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The endogenous level of cytokinins (CKs) and auxins (Auxs) was analyzed in isolated microspores and ovaries of two doubled haploid (DH) lines of triticale (× Triticosecale Wittm.) to better understand the mechanism of microspore embryogenesis (ME)—the most efficient and widely applied method of producing DHs. The responsiveness of the studied lines to ME significantly varied. ME was induced by pre-treating tillers with low temperature (4 °C for 3 weeks) alone or in combination with synthetic auxin (2,4-D), auxin inhibitor (PCIB) or melatonin (MEL) applied for 4 days before microspore isolation. Hormonal profile analyses, accompanied by ME effectiveness evaluation confirmed the multi-level crosstalk of Auxs/CKs and the specific hormonal homeostasis required for effective microspore reprogramming. It was found that triticale microspores contained mainly cis zeatin derivatives: cis-zeatin-O-glucoside (cZOG), cis-zeatin riboside (cZR) and cis-zeatin (cZ), as well as indole-3-acetic acid (IAA) and IAA-aspartate (IAAsp). Increased ME efficiency was associated with higher contents of most of the identified CKs and Auxs, as well as the higher active Aux/active CK ratio. Trans CK isoforms were detected only in ovaries, confirming their importance as a source of bioactive molecules stimulating embryogenic development. Two of the pre-treatments tested: 12.5 μM PCIB and 50 μM MEL decreased the active Aux/active CK ratio, which was accompanied by an increase in the regeneration efficiency.

The effectiveness of microspore embryogenesis (ME) is determined by a complex network of internal and environmental factors. In the present study on triticale and barley, strong positive correlation (r = 0.85) between the generation of hydrogen peroxide (H2O2) and ME effectiveness confirmed the important role of reactive oxygen species in microspore reprogramming. However, for high effectiveness of ME induction, intensive H2O2 generation had to be associated with high activity of antioxidative enzymes, superoxide dismutase and catalase. The strong seasonal effect on the physiological status of microspores revealed in the study suggests a kind of ‘biological clock’ controlling plant reproduction, crucial for microspore viability and embryogenic potential. Although the effect of various modifications of ME-inducing stress tiller pre-treatment was determined mainly by the physiological condition of microspores, at higher stress intensity positive effects induced by antioxidant molecules—reduced glutathione and its precursor, l-2-oxothiazolidine-4-carboxylic acid—were observed. High level of variation in the response to ME-inducing stress tiller pre-treatment was also revealed between the two DH lines of triticale and two cultivars of barley and among microspores isolated from subsequently developed spikes. Microspore reprogramming towards embryogenic development is initiated by the genotype-specific threshold of reactive oxygen species accumulation. However, high effectiveness of the process requires efficient system of antioxidative defence.

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) Arabidopsis thaliana (L.) Heynh. And its two mutants: (1) totally TC-deficient vte1, (2) vte4 accumulating γ-TC instead of α-TC, and (3) tmt transgenic line overaccumulating α-TC. Raman spectra revealed that salt-exposed α-TC accumulating plants were more flexible in regulating chlorophyll, carotenoid and polysaccharide levels than TC deficient mutants, while the plants overaccumulating γ-TC had the lowest levels of these biocompounds. Tocopherol composition and NaCl concentration affected xanthophyll cycle by changing the rate of violaxanthin de-epoxidation and zeaxanthin formation. NaCl treated plants with altered TC composition accumulated less oligosaccharides than WT plants. α-TC deficient plants increased their oligosaccharide levels and reduced maltose amount, while excessive accumulation of α-TC corresponded with enhanced amounts of maltose. Salt-stressed TC-deficient mutants and tmt transgenic line exhibited greater proline levels than WT plants, lower chlorogenic acid levels, and lower activity of catalase and peroxidases. α-TC accumulating plants produced more methylated proline- and glycine- betaines, and showed greater activity of superoxide dismutase than γ-TC deficient plants. Under salt stress, α-TC demonstrated a stronger regulatory effect on carbon- and nitrogen-related metabolites reorganization and modulation of antioxidant patterns than γ-TC. This suggested different links of α- and γ-TCs with various metabolic pathways via various functions and metabolic loops.

