| Wheat is the main crop grown in many areas of the world.Molecular breeding made that the breeding period is shorten,the conventional breeding's weak is made up,and the development of genetic breeding is accelerated.In recent years,with the continuous improvement of transgenic technology,the study of transgenic wheat's technology is also becoming more and more thoroughly.Therefore,studying and exploring more simple,convenient,and efficient genetic transformation techniques is very important to promote the breeding of wheat genetic engineering.As pollen cells belong to haploid and single-cell,it is an ideal receptor of plant genetic transformation.The method of pollen electroporation is made DNA into the mature pollen,and then use the conventional cross-pollination to achieve genetic transformation,it is a transgenic method combining cross-pollination and electroporation.Morever,compared with the main transgenic methods such as Agrobacterium transformation and gene gun transformation method,pollen electroporation is more simple and it costs lowly,it also eliminates the process of tissue culture.It coud avoide the poor renewable function and is not easy to form chimera,there is also no receptor species restrictions.Currently,the use of pollen electrotransformation has been widely used in many plants,and this transgenitic method will be applied in deeper research and will have a broader future.Thus,the present study intends to use the results of the above experiments and use the wheat's mature pollen as transformation receptor,and apply the method of electrotransformation,by screening and optimization the electroporation's buffer and the germination's buffer in vitro to improve the wheat pollen's viability and germination in vitro.Thereby,this experiment could optimize the electroporation system,improve the seed setting rate and improve the wheat genetic transformation efficiency of pollen using electroporation transformation.Therefore,it could lay the foundation for the establishment of a stable and practical transformation of wheat pollen electroporation technology.In addition,this experiment used electroporation method to transfer GUS gene,DREB4A and W17,which is related to drought and cold-induced,to get transgenic plants.Finally,by studying the experiment,the following aspects was concluded:(1)Through transformation of wheat pollen cells by electroporation,the study used the FDA staining and then observed directly under an optical microscope,the statistical analysis showed that the most appropriate electroporation' s buffer is Pl,and at this point the pollen viability is highest as 74.30%,and it is significantly higher than other treatments,in addition,it is 20.26%higher than the control.(2)Through comparison of the proportions of different components of the germination,buffer in vitro after electroporation of wheat pollen,the results showed that the wheat pollen germination' buffer in vitro is best suited when the treatment is C3,which is 43.22%,and the treatment is also significantly higher than other treatments,in addition,it is 27.33%higher than the control P1.(3)Using the optimized electroporation's buffer P1 and the pollen germination' buffer in vitro C3 to conduct the pollination,and this study transformed the three genes,which include reporter gene GUS and two transcription factor genes DREB4A and W17.The result showed that the seed setting rate was 3.37%on average and it was higher 2.49%compared with the control;Having obtained the seed germination leaves,thie study showed that transfer DREB4A got two transgenic plants,the conversion rate of 0.59%after PCR assay. |
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