Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize.

A novel constitutive promoter from the maize histone H2Bgene was recently identified. In this study, we characterised H2B promoter activity in both wheat and maize tissues using the gusA reporter gene and two synthetic versions of the pat (phosphinothricin acetyl transferase) selectable marker gene, namely mopat and popat. Analyses of transgenic plants showed that the H2B promoter is able to drive the expression of gusA to strong, constitutive levels in wheat and maize tissues. Using an H2B:mopat construct and phosphinothricin selection, we recovered transgenic wheat plants at efficiencies ranging from 0.3% to 7.4% (mean 1.6%), and the efficiency of selection ranged from 40% to 100% (mean 77.7%). In another application, H2B was combined with the maize Ubi-1 or the maize Adh-1 intron to drive the expression of mopat and popat. Transformation efficiencies with the Ubi-1 intron were between 1.4- to 16-fold greater than with the Adh-1 intron. However, the use of either of the introns was necessary for the recovery of transgenic plants. Mopat gave higher transformation efficiencies and induced higher levels of PAT protein in maize tissues than popat.

Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment.

Ten current European wheat varieties were transformed at efficiencies ranging from 1-17% (mean 4% across varieties) following modifications in particle bombardment and tissue culture procedures. All plants surviving phosphinothricin selection were screened for uidA and bar gene activity, and for the presence of marker gene sequences by PCR analysis. A minimum of 35% plant 'escape' frequency was achieved with selection on 4 mg l(-1) gluphosinate ammonium after shoot initiation. Mean co-transformation frequency with various genes-of-interest was 66%. The estimated number of insertions of the uidA gene in 25 lines were; 1-2 in 32%, 3-5 in 52%, and 6-8 in 16% of lines. In T(1) progenies, marker genes segregated in a Mendelian fashion in 50% of 39 lines analysed, as determined by transgene activity assays. Based on PCR analysis, it appeared that in some lines the occurrence of distorted segregation was due to poor transmission of the transgenes.

Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues.

The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to wheat (Triticum aestivum L.) scutellum and inflorescence tissue. The main factors studied were the DNA/gold precipitation process, bombardment parameters and tissue culture variables. Efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS expression in bombarded tissues. Of the parameters analysed, amount of plasmid DNA, spermidine concentration, presence of Ca++ ions, calcium chloride concentration, amount of gold particles, gold particle size, acceleration pressure, chamber vacuum pressure, bombardment distance, osmotic conditioning of tissues and type of auxin had a clear influence on transient gene expression. A bombardment procedure suitable for elite wheat varieties was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.