%), respectively. The event-specific detection method has a high degree of specificity and good sensitivity for the genetically modified rapeseed lines Z7B10, Z7B16 and Z7B22 identification.Gene transferability from transgenic rapeseed to various subspecies and varieties of B. rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa and the transgenic rapeseed Ms8×Rf3 ranged from 0.039% to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20h when pollinated with the transgenic rapeseed Ms8×Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10%-70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.Through this study we successfully obtained transgenic winter rapeseed material. And by study on crossability and survival fitness between transgenic rapeseed and B. rapa, we determinded the adventitious presence threshold of B. rapa, provided an important reference for environmental safety of transgenic rapeseed evaluation. |
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