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.

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 H₂O₂ generation, and negative correlation with catalase (CAT) activity suggest that some threshold level of H₂O₂ is important for successful ME initiation. LT tillers pre-treatment significantly increased H₂O₂ accumulation, which had a negative effect on ME effectiveness. However, even high level of H₂O₂ 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.

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.

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.

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.

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.