Androgenic responsiveness in anther (AC) and isolated microspore cultures (MC) was analysed using 15 lines of Polish winter rye (Secale cereale L.). The effect of low temperature (LT) alone or in combination with osmotic stress induced by mannitol treatment (MAN) and/or with reduced glutathione (GSH) on the effectiveness of the process was studied. Interestingly, each treatment had a different effect on microspore (mcs) vitality and capability to divide symmetrically. The first criterion for successful embryogenesis was to exceed the threshold number of at least 25% dividing microspores, which determined ‘embryogenic suspension culture’. In some configurations a spectacular effect was achieved, especially in lines highly recalcitrant to androgenesis induction. Relatively high effectiveness of androgenesis induction (up to 4.58 AS per 105 mcs per spike in MC and 21.29 AS per spike in AC) showed that the developed protocol with GSH and/or MAN tiller pre-treatments overcomes the genotypic barrier for androgenesis initiation in rye. Moreover, relatively high, spontaneous genome diploidization (55%) of regenerated plants demonstrated that the described protocols could be effectively integrated into conventional rye breeding programmes. Glutathione and mannitol break the barrier of androgenesis initiation by increasing the dividing and vital microspores ratio. This marker is useful for screening material in breeding programs utilizing DH technology.

Ten doubled haploid (DH) lines of winter barley with an increased range of freezing/drought tolerance were used to identify phytohormones involved in plant stress acclimation. Cold hardening and drought stress were applied at the most critical stages of plant development on young seedlings and heading plants, respectively. The level of the phytohormones was significantly higher at heading, more than 5-fold in respect of salicylic acid (SA) and total brassinosteroids (BRs) and 1.7-fold in the case of abscisic acid (ABA). Moreover, the spectrum of detectable BRs increased from one—homocastasterone (HCS)—found in seedlings to four BRs identified in heading plants [HCS, castasterone (CS), teasterone and dolicholide], with the last one detected for the first time in cereal species. To some extent freezing tolerance seems to be determined by native hormonal status as control seedlings of tolerant DH lines contained 1.4- and 2.3-fold lower amount of ABA and HCS and 2.3-fold higher amount of SA in comparison to freezing-sensitive ones. Such dependency was not observed in heading plants as significant variation in CS content was the only detected difference. Under stress treatments, tolerant DH lines accumulated significantly lower (75–81%) amount of ABA, which probably reflected lower stress intensity resulting from another defence strategy. In contrast, stress-induced significant almost 2-fold increase in HCS/CS and 2–3-fold decrease in SA content specific for tolerant DH lines of barley suggest the involvement of these molecules in freezing/drought defence. Detected correlations suggest their interaction with nonspecific peroxidase and low molecular weight antioxidants.

High frequency of albino plant formation in isolated microspore or anther cultures is a great problem limiting the possibility of their exploitation on a wider scale. It is highly inconvenient as androgenesis-based doubled haploid (DH) technology provides the simplest and shortest way to total homozygosity, highly valued by plant geneticists, biotechnologists and especially, plant breeders, and this phenomenon constitutes a serious limitation of these otherwise powerful tools. The genotype-dependent tendency toward albino plant formation is typical for many monocotyledonous plants, including cereals like wheat, barley, rice, triticale, oat and rye - the most important from the economical point of view. Despite many efforts, the precise mechanism underlying chlorophyll deficiency has not yet been elucidated. In this chapter, we review the data concerning molecular and physiological control over proper/disturbed chloroplast biogenesis, old hypotheses explaining the mechanism of chlorophyll deficiency, and recent studies which shed new light on this phenomenon.

High frequency of albino plant formation in isolated microspore or anther cultures is a great problem limiting the possibility of their exploitation on a wider scale. It is highly inconvenient as androgenesis-based doubled haploid (DH) technology provides the simplest and shortest way to total homozygosity, highly valued by plant geneticists, biotechnologists and especially, plant breeders, and this phenomenon constitutes a serious limitation of these otherwise powerful tools. The genotype-dependent tendency toward albino plant formation is typical for many monocotyledonous plants, including cereals like wheat, barley, rice, triticale, oat and rye - the most important from the economical point of view. Despite many efforts, the precise mechanism underlying chlorophyll deficiency has not yet been elucidated. In this chapter, we review the data concerning molecular and physiological control over proper/disturbed chloroplast biogenesis, old hypotheses explaining the mechanism of chlorophyll deficiency, and recent studies which shed new light on this phenomenon.

