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Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5–10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2). [ABSTRACT FROM AUTHOR]

Key message: Depending on the capability for stress adaptation, the role played by glutathione in microspore embryogenesis consists of both antioxidative activity and stimulation of embryo-like structure development.Abstract: The efficiency of microspore embryogenesis (ME) is determined by the complex network of internal and environmental factors. Among them, the efficient defence against oxidative stress seems to be one of the most important. The present study confirms this hypothesis showing the positive effect of glutathione—the most abundant cellular antioxidant—on ME in isolated microspore cultures of triticale (× Triticosecale Wittm.). For the first time, low temperature (LT) pre-treatment of tillers was combined with the exogenous application of glutathione and associated with the total activity of low-molecular weight antioxidants, the endogenous content and redox status of glutathione, and the effectiveness of ME. The results indicate that efficient antioxidative defence is the first, although not the only, prerequisite for effective ME. In responsive genotypes, LT alone stimulated antioxidative defence and decreased cell redox status, which was associated with increased cell viability and high frequency (ca. 20%) of microspore reprogramming. Application of glutathione had no effect either on the microspore viability or on the initial number of embryogenic microspores. However, it increased the number of embryo-like structures, probably by stimulating the next phases of its development. In recalcitrant genotypes, the main role of glutathione seems to be its participation in cell protection from oxidative stress. However, even enhanced antioxidative activity, which sustained cell viability and increased the number of embryogenic microspores, was insufficient for efficient haploid/doubled haploid plant production. Evidently, there are still other defective elements in the complex network of factors that regulate the process of ME. [ABSTRACT FROM AUTHOR]

Drought is one of the stresses that limit the yield of barley. Despite extensive studies focused on the issue, the molecular mechanism of the response to drought is still not fully understood. In our previous study, we proposed drought-induced signal perception controlled by actin filaments (AFs). To test this hypothesis, we used a chemical inhibitor of AF polarization—latrunculin B. In drought-treated barley leaves, latrunculin B induced AF depolymerization and altered gene expression (mainly those controlling AF formation), notably inhibiting the expression of HVA1 , a dehydrin encoding gene whose function in drought tolerance has been widely studied. These results suggest that AFs might be involved in water-deficit signal perception in plant cells. [ABSTRACT FROM AUTHOR]

Key message: Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. Abstract: The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11-39 nmol g DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121-424 pmol g DW) and zeatin ryboside ( cZR, 146-432 pmol g DW). Surprisingly, a relatively high level (10-64 pmol g DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of 'responsive' DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with 'recalcitrant' genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential. [ABSTRACT FROM AUTHOR]

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro -cultured microspores and on microspore-derived embryo development. In the 'mild' heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 μm, endogenous auxin content in a single cell corresponded to concentration of 1.01 μM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant. [ABSTRACT FROM AUTHOR]

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical-basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis. [ABSTRACT FROM AUTHOR]

Key message: A better understanding of androgenesis with a focus on the changes in plasma membrane fluidity and endogenous ABA content affecting embryogenesis induction in microspore suspension of B. napus. Abstract: Changes in plasma membrane fluidity (MF) and ABA content associated with androgenesis induction were under the study. Both parameters were monitored in microspores of two Brassica napus L. genotypes differing in their response to androgenic induction under heat (1 day at 32 °C). MF was assessed by DPH method. ABA content was evaluated by ELISA. Heat caused microspores' plasma membrane to become more rigid. Lower MF in microspores of 'DH 4079' (of high androgenic potential) seems to maintain proper cell protection and leads to efficient embryogenesis induction. Plasma membrane remodelling coincided with changes of ABA content in microspores and in the culture medium in both genotypes. ABA concentration (μM) and ABA content (fmol per 10 microspores or pmol g FW) were for the first time measured in microspores. ABA concentration (μM) in microspores and in the culture medium (nM) differed significantly for the genotype and the treatment. The interaction between both variables was also significant. In general, ABA content ranged from <3.5 to 87.1 fmol per 10 microspores. The highest content of ABA was detected in 'DH 4079' microspores at 32 °C. Assuming a mean microspores' radius of 10 μm, it corresponds to ABA concentration of 2.1 μM. Heat shock resulted in quantum of medium pH reduction (0.1-0.2) and increased levels of ABA in microspores and in the medium of both tested genotypes. However, heat induced increase of ABA content in microspores of non-responsive 'Campino' had no clear-cut impact, on androgenesis induction efficiency, which suggests a more complex mechanism of process initiation. [ABSTRACT FROM AUTHOR]

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique. [ABSTRACT FROM AUTHOR]

