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Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like a-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers’ asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers’ asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly fromT0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines.

The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post anthesis, the embryonic axis is removed, and the immature scutella are pre-cultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-β-farnesene (Eβf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eβf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

Unpredictable silencing of foreign genes introduced into wheat by particle bombardment transformation has been previously observed. To investigate the cause of this silencing, we selected two independent wheat lines that had been transformed via the particle gun with plasmid pAHC25 containing the genes uidA (GUS) and bar (BASTA herbicide-resistance) each controlled by the maize ubiquitin promoter-intron (Ubi1). One of these lines (WT2) had shown progressive reductions in transgenic phenotype in successive generations until GUS activity and herbicide-resistance were undetectable in all progeny of the T 3 generation tested. A second line, (WT1) exhibited the expected levels of GUS expression and herbicide-resistance up to the T 3 generation except for the loss of transgenic phenotype in a single T 1 segregant. Transgene stability, silencing and integration locus was investigated in three generations in both lines. The silencing observed in the WT2 line was found to be acting at the transcription level for both the uidA and bar genes. Analysis of transgene promoter methylation in selected T 1 , T 2 and T 3 individuals revealed a positive link between methylation status and gene silencing. The silenced T1 individual from line WT 1 was also linked to promoter methylation and insertion locus rearrangement. Fluorescence in situ hybridisation (FISH) revealed highly divergent transgene insertion loci for WT1 and WT2. WT1 was detected close to the telomere whereas the WT2 insertion was located in the region of the centromere. The effect of these findings on transgene silencing and stability are discussed.

The stability and heritability of three marker genes was investigated in a population of twelve independent transgenic cereal lines (six wheat and six tritordeum). Integration patterns, inheritance of structural transgenes and inheritance of expression were analysed in the T0 and T1 generations for all 12 lines. Transmission and expression were analysed in the T2 generation for 9 lines and in the T3 generation for the six wheat lines. Inheritance of integration patterns was highly stable, and transmission of the transgenes and inheritance of their expression followed Mendelian ratios in the majority of lines. A gradual reduction in uidA expression was observed over three generations, which was not accompanied by a similar reduction in bar expression. Some unexpected phenomena associated with transgene inheritance were also observed and are discussed.

Transient expression in plants is a valuable tool for many aspects of functional genomics and promoter testing. It can be used both to over-express and to silence candidate genes. It is also scaleable and provides a viable alternative to microbial fermentation and animal cell culture for the production of recombinant proteins. It does not depend on chromosomal integration of heterologous DNA so is a relatively facile procedure and can lead to high levels of transgene expression. Recombinant DNA can be introduced into plant cells via physical methods, via Agrobacterium or via viral vectors.

At Rothamsted Research UK, we have developed a robust protocol for the Agrobacterium-mediated transformation of elite wheat and during the last year, made over one hundred independent transgenic lines in one durum and three bread wheat varieties. We are now able to provide training or generate transgenic wheat lines as a not-for-profit service (see www.bract.org). Approximately 40% of our lines contain a single, functional T-DNA insertion. This paper describes the current status of Agrobacterium transformation of wheat and some initial molecular analysis of our lines. It also describes our plan to further increase throughput using mature seed-derived callus and other approaches

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.