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.

Enhanced channeling carbon through pathways: shikimate/chorismate, benzenoid-phenylopropanoid or 2-C-methyl-D-erythritol 4-phosphate (MEP) provides a multitude of secondary metabolites and cell wall components and allows plants response to environmental stresses. Through the biosynthetic pathways, different secondary metabolites, like tocopherols (TCs), are bind to mutual dependencies and metabolic loops, that are not yet fully understood. We compared, in parallel, the influence of α- and γ-TCs on metabolites involved in osmoprotective/antioxidative response, and physico-chemical modification of plasma membrane and cell wall. We studied Arabidopsis thaliana Columbia ecotype (WT), mutant vte1 deficient in α- and γ-TCs, mutant vte4 over-accumulating γ-TC instead of α-TC, and transgenic line tmt over-accumulating α-TC; exposed to NaCl. The results indicate that salt stress activates β-carboxylation processes in WT plants and in plants with altered TCs accumulation. In α-TC-deficient plants, NaCl causes ACC decrease, but does not change SA, whose concentration remains higher than in α-TC accumulating plants. α/γ-TCs contents influence carbohydrates, poliamines, phenolic (caffeic, ferrulic, cinnamic) acids accumulation patterns. Salinity results in increased detection of the LM5 galactan and LM19 homogalacturonan epitopes in α-TC accumulating plants, and the LM6 arabinan and MAC207 AGP epitopes in α-TC deficient mutants. Parallel, plants with altered TCs composition show decreased both the cell turgor and elastic modulus determined at the individual cell level. α-TC deficient plants reveal lower values of cell turgor and elastic modulus, but higher cell hydraulic conductivity than α-TC accumulating plants. Under salt stress, α-TC shows stronger regulatory effect than γ-TC through the impact on chloroplastic biosynthetic pathways and ROS/osmotic-modulating compounds.

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, &beta, 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.

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. Electronic supplementary material The online version of this article (10.1007/s00299-018-2362-x) contains supplementary material, which is available to authorized users.

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.

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.

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.

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−1 DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121–424 pmol g−1 DW) and zeatin ryboside (cZR, 146–432 pmol g−1 DW). Surprisingly, a relatively high level (10–64 pmol g−1 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. Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1686-4) contains supplementary material, which is available to authorized users.

7 Págs., 2 Figs., 2 Tabls. Available online: 9 November 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited., To gain a better understanding of the molecular mechanisms controlling microspore embryogenesis (ME) in triticale (×Triticosecale Wittm.), the expression patterns of 13 genes, previously identified in bread wheat to be associated with microspore-derived embryo development, were analysed. Four triticale doubled haploid (DH) lines, significantly different with respect to embryogenic potential, were studied. The gene expression profile was dissected at different points of the ME induction procedure up to the 8th day of in vitro culture (dc). RT-PCR revealed that these 13 genes were expressed during triticale ME. Variations in gene expression profiles were observed between the studied DH lines. DH28 (highly embryogenic) was the only one in which all analysed genes (Ta.TPD1-like, TAA1b, GSTF2, GSTA2, CHI3, Tad1, XIP-R1, TaAGL14, TaNF-YA7, SERK2, SERK1, TaEXPB4, TaME1) were up-regulated during the first 8dc. In the less embryogenic DH31, TAA1b, GSTA2 and TaEXPB4 were already induced on 4dc. In DH25, ME was initiated quite efficiently but soon inhibited, which coincided with the lack of gene expression (TaEXPB4, TaME1) or down-regulation (Tad1, XIP-R1, TaAGL14, TaNF-YA, SERK2, SERK1) on 8dc. In the recalcitrant DH50 line, the majority of genes were expressed at a lower level or not at all, indicating disturbances in ME initiation. In this study, the molecular mechanisms involved in triticale ME induction were analysed for the first time, laying the foundation for further characterisation of specific genes controlling microspore-derived embryo development., The work was supported by Project AGL2010-17509 from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain, the Bilateral Project CSIC (Spain)-PAS (Poland) 2010PL0006 and by COST Action FA0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI). Dr. Ewa Dubas was the recipient of a STSM fellowship from COST Action FA0903 ‘Harnessing of Repro- duction for Plant Improvement’. RA Sánchez-Díaz was the recipient of a predoctoral fellowship, from Junta Ampliación de Estudios, Consejo Superior de Investigaciones Científicas (JAE-CSIC) of Spain.

