Establishment Of The Transgenic Acceptor System In Maize Inbred Lines And The Genetic Transformation Of Barnase/Barstar Gene

It's an important approach of obtaining heterosis by using maize male sterility. Creating male sterility line by transgenic technology becomes a current research hotspot. Bamase gene and Barstar gene that express especially in plant anther are applied to creat male sterility line and restoring line, which becomes the most successful and consummative strategy. In this study, the plasmid pBBN (with Barnase and Bar gene) and pBBS(with Barstar gene and Bar gene) were transfered to maize eminent inbred lines by using the methods of particle bombardment and agrobacterium-mediated transformation. The optimal receiptant materials is embryonic callus, and in this study the constitution of medium that inducing embryonic callus from immature embryo were improved in order to establish a stable and high-efficient transformation system. The research results are as follows:1. Five maize inbred lines(18-599red, 18-599white, qi319, 178, 48-2) were induced through eight kinds of inducing mediums(N6 A . N6B. N6C. N6D, MS1. MS2. MS3, MS4). The results indicated that N6 A based on N6 medium was the best one, and 18-599hong, 18-599bai, qi319 three inbred lines were good receipant materials that had high efficiency of embryonic callus induction, but 48-2 was believed to bad material because of its low efficiency of embryonic callus induction.2. It's necessary to append 2,4-D to successive medium. The appendents of mannitol, AgNOs and pentefezol have different functions in successive medium, and they avail to the embryonic callus induction but do harm to the ability of regenerating plant, In order to achieve the optimal culture effection, these appendents had better be use 1~2 times in thesuccessive medium.3. The ability of embryonic callus induction and clone are determined by its genotype in maize inbred lines, and there are no raletive between the two facets.4. The plasmid pBBN and pBBS were transfered to maize eminent inbred lines, includes 18-599(red), 18-599(white) and qi319, by using the methods of particle bombardment and agrobacterium-mediated transformation. Using the herbicide (Basta) with concertration of 8 mg/L selected the transgenic embryonic callus, and the selested transgenic embryonic callusregenerate plants. There were 72 plants achieved, includes 23 plants transferred Barnase gene and 49 plants transferred Barstar gene. By way of avoiding getting artificial positive plants, we detected respectively several genes by PCR detection, includes purpose gene(Bamase and Barstar), maker gene(Bar), promotor(TA29). The results indicated there was 1 plant transgenic maize with Barnase gene and 2 plants transgenic maize with Barstar gene, they are 18-599(white). We further detected that three positive plants by PCR-Southern analysis, and the result was accordant with that of PCR detecting.The transgenic maize with Barnase gene expressed male sterility in the field, which showed elementarily that the foreign gene had been transferred into maize genome and expressed.