Optimization Of Regenerated System In Somatic Embryos Of Soybean And Genetic Transformation Of Bovalent Insect Resistant Genes In Soybean

The subculture ,germination and regeneration about somatic embryos of soybean were studied.Optimization of the regenerated system in somatic embryos of soybean were also carried out. The medium for subculture was MS withl0-20mg/L2,4-D,The subculture coefficiency reached 3n,The germination frequency increased with the normal somatic embryos of soybean when dry treament on somatic embryos, active carbon and cane sugar with high concentration were applied.Establishment of the concentration of filter medicament kanamycin (km).Before rooting, l00mg/L Km concentration was applied, After Rooting, Km concentration reduced to 25mg/L.The working concentration of cefotaxine (Cef) has been determined, 300mg/L Cef concentration was used initially in medium,and reduced l00mg/L each time for changing medium until reduced to 0.The factors influencing genetic transformation about somatic embryos of soybean had been studied. Mediated by Agrobacterium tumefaciens ,The suitable condition was determined as followed: injured treatment of somatic embryos of soybean,pre-culture one day and a half.the bacterial concentration reached 0.5-0.7 (OD600) ,2-3 days co-culture ,10 minutes infection time,50-100umol/L AS.While via microprojectile bombardment, The suitable conditions were designed as twice bombardment, 1100 Psi Helium gas pressure,2.5mol/L CaCl2 concentration. The transient expression of Gus gene was examined in such condition with somatic embryos of soybean.Many obvious blue spots appeared on the surface of somatic embryos of soybean after GUS dying.lt was indicated that the target gene had been transformed into soybean. PCR technique was further used to identify the resistance plants after biotic selection.5 positive plants were obtained mediated by Agrobacterium tumefaciens, Among them 2 for Dong Nong LI3,1 for CH21141, 1 for HE Feng 25,1 for Progress,Each are positive with transformation frequency 0.72%.While via microprojectile bombardment, 2 positive plants were also obseverd,The transformation frequency reached 0.50%. These positive plantlets were further analyzed by dot blotting and PCR-Southern blotting.These results demonstrated that the target gene had been transformed into the soybean genome.