Update on Multiple-Gene Expression Systems in Plants Delivery of Multiple Transgenes to Plant Cells

The use of transgenic technologies for the genetic manipulation of plant species has had a profound impact on basic plant research and biotechnology. Overexpression of heterogonous genes, for example, is widely used for the introduction of novel traits into transgenic crop plants. Overexpression can also be used in combination with down-regulation and controlled expression (e.g. induced, developmentally regulated, or tissue specific) studies as a tool for basic plant research and for functional analysis of native genes in various model plants. Two crucial components are required for the transient and/or stable expression of foreign genes in plant cells. The first is a plasmid vector capable of carrying the foreign gene-encoding sequence and the regulatory elements, i.e. promoter and terminator sequences, needed for its expression in plant cells (thus collectively referred to as an expression cassette). The second is the biological or physical methods by which the vector can be delivered into the target plant cells. With the exception of virusmediated gene expression, where the distinction between the plasmid vector and the vehicle itself is rather blurry, biolistics, polyethylene glycol, and Agrobacteriummediated gene transfer all require plasmid vectors, some of which have been specifically designed for optimal use by a particular method. Thus, for example, small and multicopy plasmids are most useful vectors for biolistics and polyethylene glycol-mediated genetic transformation where large quantities of DNA are typically used in each experiment. On the other hand, single-copy plasmids carrying certain biological features have been specifically designed for use with Agrobacterium as the transformation vehicle. In the past several decades, a remarkable variety of plasmids suitable for the cloning, transfer, and expression of foreign genes in plant cells has been developed (e.g. Simoens et al., 1986; Hajdukiewicz et al., 1994; Hamilton, 1997; Xiang et al., 1999; Hellens et al., 2000b; Earley et al., 2006; Coutu et al., 2007). While these plasmids have proven instrumental in plant biology research and biotechnology, the basic designs of many of these vectors are quite restrictive and rarely permit the cloning and transfer of more than a single target gene (excluding the selection gene, which may or may not be present on the transformation vector) as a single unit into plant cells. The growing interest in dissecting and analyzing complex metabolic pathways and the need to exploit the full potential of multigene traits for plant biotechnology (for review, see Halpin et al., 2001; Halpin and Boerjan, 2003; Capell and Christou, 2004; Tyo et al., 2007) mandate the development of new methods and tools for the delivery and stacking of multiple genes in plant cells. Here, we review some of the methods that can be used for the delivery of multiple genes into plant cells, while focusing mainly on systems that allow the assembly of multigene plant transformation vectors.