Preliminary Study On Agrobacterium Rhizogenes Mediated Genetic Transformation And Transformatic Root Regeneration Of Torenia Fournieri L.

In this study, the screening and identification of the different Agrobacterium rhizogenes strains were carried out using physiological, chemical , molecular and bioassay methods, and the vigor of different strains was also compared based on their characters; the transformation of torenia mediated by Agrobacterium rhizogenes, the hairy root formation and its regeneration were importantly studied. The purpose of this study is to provide theoretical and technical support for torenia transgenic hairy root and other molecular researches used by torenia.The comparation and identification of Agrobacterium rhizogenes strains were investigated using 3-ketolactose test, the differential acid production assay and the motility assay. The results showed that these physiological tests were quickly, sufficiently to identify the Agrobacterium rhizogenes strains; and the vigor of strains decreased in the turn of R1000, R1205, A4 and R1601. The logarithmic growth of the strains was from 16 h to 32 h. The OD600 value at 32 h from the highest to the lowest was R1000 R1205, R1601, R1022, A4 and15834. But the concentration of A4 at 40 h was higher than that of other strains. Four strains (A4, R1205, R1000, R1601) in this study were Agrobacterium rhizogenes according to PCR assay. Using cucumber seedlings as the transformatic model mediated by the strains (A4, R1205, R1000, R1601), the transformatic frequency of R1000 for cucumber seedlings was the highest (79.6%), which was followed by R1205, R1601and A4.The transformation of Torenia fournieri L mediated by Agrobacterium rhizogenes is reported. Almost all roots induced by four different bacterial strains R1000, R1601, A4 and R1205 were found to harbor the rolB gene according to the PCR assay and Southern blot analysis, the results showed that all roots that could grew on selection medium containing 250 mg/1 kanamycin were putative hairy roots.The effects of varying conditions on torenia transformation were investigated. Torenia transformation of R1000 and A4 were more efficient than that of R1025 or R1601. The optimum concentration of the strain was about 1.0(OD600 value). 2 ~3 days for co-cultivation were found to be sufficient for this transformation. The best concentration of acetosyringone was about 30 M. Silver nitrate below 4 mg/L caused little change in efficiency and higher concentration (above 10 mg/L) strongly inhibited hairy root induction. pH value increase from 4.5 to 6.5 strongly stimulated hairy root formation and higher pH value (above 7.5) inhibited it. Under the optimized condition, the transformation frequency of torenia approached 90%.No Calli were formed from any hairy root after 3 weeks culture. 7 buds were induced from 2.5% transformatic roots on 1/2 MS containing 1.0 mg/1 4-PU after 3 weeks culture. 86.7% buds surrived on 1/2 MS containing 250 mg/1 Km after 28d. Two of six buds were dead 40d after that were put on 1/2MS medium containing 0.1 mg/1 NAA, and the tall of other four buds was less 2 cm. The four buds still grew very slowly on the 1/2 MS containing 1 mg/1 GA.