Resistance to snow mould is a feature determined by multiple genes. Therefore, determining the phenotype of resistant plants is difficult as it requires an investigation over a long period of time from cold acclimation through pathogenesis. The aim of the present study was (i) to determine the characteristics of the resistant genotype and (ii) to clarify the connections between photosynthesis during cold acclimation and then pathogenesis caused by Microdochium nivale. Two inbred lines of winter rye (Secale cereale L.) differing in their susceptibility to snow mould were used in the study. After cold acclimation snow mould resistant (SMR) line was characterised by higher values of CO2 assimilation and electron transport efficiency but did not differ from snow mould susceptible (SMS) line in carboxylation rate of RuBisCO (Vcmax). Higher soluble carbohydrate accumulation, due to higher photosynthesis intensity, as well as an ABA increase at 5 days post infection (DPI) in leaves and crowns were found in SMR line during the pathogenesis period. Callose deposition was found around non-infected bundle sheets and in cortex cells at 5 DPI (at the same time point as ABA peak) only in SMR line, which probably prevented the infection of leaf initials. Early leaf initials infection in SMS line may be responsible for inhibiting leaf growth and plant regeneration after stress cessation. The results show different physiological and biochemical characteristics of the investigated lines, which can be applied in the selection of resistant genotypes and identifying genomic regions responsible for metabolic pathways increasing pathogen resistance.

The accumulation of pathogenesis-related proteins such as β-1,3-glucanases and chitinases was studied in cold induced snow mould resistance in two Polish cultivars of winter triticale, cv. Hewo and cv. Magnat that substantially differ in resistance to Microdochium nivale. The plants were pre-hardened at 12°C for 10 days and hardened at 4°C for 28 days. Subsequently, cold hardened plants were inoculated with fungal mycelium (M. nivale) and incubated at 4°C for 7 days in dark. Cold acclimatisation resulted in suppression of the total glucanase and chitinases activities in the resistant Hewo as well as sensitive Magnat cultivars that possibly coincides with altered metabolism. However, upon infection with M. nivale the chitinases were markedly induced in the cv. Hewo. At the same time, total β-1,3 glucanases activities did not seem to be affected by fungus in any of the tested triticale cultivars. The pattern and/or the activity of chitinases in plants might be indicative for the resistance/susceptibility against M. nivale.

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.

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.

Quantitative trait loci (QTLs) associated with androgenic responsiveness in triticale were analyzed using a population of 90 DH lines derived from the F1 cross between inbred line 'Saka 3006' and cv. 'Modus', which was used in a number of earlier studies on molecular mapping in this crop. Using Windows QTL Cartographer and MapQTL 5.0, composite interval mapping (CIM) and association studies (Kruskal-Wallis test; K-W) for five androgenesis parameters (androgenic embryo induction, total regeneration and green plant regeneration ability, and two characteristics describing final androgenesis efficiency) were conducted. For the studied components of androgenic response, CIM detected in total 28 QTLs which were localized on 5 chromosomes from A and R genomes. Effects of all QTLs that were identified at 2.0 or above of the LOD score explained 5.1-21.7 % of the phenotypic variation. Androgenesis induction was associated with seven QTLs (LOD between 2.0 and 5.8) detected on chromosomes 5A, 4R, 5R and 7R, all of them confirmed by K-W test as regions containing the markers significantly linked to the studied trait. What is more, K-W test revealed additional markers on chromosomes: 5A, 2BL, 7B and 5R. Both total and green regeneration ability were controlled by genes localized on chromosome 4A. Some of the QTLs that affected final androgenesis efficiency were identical with those associated with androgenic embryo induction efficiency, suggesting that the observed correlation may be either due to tight linkage or to pleiotropy. Key message Five regions of the triticale genome were indicated as revealing significant marker/trait association. Markers located in these regions are potentially useful for triticale breeding through marker-assisted selection.

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.

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.

