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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 timeefficient 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. [ABSTRACT FROM AUTHOR]

In recent years, the increasing knowledge on the molecular mechanisms underlying mineral uptake, transport, homeostasis and deposition within plants, has paved the way for a more targeted approach to improving the nutrient status of crop plants based on biotechnology. In the present paper we will briefly review existing knowledge on the distribution and transport pathways of iron in the two small grained cereals, barley and wheat, and focus on the efforts made to increase the iron content in cereals in general. However, mineral content is not the only factor of relevance for improving the nutritional status of poor populations. It is thus well documented that a number of plant components can act either as promoters or inhibitors of mineral uptake in the human digestive system (Frossard et al. J Sci Food Agric 80, 817-879 ; Brinch-Pedersen et al. J Cereal Sci 46, 308-326 ). The nutritional impact of increasing mineral content accordingly has to be seen in the context of mineral bioavailability. Finally, we will briefly report on recent data from barley, where laser capture microdissection of the different grain tissues combined with gene expression profiling has provided some insight into metal transport and deposition (Tauris et al. ). In the present paper we will provide a tentative and preliminary roadmap for iron trafficking in the barley grain. [ABSTRACT FROM AUTHOR]

Nutrients destined for the developing cereal grain encounter several restricting barriers on their path towards their final storage sites in the grain. In order to identify transporters and chelating agents that may be involved in transport and deposition of zinc in the barley grain, expression profiles have been generated of four different tissue types: the transfer cells, the aleurone layer, the endosperm, and the embryo. Cells from these tissues were isolated with the ‘laser capture microdissection’ technology and the extracted RNA was subjected to three rounds of T7-based amplification. The amplified RNA was subsequently hybridized to Affymetrix 22K Barley GeneChips. Due to the short average length of the amplified transcripts and the positioning of numerous probe sets at locations more than 400 base pairs (bp) from the poly(A)-tail, a normalization approach was used where the probe positions were taken into account. On the basis of the expression levels of a number of metal homeostasis genes, a working model is proposed for the translocation of zinc from the phloem to the storage sites in the developing grain. [ABSTRACT FROM PUBLISHER]