A Gateway-based platform for multigene plant transformation

The post-genomic era offers unrivalled opportunities for genetic manipulation of polygenic traits, multiple traits, and multiple gene products. However, remaining technical hurdles make the manipulation of multiple genes in plants difficult. Here we describe a Gateway-based vector system to enable multiple transgenes to be directly linked or fused. The vector system consists of a destination vector and two special attL-flanked entry vectors each containing an attR cassette incompatible with the attL. By multiple rounds of LR recombination reactions, which we call MultiRound Gateway, multiple transgenes can be delivered sequentially and indefinitely into the Gateway-compatible destination vector through alternate use of the two special entry vectors. In our proof-of-principle experiments we have used this vector system to construct a plant transformation vector containing seven functional DNA fragments, including a screening marker gene, two reporter genes and four matrix attachment region sequences. This system provides a platform for fully realizing the potential of plant genetic manipulation.