Obtaining Of Corn Materials With BRi Gene By Agrobacterium-mediated Transformation And Gene Gun

The development of Genetic engineering and transgenic technology, makes up theweaknesss of traditional hybrid breeding way and provides new ideas and methods for theinnovation of the germplasm resources and crop quality improvement.Agrobacterium-mediated transformation and gene gun is the main two genetic transformationmethods. This test transformed BRi gene into type II callus derived from immature embryosof the inbred lines18-599, Qi319, A188and chang7-2via Agrobacterium–mediatedtransformation and gene gun. The results were as follows:1. Genetic type is the main factor influencing the production of type II callus. Immatureembryos from the four selected inbred lines18-599, Qi319, A188and chang7-2can be allinduced. Among them, the quality of18-599induced callus was good, the number was large,and it was suitable for genetic transformation; The particles of callus derived from Qi319andchang7-2was loose, the texture was hard, and the callus quality is bad; A188callus waswhite, particle loose, and has a certain number. The concentration of ABT in medium wasadjusted to1.5mg L-1, because in this condition the root had the maximum number and theaverage root number in every tree was largest.2. Infective intensity is one of the important factors impacting on agrobacterium-mediatedtransformation efficiency. Studies on infection time and Bacteria liquid concentration showedthat the best infective intensity for18-599was OD600=0.6,10min, for Qi319was OD600=0.4,20min.3. In particle bombardment process, callus pretreatment methods, tungsten powderconsumption, plasmid concentration had a certain effect on rate of transformation. Calluswith drying2h, then high permeability processing2h had the higher transformation rate thanthat only dealing with high permeability4h; Tungsten powder concentration for100μg pergun has the highest transformation rate and lower cost; Plasmid concentration for1.5μg pergun is best.4. Positive plants had been proved by the molecule detection. The PCR detection inagrobacterium-mediated transformation showed that A188obtained94plants, among them22plants were positive ones, the transformation rate was23.4%;208plants of18-599were gotten, but only26plants were positive, the transformation rate was12.5%; All20of Qi319only detected1positive, the transformation rate was5%; Chang7-2was not acquired apositive plant. In the PCR detection of plants gaining from gene gun, among46plants of18-5993were tested positive, the transformation rate was6.52%. Qi319had10plants, noneof them was positive. The result indicated that gene gun transformation rate is low. TheRT-PCR detection showed a total of eight plants were RT-PCR positive, explainingexogenous gene expression in leaf. According to southern blotting, we won thetransformation plants.