Transfer Of Bivalent Insect Genes (Bt,CpTI) Into Soybean Mediated By Agrobacterium Tumefaciens

Genetic transformation of introducing binary insecticidal genes into soybean was studied in this paper via Agrobacterium-mediated method with the cotyledonal nodes of main cultivated soybean species in the Northeast as genetic transformation receptor.Establishing of transformation receptor system.A high frequency regeneration (above 60%) transformation receptor system was established after the research on inducing of soybean cotyledonal nodes, buds germination, roots regeneration, as well as transplant of regeneration seedlings through a series of tissue culture technologies. The result showed that plant genotype, incretion proportion of culture medium, incised part of cotyledonal nodes as well as touch mode between cotyledonal nodes and culture mediums influenced inducing rate of buds directly. (1) WY6 ( MSB+2mg.L-1 6BA +0.2mg/L IBA) culture medium was the most suitable medium to multiple shoots in which nine species had highest inducing rate among twelve species in trials after selecting ten culture mediums. (2) Five higher frequency regeneration genotypes selected from soybean varieties in Northeast were used as receptor materials of genetic transformation. (3) Suitable material parts of cotyledonal nodes with one-third cotyledonal nodes and inoculation manners of inserting culture medium were made certain. (4) The kinds and concentrations of incretions were confirmed by exploring culture mediums for the length of buds and regeneration of roots.Agrobacterium-mediated method The higher regeneration frequency Jinlin 43 and Hefeng 25 species as receptor materials were selected. The explants of cotyledonal nodes were transformed by Agrobaterium tumefaciens LBA4404 with plasmid pGB11214ABC .Firstly, the conditions of Agrobacterium transformation were optimized. Through the experiment, the result showed that 200mg/L kanamycin was suitable and the concentration of bacteria liquid was OD600= 0.5-0.7. The time of inoculation between Agrobaterium and cotyledonal nodes was ten minutes. The concentration of acetosyringone was 100 mol/L and 2d time ofco-culture was optimal.30.1% resistant plants were obtained through selecting with kanamycin, then 11 plants positive seedlings were gained by PCR and Southern examination. The results were as follows: (1) Three factors, which affected the efficiency of genetic transformation of plants by Agrobacterium-mediated method, respectively were plant factors ( genotype, recept state, etc. ), agrobacterium factor ( bacterium species, active concentration, etc.) and outer factor (the time Two side factors were considered with using concentration of antibiotics. Firstly, whether it worked as selection or sterilization or not. Secondly, antibiotics influenced on the growth and develop of soybean itself. Optimal concentration of kanamycin was 200mg/L and that of Cef was 500mg/L with using cotyledonal nodes o of inoculation, the concentration of AS and the time of co-culture, etc. ) . (2) f explants in terms of above two facets in this experiment.