The Genome Regions Associated with Abiotic and Biotic Stress Tolerance, as Well as Other Important Breeding Traits in Triticale

This review article presents the greatest challenges in modern triticale breeding. Genetic maps that were developed and described thus far, together with the quantitative trait loci and candidate genes linked to important traits are also described. The most important part of this review is dedicated to a winter triticale mapping population based on doubled haploid lines obtained from a cross of the cultivars ‘Hewo’ and ‘Magnat’. Many research studies on this population have focused on the analysis of quantitative trait loci regions associated with abiotic (drought and freezing) and biotic (pink snow mold and powdery mildew) stress tolerance as well as related to other important breeding traits such as stem length, plant height, spike length, number of the productive spikelets per spike, number of grains per spike, and thousand kernel weight. In addition, candidate genes located among these regions are described in detail. A comparison analysis of all of these results revealed the location of common quantitative trait loci regions on the rye chromosomes 4R, 5R, and 6R, with a particular emphasis on chromosome 5R. Described here are the candidate genes identified in the above genome regions that may potentially play an important role in the analysis of trait expression. Nevertheless, these results should guide further research using molecular methods of gene identification and it is worth extending the research to other mapping populations. The article is also a review of research led by other authors on the triticale tolerance to the most current stress factors appearing in the breeding.


Introduction
Triticale (× Triticosecale Wittmack), a man-made cereal species, developed by crossing wheat (Triticum aestivum L.) and rye (Secale cereale L.) was first described in 19th century by Scottish botanist A. Stephen Wilson [1]. The hybrid created by this crossing was originally an octoploid with AABBDDRR genome with a base genomic construction x = 7 [2]. Initially, this crop was created as a species that combined new agronomic, morphological, and utility features [1]. Over time, various types of triticale with different ploidy levels and chromosomal constitutions have been created and evaluated, and currently, a cultivated hexaploid triticale that belongs to the Poaceae family contains a genomic constitution of 2n = 6x = 42 with the AABBRR genome [3]. It combines valuable traits such as high fertility and grain quality received from wheat with higher stress tolerance obtained from rye [1,4,5]. Up to now, triticale exhibits better drought [6], aluminum [7], freezing [8] as well as waterlogging [9] tolerance than wheat. All of these qualities make triticale a valuable and well-established crop that is cultivated in many European countries such as Poland, Germany, Spain, and France [10]. Triticale grain is mainly used as an animal food, but is also used for human food consumption as well as in bioethanol and biofuel production [3,11,12].
This crop is also a valuable genetic bridge for transferring eligible genes among the rye and wheat genetic pool using molecular breeding techniques [4,13,14]. Quantitative the 'Grenado' × 'Zorro' DH population was created mainly based on DArT-seq and DArT markers. This map was composed of 1891 unique markers ordered to 21 linkage groups with a total map length of 5249.9 cM and marker saturation of 2.8.
When comparing the total marker number of all triticale genetic maps, the results were very different. The highest number of markers assigned to the A and B linkage group was found in the DH 'Hewo' × 'Magnat' line mapping population [21] while for the R group in the DH 'Saka' × 'Modus' line mapping population [20]. Additionally, the A group was previously described by Tyrka et al. [20,21], Karbarz et al. [22], Wąsek et al. [23], and Dyda et al. [16] as the one with the lowest number of markers assigned, regardless of the marker type used in the map construction.
Knowledge of the position of the genomic regions linked with an important trait may help with candidate gene identification. Such genes that encode multiple proteins can be widely used in modern molecular breeding programs, but so far, not many studies have described such genes identified in crops. In triticale, the information of important candidate genes is still limited [16,22,23,28,32,45].

Low Temperature Tolerance
The ability of cereal seedlings to survive the winter (i.e., winter hardiness) depends on the plant's ability to tolerate a wide range of environmental stresses such as freezing, changing temperatures and climate, low light intensity, desiccation, wind, snow cover, icing, and various winter-related diseases [46].
There is a large variation in triticale freezing tolerance. A lower/higher level of this feature may be related to the genetic hexaploid structure, where part of the chromosomes of the D wheat subgenome and R rye subgenome contains genes important for winter hardiness. Thus, selection for freezing tolerance in triticale seems to be very important for further expansion of this crop area. A strong interaction between the frost tolerance of the genotype and the environment was also observed in triticale [23]. Freezing induces complex and gradual changes in the photosynthetic apparatus [47,48]. In winter, the photosynthetic apparatus is damaged not only by freezing, but also by the photoinhibition of photosynthesis. Tolerance to cold-induced photoinhibition appears to be closely related to freezing tolerance, partly due to the common mechanisms of acclimatization to both stresses [49].
Temporary warming in winter can cause a reduction in snow cover and deacclimatization, ultimately reducing winter hardiness [46,50]. The risk of frost infestation is related to the number of days with a daily minimum temperature below the set freeze tolerance level (−20 • C) on days without continuous snow cover [8].
As has been shown by many years of research, low temperature may increase the triticale tolerance to stresses that coexist with cold in winter, which could remain persistent during the following early spring period and even at the plant adult stage. Tolerance to freezing [23] and pink snow mold caused by Microdochium nivale [32] appears after a cold-hardening period and it is an essential, genotype-dependent, complex quantitative trait for wintering.

