| In order to establish an optimized system for callus induction, regeneration and high-efficient genetic transformation of wheat variety Alondra's, the effects of culture medium, hormones on the callus induction and plant regeneration of immature embryo were studied. It is found that when using the N6 medium, the use of 3 mg.L-12,4-D together with 1000 mg.L-1 CH got the best results for callus induction, and use of 4 mg.L-1ZT without any IAA had the highest regeneration frequency. The Vector pCAMBIA1301-220.6 was constructed and transformed into Alondra's by particle bombardment. Thirty plants with hygromycin-resistance were selected and regenerated. Among them,5 were identified to be positive by PCR, with a transformation frequency as 0.5%. The establishment of the system further enriched the wheat genotype for transformation, and this will be helpful for both the genetic improvement and gene function analysis in various background by genetic transformation.Agrobacterium-mediated transformation system has advantages for its singly copy insertion and stable expression of the transgene. However, in wheat, this system is low repeatable and the transformation efficiency is relatively low compared with other methods. This limits the its widely used in wheat transformation. In order to optimize the agrobacterium-mediated genetic transformation in wheat, using the callus induced from immature embryo of wheat varieties Yangmai 15 and Alondra's as materials and using the transient expression array of the Gus gene, main factors affecting the transformation efficiency was studied. The results showed that the optimized condition for the above two varieties were different under the same treatment. The best condition for Agrobacterium-mediated transformation in Yangmai 15 are:Firstly, pre-cultured without acetosyringone, then infected for 10 min with Agrobacterium tumefaciens bacterial at a concentration of OD600=0.2 and acetosyringone at a concentration of 100μmol/L, finally co-cultured for 1 day at the temperature of 25℃; Under the similar condition, the most transient expression frequency of Gus gene was obtained in Alondra's when infecting with Agrobacterim tumefaciens bacterial at a concentration of OD600=0.5 and then co-culturing for 2 days.Oxidative stress is one of the major factors causing injury to plants exposed to environmental stresses. Cells of plants will produce reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion radical, and hydroxyl radicals when encountering injurious environments. The sweep off ROS is an mechanism in plant for the release of hurt from injuries. Genetic transformation is an important alternative for improving ROS resistance level, and the cloning of related genes is critical for this approach. In our lab, using a isogenic line with powdery mildew resistance and its recurrent parent Yangmai 5, a SSH library induced by Erysiphe graminis DC.f.sp. tritici was constrcted. A full-length cDNA gene coding the ascorbate peroxidase was cloned and designated as Ta-APX.To characterize the function of this gene in ROS and powdery mildew resistance, a expression vector pAHC-Ta-APX was constructed and Ta-APX was transformed into wheat variety Yangmai 158 by microproject-bombardment. After two rounds of herbicide bialaphos selection and regeneration, we obtained 38 regenerated plants. These herbicide-resistant plants were further identified by PCR using primers of the Bar gene and the target gene, and 10 transgenic plants were identified. The To derived T1 progenies were identified by PCR using the primer of the target gene, and the result showed that all the T1 lines segregated with different segregation ratios. This indicated that the To plants were heterozygous for the Ta-APX, and the copy number were different from one line to another. The T1 progenies were evaluated for powdery mildew and scab resistance by artificial inoculation under the field condition, and all the T1 lines showed improved resistance at different level. However, the resistance level of different T1 lines showed significant difference, and there was also segregation for resistance with the same T1 lines, which is in accordance with the PCR results. The APX enzyme activity of two lines. T0-8 and T0-1, were further analyzed, the results showed that the enzyme activity was higher in the transgenic plants than that in the non-transgenic plants at all the analyzed time points in both treatments of powdery mildew and PEG6000,indicating that the transgenic plants improved their ability to regulate the enzyme activity under the environmental stresses, and this might contribute to their improved plant pathogenic resistance. The major agronomic traits of the T1 transgenic plants were also investigated. Compared with the control, the transgenic lines showed no significant difference for all the investigated traits except plant height. |
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