T-STRAND INTEGRATION IN MAIZE PROTOPLASTS AFTER CODELIVERY OF A T-DNASUBSTRATE AND VIRULENCE GENES

We describe a plant protoplast transformation method that provides tra nsformants with a simple pattern of integration of a foreign gene, The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without vir E2, together with a target plasmid containing a gene of interest flank ed by Agrobacterium T-DNA border repeat sequences of 25 bp, We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integrati on events was about 20-35% of the total number of transformants. The a ddition of virE2 doubled the transformation efficiency. The method des cribed here is of sufficient efficiency and simplicity to be useful fo r the production of transgenic plants with single-copy well-defined tr ansgenic inserts.