Isolated microspores and pollen suspension of Brassica napus 'Topas' cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a 'comet-tail' form. These GC 'tail' MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a 'whole-mount' immunodetection technique and confocal laser-scanning microscopy was essential. [ABSTRACT FROM AUTHOR]

Abstract: Pink snow mould caused by Microdochium nivale can lead to serious yield loss in many winter cereals and turf grasses. The study examined the infection mechanism in four inbred lines and one cultivar of rye (Secale cereale L.) with different sensitivities to this pathogenic fungus. Fungus development was analyzed at the early stages of the infection, up to the 13th day after inoculation, in hardened and non-hardened seedlings, using light/fluorescence microscopy. The cytological analysis was supplemented with a spectrophotometrical assay of the phenolics. Significantly higher phenolic content was revealed in the leaves of the lines more resistant to M. nivale, as compared to those which are more sensitive. [Copyright &y& Elsevier]

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A 'mild' heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3-8 celled. [ABSTRACT FROM AUTHOR]

Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors. Haploids/doubled haploids (DHs) derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering. However, the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined. Here, RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis (ME) in barley cultivars Golden Promise and Igri, which exhibit contrasting responsiveness to microspore-derived plant formation. Our experiments revealed fundamental regulatory networks, specific groups of genes, and transcription factor (TF) families potentially regulating the developmental switch. We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME. Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition. [ABSTRACT FROM AUTHOR]

• Modulation of androgenesis-inducible stress intensity causes changes in the activity of the β-1,3-glucanases, chitinases and AGP in rye. • ∼26 kDa and some acidic isoforms of β-1,3-glucanases are anther specific. • β-1,3-Glucanases and chitinases (GH18 and GH19) are highly active in anthers. • JIM 4 and JIM 13 are detectable in anthers/microspores. • JIM 13 is required to trigger microspores towards embryogenesis. This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, β-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of β-1,3-glucanases and chitinases. Some isoforms of β-1,3-glucanases, namely several acidic isoforms of about 26 kDa; appeared to be anther specific. Chitinases were well represented but were less variable. RT-qPCR revealed that the cold-responsive chitinase genes Chit1 and Chit2 were expressed at a lower level in the microspores and whole anthers while the cold-responsive Glu2 and Glu3 were not active. The stress pre-treatments modifications promoted the AGP accumulation. An apparent dominance of some AGP epitopes (LM2, JIM4 and JIM14) was detected in the androgenesis-responsive rye line. An abundant JIM13 epitopes in the vesicles and inner cell walls of the microspores and in the cell walls of the anther cell layers appeared to be the most specific for embryogenesis. [ABSTRACT FROM AUTHOR]

• Heavy metals induce programmed cell death (PCD) in Viola tricolor cells. • Papain-like cysteine proteases (PLCPs) but not caspase-3-like enzyme are involved in heavy metal-induced PCD. • E-64-d, a PLCPs inhibitor, prevents the PCD pathway increasing cell viability after heavy metal treatment. Programmed cell death (PCD) plays a crucial role in plant development (dPCD) and is also activated under the influence of environmental factors (ePCD). As the mechanism of PCD under heavy metal stress is poorly understood, a suspension culture of cells from the facultative metallophyte Viola tricolor was used as a model to study the role of caspase-like proteases in PCD in plant cells under heavy metal stress. Cell suspension cultures treated with Zn or Pb (2000 μM for 72 h) were subjected to TUNEL assay and ligand blotting using a biotinylated inhibitor of caspase-like proteases (biotin-xVAD-fmk). The results from the TUNEL assay showed that the frequency of PCD events was significantly higher in the metal-treated suspended cells (6.8–19.6 % vs 21.8–30.1 %, Zn or Pb, respectively) than it was in the control cells (7.1–9.1 %). The results from the biotinylated inhibitor blot analysis revealed that papain-like cysteine proteases (PLCPs) were involved in heavy metal-induced PCD. Exogenous application of the PLCPs inhibitor E-64-d at a concentration of 50 μM together with Zn or Pb remarkably increased cell viability. This is the first report on the role of the PLCPs in ePCD in plant cells under heavy metal stress. [ABSTRACT FROM AUTHOR]

