Culture And Exogenous Gene Transformation Of Suspension Cells In Soybean

Soybean (Glycine max (L.) Merrill), as an important crop, occupies an importantposition in food production for its high nutritional value that richness in protein andvegetable oil as well as a variety of physiological beneficial components. With humanpopulation growth and improvement of living standards, people have higherrequirements on the yield and quality of soybeans. Since the success of the firsttransgenic soybeans in1988, many researchers conducted gene transformation withsoybean and tried to transgene into soybean cell in order to improve quality of soybeanvariety, but fewer people succeed. It is still a research hot point for domestic and foreignscholars to establish a stable and efficient genetic transformation system to achievesoybean varieties with higher quality and yield.Boron is an essential trace element for plant growth and development and plays animportant role in maintaining the normal structure and function of plant cell walls andother functions. In this experiment, a soybean suspension cells was initially establishedwith the callus induced from the hypocotyl of sterile seedling of Jilinxiaoli7soybean.And then the influences of concentrations of boric acid on the growth of suspension cellcultures (fresh weight) were determined and the transformation of exogenous GUS geneto the cells was conducted by using Agrobacterium mediation. The transformationefficiencies influenced by sonication duration and boric acid concentration were mainlyinvestigated through histochemical method in order to find an appropriate genetictransformation way. The positive transformed cell clumps were further identified byPCR technique and Southern blot hybridization.The results showed that:1. Soybean hypocotyl was better than the cotyledon nodeas explants to induce callus which was large in size, yellow in color and loose instructure for easily establishing a stable soybean suspension cells, and the optimummedium was MSB+2,4-D0.8mg/L+KT0.6mg/L.2. During soybean suspension cellculture process, the soybean suspension cells was shortest in transition period and mainly uniform spherical in shape under the normal concentration of boric acidconcentration (6.2mg/L); when slightly increasing the concentration up to10mg/L, thetransition period of cells became longer but the maximum growth was higher than thatof normal boric acid concentration; however, higher or lower boron acid medium wouldhad an inhibition effect on soybean cell growth especially at a high level boron of morethan100mg/L and the cells became abnormal like a bar shape.3. Foragrobacterium-mediated genetic transformation of soybean suspension cells, thenumber of cell clumps of GUS staining was25with the assist of3min sonication,which had little deviation and good stability compared to other sonication times.Moreover, the appropriate increase in boric acid concentration (10~30mg/L) couldimprove the transformation efficiency of the cell suspension, which was also helpful tothe screening of the transformant in the transformation process. Twenty eighttransformants were obtained in1mg transformed cells under30mg/L boric acidconcentration with the assistance of3min sonication.