Abstract
Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.
Highlights
Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement
The discovery of RNA interference (RNAi) triggered by doublestranded RNA paved the way for the high-throughput production of loss-of-function mutants for functional genomics in plants, including cereals (Waterhouse et al, 1998)
The set of binary destination vectors for cereals described here allows the constitutive expression of RNAi sequences under the control of the double-enhanced CaMV 35S promoter (d35S) promoter, as well as of the ZmUbi1 and the OsAct1 promoter
Summary
Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley. A number of GATEWAY-based binary vector sets for plant functional genomics have been developed, thereby allowing overexpression or knock-down of effector genes, expression of fusion proteins (Karimi et al, 2002; Curtis and Grossniklaus, 2003; Chung et al, 2005; for review, see Earley et al, 2006), and transformation of multiple genes (Chen et al, 2006).
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