Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

early maturity 7 promotes early flowering by controlling the light input into the circadian clock in barley.

Breeding for variation in photoperiod response is crucial to adapt crop plants to various environments. Plants measure changes in day length by the circadian clock, an endogenous timekeeper that allows plants to anticipate changes in diurnal and seasonal light-dark cycles. Here, we describe the early maturity 7 (eam7) locus in barley (Hordeum vulgare), which interacts with PHOTOPERIOD 1 (Ppd-H1) to cause early flowering under non-inductive short days. We identify LIGHT-REGULATED WD 1 (LWD1) as a putative candidate to underlie the eam7 locus in barley as supported by genetic mapping and CRISPR-Cas9-generated lwd1 mutants. Mutations in eam7 cause a significant phase advance and a misregulation of core clock and clock output genes under diurnal conditions. Early flowering was linked to an upregulation of Ppd-H1 during the night and consequent induction of the florigen FLOWERING LOCUS T1 under short days. We propose that EAM7 controls photoperiodic flowering in barley by controlling the light input into the clock and diurnal expression patterns of the major photoperiod response gene Ppd-H1.

RING/U-box E3 protein BIR1 interacts with and ubiquitinates barley growth repressor BROAD LEAF1.

Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.

The Arabidopsis Mitochondrial Nucleoid-Associated Protein WHIRLY2 Is Required for a Proper Response to Salt Stress.

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.

HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Anatomical insights into the vascular layout of the barley rachis: implications for transport and spikelet connection.

BACKGROUND AND AIMS: Vascular patterning is intimately related to plant form and function. Here, using barley (Hordeum vulgare) as a model, we studied the vascular anatomy of the spike-type inflorescence. The main aim of the present work was to clarify the relationship between rachis (spike axis) vasculature and spike size, to define vascular dynamics and to discuss the implications for transport capacity and its interaction with the spikelets. METHODS: We used serial transverse internode sections to determine the internode area, vascular area and number of veins along the rachis of several barley lines. KEY RESULTS: Internode area and total vascular area show a clear positive correlation with spike size, whereas the number of veins is only weakly correlated. The lateral periphery of the rachis contains large mature veins of constant size, whereas the central part is occupied by small immature veins. Spikelet-derived veins entering the rachis often merge with the immature rachis veins but never merge with the mature veins. An increase in floret fertility through the conversion of a two-rowed barley into an isogenic six-rowed line, in addition to a decrease in floret fertility owing to enhanced pre-anthesis tip degeneration caused by the mutation tip sterile 2.b (tst2.b), significantly affected vein size but had limited to no effects on the number of veins or internode area. CONCLUSIONS: The rachis vasculature is the result of a two-step process involving an initial layout followed by size adjustment according to floret fertility/spike size. The restriction of large mature vessels to the periphery and that of small immature vessels to the centre of the rachis suggests that long-distance transport and local supply to spikelets are spatially separated processes. The identification of spikelet-derived veins entering the rachis without fusing with its vasculature indicates that a vascular continuity between rachis and spikelets might be non-essential.

Barley HISTIDINE KINASE 1 (HvHK1) coordinates transfer cell specification in the young endosperm.

Cereal endosperm represents the most important source of the world's food; nevertheless, the molecular mechanisms underlying cell and tissue differentiation in cereal grains remain poorly understood. Endosperm cellularization commences at the maternal-filial intersection of grains and generates endosperm transfer cells (ETCs), a cell type with a prominent anatomy optimized for efficient nutrient transport. Barley HISTIDINE KINASE1 (HvHK1) was identified as a receptor component with spatially restricted expression in the syncytial endosperm where ETCs emerge. Here, we demonstrate its function in ETC fate acquisition using RNA interference-mediated downregulation of HvHK1. Repression of HvHK1 impairs cell specification in the central ETC region and the development of transfer cell morphology, and consecutively defects differentiation of adjacent endosperm tissues. Coinciding with reduced expression of HvHK1, disturbed cell plate formation and fusion were observed at the initiation of endosperm cellularization, revealing that HvHK1 triggers initial cytokinesis of ETCs. Cell-type-specific RNA sequencing confirmed loss of transfer cell identity, compromised cell wall biogenesis and reduced transport capacities in aberrant cells and elucidated two-component signaling and hormone pathways that are mediated by HvHK1. Gene regulatory network modeling was used to specify the direct targets of HvHK1; this predicted non-canonical auxin signaling elements as the main regulatory links governing cellularization of ETCs, potentially through interaction with type-B response regulators. This work provides clues to previously unknown molecular mechanisms directing ETC specification, a process with fundamental impact on grain yield in cereals.

