An Agrobacterium tumefaciens-mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective than octopine-type strain for corn multi-shoot tissues transformation. The average frequency of Glucuronidase (GUS)-positive explants obtained from 14 corn genotypes ranged from 36% to 76%. L-proline (0.7 g L-1) in the co-culture medium apparently improved the frequency of transformation. The newly initiated multi-shoot tissues were most responsive to Agrobacterium infection. A positive correlation was found between multi-shoot tissue susceptibility to Agrobacterium and the proportion of cells in G1 phase. Transformants were identified by reverse transcription Polymerase Chain Reaction (PCR) and by southern blot hybridization assays. The frequency of transformants was approximately 2% based on the number of multi-shoot explants co-cultivated with Agrobacterium.
Keywords: agrobacterium tumefaciens-mediated transformation; corn; multiple shoots; plant regeneration.