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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)., (© 2021. The Author(s).)

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., (Copyright © 2019. Published by Elsevier B.V.)

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. 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.

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., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

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]

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]

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]

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]