Molecular Farming in Seed Crops: Gene Transfer into Barley (Hordeum vulgare ) and Wheat (Triticum aestivum ).

The production of recombinant proteins in seed crops has a long history and cereal grains are now one of the platforms in commercial use. Specific advantages include excellent storage properties, a well-developed endomembrane system with a high biosynthetic capacity and well-established cultivation procedures worldwide. However, the production of transgenic cereals is a time-consuming procedure and the lack of efficient transformation systems is still a significant bottleneck. Barley can be transformed at high efficiency but the protocols are genotype-dependent. Wheat is generally more challenging to transform, but considerable progress has been made in enhancing transformation efficiencies and in controlling transgene expression. In this chapter, we describe and discuss standard procedures for generating transgenic barley and wheat for the production of recombinant proteins.

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.

Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions in Barley and Generation of Homozygous Edited Lines via Embryogenic Pollen Culture.

The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.

Heritable Genomic Fragment Deletions and Small Indels in the Putative ENGase Gene Induced by CRISPR/Cas9 in Barley.

Targeted genome editing with the CRISPR/Cas9 system has been used extensively for the selective mutation of plant genes. Here we used CRISPR/Cas9 to disrupt the putative barley (Hordeum vulgare cv. "Golden Promise") endo-N-acetyl-ß-D-glucosaminidase (ENGase) gene. Five single guide RNAs (sgRNAs) were designed for different target sites in the upstream part of the ENGase coding region. Targeted fragment deletions were induced by co-bombarding selected combinations of sgRNA with wild-type cas9 using separate plasmids, or by co-infection with separate Agrobacterium tumefaciens cultures. Genotype screening was carried out in the primary transformants (T0) and their T1 progeny to confirm the presence of site-specific small insertions and deletions (indels) and genomic fragment deletions between pairs of targets. Cas9-induced mutations were observed in 78% of the plants, a higher efficiency than previously reported in barley. Notably, there were differences in performance among the five sgRNAs. The induced indels and fragment deletions were transmitted to the T1 generation, and transgene free (sgRNA:cas9 negative) genome-edited homozygous ENGase knock outs were identified among the T1 progeny. We have therefore demonstrated that mutant barley lines with a disrupted endogenous ENGase and defined fragment deletions can be produced efficiently using the CRISPR/Cas9 system even when this requires co-transformation with multiple plasmids by bombardment or Agrobacterium-mediated transformation. We confirm the specificity and heritability of the mutations and the ability to efficiently generate homozygous mutant T1 plants.

Cell layer-specific distribution of transiently expressed barley ESCRT-III component HvVPS60 in developing barley endosperm.

The significance of the endosomal sorting complexes required for transport (ESCRT)-III in cereal endosperm has been shown by the identification of the recessive mutant supernumerary aleurone layer1 (SAL1) in maize. ESCRT-III is indispensable in the final membrane fission step during biogenesis of multivesicular bodies (MVBs), responsible for protein sorting to vacuoles and to the cell surface. Here, we annotated barley ESCRT-III members in the (model) crop Hordeum vulgare and show that all identified members are expressed in developing barley endosperm. We used fluorescently tagged core ESCRT-III members HvSNF7a/CHMP4 and HvVPS24/CHMP3 and the associated ESCRT-III component HvVPS60a/CHMP5 for transient localization studies in barley endosperm. In vivo confocal microscopic analyses show that the localization of recombinantly expressed HvSNF7a, HvVPS24 and HvVPS60a differs within barley endosperm. Whereas HvSNF7a induces large agglomerations, HvVPS24 shows mainly cytosolic localization in aleurone and subaleurone. In contrast, HvVPS60a localizes strongly at the plasma membrane in aleurone. In subaleurone, HvVPS60a was found to a lesser extent at the plasma membrane and at vacuolar membranes. These results indicate that the steady-state association of ESCRT-III may be influenced by cell layer-specific protein deposition or trafficking and remodelling of the endomembrane system in endosperm. We show that sorting of an artificially mono-ubiquitinated Arabidopsis plasma membrane protein is inhibited by HvVPS60a in aleurone. The involvement of HvVPS60a in different cell layer-specific trafficking pathways, reflected by localization of HvVPS60a at the plasma membrane in aleurone and at the PSV membrane in subaleurone, is discussed.

Fusion, rupture, and degeneration: the fate of in vivo-labelled PSVs in developing barley endosperm.

Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins. The endosperm of barley contains hordeins, which are ultimately deposited within protein storage vacuoles (PSVs). These organelles have been characterized predominantly by the histochemical analysis of fixed immature tissue samples. However, little is known about the fate of PSVs during barley endosperm development, and in vivo imaging has not been attempted in order to gain further insight. In this report, young seeds were followed through development to characterize the dynamic morphology of PSVs from aleurone, subaleurone, and central starchy endosperm cells. TIP3-GFP was used as a PSV membrane marker and several fluorescent tracers were used to identify membranes and monitor endomembrane organelles in real time. Whereas the spherical appearance of strongly labelled TIP3-GFP PSVs in the aleurone remained constant, those in the subaleurone and central starchy endosperm underwent substantial morphological changes. Fusion and rupture events were observed in the subaleurone, and internal membranes derived from both the tonoplast and endoplasmic reticulum were identified within these PSVs. TIP3-GFP-labelled PSVs in the starchy endosperm cells underwent a dramatic reduction in size, so that finally the protein bodies were tightly enclosed. Potential desiccation-related membrane-altering processes that may be causally linked to these dynamic endomembrane events in the barley endosperm are discussed.

The elimination of a selectable marker gene in the doubled haploid progeny of co-transformed barley plants.

Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.

phiC31 integrase-mediated site-specific recombination in barley.

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1), even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

Telomere-mediated truncation of barley chromosomes.

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.

Induction of telomere-mediated chromosomal truncation and stability of truncated chromosomes in Arabidopsis thaliana.

Minichromosomes possess functional centromeres and telomeres and thus should be stably inherited. They offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Segregating independently of host chromosomes, they provide a platform for accelerating plant breeding. Following a top-down approach, we truncated endogenous chromosomes in Arabidopsis thaliana by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of A. thaliana telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into the natural tetraploid A. thaliana accession Wa-1, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by DNA gel blot analysis, PCR (polymerase chain reaction), and fluorescence in situ hybridisation. The addition of new telomere repeats in this process could start alternatively from within the T-DNA-derived telomere repeats or from adjacent sequences close to the right border of the T-DNA. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.