Flooding induction of alcohol dehydrogenase in shoots and roots of barley seedlings

Barley (Hordeum vulgare) seedlings were exposed to flooding and activities of alcohol dehydrogenase (ADH) and their isoform profiles were determined. The flooding increased ADH activities in shoots and roots of the seedlings. By day 3, the activity increased to 4 and 3 times that of the initial level for the shoots and the roots, respectively. Only two bands of ADH isoform were found in the shoots and the roots of non-induced seedling, whereas five bands were identified in those of induced seedlings.     

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12–16 nmol min⁻¹ mg⁻¹ protein for both species. The anaerobic ATP synthesis rate was 7–9 nmol min⁻¹ mg⁻¹ protein for barley and 15–17 nmol min⁻¹ mg⁻¹ protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3–5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Isolation and culture of barley megasporocyte protoplasts

An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were dissected out of ovaries under aseptic conditions, subjected to a brief enzymatic digestion, and then transferred to a modified Kao medium with 90 g/l sucrose and 20 mM CaCl₂. A small incision was made with a scalpel through the softened epidermal cell layer of the nucellus and the megasporocyte could then be liberated into the medium by applying gentle pressure on the nucellus. The megasporocyte appeared to be completely devoid of a wall and changed its in situ pyriform shape to completely spherical when extruded into the medium. Four to nine protoplasts could typically be isolated per spike. Protoplasts cultured in medium degenerated after a few days. Viability was dramatically improved if protoplasts were co-cultivated with barley microspores undergoing microspore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts stained with 4,6-diamidino-2-phenylindole (DAPI) or aniline blue revealed that the protoplasts remained uninuclear and reformed their callose wall.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.     

Genetic analyses of rDNA spacer-length variation in barley

Summary The genetic mechanism controlling the inheritance of single and multiple spacer-length variant (slv) phenotypes in barley was investigated in six F₂ segregating populations. The results indicated that two independently assorting loci, each with co-dominant alleles, govern genetic variability for rDNA in barley regardless of the number of bands expressed by a given phenotype. The following chromosomal locations are proposed: sl variants 1, 4, 5, 6, and 7 on chromosome 7, and sl variants 7, 8, 9, 12, and 13 on chromosome 6; sl variant 7 is thus located on both of the chromosomes.     

Ethylene influences green plant regeneration from barley callus

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from ‘Golden Promise’, the best regenerator, and lowest from ‘Morex’ and ‘DH-20’, the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8–10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5–10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8–14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F₂ population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.     

Contact with ballotini (glass spheres) stimulates exudation of iron reducing and iron chelating substances from barley roots

Contact between roots and Fe-containing solid substrate is known to facilitate acquisition of iron by plants, but the actual mechanism of this contact effect is not yet clear. This study was undertaken to evaluate the effect of root contact with ballotini (glass spheres) on exudation of substances capable of reducing or chelating insoluble Fe(III) compounds by the roots of barley (Hordeum vulgare L. cv. Europa) seedlings. Seedlings with roots encountering mechanical impedance (i.e., in contact with ballotini) produced more lateral roots than the seedlings with unimpeded (i.e., freely suspended) roots in the nutrient solution. Nutrient solution bathing roots in contact with ballotini showed higher concentrations of Fe(III)-chelating (83% on day 7) and Fe(III)-reducing (107% on day 12) substances than solutions bathing unimpeded roots. The pH of all solutions rose continuously during the course of the experiment but was always lower (by a nonsignificant degree) in the solutions with roots in contact with ballotini than in those with unimpeded roots. The data indicate that under natural soil conditions the amount of Fe-chelating and Fe-reducing root exudates may be higher than is usually measured from roots of terrestrial plants artificially suspended in nutrient solution.     

Ribosomal DNA polymorphisms and the Oriental-Occidental genetic differentiation in cultivated barley

Summary A total of 289 accessions of cultivated barley were assayed for ribosomal DNA (rDNA) polymorphisms. These accessions comprised four independent samples: (1) 79 entries from China, (2) 59 accessions from Ethiopia, (3) 59 entries from Tibet and (4) 92 entries representing 36 barley growing countries of the world (referred to as world sample). In all, 17 rDNA phenotypes (genotypes) were observed, which were composed 10 alleles at two rDNA loci, Rrn1 and Rrn2. The world sample contained the largest number of phenotypes and alleles and also demonstrated the highest level of diversity. Ribosomal DNA phenotypes 104, 112 and 107, 112 occurred at high frequencies worldwide. Allele 112 was the predominant allele of Rrn1 in all four samples, and 104 and 107 were the two major alleles of Rrn2 worldwide. The distributions of rDNA genotypes and alleles demonstrated a clear differentiation of two distinct barley groups: an Oriental group represented by the samples from China and Tibet, which is characterized by allele 107 at the Rrn2 locus (rDNA phenotype 107, 112); and an Occidental group, represented by Ethiopian and world samples, which is comprised mostly of allele 104 at the Rrn2 locus (rDNA phenotype 104, 112). The results also raised new questions concerning the phylogeny and evolution of cultivated barley.     

In vitro culture selection increases glyphosate tolerance in barley

In vitro culture of barley calluses has been used to produce plants with increased glyphosate tolerance. Calluses from immature embryos of barleyHordeum vulgare L. (Jeff) were cultured on Murashige and Skoog medium with 10^-6, 10^-5, 10^-4, 5×10^-4, 10^-3, or 10^-2M glyphosate for one, four or thirty months. Plants were regenerated from calluses maintained in glyphosate medium at 10^-6, 10^-5 or 10^-4M for four months, at 10^-5 or 5×10^-4M for one month and at 10^-5M for thirty months. The progeny of each regenerated plant was analyzed for response to glyphosate. Some progenies showed increased tolerance to glyphosate.     