For a better understanding of the physiological background of microspore embryogenesis (ME), the protein profile was analyzed in four winter triticale DH lines, which show extremely different embryogenic potential. The analysis were conducted with anthers at the phase of development optimal for ME induction and then after low temperature (LT, 3 weeks at 4°C) ME-inducing tillers treatment. The sub-proteome of anthers was mapped by two-dimensional gel electrophoresis (2- DE). The protein species significantly more abundant (at least 2-fold) in responsive DH lines after LT treatment were chosen for identification by MALDI-TOF/TOF analysis. In total, 31 protein species were successfully identified as involved in the determination of microspore competence, stress response and in the regulation of ME induction. Microspore competence required sufficient energy supply and efficient system of cell protection that determine survival under prolonged LT stress treatment. LT stress was associated with increased accumulation of proteins typical for cell defence against oxidative stress (e.g., L-ascorbate peroxidase), chaperons (e.g., HSP70) and other enzymes/factors ensuring protein biosynthesis, stability and active cell divisions. Also here, effective cell defence required undisturbed energy supply. Among proteins that accumulated differentially in accordance with microspore embryogenic potential again the most important role seems to be played by the enzymes ensuring energy production and determining ability of plant stress adaptation. Two protein species (enolase, 12S storage protein), proposed earlier as candidates for markers of embryogenesis in other in vitro plant culture systems confirmed their utility for triticale anther cultures. [ABSTRACT FROM AUTHOR]

High frequency of albino haploids/doubled haploids (DHs), regenerated in androgenic cultures is one of the major obstacles that limit incorporation of DHs technology into cereal breeding programs. Therefore, quantitative trait loci (QTL) associated with albino plant production in triticale anther cultures were analyzed using the population of 90 DH lines derived from F1 cross 'Saka 3006' × 'Modus'. Composite interval mapping (CIM) and single marker analysis (SMA) in Windows QTL Cartographer ver 2.5 were used to localize the major QTLs. CIM method revealed seven QTLs with LOD scores between 2.9 and 5.6 on five chromosomes from B to R subgenomes (3B, 4B, 4R, 5R and 7R). Effects of all QTLs explained 8.3-17.6 % of the phenotypic variation and were confirmed by SMA analysis. Additionally SMA revealed another seven markers on chromosomes: 2AL.2BL, 3B, 2BS.6AL, 2RS.3R and 4R associated with QTL for albino plant regeneration (p < 0.01). The additional experiment with ten DH lines varied significantly in their androgenic responsiveness was conducted to analyze the changes in the level of oxidative stress, antioxidative system activity and endogenous hormonal balance associated with androgenesis-inducing low temperature stress treatment (3 weeks at 4 °C). The correlation analysis between albino/green plant regeneration ability and analyzed traits were performed by using Spearman Rank test (p ≤ 0.05). Revealed associations may suggest that some level of oxidative stress is necessary for transition from a non-photosynthetic proplastids to the functional chloroplasts. On the other hand, the efficient antioxidative enzyme system and endogenous hormonal balance are also very important. Key message: Fourteen chromosome regions were indicated to control albino plant formation during triticale anther culture. Additionally, reactive oxygen species (ROS) generation, antioxidative system activity and hormonal balance were discussed as determinants in androgenesis. [ABSTRACT FROM AUTHOR]

One of the main criterions in turfgrass breeding selection is good wear tolerance, which means the plant's ability to withstand forces that crush leaves, stems, crowns and roots. In the present study the relationship between the anatomical and morphological traits and wear tolerance of 37 cultivars of seven turfgrass species was investigated. The wear tolerance of turf was determined using a Brinkman traffic simulator during a field trial over a two-year period from 2012 to 2013. The experimental plots were established with three replications in a randomized block design. The anatomical and morphological characteristics of the above-ground part of grasses were analyzed. Based on the quantitative data obtained, a new index reflecting turf appearance, and in particular turf cover (TCI), quality (TQI) and shoot density (SDI) has been proposed. These new wear tolerance indexes created could be a very helpful and useful tool for breeding and research purposes. Standardized trait mean values were used to perform principal component and hierarchical cluster analysis. The relationship between leaf parameters and turf indexes was analyzed using multiple linear regression in a stepwise procedure. According to the TCI, the most wear-resistant cultivars were those of Lolium perenne , Ilso Nira and Oxiana (−0.04 in average). As turf quality was considered, all cultivars of L. perenne and Poa pratensis were characterized as high quality after BTS compaction with the TQI below 0.1. The shoot density of untreated turf cultivars varied in a wide range from 2.23 to 6.18 shoots per cm 2 . For most of the cultivars the BTS compaction reduced shoot density by 27% on average. The increase in the number of vascular bundles, leaf width, leaf cross-section area and leaf angle were associated with better wear tolerance. Wider leaves with a higher number of vascular bundles together with the higher angle between the leaf and tiller axis, improved the wear resistance. It was also observed that higher tillering does not favour wear tolerance. This information concerning the relationships between turf traits and wear tolerance is of use to the plant breeder as a tool for the selection of appropriate turfgrass cultivars. [ABSTRACT FROM AUTHOR]