The nucleoid-associated protein WHIRLY1 is required for the coordinate assembly of plastid and nucleus-encoded proteins during chloroplast development.

MAIN CONCLUSION: Chloroplasts deficient in the major chloroplast nucleoid-associated protein WHIRLY1 have an enhanced ratio of LHCs to reaction centers, indicating that WHIRLY1 is required for a coordinate assembly of the photosynthetic apparatus during chloroplast development. Chloroplast development was found to be delayed in barley plants with an RNAi-mediated knockdown of WHIRLY1 encoding a major nucleoid-associated protein of chloroplasts. The plastids of WHIRLY1 deficient plants had a reduced ribosome content. Accordingly, plastid-encoded proteins of the photosynthetic apparatus showed delayed accumulation during chloroplast development coinciding with a delayed increase in photosystem II efficiency measured by chlorophyll fluorescence. In contrast, light harvesting complex proteins being encoded in the nucleus had a high abundance as in the wild type. The unbalanced assembly of the proteins of the photosynthetic apparatus in WHIRLY1-deficient plants coincided with the enhanced contents of chlorophyll b and xanthophylls. The lack of coordination was most obvious at the early stages of development. Overaccumulation of LHC proteins in comparison to reaction center proteins at the early stages of chloroplast development did not correlate with enhanced expression levels of the corresponding genes in the nucleus. This work revealed that WHIRLY1 does not influence LHC abundance at the transcriptional level. Rather, WHIRLY1 in association with nucleoids might play a structural role for both the assembly of ribosomes and the complexes of the photosynthetic apparatus.

Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress in barley (Hordeum vulgare L.).

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression. Comparison of wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1 showed, that the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress. The results support a central role of WHIRLY1 in retrograde signaling and also underpin a so far underestimated role of microRNAs in this process.

Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1.

WHIRLY1 in barley was isolated as a potential regulator of the senescence-associated gene HvS40. In order to investigate whether the plastid-nucleus-located DNA/RNA-binding protein WHIRLY1 plays a role in regulation of leaf senescence, primary foliage leaves from transgenic barley plants with an RNAi-mediated knockdown of the WHIRLY1 gene were characterized by typical senescence parameters, namely pigment contents, function and composition of the photosynthetic apparatus, as well as expression of selected genes known to be either down- or up-regulated during leaf senescence. When the plants were grown at low light intensity, senescence progression was similar between wild-type and RNAi-W1 plants. Likewise, dark-induced senescence of detached leaves was not affected by reduction of WHIRLY1. When plants were grown at high light intensity, however, senescence was induced prematurely in wild-type plants but was delayed in RNAi-W1 plants. This result suggests that WHIRLY1 plays a role in light sensing and/or stress communication between chloroplasts and the nucleus.

Stable gene replacement in barley by targeted double-strand break induction.

Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley.

Increasing abscisic acid levels by immunomodulation in barley grains induces precocious maturation without changing grain composition.

Abscisic acid (ABA) accumulates in seeds during the transition to the seed filling phase. ABA triggers seed maturation, storage activity, and stress signalling and tolerance. Immunomodulation was used to alter the ABA status in barley grains, with the resulting transgenic caryopses responding to the anti-ABA antibody gene expression with increased accumulation of ABA. Calculation of free versus antibody-bound ABA reveals large excess of free ABA, increasing signficantly in caryopses from 10 days after fertilization. Metabolite and transcript profiling in anti-ABA grains expose triggered and enhanced ABA-functions such as transcriptional up-regulation of sucrose-to-starch metabolism, storage protein synthesis and ABA-related signal transduction. Thus, enhanced ABA during transition phases induces precocious maturation but negatively interferes with growth and development. Anti-ABA grains display broad constitutive gene induction related to biotic and abiotic stresses. Most of these genes are ABA- and/or stress-inducible, including alcohol and aldehyde dehydrogenases, peroxidases, chaperones, glutathione-S-transferase, drought- and salt-inducible proteins. Conclusively, ABA immunomodulation results in precocious ABA accumulation that generates an integrated response of stress and maturation. Repression of ABA signalling, occurring in anti-ABA grains, potentially antagonizes effects caused by overshooting production. Finally, mature grain weight and composition are unchanged in anti-ABA plants, although germination is somewhat delayed. This indicates that anti-ABA caryopses induce specific mechanisms to desensitize ABA signalling efficiently, which finally yields mature grains with nearly unchanged dry weight and composition. Such compensation implicates the enormous physiological and metabolic flexibilities of barley grains to adjust effects of unnaturally high ABA amounts in order to ensure and maintain proper grain development.

The fungal core effector Pep1 is conserved across smuts of dicots and monocots.

