Barley Pollen Research Articles

An account of chromosomal damage in PMCs of stripe rust infected barley

A study, on the effects of natural mild fungal infection on meiosis of Barley Pollen Mother Cells (PMCs), was done in order to analyze the chromosomal damage elicited by the pathogenic conditions. A pattern similar to common mutagens, of reduction in mitotic index and chiasma frequency, as well as, production of various aberrations that demonstrate chromosomal damage, was observed. The most common abnormalities were un-orientation and other spindle related aberrations, as well as stickiness and clumping of chromosomes. The disease induced a reduction in pollen viability as compared to the control plants. The results were compared with those of a high dose of a known mutagen ie gamma rays in order to draw commonalities between the two conditions.

Auxin boosts energy generation pathways to fuel pollen maturation in barley

Pollen grains become increasingly independent of the mother plant as they reach maturity through poorly understood developmental programs. We report that the hormone auxin is essential during barley pollen maturation to boost the expression of genes encoding almost every step of heterotrophic energy production pathways. Accordingly, auxin is necessary for the flux of sucrose and hexoses into glycolysis and to increase the levels of pyruvate and two tricarboxylic (TCA) cycle metabolites (citrate and succinate). Moreover, bioactive auxin is synthesized by the pollen-localized enzyme HvYUCCA4, supporting that pollen grains autonomously produce auxin to stimulate a specific cellular output, energy generation, that fuels maturation processes such as starch accumulation. Our results demonstrate that auxin can shift central carbon metabolism to drive plant cell development, which suggests a direct mechanism for auxin's ability to promote growth and differentiation.

High-throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM.

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCRTM -based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.

Preparation of Barley Pollen Mother Cells for Confocal and Super Resolution Microscopy.

Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (Hordeum Vulgare)have provided new insights into the control of crossing over in large genome species. A major achievement in these studies has been the use of cytological procedures to follow meiotic events. This protocol provides detailed practical steps required to perform immunostaining of barley meiocytes (pollenmother cells) for confocal or structured illumination microscopy.

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

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 24h 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.

Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley.

The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.

The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis.

Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well-established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five-fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every 4 days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore, the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species.

Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging.

Plants display a remarkable capacity for cellular totipotency. An intriguing and useful example is that immature pollen cultured in vitro can pass through embryogenic development to form haploid or doubled haploid plants. However, a lack of understanding the initial mechanisms of pollen embryogenesis hampers the improvement and more effective and widespread employment of haploid technology in plant research and breeding. To investigate the cellular dynamics during the onset of pollen embryogenesis, we used time-lapse imaging along with transgenic barley expressing nuclear localized Green Fluorescent Protein. The results enabled us to identify nine distinct embryogenic and non-embryogenic types of pollen response to the culture conditions. Cell proliferation in embryogenic pollen normally started via a first symmetric mitosis (54.3% of pollen observed) and only rarely did so via asymmetric pollen mitosis I (4.3% of pollen observed). In the latter case, proliferation generally originated from the vegetative-like cell, albeit the division of the generative-like cell was observed in few types of pollen. Under the culture conditions used, fusion of cell nuclei was the only mechanism of genome duplication observed.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.

Relationship of environmental changes in central Sri Lanka to possible prehistoric land-use and climate changes

A radiocarbon-dated pollen-analysed peat sequence from the Horton Plains (> 2000 m a.s.l.), in central Sri Lanka, together with physical and chemical parameters (organic carbon, mineral magnetics, carbon isotopes and phytoliths), indicates major environmental changes during the last 24,000 years. The results suggest that a mobile life form, i.e. a hunter–forage culture, predominated in an open landscape, associated with xerophytic vegetation, e.g. Chenopodium spp. at ∼ 17.5 ka BP. Incipient management of cereal plants and slash-and-burn techniques seem to have prevailed between 17.5 and 13 ka BP, which was indigenous and associated with grazing. Evidence of systematic cereal cultivation in the form of oat and barley pollen grains is found from the late Pleistocene (∼ 13 ka BP). This is the earliest evidence of farming activities noted in Sri Lanka as well as in south Asia. After 13 ka BP, cereal cultivation was associated with an increase in humidity. With a later abrupt increase in aridity, agricultural land-use decreased from ∼ 8 to ∼ 3.6 ka BP, when the area appears to have been almost deserted. After a severe middle Holocene arid phase (i.e. 5.4–3.6 ka BP), the agricultural activity with a limited extension was again initiated by ∼ 2.9 ka BP. During the next ∼ 900 years, cultivation ceased allowing the upper montane rain forest to dominate. Between 0.2 and 0.15 ka BP, new phases of agricultural activities were undertaken and potato cultivation took place lately, between 1950 and 1969 AD.

Confocal potassium imaging: Giving new insight into potassium concentrated at the aperture area of barley ( Hordeum vulgare L.) pollen

Potassium (K) presence at the aperture area of barley ( Hordeum vulgare L.) pollen was studied by using the potassium sensitive fluorescent dye potassium-binding benzofuran isophthalate (PBFI) and confocal laser scanning microscopy. The presence of heavy K fluorescence was detected only at the aperture area of the pollen.

Does barley (Hordeum vulgare L.) pollen swell in fractions of a second?

Does barley (Hordeum vulgare L.) pollen swell in fractions of a second?

Does barley ( Hordeum vulgare L.) pollen swell in fractions of a second?

