A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions.

BACKGROUND: The 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. RESULTS: A 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. CONCLUSIONS: The 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.

Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield.

Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1-T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20-30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance.

Biolistic transformation of wheat with centrophenoxine as a synthetic auxin.

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.

Agrobacterium-mediated transformation of barley (Hordeum vulgare L.).

Barley biotechnology requires efficient genetic engineering tools for producing transgenic plants necessary for conducting reverse genetics analyses in breeding and functional genomics research. Agrobacterium-mediated genetic transformation is an important technique for producing barley transgenics with simple low-copy number transgenes. This chapter reports a refined protocol for the systematic high-throughput transformation of the advanced Australian spring barley breeding line WI4330.

Characterization of the wheat gene encoding a grain-specific lipid transfer protein TdPR61, and promoter activity in wheat, barley and rice.

The TaPR61 gene from bread wheat encodes a lipid transfer protein (LTP) with a hydrophobic signal peptide, predicted to direct the TaPR61 protein to the apoplast. Modelling of TaPR61 revealed the presence of an internal cavity which can accommodate at least two lipid molecules. The full-length gene, including the promoter sequence of a TaPR61 orthologue, was cloned from a BAC library of Triticum durum. Quantitative RT-PCR analysis revealed the presence of TaPR61 and TdPR61 mainly in grain. A transcriptional TdPR61 promoter-GUS fusion was stably transformed into wheat, barley, and rice. The strongest GUS expression in all three plants was found in the endosperm transfer cells, the embryo surrounding region (ESR), and in the embryo. The promoter is strong and has similar but not identical spatial patterns of activity in wheat, barley, and rice. These results suggest that the TdPR61 promoter will be a useful tool for improving grain quality by manipulating the quality and quantity of nutrient/lipid uptake to the endosperm and embryo. Mapping of regions important for the promoter function using transient expression assays in developing embryos resulted in the identification of two segments important for promoter activation in embryos. The putative cis-elements from the distal segment were used as bait in a yeast 1-hybrid (Y1H) screen of a cDNA library prepared from the liquid part of the wheat multinucleate syncytium. A transcription factor isolated in the screen is similar to BES1/BLZ1 from Arabidopsis, which is known to be a key transcriptional regulator of the brassinosteroid signalling pathway.

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice.

An 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.

Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors.

Transcription 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.

Defensin promoters as potential tools for engineering disease resistance in cereal grains.

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.

Characterization of the wheat endosperm transfer cell-specific protein TaPR60.

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.

Expression of active Streptomyces phage phiC31 integrase in transgenic wheat plants.

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.