Developments and prospects for doubled haploid wheat.

Doubled haploid production is a valuable biotechnology that can accelerate the breeding of new wheat varieties by several years through the one-step creation of 100% homozygous plants. The technology also plays important role in studying the genetic control of traits in wheat, in marker-assisted selection, in genomics and in genetic engineering. In this paper, recent advances in androgenesis and gynogenesis techniques, emphasizing predominantly the in vitro culture phase, as well as the emerging innovative approaches in researching and producing wheat doubled haploids are reviewed. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing, that allows targeted mutagenesis and gene targeting, is being tested extensively as a powerful and precise tool to induce doubled haploids in wheat. The review provides the reader with recent examples of gene modifications in wheat to induce haploidy.

Genetic Modification for Wheat Improvement: From Transgenesis to Genome Editing.

To feed the growing human population, global wheat yields should increase to approximately 5 tonnes per ha from the current 3.3 tonnes by 2050. To reach this goal, existing breeding practices must be complemented with new techniques built upon recent gains from wheat genome sequencing, and the accumulated knowledge of genetic determinants underlying the agricultural traits responsible for crop yield and quality. In this review we primarily focus on the tools and techniques available for accessing gene functions which lead to clear phenotypes in wheat. We provide a view of the development of wheat transformation techniques from a historical perspective, and summarize how techniques have been adapted to obtain gain-of-function phenotypes by gene overexpression, loss-of-function phenotypes by expressing antisense RNAs (RNA interference or RNAi), and most recently the manipulation of gene structure and expression using site-specific nucleases, such as CRISPR/Cas9, for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to increase yield, improve nutritional and health-promoting qualities in wheat, and enhance the crop's resistance to various biotic and abiotic stresses.

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.

Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification.

Plants respond to abiotic stresses by changes in gene regulation, including stress-inducible expression of transcriptional activators and repressors. One of the best characterized families of drought-related transcription factors are dehydration-responsive element binding (DREB) proteins, known as C-repeat binding factors (CBF). The wheat DREB/CBF gene TaRAP2.1L was isolated from drought-affected tissues using a dehydration-responsive element (DRE) as bait in a yeast one-hybrid screen. TaRAP2.1L is induced by elevated abscisic acid, drought and cold. A C-terminal ethylene responsive factor-associated amphiphilic repression (EAR) motif, known to be responsible for active repression of target genes, was identified in the TaRAP2.1L protein. It was found that TaRAP2.1L has a unique selectivity of DNA-binding, which differs from that of DREB activators. This binding selectivity remains unchanged in a TaRAP2.1L variant with an inactivated EAR motif (TaRAP2.1Lmut). To study the role of the TaRAP2.1L repressor activity associated with the EAR motif in planta, transgenic wheat overexpressing native or mutated TaRAP2.1L was generated. Overexpression of TaRAP2.1L under constitutive and stress-inducible promoters in transgenic wheat and barley led to dwarfism and decreased frost tolerance. By contrast, constitutive overexpression of the TaRAP2.1Lmut gene had little or no negative influence on wheat development or grain yield. Transgenic lines with the TaRAP2.1Lmut transgene had an enhanced ability to survive frost and drought. The improved stress tolerance is attributed to up-regulation of several stress-related genes known to be downstream genes of DREB/CBF activators.

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.

Application of next-generation sequencing technology to study genetic diversity and identify unique SNP markers in bread wheat from Kazakhstan.

BACKGROUND: New SNP marker platforms offer the opportunity to investigate the relationships between wheat cultivars from different regions and assess the mechanism and processes that have led to adaptation to particular production environments. Wheat breeding has a long history in Kazakhstan and the aim of this study was to explore the relationship between key varieties from Kazakhstan and germplasm from breeding programs for other regions. RESULTS: The study revealed 5,898 polymorphic markers amongst ten cultivars, of which 2,730 were mapped in the consensus genetic map. Mapped SNP markers were distributed almost equally across the A and B genomes, with between 279 and 484 markers assigned to each chromosome. Marker coverage was approximately 10-fold lower in the D genome. There were 863 SNP markers identified as unique to specific cultivars, and clusters of these markers (regions containing more than three closely mapped unique SNPs) showed specific patterns on the consensus genetic map for each cultivar. Significant intra-varietal genetic polymorphism was identified in three cultivars (Tzelinnaya 3C, Kazakhstanskaya rannespelaya and Kazakhstanskaya 15). Phylogenetic analysis based on inter-varietal polymorphism showed that the very old cultivar Erythrospermum 841 was the most genetically distinct from the other nine cultivars from Kazakhstan, falling in a clade together with the American cultivar Sonora and genotypes from Central and South Asia. The modern cultivar Kazakhstanskaya 19 also fell into a separate clade, together with the American cultivar Thatcher. The remaining eight cultivars shared a single sub-clade but were categorised into four clusters. CONCLUSION: The accumulated data for SNP marker polymorphisms amongst bread wheat genotypes from Kazakhstan may be used for studying genetic diversity in bread wheat, with potential application for marker-assisted selection and the preparation of a set of genotype-specific markers.

Complex regulation by Apetala2 domain-containing transcription factors revealed through analysis of the stress-responsive TdCor410b promoter from durum wheat.

Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.

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.