The secreted fungal effector Pep1 is essential for penetration of the host epidermis and establishment of biotrophy in the Ustilago maydis-maize pathosystem. Previously, Pep1 was found to be an inhibitor of apoplastic plant peroxidases, which suppresses the oxidative burst, a primary immune response of the host plant and enables fungal colonization. To investigate the conservation of Pep1 in other pathogens, genomes of related smut species were screened for pep1 orthologues. Pep1 proteins were produced in Escherichia coli for functional assays. The biological function of Pep1 was tested by heterologous expression in U. maydis and Hordeum vulgare. Pep1 orthologues revealed a remarkable degree of sequence conservation, indicating that this effector might play a fundamental role in virulence of biotrophic smut fungi. Pep1 function and its role in virulence are conserved in different pathogenic fungi, even across the monocot-dicot border of host plants. The findings described in this study classify Pep1 as a phylogenetically conserved fungal core effector. Furthermore, we documented the influence of Pep1 on the disease caused by Blumeria graminis f. sp. hordei which is a non-smut-related pathosystem.

Abscisic acid flux alterations result in differential abscisic acid signaling responses and impact assimilation efficiency in barley under terminal drought stress.

Abscisic acid (ABA) is a central player in plant responses to drought stress. How variable levels of ABA under short-term versus long-term drought stress impact assimilation and growth in crops is unclear. We addressed this through comparative analysis, using two elite breeding lines of barley (Hordeum vulgare) that show senescence or stay-green phenotype under terminal drought stress and by making use of transgenic barley lines that express Arabidopsis (Arabidopsis thaliana) 9-cis-epoxycarotenoid dioxygenase (AtNCED6) coding sequence or an RNA interference (RNAi) sequence of ABA 8'-hydroxylase under the control of a drought-inducible barley promoter. The high levels of ABA and its catabolites in the senescing breeding line under long-term stress were detrimental for assimilate productivity, whereas these levels were not perturbed in the stay-green type that performed better. In transgenic barley, drought-inducible AtNCED expression afforded temporal control in ABA levels such that the ABA levels rose sooner than in wild-type plants but also subsided, unlike as in the wild type , to near-basal levels upon prolonged stress treatment due to down-regulation of endogenous HvNCED genes. Suppressing of ABA catabolism with the RNA interference approach of ABA 8'-hydroxylase caused ABA flux during the entire period of stress. These transgenic plants performed better than the wild type under stress to maintain a favorable instantaneous water use efficiency and better assimilation. Gene expression analysis, protein structural modeling, and protein-protein interaction analyses of the members of the PYRABACTIN RESISTANCE1/PYRABACTIN RESISTANCE1-LIKE/REGULATORY COMPONENT OF ABA RECEPTORS, TYPE 2C PROTEIN PHOSPHATASE Sucrose non-fermenting1-related protein kinase2, and ABA-INSENSITIVE5/ABA-responsive element binding factor family identified specific members that could potentially impact ABA metabolism and stress adaptation in barley.

A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species.

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by its parthenogenetic development into offspring that are genetic clones of the mother plant. Fertilization (i.e. pseudogamy) of the central cell is important for the production of a functional endosperm with a balanced 2:1 maternal:paternal genome ratio. Here, we present the APOLLO (for apomixis-linked locus) gene, an Aspartate Glutamate Aspartate Aspartate histidine exonuclease whose transcripts are down-regulated in sexual ovules entering meiosis while being up-regulated in apomeiotic ovules at the same stage of development in plants of the genus Boechera. APOLLO has both "apoalleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sexalleles." All apomictic Boechera spp. accessions proved to be heterozygous for the APOLLO gene (having at least one apoallele and one sexallele), while all sexual genotypes were homozygous for sexalleles. Apoalleles contained a 20-nucleotide polymorphism present in the 5' untranslated region that contains specific transcription factor-binding sites for ARABIDOPSIS THALIANA HOMEOBOX PROTEIN5, LIM1 (for LINEAGE ABNORMAL11, INSULIN1, MECHANOSENSORY PROTEIN3), SORLIP1AT (for SEQUENCES OVERREPRESENTED IN LIGHT-INDUCED PROMOTERS IN ARABIDOPSIS THALIANA1), SORLIP2AT, and POLYA SIGNAL1. In the same region, sexalleles contain transcription factor-binding sites for DNA BINDING WITH ONE FINGER2, DNA BINDING WITH ONE FINGER3, and PROLAMIN BOX-BINDING FACTOR. Our results suggest that the expression of a single deregulated allele could induce the cascade of events leading to asexual female gamete formation in an apomictic plant.

A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells.

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

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.

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.