Establishment Of Transformation System In Chieh Qua And Transformtion Of The OsOxO1Gene

Chieh-qua (Benincasa hispida Cogn. var. chieh-qua How) is one of the special vegetable crops in South China. As well as the production area expanding, the occurrence of diseases is becoming more and more serious. However, considering its narrow genetic background, the breeding of Chieh-qua, especially for disease-resistant breeding, develops slowly in these years. In view of the function of transgenic technology in innovation of the germplasm resources, the exploration of efficient transgenic technology is necessary. In this research, different receptor tissues of Chieh-qua were used as materials to establish transformation system that was mediated by Agro bacterium. The main resμLts were as follows:1. Based on the existing optimization system for regeneration, Transformation system mediated by Agro bacterium was established using semi species as explants. In order to grasp the transformation system, the best concentrations of kanamycin screening and bacteriostatic antibiotic cephalosporin antibiotic were proved to be45mg/L and500mg/L. by this way, finally obtained9strains Kan resistant plant,and5strains of all were detected to be positive plants by the PCR detection; the conversion rate was0.125%.2. In the research, the inbred varietie of chieh-qua ’A39’ was used as the materials, the experiment optimized the transform system of insitu conversion methods.and also inquiry the influence for the growth and development of receptors by different processing on the transformation of chieh-qua, established the detailed preliminary transformation process for them, The experimental resμLts showed that, The method dipping the scathing seeds with the Agro bacterium(OD600=0.4) successfμLly get one transformed plant,and the best dipping time was12h, in which the conversion rate was0.2%; Growth point transformation method dialed the growing point of seedlings with the same Agro bacterium, The injection method had none transformed plants, and the dipping method got3transformed plants, the conversion rate was0.69%.3. Using the oxalate oxidase activity test、oxalic acid tolerance test、PCR and Southern blot test to detect and analysis the pattern that the objective gene integrated into the genome of Chieh qua. Showed that the OsOxO1gene had successfμLly expressed and the plant had patience to oxalic acid. However, resμLts of Southern blot and PCR show that some positive plants have the specific band, and most of them were by the low amount copy. Only the plant getting form dipping the scathing seeds with the Agro bacterium was the single copy. The resμLts preliminarily show that OxO gene had been integrated into the plant genome.