Transformation Of Linear Gene Cassette Into Aarabidopsis Thaliana By Floral-dip

Transgenic site of floral-dip method is female ovule, the process of this method does not require tissue culture and direct access to genetically modified seeds. Compared with traditional crop breeding, this method has many advantages such as short cycle, simple operation and high transgenic frequency. Agrobacterium tumefaciens mediated floral-dip method has successfully applied on some cruciferous plants. However, backbone sequence and resistant marker could affect the biological safety of transgenic plants. Therefore, we transferred a vector backbone-free gene cassette into Arabidopsis thaliana genome by floral-dip method, and our purpose was to explore a reliable transformation system without tissue culture, vector backbone and selectable marker.This study takes arabidopsis thaliana as material and NPTâ…¡gene as target gene, as it is time-saving and labor-saving to screen transformants of arabidopsis thaliana with kanamycin. We constructed a vector backbone-free and selectable marker-free linear NPTâ…¡gene cassette (nos promoter, NPTâ…¡open reading frame, and nos terminator, flanked by 25bp T-DNA borders), then transfer the cassette into arabidopsis thaliana by floral-dip method, the transformation solution is 1/2 MS+0.05% surfactant Silwet L-77+5% sucrose. The key features of this method centered on the subsequent application of DNA solution directly onto the flower bud. Transformation of arabidopsis thaliana with agrobacterium tumefaciens strain GV3101-pBI121 and plasmid pBI121 are used as controls, the three ways were compared on transgenic frequency, integration characteristics and genetic stability.The seeds were selected aseptically with kanamycin, the results of many independent repeat transformations showed that average transgenic frequency of agrobactrium tumefaciens, plasmid pBI121 and linear gene cassette were 0.72%,0.19% and 0.08%. The reproducibility of transformation of linear gene cassette is poor, and five of nine times repeat experiments get positive plants. PCR analysis of resistant plant showed the existence of NPTâ…¡gene in arabidopsis thaliana. Southern blot analysis of transgenic plants showed that one to four independent sites in the samples transformed with agrobacterium tumefaciens, and for the samples transformed with plasmid and linear fragment, independent sites were respectively two and one respectively. For the three ways, genetic analysis indicated that part of the plant showed a mendelian segregation for the NPTâ…¡gene.This study transfer a marker-free and vector-free gene cassette into arabidopsis thaliana genome by flower-dip, establish a new transformation system that non-needs tissue culture, selectable marker and vector backbone for Cruciferae. This method would be applied to some particular genes, such as the genes related with color or shape of plants, then transformed plants could be identified through change of color, shape or physiological screening. If only changing a physiological or biochemical property can improve the quality of receptor plants, we can get transgenic plants with biological safety through transformation of linear gene cassette into plants by floral-dip method. The system needs further improvement to enhance the transformation rate and stability, but we believe that it might become a favorable choice for vector-and marker-free transformation of Cruciferae.