Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis.

BACKGROUND: Barley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive. RESULTS: This study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity. CONCLUSIONS: We identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants.

Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells: one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.

Agrobacterium-Mediated Transformation of Barley Immature Embryos.

Barley transformation is an essential tool for a range of functional genomics studies as well as for future crop improvement. The demand for efficient crop transformation systems continues to grow, with new genome editing technologies adding to that demand. Here we describe an efficient and routine transformation protocol for the spring barley Golden Promise, based on Agrobacterium-mediated inoculation of immature embryos. This protocol has been widely used for overexpression and RNAi applications and more recently for CRISPR/Cas9 mediated genome editing. Average transformation efficiencies of 25% can be easily achieved.

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.

Differential Expression Profiling of Microspores During the Early Stages of Isolated Microspore Culture Using the Responsive Barley Cultivar Gobernadora.

In barley, it is possible to induce embryogenesis in the haploid and uninucleate microspore to obtain a diploid plant that is perfectly homozygous. To change developmental fates in this fashion, microspores need to engage in cellular de-differentiation, interrupting the pollen formation, and restore totipotency prior to engaging in embryogenesis. In this work, we used the barley cultivar Gobernadora to characterize the transcriptome of microspores prior to (day 0) and immediately after (days 2 and 5) the application of a stress pretreatment. A deep RNA-seq analysis revealed that microspores at these three time points exhibit a transcriptome of ∼14k genes, ∼90% of which were shared. An expression analysis identified a total of 3,382 differentially expressed genes (DEGs); of these, 2,155 and 2,281 DEGs were respectively identified when contrasting expression at days 0 and 2 and at days 2 and 5. These define 8 expression profiles in which DEGs share a common up- or down-regulation at these time points. Up-regulation of numerous glutathione S-transferase and heat shock protein genes as well as down-regulation of ribosomal subunit protein genes was observed between days 0 and 2. The transition from microspores to developing embryos (days 2 vs. 5) was marked by the induction of transcription factor genes known to play important roles in early embryogenesis, numerous genes involved in hormone biosynthesis and plant hormonal signal transduction in addition to genes involved in secondary metabolism. This work sheds light on transcriptional changes accompanying an important developmental shift and provides candidate biomarkers for embryogenesis in barley.

Method for hull-less barley transformation and manipulation of grain mixed-linkage beta-glucan.

Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-ß-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition.

Induced dedifferentiation of barley (Hordeum vulgare L.) embryonic cells and its relationship with agronomic traits.

This study was carried out to investigate the response of 42 Iranian and European barley (Hordeum vulgare L.) cultivars to induced dedifferentiation of embryonic cells via immature embryo culture and understand the relationship between embryo culture characters and agronomic traits. The cultivars were evaluated for dedifferentiation of embryonic cells or callus induction from immature embryo culture based on a completely randomized design with unequal replication. Immature embryos were placed scutellum down on cell dedifferentiation medium based on MS and supplemented with 2.5 mg/l 2,4-D. The developed calli were transferred to MS regeneration medium with different concentrations and combinations of plant growth regulators. The results of group comparisons showed that Iranian cultivars were greater than European cultivars regarding callus growth rate, callus primary diameter and total regenerated plantlets. The path correlation analysis revealed that grain width and kernel filling period had the highest positive and negative direct effects on embryo culture traits, respectively. Clustering cultivars based on the embryo culture characters and agronomic traits divided the cultivars into three groups. The third group consisted of the cultivars which all of them were with the highest mean for flag leaf length, days to anthesis, grain yield, callus growth rate and callus primary diameter. Mantel test revealed a negative (-0.101) and significant correlation (P<0.01) between embryo culture characters and agronomic traits. The significant relationships between few numbers of embryo culture characters and agronomic traits confirm that these characteristics could be genetically dependent and also tissue culture characters can be estimated from agronomic data.

Dynamics of post-translationally modified histones during barley pollen embryogenesis in the presence or absence of the epi-drug trichostatin A.

