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Among various methods stimulating biological progress, double haploid (DH) technology, which utilizes the process of microspore embryogenesis (ME), is potentially the most effective. However, the process depends on complex interactions between many genetic, physiological and environmental variables, and in many cases, e.g., winter wheat, does not operate with the efficiency required for commercial use. Stress associated with low-temperature treatment, isolation and transfer to in vitro culture has been shown to disturb redox homeostasis and generate relatively high levels of reactive oxygen species (ROS), affecting microspore vitality. The aim of this study was to investigate whether controlled plant growth, specific tiller pre-treatment and culture conditions could improve the potential of microspores to cope with stress and effectively induce ME. To understand the mechanism of the stress response, hydrogen peroxide levels, total activity and the content of the most important low-molecular-weight antioxidants (glutathione and ascorbate), as well as the content of selected macro- (Mg, Ca, NA, K) and micronutrients (Mn, Zn, Fe, Cu, Mo) were determined. These analyses, combined with the cytological characteristics of the microspore suspensions, allowed us to demonstrate that an increased microspore vitality and stronger response to ME induction were associated with higher stress resistance based on more efficient ROS scavenging and nutrient management. It was shown that a modified procedure, combining a low temperature with mannitol and sodium selenate tiller pre-treatment, reduced oxidative stress and improved the effectiveness of ME in winter wheat lines. [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]

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]

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]

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]

Plant growth regulator (PGR) crosstalk and interaction with the plant’s genotype and environmental factors play a crucial role in microspore embryogenesis (ME), controlling microspore-derived embryo differentiation and development as well as haploid/doubled haploid plant regeneration. The complexity of the PGR network which could exist at the level of biosynthesis, distribution, gene expression or signaling pathways, renders the creation of an integrated model of ME-control crosstalk impossible at present. However, the analysis of the published data together with the results received recently with the use of modern analytical techniques brings new insights into hormonal regulation of this process. This review presents a short historical overview of the most important milestones in the recognition of hormonal requirements for effective ME in the most important crop plant species and complements it with new concepts that evolved over the last decade of ME studies. [ABSTRACT FROM AUTHOR]

To verify the hypothesis that cell redox status regulates the process of microspore embryogenesis (ME), reactive oxygen species (ROS) generation and the activity of enzymatic and non-enzymatic antioxidants were analyzed in eight doubled haploid lines of triticale with significant differences in embryogenic potential. The analyses were performed in anthers excised from freshly cut tillers (control) and from low temperature (LT) pre-treated tillers (3 weeks at 4 °C) in which ME has been initiated. Significant associations between ME effectiveness and the variables studied were found. In control cultures, high superoxide dismutase (SOD) activity appeared crucial for microspore viability. On the other hand, positive though non-linear correlation between ME effectiveness and HO generation, and negative correlation with catalase (CAT) activity suggest that some threshold level of HO is important for successful ME initiation. LT tillers pre-treatment significantly increased HO accumulation, which had a negative effect on ME effectiveness. However, even high level of HO did not endanger cell viability as long as the cells exhibited high activity of ROS-decomposing enzymes (SOD, CAT and POX). The ability to sustain antioxidative enzyme activity under cold stress in the dark was another important requirement for high effectiveness of ME, allowing for the generation of the signal initiating microspore reprogramming and simultaneously protecting the cells from the toxic effects of ROS production. The role of antioxidative enzymes cannot be replaced even by high activity of non-enzymatic antioxidants. In conclusion, genetically controlled but environmentally modified cell tolerance to oxidative stress seems to play an important role in triticale ME. [ABSTRACT FROM AUTHOR]

QTLs associated with ABA content in triticale anthers were analysed using the population of 72 DH lines derived from F1 cross between inbred line 'Saka 3006' and cv. 'Modus'. Using Windows QTL Cartographer and MapQTL 5.0 softwares, composite interval mapping (CIM) and association analysis (Kruskal-Wallis test) for ABA content before and after low temperature treatment inducing androgenic development were conducted. The concentration of ABA in freshly cut tillers varied from 0.8 to 2.2 nmol g DW. Intensive ABA accumulation (up to 5.3 nmol g DW) was observed in response to low temperature stress. No correlation between ABA concentration and androgenesis induction efficiency was detected, whereas negative correlation (up to r = −0.33) was found between concentration of ABA in anthers collected from cold treated tillers and regeneration ability. CIM analysis indicated one QTL localized on chromosome 5A (LOD = 2.5; R = 9.3 %) associated with ABA concentration in unstressed plants and four QTLs on chromosomes 2A, 1B and 5R (LOD 2.6-5.7; R = 9.5-24 %) regulating ABA accumulation in response to low temperature stress. The Kruskal-Wallis test revealed significant ( p < 0.01) association with markers localized on chromosome 4A, 5A and 2RS.3R for unstressed plants and on chromosomes 3A, 3B, 5B and 2RS.3R for low temperature treated plants. [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]

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]

ording to regular reports, one of the most serious diseases of winter cereal and grass varieties in moderate and cold climatic areas is pink snow mould caused by Microdochium nivale. Currently, the resistance of the economically important cereal species as triticale is not satisfactory. Moreover, there is no efficient strategy of protection against this pathogen and the understanding of plant resistance mechanisms is rather poor. Presented paper for the first time shows the cytological analysis of M. nivale infection in model triticale varieties by the use of fluorescent and light microscopy in combination with fluorescent dyes and hydrogen peroxide staining. Both, the infection level and the dynamic of the process varied for tested genotypes confirming the field and laboratory data of their different resistance to this pathogen. Moreover, our analysis showed that in both cultivars cold-hardening of seedlings delayed the mycelium growth. In both cultivars, hyphal walls and fungal penetration sites were visualized in crowns, leaf sheaths and leaves of hardened and non-hardened inoculated seedlings. For the first time the presence of the haustoria produced by M. nivale was confirmed in those tissues. Single infection hyphae usually penetrated into the host tissues via stomatal apparatuses were accompanied by the efflux of hydrogen peroxide. The data show a great potential of fluorescence techniques in studying the host plant-pathogen interactions providing a better insight into plant defence reactions that may allow elaboration of the efficient breeding strategies aimed at increasing resistance to this pathogenic fungus. [ABSTRACT FROM AUTHOR]

Enhanced antioxidant defence plays an essential role in plant survival under stress conditions. However, excessive antioxidant activity sometimes suppresses the signal necessary for the initiation of the desired biological reactions. One such example is microspore embryogenesis (ME)—a process of embryo-like structure formation triggered by stress in immature male gametophytes. The study focused on the role of reactive oxygen species and antioxidant defence in triticale (×Triticosecale Wittm.) and barley (Hordeum vulgare L.) microspore reprogramming. ME was induced through various stress treatments of tillers and its effectiveness was analysed in terms of ascorbate and glutathione contents, total activity of low molecular weight antioxidants and activities of glutathione–ascorbate cycle enzymes. The most effective treatment for both species was a combination of low temperature and exogenous application of 0.3 M mannitol, with or without 0.3 mM reduced glutathione. The applied treatments induced genotype-specific defence responses. In triticale, both ascorbate and glutathione were associated with ME induction, though the role of glutathione did not seem to be related to its function as a reducing agent. In barley, effective ME was accompanied by an accumulation of ascorbate and high activity of enzymes regulating its redox status, without direct relation to glutathione content. [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]

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]

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]

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]

• 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]