The accumulation of abscisic acid (ABA) and the activities of antioxidative enzymes along with cell metabolic activity were monitored during androgenesis induction in triticale (×Triticosecale Wittm.). Tested cultivars ‘Mieszko’ and ‘Wanad’ were selected due to their significantly different responses to androgenic induction. Significant variation was observed in respect of superoxide dismutase activity and endogenous ABA content in anthers isolated from freshly cut tillers. For both cultivars, tillers pretreatment with low temperature decreased peroxidase activity by 36%, highly accelerated respiration rate and reduced heat production. At the same time, the level of ABA in ‘Mieszko’ was increased to the level measured in ‘Wanad’. This effect was associated with higher microspore culture viability and increased stress tolerance in ‘Mieszko’. Low temperature and metabolic starvation during 4-day anther preculture did not influence activities of antioxidative enzymes, while it resulted in slight decrease in respiration rate and heat emission. The importance of these changes for effective androgenesis induction is discussed.

Reason for Erratum: In the original article 2 of the references were cited incorrectly for the name of Teixeira da Silva, J. A. Below are the correct citations: Ahmadi, B., Alizadeh, K., and Teixeira da Silva, J. A. (2012). Enhanced regeneration of haploid plantlets from microspores of Brassica napus L. using bleomycin, PCIB, and phytohormones. Plant Cell Tissue Organ Cult. 109, 525–533. doi: 10.1007/s11240-012-0119-8 Ahmadi, B., Shariatpanahi, M. E., and Teixeira da Silva, J. A. (2014). Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult. 116, 343–351. doi: 10.1007/s11240-013-0408-x This error does not change the scientific conclusions of the article in any way. The original article has been updated.

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.

The fungus Leptosphaeria maculans that causes blackleg can attack winter rape throughout the entire growing season. Therefore, appropriate treatments were designed to simulate environmental factors that may occur under field conditions. The callus tissue of winter rape was treated with abiotic stress in the form of cold (2 °C) and temperature change (2/20 °C and 20/2 °C), and biotic stress in the form of secondary metabolites of L. maculans. The following parameters were analyzed: total phenolics, the activity of l-phenylalanine ammonia lyase (PAL), catalase, respiration intensity, heat emission, and metabolic activity determined by means of 2,3,5-triphenyl-tetrazolium chloride (TTC) assay. Additionally, the changes in the electrical potential, known as a sensitive indicator of biochemical changes in the callus tissue, were measured. The obtained results seem to indicate rather increased stress, caused by temperature (2 or 20 °C) or its changes (2/20 °C, 20/2 °C) and the secondary metabolites of L. maculans than a cross reaction. The factor that most strongly affected the metabolism of winter rape callus tissue was the temperature change, which masked the changes caused by the fungal secondary metabolites or intensified their adverse effects. The only parameters that could be regarded as reliable indicators of the callus tissue response to the elicitation with secondary metabolites of L. maculans were catalase activity and respiration intensity. Additionally, a protective function of low temperature involved a reduction in catalase activity, resulting in increased level of reactive oxygen species (ROS), key molecules inducing the defense mechanisms during pathogenesis. Only the changes in l-phenylalanine ammonia lyase (PAL) activity and the accompanying changes in the content of phenolic compounds can be considered as beneficial effects of low temperature (2 °C) on a more efficient callus acclimation to the biotic stress.

Changes in the membrane lipid and sterols content and composition were studied during induction and differentiation in callus cultures of Brassica napus var. oleifera. Callus induction was associated with an increase of DGDG content, significant changes in fatty acids composition of all lipid fractions and increased degree of lipid unsaturation. The membrane lipid composition of tissue at different degrees of differentiation was found to vary significantly, particularly two weeks after transfer of callus to regeneration medium. The main differences concerned the content and composition of galactolipids. Curiously in many cases, these differences declined during subsequent culture, in spite of the morphogenesis process which was in progress. Another result of differentiation was the change in free sterol composition: in shoot regenerating calli the content of stigmasterol had rose whereas the accumulation of campesterol decreased. Even though observed changes in membrane properties may not play a role in morphogenesis they are nevertheless useful as developmental markers and can be invaluable in understanding biochemical basis of morphogenesis.

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.

