Estabishment Of In Planta Transformation System Of Maize And Preliminary Analysis Of Transformation Mechanism

In recent years, Agrobacterium-mediated in planta transformation has been widely used in Arabidopsis thaliana, chinese cabbage,rape,and alfalfa. However, the application in monocotyledon such as maize genetic transformation is rarely reported. The leading maize lnbred line Zheng58was used as receptor in this study, used orthogonal design, in order to set up the system of in planta transformation that is stable and efficient. Meanwhile, optimum conditions to clear up transformation system. Then, the transient expression of GUS gene based on this transformation system was used to study the problems where Agrobacterium to enter the inflorescence and the pathway of infection for Agrobacterium，w hich can provide rich materials and technical support for maize transformation. Main findings are as follows:1. The establishment of the transfomation system. Process steps are as follows: A. The cultivation of Agrobacterium tumefaciens and the preparation of transformation buffer B. In planta transformation: The culture medium was extracted by syring and inserted between the bract and the ear, then pushed it into cob at the beginning of flowering(the female inflorescence hold out3-5cm and no male inflorescence blossom out). The seeds were harvested after mature C. Herbicide resistance screening D. Molecule detection in resistant plant by PCR.2. Optimize the main factors that affect transformation by orthogonal design and confirm optimal parameters for the transformation system. The results show that: Inclusion of silwet L-77has the most impact to the result of transformation, the next is OD value of bacteria. The optimum condition for transformation is that Agrobacterium suspension(OD=0.9),30g/L of inclusion of sucrose and addition of0.03%silwet L-77, and the PCR positive rate reaches to2.38%.3. GUS gene transient expression is used to research the problems infection pathway and position by histochemical staining of plants. The results indicated that: GUS staining was observed on ovary tissue at the earliest, also other tissues such as rachilla and rachis can be observed at the later stage and the GUS staining can be found in young embryo at last, show that: the ovary tissue was infected at the earliest by Agrobacterium, other tissues can be transformed to obtain the transformational embryo GUS gene transient expression from upper inflorescence is higher than bottom showed that transformed cases occurred in the begining of flowering. The conversion rate can be incresed by early infection.These results provided experimental basis that is very important for the method of in planta transformation of Maize.