Binary Vectors,Agrobacterium Cells,Functional Vir Region,T-DNA Region,Plant Genomes

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	Binary Vectors - A binary vector consists of a pair of plasmids of which one plasmid contains disarmed T-DNA sequences (at least the left and right borders of T-DNA must be present), while the other contains vir region, and ordinarily lacks the entire T-DNA including the borders. The plasmid containing disarmed T-DNA is called mini-Ti or micro-Ti, e.g., Binl9 , and has the origins for replication in both E. coli and Agrobacterium. The DNA insert is integrated within the T-region of mini-Ti, and the recombinant mini-Ti is cloned in E. coli.
Transfer of recombinant mini- Ti from E. coli into Agrobacterium is achieved either by a three way cross or by direct transformation of an Agrobacterium strain containing the helper Ti plasmid. Alternatively, the mini-Ti itself may be capable of conjugational transfer. The 'helper' Ti plasmid is a Ti plasmid having a functional vir region but lacking the T-DNA region, including the border sequences. pAL4404 helper Ti plasmid is derived from the wild type pTiAch5 by deletion of the entire T-region. The recombinant mini-Ti Binl9 is introduced into Agrobacterium cells containing pAL4404 either by conjugation or direct transformation.
The vir genes present in 'helper' pAL4404 induce the transfer of T-DNA (containing the DNA insert) of the mini-Ti Binl9 into the plant cells. The transformed plant cells can be selected on kanamycin medium due to the gene neo present within the T-DNA. The binary system avoids the transfer into plant genomes of unnecessary sequences, which occurs in the case of co-integrate vectors. Although the maximum size of DNA insert acceptable by T-DNA appears to be greater than 50 kb.