Microspore cultures as donor tissue for the initiation of embryogenic cell suspensions in barley

We have initiated embryogenic cell suspension cultures of barley (Hordeum vulgare L.) Igri from isolated microspore cultures. Data were obtained on the time required for establishment, frequency of establishment, i.e. number of calluses out of the total number of initiations giving rise to suspensions, and embryogenic capacity of the suspension cultures. For comparison, establishment of embryogenic cell suspensions from callus derived from immature zygotic embryos of Igri, Dissa and Golden Promise was also carried out. The results revealed that embryogenic suspension cultures were established in half the time and with a seven-fold higher frequency from microspore cultures than from zygotic embryo-derived calluses. The suspension cultures were still capable of embryo formation after two years. However, only albino plantlets were regenerated. For comparison, long term callus cultures derived from microspores, anthers and zygotic embryos were established. From the anther and zygotic embryo-derived callus cultures green plants were continuously regenerated, whereas the microspore-derived callus cultures lost this ability after the second subculture.     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

Cd-induced system of defence in the garlic root meristematic cells

Studies on cadmium effects in the root meristematic cells of Allium sativum L. were carried out using electron microscopy in order to explain the possible mechanisms of garlic seedlings’ tolerance to Cd stress. Seedlings were treated with 0.01, 0.10 and 1.00 mM CdCl₂ solutions for 0.5, 1, 2, 4, 8, 10, 12, 24 and 48 h, respectively. The results indicated that cell walls, plasma membrane and main organelles actively participated in Cd detoxification and tolerance at low Cd concentrations. Once excessive Cd ions entered the cytosol, a defence mechanism becomes activated, protecting the cells against cadmium toxicity. However, under high Cd content in cells, the cell structure was damaged, even leading to cells death.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

Sequences variation and classification of B-hordein genes in hull-less barley from the Qinghai-Tibet Plateau

The goal of this study is to understand the evolution relationship of the members of the B-hordein gene family in hull-less barley by analysis of their structure and to explore their utility in grain quality improvement. Six copies of the B-hordein gene (Hn1-Hn3, Hn7-Hn9) were cloned from six hull-less barley cultivars collected from Qinghai-Tibet Plateau and molecularly characterized. Comparison of their predicted polypeptide sequences with the published data suggested that they all share the same basic protein structures. In addition, we found that the C-terminal end sequences of all B-hordeins shared a similar feature. In the six clones and the other three published genes (Hn4, Hn5, and Hn6) from hull-less barley, Hn2 and Hn7 contained the identical C-terminal end sequence DIMPVDFWH. Hn3, Hn4, Hn5, Hn8 and Hn9 also shared the common sequence DIMPPDFWH, which was similar to that of a B-hordein reported previously. Both Hn1 and Hn6 exhibited differences in their C-terminal end sequences, and they clustered into different subgroups. The B-hordeins with identical C-terminal end sequences were clustered into the same subgroup, so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide. Phylogenetic analysis also indicated that there is a relatively weak identity between our predicted B-hordeins and those reported from H. chilense and H. brevisubulatum. All of our nine predicted B-hordeins were clustered together and other B-hordeins formed another cluster. The possible use of these genes in relation to barley quality is discussed. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 1, pp. 63–70. The text was submitted by the authors in English     

Genetic mapping of the barley nitrate reductase-deficient nar1 and nar2 loci

Summary The nar2 locus that codes for a protein involved in molybdenum cofactor function in nitrate reductase and other molybdoenzymes was mapped to barley chromosome 7. F₂ genotypic data from F₃ head rows indicated nar2 is located 8.4±2.1 and 23.0± 4.6 cm from the narrow leaf dwarf (nld) and mottled seedling (mt2) loci, respectively. This locates the nar2 locus at 54.7±3.1 cm from the short-haired rachilla (s) locus near the centromere of chromosome 7. Close linkage of nar2 with DDT resistance (ddt) and high lysine (lys3) loci was detected but could not be quantified due to deviations from the individual expected 121 segregations for the ddt and lys3 genes. Southern blots of wheat-barley addition lines probed with a nitrate reductase cDNA located the NADH : nitrate reductase structural gene, nar1, to chromosome 6.Scientific Paper No. 7762. College of Agriculture and Home Economics Research Center, Washington State University, Project No. 0745. This investigation was supported in part by United States Department of Agriculture Grant No. 86-CRCR-1-2004     

Cytogenetics of in vitro cultured somatic cells and regenerated plants of barley (Hordeum vulgare L.)

Summary Cytogenetic analysis of immature embryoderived calli and regenerated plants of barley has demonstrated high heterogeneity of callus cultures and significant differences in cytogenetic processes between different callus lines. Regenerated plants usually have a normal chromosome complement (2n=14). Tetraploid plaints occur with a frequency of 1%. No chromosome aberrations have been detected by Feulgen staining. The phenomenon of chromosome stickiness recorded from the 2nd day of culture was discovered in a majority of callus lines as well as the phenomena of chromatin hypercondensation and chromosome supercoiling. A possible contribution of cytogenetic and molecular processes to somaclonal variation is discussed.