Fungal Infection Tolerance after Cold-Acclimation
Tolerance to low temperature and diseases caused by the fungi infecting seedlings in the cold is an essential trait for triticale overwintering. Long-term studies indicate that after long-term exposure to low temperature (acclimation, hardening), cereal seedlings acquire genotype-dependent cross-tolerance to other later stresses. Low temperature (4 weeks at 4 • C) may increase the tolerance of triticale seedlings to stresses coexisting with cold in winter, which may persistently follow throughout the early spring period and even in the adult phase of the plant [16,23,28,32,51,52]. Tolerance to pink snow mold caused by M. nivale appears after a period of hardening with low temperature and is a genotype-dependent, complex feature significant for wintering seedlings [23,31,32,52,53]. Furthermore, Dyda et al. [31] presented new insights into the mechanism of triticale resistance to M. nivale. Their experiment with three different M. nivale strains and three different infection assays showed that plants that maintained a higher maximum quantum efficiency of PSII showed, at the same time, less leaf damage upon infection. M. nivale can establish necrotrophic or biotrophic interactions with the susceptible or resistant genotypes, respectively. The genetic regions associated with PSII functioning and resistance, together with a wide range of PSII-and resistance-related genes, were found on chromosomes 4 and 6. In addition, it was confirmed that the structural and functional integrity of the plant are required factors to meet the energy demand of infected cells, photosynthesis-dependent systemic signaling, and defense responses [31].
The proteomic profiling allowed for the identification of candidate proteins associated with the cold acclimation of triticale seedlings [53] as well as the tolerance to freezing and pink mold infection [52]. The content of individual proteins was analyzed by two-dimensional gel electrophoresis (2-DE) and matrix-enhanced time-of-flight laser desorption/ionization (SELDI-TOF). Low temperature exposure of seedlings only caused quantitative changes in the leaves of both cultivar parents, causing an increase or decrease in the abundance of the proteins with a molecular weight of 4-50 kDa. Among the proteins accumulated under the influence of cold in the leaves of the tolerant cultivar 'Hewo', two thioperoxidases (antioxidant proteins specific for chloroplastic thiols) as well as a subunit of mitochondrial ATP synthase and ADP-binding resistance protein were identified [53]. On the other hand, in low-temperature hardened seedlings of this genotype, a reduced level of the small subunit RuBisCO and the PW9 subunit of peroxidase 10 was observed. Simultaneous SELDI-TOF analysis revealed several proteins of low weight with increased concentration in cold-exposed plants of the tolerant genotype versus the sensitive one. Non-gel protein profiling in triticale seedlings was performed by high-performance liquid chromatography coupled with mass spectrometry (LC-MS) and Raman spectroscopy [52]. Seedlings of doubled haploid (DH) lines selected from the 'Hewo' × 'Magnat' mapping population with extreme tolerance/susceptibility to freezing and M. nivale infection were used in these studies. These untargeted methods led to the detection of twenty-two candidate proteins that accumulated under the influence of low temperature in the most tolerant seedlings in relation to the susceptible ones, classified as biomolecules involved in protein biosynthesis, response to various stimuli, energy balance, response to oxidative stress, protein modification, membrane construction, and anthocyanin synthesis. Additionally, in seedlings of the most frost-tolerant line and M. nivale, hardening resulted in a decrease in the content of carotenoids and chlorophyll. Moreover, a decrease was detected in the intensity of the spectra characteristic for carbohydrates and an increase in the intensity spectra characteristic of the protein compound. Both tested lines-tolerant and sensitive to freezing and M. nivale infection-showed different stress responses in the characteristic phenolic components [52].
Zur et al. [54] showed that the 'Hewo' and 'Magnat' response to cold treatment and M. nivale infection affected the accumulation of b-1,3-glucanase and chitinase. The results indicate that both glucanhydrolases were substantially suppressed as the result of cold treatment in both cultivars due to altered metabolism. On challenge with M. nivale, 'Hewo' showed a marked increase in chitinase while none of the cultivars showed a change in glu-canase, confirming the role of chitinases in resistance against M. nivale [54]. Gawrońska and Gołębiowska [55] identified biochemical markers that are potentially involved in increased resistance against M. nivale. It was shown that the triticale genotype and seedling treatment may influence the level of TBARS, which is well-known as a marker of oxidative stress in response to different abiotic and biotic factors. Plant resistance after cold hardening [56], cold-enhanced gene expression at seedling stage [57], cold-modulated small protein abundance at the seedling stage [53], presence and concentration of free and cell wall-bound phenolic acids [51], cold-modulated leaf compounds [52], cold-induced changes in cell wall stability [58], and changes in the physical and chemical leaf properties [59] were tested on the 'Hewo' × 'Magnat' population after M. nivale infection and provided new insights on triticale cold-acclimation as well as plant vs. pathogen interactions.
In addition to the aforementioned pink snow mold tolerance studies, Karbarz et al. [22] and Dyda et al. [16,28] described triticale adult-plant resistance to B. graminis infection measured at the adult plant stage in the field after isolate mixture inoculation and after natural field infection, accordingly. Using the area under disease progress curve (AUDPC), maximum disease severity (MDS), and the average value of powdery mildew infection (avPM) methods, many new QTL regions and candidate genes associated with powdery mildew resistance were described for the first time and presented. Such regions, after careful validation in available triticale varieties and lines, can be used in marker-assisted selection (MAS) and the pyramiding of adult-plant resistance (APR) genes to PM in triticale breeding that can assist molecular breeding programs.