Microscopic studies revealed that freshly collected six-rowed barley ( Hordeum vulgare L. cv. Baegdong) pollen swelled in 600±50 milliseconds (ms) (0.6±0.05 s) upon hydration with distilled water. The half time ( T 1/2) of complete swelling of pollen was 200 ms, which gave values of 0.14×10 −5 m s −1 MPa −1 for hydraulic conductivity ( Lp). This value was higher than usually reported for higher plant cells. It was hypothesized that potassium (K) may be involved in creating osmotic gradient, resulting in speedy uptake of water and ultimately rapid swelling of pollen. Macallum’s solution was used to stain K in pollen. It was found that K concentrated in the aperture area of pollen. However, breaking of pollen further revealed that heavy stains of K were not only found at the aperture wall of pollen but also found at the part of cytoplasmic area that attached with the aperture. Furthermore, energy dispersive X-ray analysis (EDX) verified staining results of K at the aperture area of pollen. These results demonstrate that barley pollen swell in fractions of a second. The high hydraulic conductivity of pollen membrane could be responsible for rapid movement of water through membrane. These results also suggest that there could be a possible role of K located at the aperture area, in rapid swelling of pollen by creating osmotic gradient across membrane, which is the prerequisite of rapid protrusion of pollen tube.

Ontogenetic events in androgenesis of Brazilian barley genotypes.

This paper describes a simple procedure for obtention of barley (Hordeum vulgare L.) doubled haploid plants from Brazilian hybrid genotypes. Anatomical and histological examinations showed the reversion of barley pollen to an sporophytic mode of development. A sequence of mitosis led to the formation of multicellular pollen grains. Regeneration of plants occurred either by direct embryogenesis or callus formation followed by differentiation through direct embryogenesis or organogenesis. Plants were formed in the same medium used for induction dispensing an additional regeneration step. This procedure makes doubled haploid production simpler and faster. Plantlets were transferred to another medium for rooting and after that planted in pots with vermiculite and nutrient solution.

Cloning and expression pattern of Hor v 9, the group 9 pollen isoallergen from barley

In this study we report the cloning, sequence, and characterization of Hor v 9 allergen cDNAs from barley ( Hordeum vulgare) pollen. Structural homologues of Kentucky bluegrass ( Poa pratensis) group 9 pollen allergens were identified in a cDNA library of barley pollen expressed mRNAs. The Hor v 9 cDNA clone (hvp9742) contained an open reading frame encoding 313 amino acids which included a putative 27-residue signal peptide and one asparagine sequon for glycosylation. The mRNA corresponding to clone hvp9742 was produced abundantly in pollen during the late stages of anther development. The protein encoded by clone hvp974 was synthesized as a fusion protein in the E. coli expression vector pMAL. Immunoblots using antibodies to this recombinant allergen, rHor v 9, showed that Hor v 9 protein accumulated during pollen development and was produced maximally at pollen maturity. Using these antibodies, we also provide evidence that Hor v 9 protein localized to the extracellular matrix of mature pollen. Southern blots suggested that Hor v 9 allergens exist as multiple isoforms in barley. Sequence comparisons showed that the Hor v 9 cDNA clones were also homologous to group 5 allergens of Timothy grass ( Phleum pratense) pollen and canary grass ( Phalaris aquatica) pollen, and the group 9 allergen of ryegrass ( Lolium perenne) pollen.

Pollen allergen homologues in barley and other crop species.

Pollen from 10 agricultural plant species was surveyed for the presence of proteins crossreactive with group I, group IV and group IX allergens. Barley (Hordeum vulgare), maize (Zea mays), rye (Secale cerale), triticale (xTriticosecale cereale), oats (Avena sativa), Canola (Brassica napus) and sunflower (Helianthus annus) pollens contained numerous allergen cognate proteins. Northern blot analysis of barley pollen RNA revealed the presence of group I and group IX allergen transcripts. The barley pollen cDNA hvp9742, and three other cloned allergens: phlenum protense (Phl p) V, Phl p Va and Lolium perenne (Lol p) 1b, were demonstrated to have extensive nucleotide and amino acid sequence similarity to the Poa p IX isoallergens. It was concluded that hvp9742 represents a Poa p IX isoallergen homologue expressed by barley pollen, and was therefore designated Hor v IX. It is further shown that the most highly conserved domains of all seven proteins, including Hor v IX, map to previously defined Poa p IX antibody binding epitopes.

The Effects of Genotype and Mannitol Pretreatment on High Frequency Androgenesis in Barley

Since the success in induction of barley pollen plants through anther culture was achieved by Clapham in 1973, a great amount of efforts have been further made on the study. In the past 20 years, the green plant production has been increased by 100-fold by improving culture methods and media etc. However, there still exist a lot of theoretical and applied problems, for example, the green plantlet yield per anther for most materials except some

Influence of combined alpha, beta and gamma radionuclide contamination on the frequency of waxy-reversions in barley pollen

The mutagenic effectiveness of chronic, combined alpha-, beta-, and gamma-irradiation caused by radionuclide contamination of the biogeocenosis was studied in comparison with chronic gamma-irradiation from an external 60Co source. The waxy-test on barley pollen proved to be sufficiently sensitive to evaluate the increase in waxy-reversion frequencies in plants exposed to a 3 mSv dose. A dose of 50–200 mSv from external irradiation was needed to double the mutation rate in barley pollen. Moreover, the smaller doses (dose rates) were more harmful than the higher doses if measured as the increase in mutation frequency per unit of irradiation. Higher genotoxicity was observed from combined irradiation due to radionuclide contamination than from similar doses from an external gamma source. We concluded that mutagenic effects in plant cells, in the case of radionuclide contamination of the biogeocenosis, could not be extrapolated only on the basis of measured dose, but separate investigations should be carried out to establish the genotoxicity of specific radionuclide mixtures and possible toxic chemical synergisms.

The use of pollen irradiation in barley breeding

Second generation progenies (M2) derived from crosses using barley pollen irradiated at 500, 1,000 1,500 and 2,000 rads increasingly resembled the maternal parent. 'Golden Promise', but contained some characteristics derived from the paternal parent, 'Magnum'.