Establishment Of A Vector And Selectable Marker-free Transformation Method For Wheat

Along with the wide application of transgenic technology recently, the biosafety of genetically modified (GM) crops attracted more attention than before. The selectable maker gene and vector backbone sequences are two important factors, which affect estimating the biosafety of GM crops. Now, vector and selectable marker-free transformants could not be obtain by Agrobacterium-mediated transformation and particle bombardment easily. However, the pollen tube pathway method was hopefully to solve the difficulty, although its transformation frequency was low and the transformation result could not be repeated well.In this study, to establish a repeatable and efficient vector and selectable marker-free transformation method for wheat, the ovary-drip method was presented based on the study of pollen tube pathway method, and the minimal gene cassette (ubiquitin promoter, smgfp open reading frame, nos terminator and T-DNA border sequence) was used for transformation simultaneously.The possible factors, which could affect pollen tube pathway method for wheat transformation, were studied firstly, including the observation of pollen tube growth and the trace of exogenous DNA with FITC (Fluorescein Isothiocyanate) label. The effect of different transformation location on the route of exogenous DNA entering into wheat ovary, the effect of transformation time and solution on the efficiency of exogenous DNA entering into wheat embryo sacs were observed in the progress of tracing exogenous DNA. Results showed that the pollen tube could grow into the ovary 20min after pollination; it was estimated that exogenous DNA entered into embryo sacs through the pollen-tube pathway; transformation location could affect the route of exogenous DNA entering into wheat ovary, because of the large cut, more exogenous DNA could enter; higher probability of exogenous DNA entering into embryo sacs was obtained when transformation was conducted 45 and 60 minutes after pollination; compared to TE solution, using 0.05%Silwet L-77 plus 5%sucrose solution as the transformation solution could shorten the time of exogenous DNA entering into embryo sacs.Based on the above results, a method named ovary-drip was presented. More PCR positive plants could obtain by ovary-drip (4.5%) than that by pollen tube pathway (2.0%), when transformation was conducted. Then the transformation time and solution of ovary-drip method was optimized. The result proved that higher frequency of PCR positive plants (8.3%) could be obtained when DNA was solved in 0.05%Silwet L-77 plus 5%sucrose, and transformation was implemented 60～90min after pollinations. Southern blot analysis indicated that the exogenous DNA was integrated into the wheat genome. The RT-PCR and Northern blot analysis showed exogenous DNA could be transcripted normally. The fluorescence detection confirmed smGFP could be expressed in the transformants.In conclusion, an efficient vector and selectable marker-free transformation method for wheat named ovary-drip was established.