Beet And Transgenic Plant Regeneration System Influencing Factors

Sugar beet which provides approximately 30% of the world's sugar, is one of Chinese two major sugar crops. Since high degree of sugar beet self-incompatibility, it often leads to loss of fine individual genotype, and is hard to keep good breeding material for breeding. Nevertheless, the development of plant biotechnology has opened up a new way of sugar beet breeding. In this process, the establishment of sugar beet tissue culture regeneration and rapid propagation system, based on genetic engineering characterized by the recombinant DNA.In this study, diploid sugar beet were used to establish a sugar beet plant regeneration system. The foreign gene (OsAPX3) responsive to stress was ligated to plant expressing vector, and was transformed into sugar beet. Meanwhile, the experiment compared a series of conditions and methods of agrobacterium transformation. The major findings are as follows:1. The establishment of sugar beet regeneration systemThrough the analysis of different factors influencing regeneration, it can be summarized as follows:the direct regeneration medium is MS+6-BA 0.5 mg/L+NAA 0.5 mg/L; the petiole explants have higher recycling rate; the sugar beet genotype no.9 has higher direct regeneration; the callus induction medium is MS+6-BA 0.5 mg/L+ NAA 2.0 mg/L; the callus subculture medium is MS+6-BA 0.5 mg/L+NAA 1.0 mg/L +IAA 1.0 mg/L; Callus differentiation medium is MS+6-BA 0.5 mg/L+ZT 0.5 mg/L +NAA 0.25 mg/L.2. Optimization of sugar beet transgenic systemThe experiment compared and analyzed the factors which influenced agrobacterium transformation, anddetermined Km-transformant selection agent as concentration of 100 mg/L; the optimal infection bacteria concentration was OD600= 0.3 bacteria, and the optimal infection time was lOmin; the optimal days of explant co-culturing with agrobacterium was 4days; the optimum concentration of acetosyringone added was 100μmol/L. 3. Aquirment of OsAPX3 transgenic sugar beet plants.Rice OsAPX3 was transformed to sugar beet by using agrobacterium-mediated method. It obtained 27 Km resistant plants. By extraction of genomic DNA of all these seedlings, PCR detection was performed and 6-positive plants were found, PCR positive rate was 22%.