Optimization Of Agrobacterium Tumefaciens-mediated Transformation System Of Maize Calli And Preliminary Study On Creating New Transgenic Male Sterile Lines In Maize By Genetic Engineering Method

Transformation of foreign gene into plants is highly efficiency in altering its characters. Agrobacterium tumefaciens-mediated transformation is the preferably approach in transgenic research due to its large transformed segments, less copies and stability of inheritance.In the study, we optimized the protocols of Agrobacterium tumefaciens-mediated transformation for maize embryonic calli using elite maize inbred lines Qi319 and R18-599 as the origin of explants. Two promoter sequences, Zm13 and NTPp13 were also isolated from maize and tobacco, respectively, and analyzed their specific expression. In addition, we isolated a deacetylase gene areE from Escherichia coli (E. coli) JM101, and constructed a male sterile chimeric gene for transformation of maize inbred line Qi319. With this system, we expect to acquire male sterile lines of maize, and develop good materials for utilization of heterosis in maize. The main results were summarized as follows:1. Optimized protocols of Agrobacterium-mediated transformation for maize embryonic calli and plant regeneration were developed. The results indicated that the following conditions were outstanding for the improvement of transformation efficiency of maize embryonic calli. The optimized conditions were 0.1% of Tween 20 in inoculation medium, the concentration of OD₆₀₀ 0.4 of the liquid inoculation medium, 10 min of inoculation, 200 uM of acetosyringone (AS) and 200 mg/L of L-cysteine in co-cultivation medium, the culture condition of 22 °C and pH 5.2 in the course of co-cultivation.2. Three Agrobacterium strains of C58, LBA4404 and EHA105 were used to inoculate maize inbred lines Qi319 and R18-599, respectively. The results indicated that the transformation efficiencies including the efficiencies of GUS transient expression and the regenerated resistant calli were significant difference between six combinations of bacteria strains and maize inbred lines. EHA105-Qi 319 was the optimized combination for transformation, its average GUS transient expression efficiency was 55.5%, and some up to 71.1%. The efficiency of average regenerated resistant calli was 14.4%, and some up to 20%. The optimized resistant threshold of transformed calli to hygromycin was 10 mg/L.3. Twenty-two resistant seedlings were regenerated after screened under 10 mg/L hygromycin. PCR analysis was conducted with those generated resistant T₀ plants and the efficiency of positive plants was 50%. Histochemical GUS assay was conducted with those PCR positive and control plants. The results indicated that intense blue staining was observed in the leaves of all PCR positive plants as expected, and no GUS expression was observed in the negative and wild type maize plants. The results proved that the foreign GUS gene was expressed in the T₀ generated maize plants and also confirmed the...