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Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Sugarcane is a crop with great potential for metabolic engineering, but progress has been limited by highly efficient transgene silencing. The potential exists to utilize efficient gene silencing in molecular improvement through down-regulation of sugarcane genes. However, sugarcane is highly polyploid and heterozygous, which might complicate efforts to employ transgene-mediated silencing of endogenous genes. To explore this issue, we tested in sugarcane a construct designed for hairpin-mediated silencing of the phytoene desaturase (PDS) gene in sugarcane. The hairpin construct was designed based on the sugarcane PDS EST collection containing multiple alleles, to down-regulate the suite of PDS alleles. Three out of four plants transformed using the PDS hairpin construct showed detectable hairpin transcripts when analyzed by northern analysis, and displayed the photo-bleaching phenotype characteristic of PDS knockout lines. There was near-complete reduction in the levels of endogenous PDS transcripts in leaf tissue, indicating efficient hairpin-mediated down-regulation despite the highly polyploid and heterozygous sugarcane genome. Hairpin-mediated gene silencing should therefore be a powerful tool for the molecular improvement of this important crop.

The efficient use of transgenic technology for the improvement of phosphorus (P) nutrition in crop plants may require the expression of introduced genes only under conditions of phosphate deficiency and only in specific tissues. We have investigated the use of the promoter from the Arabidopsis Pht1;2 phosphate transporter to drive the expression of a secretable Aspergillus niger phytase gene only in the root epidermis of phosphate-deprived plants. Deletion analysis of this promoter indicated that, while the 933 bp upstream of the start codon is sufficient for root-specific and phosphate-responsive reporter gene expression, the 2000 bp upstream of the start codon are required for maximum expression levels. We show that transgenic Arabidopsis plants in which this 2000 bp promoter drives the Aspergillus phytase gene secrete phytase enzyme only from roots when grown on medium containing low phosphate concentrations, and that this transgene enables the plants to grow on medium containing phytate as a sole P source. The growth rates and shoot P concentrations of these plants were similar when grown on phytate or phosphate as the P source, and were similar to transgenic lines in which the phytase was driven by the constitutive CaMV 35S promoter. Our results demonstrate the potential usefulness of the promoter from the Pht1;2 phosphate transporter for driving genes that may improve crop P uptake and nutrition.

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

This chapter contains sections titled: - Incidence and Practical Significance of Transgene Silencing - Factors Influencing Transgene Silencing - Box 12.1. An RNAi primer - Mechanisms of Transgene Silencing - Strategies to Avoid Transgene Silencing - Conclusions and Future Prospects - Box 12.2. Avoiding transgene silencing.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.