Another early problem with Agrobacterium-mediated transformation was the inability to transfer large DNA fragments to the plant genome. Although a maximum size limit for T-DNA gene transfer was not determined, the transfer of foreign DNAs larger than 30 kbp was not routinely achievable until about five years ago. This prevented the transfer of large genes or T-DNA sequences containing multiple genes. The problem was addressed by Hamilton and colleagues, who developed a novel binary vector system in which the T-DNA vector was based on a bacterial artificial chromosome and the vir component expressed higher levels of virG and virE. This bibac (binary BAC) vector facilitates the transfer of up to 150 kbp of DNA to the plant genome, allowing the introduction of gene libraries into plants and the simultaneous introduction of many genes on a cointegrate vector (34).
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