Study On Establishment Of Plant Regeneration System And Transformation Of Chi-linker-Glu Gene By Agrobacterium Tumefaciens From Maize

Maize (Zea Mays,L.) is a widely grown cereal crop and the most important fodder crop in the world today. It is very important for engineered breeding for maize to establish an Agrobacterium-mediated transformation system for maize.This dissertation deals with regeneration and transformation of maize inbred lines.A system of high frequency regeneration and Agrobacterium-mediated transformation of maize inbred lines was established and some PPT-resistant calli and plantlets of maize inbred lines were obtained successfully.The establishment of regeneration and Agrobacterium-mediated transformation of maize inbred lines can provide the important references for further research. The main conclusions of this study are as follows:1.Immature embryos have been frequently used as an explant source in maize tissue culture and genetic transformation.In the study immature embryos of maize inbred lines 87-1,Zong3,137 and K12 were used as explants to optimize the regeneration medium by the orthogonal test method and dissecting effect of single factor on in vitro culture.The results showed that 2,4-D was leading role in the primary callus induction frequency on N6B5 medium.But L-pro,L-Asn and 2,4-D led to a significantly increase the embryogenic callus induction while CH bated it. The effect of subculture times on the frequency of plant regeneration was also significant,and the frequency of plant regeneration was highest at one or two time subculture.N6B5 medium supplemented with 10g/L mannitol reduced the browning rate of calli.1/2MS medium supplemented with 0.2mg/LKT was benefit to the generation of root. Through the system the frequency of plant regeneration reached 55.2～18.3%.2. In the dissertation,an efficient transformation system was developed for maize inbred lines 87-1,Zong3,137 using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency.The results showed that inclusion of acetosyringone(150μmol/L)in both infection medium and cocultivation medium led to a significantly increase in the transformation efficiency.Improved transformation efficiencies were obtained when immature embryos were incubated with Agrobacterium suspension cells(optical density,OD600=0.6)for 15 min in the infection medium.Optimized cocultivation was performed in the acidic medium(pH5.4)at 22℃in the dark for 60 hours.It improved transformation efficiency that type II calli was treated through high osmotic medium supplemented with 60g/L mannitol for 4 hours before inoculation.Delaying selection was beneficial to the survival of resistant calli.Using the optimized transformation procedure, PCR-positive transgenic plants were obtained from the 3 elite inbred lines. Expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. This system should facilitate the introduction of agronomically important genes into commercial genotypes.