Introgression of genes from alien species into crop plants could be achieved through distant hybridisation aided by tissue culture-based embryo rescue techniques. Beside this, in vitro mutagenesis, gametoclonal/somaclonal variation and transgenesis are the other tools which can generate additional variability. However, all these tissue culture-based tools require totipotent tissues. The direct regeneration of plants from an explant without a callus stage via organogenesis or somatic embryogenesis is the quickest path for micropropagation. Because of their speed and low costs of culture phase and the fidelity of the genotype in the cloned progeny, systems with direct somatic embryogenesis or organogenesis are often recommended and subjected to transformation. On the other hand, most micropropagation procedures with a callus stage can be applied as a basis for transformation, and the fresh friable calli can be directly used as the transformation target. Cell and microspore suspension cultures have also been seen as the ideal targets for genetic transformation due to the large amount of homogenous material, easy selection of the targeted cells and less chances of chimeric regeneration, while protoplasts due to exposed plasma membrane can introduce foreign DNA very easily and therefore form the ideal targets for generating unique and novel plants. This chapter discusses various plant regeneration methods and the factors affecting them towards achieving alien gene transfer in crop plants.

Miscanthus ×giganteus Greef et Deu. (Poaceae), a hybrid of Miscanthus sinensis and M. sacchariflorus native to Japan, is an ornamental and a highly lignocellulosic bioenergy crop, cultivated in the European Union as an alternative source of energy. This grass reproduces exclusively vegetatively, by rhizomes or via expensive in vitro micropropagation. The present study was aimed at finding the barriers that prevent sexual seed production, based on detailed embryological analyses of the whole generative cycle, including microsporogenesis, pollen viability, megasporogenesis, female gametophyte development, and embryo and endosperm formation. Sterility of M. ×giganteus results from abnormal development of both male and female gametophytes. Disturbed microsporogenesis (laggard chromosomes, univalents, micronuclei) was further highlighted by low pollen staining. The frequency of stainable pollen ranged from 13.9% to 55.3% depending on the pollen staining test, and no pollen germination was observed either in vitro or in planta. The wide range of pollen sizes (25.5-47.6 μm) clearly indicated unbalanced pollen grain cytology, which evidently affected pollen germination. Only 9.7% of the ovules developed normally. No zygotes nor embryos were found in any analyzed ovules. Sexual reproduction of M. ×giganteus is severely hampered by its allotriploid (2n=3x=57) nature. Hybrid sterility, a strong postzygotic barrier, prevents sexual reproduction and, therefore, seed formation in this taxon.

Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high--32 degrees C or low--5 degrees C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5 degrees C) or efficiency of embryogenesis induction (at 32 degrees C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.

The aim of the presented methodical experiments was 1) the evaluation if callus of winter oilseed rape (Brassica napus var. oleifera L.) initiates a defence reaction to fungal elicitors: pectinase (polygalacturonase) or chitosan, and 2) the choice of the elicitor doses, which evoke the strongest tissue reaction. The results obtained will be used in the next experiments relating the studies of pathogenesis mechanisms proceeding in rape plants infected by necrotrophic fungi. The defence response was estimated on the basis of changes in electrolyte leakage from cells, metabolic efficiency, phenolic content and catalase activity. In the experiment pectinase was used at concentration of 3, 8, 16, 133 and 166 µl per 1 cm3 of culture medium while chitosan at: 25, 50, 75 and 100 µg·cm−3. Both elicitors increased cell membrane permeability: pectinase at the doses equal or greater to 16 µl·cm−3 while chitosan of 25 µg·cm−3. The greatest metabolic inefficiency was observed in calli elicited with 16 µl·cm−3 pectinase and with chitosan of 100 µg·cm−3. The decrease in phenolic content was noted under influence of most doses of both elicitors. The highest catalase activity was evoked by pectinase of 8 µl·cm−3 and chitosan of 75 and 100 µg·cm−3. The results indicated that 8–16 µl·cm−3 of pectinase and 100 µg·cm−3 of chitosan caused the strongest defence reaction of oilseed rape tissue.

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 ofB. 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 104 microspores or pmol g−1 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

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 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 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.

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−1 DW. Intensive ABA accumulation (up to 5.3 nmol g−1 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; R2 = 9.3 %) associated with ABA concentration in unstressed plants and four QTLs on chromosomes 2A, 1B and 5R (LOD 2.6–5.7; R2 = 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.