| Although the selection marker gene makes it more convenient for genetic transformation of plants,but once obtained transformed plants, the presence of the marker gene will bring potential environmentalsafety and food safety issues. Therefore, it is the very prerequisite for the development of plant geneticengineering that how to build an effective transformation system to delete screening marker gene, andnurture plants without selectable marker gene. The technology of gene gun has two advantages: norestricted in species, and which can trransforme three or more genes. Therefore, this study conducted usingparticle bombardment transformation to co-transformed the resilience of genes DREB1A, DREB3A and theherbicide resistance genes bar into wheat, comparing the Influence of the different methods to conversionefficiency; by tracking the detection of T2transgenic plants to analysis the separation law of target genesand selectable marker gene. Providing the basis for co-transformation method to get marker-free transgenicwheat. The main results of this study are as follows:(1) Using gene gun technology to co-transforme DREB3A, DREB1A with the selectable marker genebar respectively: DREB3A+bar transformed into Xiaoyan328, DREB3A+DREB1A transformed intoXiaoyan328, DREB1A+bar transformed into Zhoumai27, DREB3A+DREB1A+bar transformed intoZhoumai27. After tissue culture there were184,111,32,111regenerated plants respectively,and thentransplanted them into the field.(2)After detection on T0regenerated plants by PCR, the results of its statistical got the plants ofREB3A+bar co-transformation were3, the plants of DREB3A+DREB1A co-transformation were4, theplants DREB1A+bar co-transformation were3, the plants of DREB3A+DREB1A+barco-transformation were2, the co-transformation rate is11.11%to75.00%, the total conversion rate ofDREB1A or DREB3A is0.30%~2.89%. These results suggests that the impact of different methods fortransformation is quite remarkable. In the process of transformation has not only the co-transformed plants,but also a part directly obtained T0generation transgenic plants containing only the target gene.(3) DREB4A and bar genes, W17and bar genes were respectively co-transformed, the plants of T2generation were detected by PCR, there are25plants containing only the target gene DREB4A; and5plants of transgenic wheat containing only the target gene W17. Separated frequency of DREB4A+/bar-genotype is42.38%, W17+/bar-genotype is3.25%. These results indicate that transgenic plants obtainedby co-transformation method can separate in posterity,then we can filter the selectable marker-freetransgenic plants. These experimental results laid the foundation for further improving the conversion efficiency ofwheat, and for obtaining marker-free transgenic plants. |
