Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar.

Most maize disease are caused by fungi pathogens,such as northern leaf blight,southern leaf blight,ear and stalk rots,and so on.In normal years,this disease makes maize yield lost by 10% and this percentage may be increased by 30%-40% in its popular years.However,it is very difficult for conventional breeding to develop an elite maize material with the resistance to all the fungi disease.Therefore,we can use transgenic method to transfer some genes related to the resistance of these disease into maize in order to obtain resistance materials.Chitinase and β-1,3-glucanase are two important enzymes which play a key role in the hydrolysis reaction of fungal cell wall,so it is useful in the control of fungi disease,meanwhile,the herbicide resistant gene bar are usually used as a selectable marker in maize transformation.The purpose of this research is to introduce the fused gene of chitinase and β-1,3-glucanase and bar as well into elite maize inbred Zheng58 for improving its resistance to fungi disease.The wounded shoot apical meristem(SAM) of germinated seedlings were used as the material for Agrobacterium mediated transformation.We report here: 1) An optimized transformation system for Agrobacterium mediated transformation of shoot apical meristem,using the optimal concentration of bacterial culture(the value of OD600 was 0.6) for infection,addition of 150 μmol/L acetosyringone(AS) in the bacterial suspension,the whole infection process was carried out in a vacuum desiccators with a negative pressure of 50 kPa for 12 minutes.2) Identified by herbicide screening procedure and PCR detection,13 transformed plants were obtained among the 32 herbicide-resistant plants,and the overall transformation was 2.6%.The preliminary evidences showed that the foreign genes had been introduced into the maize genome.This method circumvented the long period of tissue culture step and limitation of different seasons,in addition,many elite inbred lines which are recalcitrant in callus induction can be efficiently transformed by this method.