Diverse processes leading to doubled haploid (DH) plant production, such as microspore embryogenesis, gynogenesis, and distant hybridization followed by genome elimination, are based on the unique ability of plant cells to form haploid embryos without fertilization. All of these are possible because of various in vitro culture systems that enable the growth and development of tissues or single cells outside of the parental organism. The possibility of re-directing cell development from its original pathway to embryogenesis brings several benefits to many research areas, but the most important is the possibility of its implementation in breeding programs. This review summarizes the achievements of Polish research groups in studies of the mechanisms of haploid/DH embryo development and demonstrates the practical applications of these systems in basic studies and plant breeding. It shows the results of studies on economically important crops including barley (Hordeum vulgare L.), oilseed rape (Brassica napus L.), triticale (Ã--Triticosecale Wittm.), oat (Avena sativa L.), rye (Secale cereale L.), sugar beet (Beta vulgaris ssp. vulgaris L.), and some vegetable species, including carrot (Daucus carota L.), onion (Allium cepa L.), red beet (Beta vulgaris L.), and members of the Brassicaceae. [ABSTRACT FROM AUTHOR]

Chromosomes and nuclear DNA amount were analyzed in leaf tissues of Luzula nivea, Luzula luzuloides, and Luzula multiflora. Intra- and interspecific karyological variability was stated. Chromosome numbers in diploids ranged 2n = 8-24 in L. nivea and L. luzuloides and 2n = 12-84 in hexaploid L. multiflora. Karyological variability resulted mainly from chromosome fission (agmatoploidy) and aneusomaty; chromosome fusion (symploidy) and polyploidy were also involved. Flow cytometric determination of nuclear genome size using propidium iodide staining gave values of 1.584 pg in L. luzuloides, 1.566 pg in L. nivea, and 3.034 pg in L. multiflora. Variability in relative nuclear genome size within species was measured by 4',6-diamidino-2-phenylindole staining. In contrast with previous reports, variability was fairly small and ranged from 1.796 to 1.864 pg in L. luzuloides, from 1.783 to 1.847 pg and from 1.737 to 1.808 pg in two populations (S and F) of L. nivea, respectively, and from 3.125 to 3.271 pg in L. multiflora. An intraplant (interleaf) genome size variation was also observed and its possible causes are discussed. [ABSTRACT FROM AUTHOR]

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, β-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley. [ABSTRACT FROM AUTHOR]

The present study investigated drought-induced changes in proteome profiles of ten DH lines of winter barley, relatively varied in water deficit tolerance level. Additionally, the parameters describing the functioning of the photosynthetic apparatus and the activity of the antioxidative system were analysed. Water deficit (3-week growth in soil with water content reduced to ca. 35%) induced significant changes in leaf water relations and reduced photosynthetic activity, probably due to decreased stomatal conductance. It was associated with changes in protein abundance and altered activity of antioxidative enzymes. From 47 MS-identified proteins discriminating more tolerant from drought-sensitive genotypes, only two revealed distinctly higher while seven revealed lower abundance in drought-treated plants of tolerant DH lines in comparison to sensitive ones. The majority were involved in the dark phase of photosynthesis. Another factor of great importance seems to be the ability to sustain, during drought stress, relatively high activity of enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Low molecular weight antioxidants seem to play less important roles. Our findings also suggest that high tolerance to drought stress in barley is a constitutively controlled trait regulated by the rate of protein synthesis and their activity level. Biological significance According to our knowledge, this is the first comparative proteomic analysis of drought tolerance performed for the model set of several winter barley doubled haploid (DH) lines. We analysed both the drought impact on the protein pattern of individual winter barley DH lines as well as comparisons between them according to their level of drought tolerance. We have identified 47 proteins discriminating drought-tolerant from drought-sensitive genotypes. The majority was involved in the dark phase of photosynthesis. Another factor of great importance in our opinion seems to be the ability to sustain, during drought stress, relatively high activity of antioxidative enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Our findings also suggest that high tolerance to drought stress in barley is a constitutively-controlled trait regulated by the rate of protein synthesis and their activity level. [ABSTRACT FROM AUTHOR]

The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important. The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CS m ). Additionally, the analysis of antioxidative enzyme activities suggests the importance of H 2 O 2 as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, H 2 O 2 generation during cold hardening treatment was accompanied by more stable activity of catalase, H 2 O 2 -decomposing enzyme. Significance In the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition. [ABSTRACT FROM AUTHOR]

