SummaryTranscription factors have been shown to control the activity of multiple stress response genes in a coordinated manner and therefore represent attractive targets for application in molecular plant breeding. We investigated the possibility of modulating the transcriptional regulation of drought and cold responses in the agriculturally important species, wheat and barley, with a view to increase drought and frost tolerance. Transgenic wheat and barley plants were generated showing constitutive (double 35S) and drought-inducible (maize Rab17) expression of the TaDREB2 and TaDREB3 transcription factors isolated from wheat grain. Transgenic populations with constitutive over-expression showed slower growth, delayed flowering and lower grain yields relative to the nontransgenic controls. However, both the TaDREB2 and TaDREB3 transgenic plants showed improved survival under severe drought conditions relative to nontransgenic controls. There were two components to the drought tolerance: real (activation of drought-stress-inducible genes) and 'seeming' (consumption of less water as a result of smaller size and ⁄ or slower growth of transgenics compared to controls). The undesired changes in plant development associated with the 'seeming' component of tolerance could be alleviated by using a drought-inducible promoter. In addition to drought tolerance, both TaDREB2 and TaDREB3 transgenic plants with constitutive over-expression of the transgene showed a significant improvement in frost tolerance. The increased expression of TaDREB2 and TaDREB3 lead to elevated expression in the transgenics of 10 other CBF ⁄ DREB genes and a large number of stress responsive LEA ⁄ COR ⁄ DHN genes known to be responsible for the protection of cell from damage and desiccation under stress.

The TaPR60 gene from bread wheat encodes a small cysteine-rich protein with a hydrophobic signal peptide, predicted to direct the TaPR60 protein to a secretory pathway. It was demonstrated by heterologous expression of recombinant TaPR60 protein that the signal peptide is recognized and cleaved in yeast cells. The full-length gene including promoter sequence of a TaPR60 orthologue was cloned from a BAC library of Triticum durum. A transcriptional promoter-GUS fusion was stably transformed into wheat, barley and rice. The strongest GUS expression in wheat and barley was found in the endosperm transfer cells, while in rice the promoter was active inside the starchy endosperm during the early stages of grain filling. The TaPR60 gene was also used as bait in a yeast two-hybrid screen. Five proteins were identified in the screen, and for some of these prey proteins, the interaction was confirmed by co-immunoprecipitation. The signal peptide binding proteins, TaUbiL1 and TaUbiL2, are homologues of animal proteins, which belong to proteolytic complexes, and therefore may be responsible for TaPR60 processing or degradation of the signal peptide. Other proteins that interact with TaPR60 may have a function in TaPR60 secretion or regulation of this process. Examination of a three dimensional model of TaPR60 suggested that this protein could be involved in binding of lipidic molecules.

HighlightConstitutive overexpression of NF-YB in transgenic wheat leads to a 20–30% increase in grain yield compared with wild-type plants (cv. Gladius) without negative changes in seed size or number.

Summary Engineering of plant protection in cereals requires well characterized tissue‐specific and wounding/pathogen‐inducible promoters for targeted expression of pathogen responsive and resistance genes. We describe the isolation of seven wheat and rice defensin genes expressed in early developing grain and during grain germination, two developmental stages that are particularly vulnerable to pathogens and insects. Comparison of three‐dimensional (3D) models of these rice and wheat PRPI defensins indicated variations in spatial architectures that could reflect their functional diversities. Wheat and rice were stably transformed with promoter–GUS fusion constructs and the spatial and temporal activities of four promoters were studied using whole‐mount and histological assays. PRPI promoters were active before and at anthesis in both transgenic wheat and rice with activity mainly in the ovary. In rice, GUS activity was also observed in vascular tissue of the lemma, palea and anthers. After fertilization, GUS was strongly expressed in the outer cell layers of the pericarp and in the main vascular bundle of the grain. During, and a short time after, seed germination, wheat promoters were active in transgenic rice embryos, roots and/or coleoptiles. All wheat and rice promoters were strongly induced by wounding in leaf, stem and grain of transgenic rice plants. These results suggest that PRPI promoters will be useful for specific targeting and accumulation of proteins conferring resistance to pathogens in vulnerable tissues of developing and germinating grain.

Site-specific recombination systems are becoming an important tool for the genetic modification of crop plants. Here we report the functional expression of the Streptomyces phage-derived phiC31 recombinase (integrase) in wheat. T-DNA constructs containing a phiC31 integrase transgene were stably transformed into wheat plants via particle gun bombardment. A plant-virus-based assay system was used to monitor the site-specific recombination activity of the recombinant integrase protein in vivo. We established several independent doubled haploid (DH) inbred lines that constitutively express an active integrase enzyme without any apparent detrimental effects on plant growth and development. The potential of phiC31 integrase expression in crop plants related to transgene control technologies or hybrid breeding systems is discussed.

BackgroundThe relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating.ResultsA simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results.ConclusionsThe DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.

SummaryAn HD-Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD-Zip IV family. The 3¢ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full-length gene containing a 3-kb-long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter-GUS fusion constructs in transgenic wheat, barley and rice plants. Whole-mount and histochemical GUS staining patterns revealed grain-specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm-dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.

Cereal crops, including bread wheat (Triticum aestivum L.), are an important staple food worldwide. With a growing global population, it is evident that current crop production will not meet the rising demands being placed on modern agriculture. Efforts to improve crop yield and stress-tolerance by traditional breeding are labor intensive, time consuming, and highly dependent upon the ability to capture existing and novel genetic variation from a restricted genetic pool. Genetic engineering of crop species is one of several alternatives to traditional breeding for the introduction of novel genetic variation. This recently established technology has proved useful for the introduction of novel traits like pest resistance and herbicide tolerance. As a universal tool for genetic transformation, the Biolistic Gene Gun allows for the genomic integration of novel gene sequences from various sources into a whole host of living organisms.In this chapter, we present a novel and detailed protocol for the Biolistic Transformation of bread wheat that uses the pharmaceutical compound, Centrophenoxine (CPX). The application of CPX as the main auxin-like plant growth regulator in cereal genetic transformation replaces the potent but more toxic herbicide 2,4-D.