Introduction and expression of multiple transgenes is frequently required for 
basic and applied studies. However, at present, multigene transformation is very 
difficult due to technical limitations of existing methods. Here, we describe a 
vector system for efficient multigene assembly and transformation. The system 
consists of a transformation-competent artificial chromosome (TAC)-based 
acceptor vector together with two donor vectors. By exploiting the CreloxP 
recombination system and homing endonucleases, multiple rounds of gene assembly 
cycling were carried out with alternate use of the donor vectors, and multiple 
genes were sequentially delivered into the TAC vector. With this system, we 
created constructs containing as many as 10 foreign DNA fragments. Multiple 
genes, including six resistant genes stacked in a construct, were transferred 
into rice genome by Agrobacterium-mediated transformation. This system extends 
the repertoire of molecular genetic studies and biotechnological endeavors by 
enabling simultaneous manipulation of multiple genes.