KEY MESSAGE: Improving pollen embryogenesis. Despite the agro-economic importance of pollen embryogenesis, the mechanisms underlying this process are still poorly understood. We describe the dynamics of chromatin modifications (histones H3K4me2, H3K9ac, H3K9me2, and H3K27me3) and chromatin marks (RNA polymerase II CDC phospho-Ser5, and CENH3) during barley pollen embryogenesis. Immunolabeling results show that, in reaction to stress, immature pollen rapidly starts reorganizing several important chromatin modifications indicative of a change in cell fate. This new chromatin modification pattern was accomplished within 24 h from whereon it remained unaltered during subsequent mitotic activity. This indicates that cell fate transition, the central element of pollen embryogenesis, is completed early on during the induction process. Application of the histone deacetylase inhibitor trichostatin A stimulated pollen embryogenesis when used on pollen with a gametophytic style chromatin pattern. However, when this drug was administered to embryogenic pollen, the chromatin markers reversed toward a gametophytic profile, embryogenesis was halted and all pollen invariably died.

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds.

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.

Transcriptome analysis reveals translational regulation in barley microspore-derived embryogenic callus under salt stress.

KEY MESSAGE: Transcriptome analysis of barley embryogenic callus from isolated microspore culture under salt stress uncovered a role of translation inhibition and selective activation of stress-specific proteins in cellular defense. Soil salinity is one of the major abiotic stresses which constrains the plant growth and reduces the productivity of field crops. In this study, it was observed that the salt stress in barley isolated microspore culture impacted not only on the quantity of embryogenic callus but also on the quality for later differentiation. The barley microspore-derived embryogenic callus, a transient intermediate form linked cells and plants, was employed for a global transcriptome analysis by RNA sequencing to provide new insights into the cellular adaptation or acclimation to stress. A total of 596 differentially expressed genes (DEGs) were identified, in which 123 DEGs were up-regulated and 473 DEGs were down-regulated in the embryogenic callus produced from microspore culture under salt stress as compared to the control conditions. KEGG pathway analysis identified 'translation' (27 DEGs; 12.56 %) as the largest group and followed by 'folding, sorting and degradation' (25 DEGs; 11.63 %) in 215 mapped metabolic pathways. The results of RNA-Seq data and quantitative real-time polymerase chain reaction validation showed that the genes related to translation regulation (such as eIF1A, RPLP0, RPLP2, VARS) were down-regulated to control general protein synthesis, and the genes related to endoplasmic reticulum stress response (such as small heat shock protein genes) were selectively up-regulated against protein denaturing during microspore embryogenesis under continuous salt stress. These transcriptional remodeling might affect the essential protein synthesis for the cell development to fulfill totipotency under salt stress.

Involvement of Alternative Splicing in Barley Seed Germination.

Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3' splicing (34%-45%), intron retention (32%-34%) and alternative 5' splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm.

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-ß-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-ß-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels.

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.

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos.

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars.

A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Carotenoids of aleurone, germ, and endosperm fractions of barley, corn and wheat differentially inhibit oxidative stress.

The antioxidant potential of carotenoids from aleurone, germ, and endosperm fractions of barley, corn, and wheat has been evaluated. HPLC analysis confirmed the presence of lutein and zeaxanthin carotenoids (nd-15139 µg/kg) in extracts of cereal grain fractions. The antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorbance capacity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays revealed significantly higher (P<0.001) antioxidant activity in the germ than in the aleurone and endosperm fractions. Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,2'azobis (2-amidinopropane)dihydrochloride (AAPH)-induced cell loss was effectively reduced by preincubating Caco-2, HT-29, and FHs 74 Int cells with carotenoid extracts. Moreover, carotenoid extracts reduced (P<0.001) AAPH-induced intracellular oxidation in the cell lines, suggesting antioxidant activity. Of the 84 antioxidant pathway genes included in microarray array analysis (HT-29 cells), the expressions of 28 genes were enhanced (P<0.05). Our findings suggest that carotenoids of germ, aleurone, and endosperm fractions improved antioxidant capacity and thus have the potential to mitigate oxidative stress.