UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Abstract

Abstract Background Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids. Results By application of the USER™ system, we engineered a collection of binary vectors, termed UCE (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient expression. Conclusions The USER™ technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction.