Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat.

Constitutive promoters are widely used to functionally characterise plant genes in transgenic plants, but their lack of specificity and poor control over protein expression can be a major disadvantage. On the other hand, promoters that provide precise regulation of temporal or spatial transgene expression facilitate such studies by targeting over-expression or knockdown of target genes to specific tissues and/or at particular developmental stages. Here, we used the uidA (beta-glucuronidase, GUS) reporter gene to demonstrate that the barley Hvhsp17 gene promoter can be induced by heat treatment of 38-40 °C for 1-2 h in transgenic wheat. The GUS enzyme was expressed only in those tissues directly exposed to heat and not in neighbouring leaf tissues. The induction of HSP::GUS was demonstrated in all organs and tissues tested, but expression in older tissues was lower. Generally, proximal root sections showed less GUS activity than in root tips. This heat-inducible promoter provides the ability to investigate the function of candidate genes by overexpression or by down-regulation of target gene expression (for example by RNAi) in selected tissues or developmental stages of a transgenic plant, limited only by the ability to apply a heat shock to the selected tissues. It also allows the investigation of genes that would be lethal or reduce fertility if expressed constitutively.

The maize Activator/Dissociation system is functional in hexaploid wheat through successive generations.

The aim of the present study was to provide useful background information and evidence of the functionality of the maize Activator/Dissociation (Ac/Ds) system in hexaploid wheat. Two transgenic parental wheat lines, one harbouring the immobilised Ac element (iAc) and the other the Ds element (pUbi[Ds-uidA]bar), were crossed. Transient GUS assays confirmed that the iAc transposase is active in hexaploid wheat. Selected F1 and F2 lines were analysed by PCR using primers specific to Ac, uidA and bar genes. The primer pair Ubi/bar-tag was used to detect excision of the Ds-uidA sequence, which occurred at a frequency of 39% in the F1 generation. Lines free of Ac and showing evidence of Ds excision were subject to Southern analysis, which indicated that at least one transposition event might have occurred in these lines. Although more evidence is required to unequivocally support the reintegration of the Ds element in the wheat genome, the evidence presented here nevertheless demonstrates the effectiveness and potential value of using this system to tag genes in wheat.

Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes.

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T3, T4, T5) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and "added" subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.