The breeding for resistance against fungal pathogens in winter triticale ( Triticosecale Wittm.) continues to be hindered by a complexity of the resistance mechanisms, strong interaction with environmental conditions, and dependence on the plant genotype. We showed, that temperature below 4 °C induced the plant genotype-dependent resistance against the fungal pathogen Microdochium nivale . The mechanism involved, at least, the adjustment of the reactions in the PSII proximity and photoprotection, followed by an improvement of the growth and development. The genotypes capable to develop the cold-induced resistance, showed a higher maximum quantum yield of PSII and a more efficient integration of the primary photochemistry of light reactions with the dark reactions. Moreover, induction of the photoprotective mechanism, involving at least the peroxidases scavenging hydrogen peroxide, was observed for such genotypes. Adjustment of the photosynthesis and stress acclimation has enabled fast plant growth and avoidance of the developmental stages sensitive to fungal infection. The same mechanisms allowed the quick regrow of plants during the post-disease period. In contrast, genotypes that were unable to develop resistance despite cold hardening had less flexible balancing of the photoprotection and photoinhibition processes. Traits related to: photosynthesis-dependent cold-acclimation and cold-induced resistance; biomass accumulation and growth; as well as protection system involving peroxidases; were integrated also at a genetic level. Analysing 95 lines of the mapping population SaKa3006 × Modus we determined region on chromosomes 5B and 7R shared within all tested traits. Moreover, similar expression pattern of a set of the genes related to PSII was determined with the metaanalysis of the multiple microarray experiments. Comparable results for peroxidases, involving APXs and GPXs and followed by PRXs, indicated a similar function during cold acclimation and defense responses. These data provide a new insight into the cross talk between cold acclimation and cold-induced resistance in triticale, indicating a key role of photosynthesis-related processes. [ABSTRACT FROM AUTHOR]

Snow mould caused by Microdochium nivale (Fr.) Samuels & Hallett is the most widespread seedling disease in winter cereals. Due to the complexity of the resistance mechanisms, a poorly understood genetic background and strong interaction with winter weather conditions, it is difficult to assess the resistance of triticale cultivars via conventional inoculation methods. Genetic resistance is the most economical and environmental friendly way to control M. nivale infection; therefore, the objective of this study was to detect the quantitative trait loci (QTLs) associated with resistance components of winter triticale in a mapping population derived from a cross of the ‘Modus’ (partly resistant) and ‘SaKa 3006’ (sensitive) varieties. High-resolution mapping was conducted by using 1518 molecular markers (diversity arrays technology, simple sequence repeat and amplified fragment length polymorphism). Partial resistance components assessed in this study, i.e. candidate QTLs, were detected on chromosomes 1B, 2A, 3A, 3B, 5A, 5B, 6A, 6B and 7B, whereas QTLs describing overall seedling vitality in non-infected control plants were located on chromosomes 1B, 2B, 3A, 5A, 7B and 7R. [ABSTRACT FROM AUTHOR]

The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis. [ABSTRACT FROM PUBLISHER]

• Responsive and recalcitrance lines of triticale differ with respect to phenotypic, genetic and epigenetic features. • There is a link between the formation of DNA damage and cytidine-analogs induced DNA de-methylation. • The application of 5-azacytidine increases the efficiency of microspore embryogenesis in the highly-responsive line. Microspores exposed to some stress factors may display cell totipotency and could be reprogrammed towards embryogenic development. Plant breeding and genetic engineering widely use haploids/doubled haploids (DHs) derived from in vitro -cultured microspores, but the mechanism of this process remains poorly understood. Recently published data suggest that microspore embryogenesis (ME) is accompanied by changes in DNA methylation and chromatin reorganization. Here, we used two triticale DH lines (DH19 and DH28), significantly different with respect to embryogenic potential. To change DNA methylation levels, we applied two cytosine-analogs: 5-azacytidine (AC) and 2′-deoxy-5-azacytidine (DAC) treatments. We found that chemically-induced DNA demethylation caused chromatin relaxation and dysregulation of marker genes (TaTPD1-like, GSTF2, GSTA2, CHI3, Tad1, TaNF-YA7, SERK2, TaME1) related to ME. Both drugs showed significant cytotoxicity in a dose-dependent manner. We noticed that lines varied in terms of overall DNA methylation levels and responded in a different way to hypomethylation caused by the drugs. DH19 (low embryogenic) after inhibitors treatment, showed higher microspore viability, but its recalcitrancy was not overcome. For highly embryogenic DH28, we noted significantly higher effectiveness of embryo-like structure production and plant regeneration. In summary, our study provides new insight into the role of DNA methylation in ME initiation. They suggest potential benefits resulting from the utilization of epigenetic inhibitors to improve the process of DHs production. [ABSTRACT FROM AUTHOR]