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The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless of their sequence. In particular, the Gateway system was designed to join fragments in a predefined order, orientation, and reading frame. The recent development of transformation vectors and large-scale clone resources amply demonstrate that plant researchers have adopted the Gateway platform and that it will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts.Agrobacterium tumefaciens binary vectors are widely used for plant transformation. They vary in size, origin of replication, bacterial selectable markers, T-DNA borders, and overall structure. Binary vectors are cumbersome to handle in conventional cloning schemes involving DNA restriction and ligation reactions, and substantial efforts have been invested in the creation of smaller vectors with a choice of unique restriction sites within the T-DNA region (Hajdukiewicz et al., 1994;Hellens et al., 2000a;Goderis et al., 2002;Tzfira et al., 2005; http://www.cambia.org/). But the recent introduction of robust site-specific recombinational cloning methods has greatly facilitated the construction of expression units in a large variety of in vivo and in vitro systems (Marsischky and LaBaer, 2004). In particular, the Gateway technology developed originally by researchers at Life Technologies, Inc. , and now commercialized by Invitrogen, has been endorsed by a large community and compatible vectors have been created for most applications requiring the creation of recombinant DNA molecules. This review gives a summary of the site-specific Gateway recombinational cloning system and presents related vectors generated by different plant research laboratories. BASIC PRINCIPLES OF GATEWAY RECOMBINATIONAL CLONINGThe Gateway system takes advantage of the sitespecific recombination reactions enabling the bacteriophage l to integrate and excise itself in and out of a bacterial chromosome (for review, see Katzen, 2007). Gateway protocols rely essentially on the BP and LR clonase reactions . The BP reaction is catalyzed by the BP Clonase II enzyme mix that consists of the phage integrase and the integration host factor (Fig. 1A). The BP clonase mix transfers a DNA fragment of interest, for example a PCR product, flanked by two attB sites into a donor vector (pDONR) carrying two attP sites. After recombination of the matching attB and attP sites, the DNA fragment is inserted into the donor backbone, resulting in an entry clone (pENTR), and is flanked by two attL sites. Entry clones can also be assembled by restriction and ligation of DNA fragments in vectors in which multiple cloning sites are flanked by attL sites. In most cases, entry clones are by themselves not directly useful because attL sites are too long (...