Studies On Transformation Of Lespedeza Bicolor By Antisense 4CL Gene

Lespedeza bicolor is a sort of perennial deciduous shrub with high adaptability and tolerance against adversity.It is a good feed of plant which belongs to legume.Besides,it has a great deal of potential values.4CL is a key enzyme during the systhesis process of lignin.We can decrease the content of ligin of Lespedeza bicolor by transferring antisense gene of 4CL into its genomic.It is with a remarkable significance for quality improving of Lespedeza bicolor feeds by decreaing the content of lignin.In this text we studied many factors impacting the transformation process of Lespedeza bicolor, got a optimized transformation system and transformed antisense 4CL gene into Lespedeza bicolor. Results as follows:1.Anitbiotic sensitivity:In the stage of cotyledon node,the proper concentration of kanamycin and G418 were oppositely 20mg/Land 7.5 mg/L;In the multiplication system,the proper concentration of kanamycin and G418 were 150 mg/L and 15 mg/L separately;During the rooting stage,the proper concentration of kanamycin and G418 were 20 mg/L and 5 mg/L correspondingly; the appropriate concentration of cephalothin was 0～300 mg/L used in each stage.And when the concentration of cephalothin reached 200～300 mg/L,it can inhibit growing of agrobacterium LBA4404 and C58 considerably.2.Eastablishment and optimization of transformation system:Pre-culture time was 48h,after that the material was infected by agrobacterium solution with a OD₆₀₀ value range of 0.4～0.6,infection time is 20～25 minutes and then co-cultured them for 2d.When we set the delayed choice time at 3d, the transformed efficiency could be highly improved.3.Molecular indentification and morphological observation of transformed plant:We got two resistant plants into which foreign gene has been inserted,this could be proved by PCR,PCR -Southern and Sounthem blotting detection.The DBH of transformed plant increased remarkably,the shape and leaf color is normal but the single leaf area is larger than that of control group.The growing rate of transformed plant is slower than that of control group,so the resistance decreased.There was a significant difference between transformed and untransformed plants after staining by phloroglucinol. So we can conclude that the content of lingin of transformed decreased indeedly.