Importance of the DH 'Hewo' × 'Mangat' Mapping Population
Our meta-analysis was mainly focused on the 'Hewo' × 'Magnat' mapping population composed of 89 doubled haploid (DH) lines that were derived by the anther method described in detail by Wędzony [60]. All lines were derived from the F 1 hybrid of two triticale cultivars, 'Hewo' (Strzelce Plant Breeding, IHAR Group Ltd., Poland) was used as a female parent and 'Magnat' (DANKO Plant Breeders Ltd., Poland). Both parental cultivars showed a different response to multiple biotic and abiotic factors.
Over the years, the DH 'Hewo' × 'Magnat' line population has been used as a model to test various traits and plant responses at different stages of development and under both natural and controlled conditions. These studies were conducted at the anatomical, physiological, biochemical, and/or genetic levels. The first genetic map constructed for the DH 'Hewo' × 'Magnat' lines enabled a new approach of research on this population. The map was 4997.4 cM long and composed of 3539 markers in total (842 DArT, 2647 SNP-DArT, and 50 SSR markers), which were ordered into 20 linkage groups assigned to the A (7), B (7), and R (6) subgenomes [21].
The results showed the quantitative trait loci (QTL) associated with many traits measured in natural and controlled conditions at the seedling and adult plant stage. Tolerance to drought [34,35], M. nivale infection [31,32], and freezing [23] were tested at the seedling stage after vernalization in a cool chamber at 4 • C under controlled conditions. Additionally, tolerance to the powdery mildew caused by Blumeria graminis [28] was tested in natural field conditions at the adult stage and morphological traits such as stem length, plant height, spike length, number of productive spikelets per spike, number of grains per spike, and thousand kernel weight [38] were determined at the adult plant stage along with the identification of QTL. Additionally, for the above mappings, multiple candidate genes were identified and described for the first time.

The Comparative Analysis of the Genomic Results Obtained from the DH 'Hewo' × 'Magnat' Mapping Population
In subgenome A, the QTL regions on four wheat-derived chromosomes were identified; the trait loci were located in different regions of chromosomes 2A, 4A, 5A, and 7A ( Figure 1). In subgenome B, the QTL regions on six wheat-derived (1B, 2B, 3B, 4B, 6B, and 7B) chromosomes were identified and the trait loci were located in different regions ( Figure 2). In subgenome R, the QTL regions on six rye-derived chromosomes (1-6R) have been identified. The trait loci were located in different regions of 1R, 2R, and 3R chromosomes ( Figure 3). QTL regions common to two or more traits have been detected on 4R, 5R, and 6R ( Figure 3). The common region for the greatest number of traits was identified on 5R between 0 and 37 cM ( Figure 4). According to the flanking marker sequences, this region was estimated between 865 109 880 and 874 163 401 kb (9 Mb long). The QTL regions, together with the candidate genes related to the studied traits of the DH 'Hewo' × 'Magnat' mapping population, are presented in Table 1. In the 5R region common for different QTL, identified between 0 and 37 cM of this chromosome, candidate genes were in silico identified here with the method described previously [32]. All candidate genes had a positive additive effect from cv. 'Hewo' and included six candidate records encoding the predicted (1) chloroplastic FAF-like protein; (2) F-box/FBD/LRR-repeat protein; (3) Myosin-10-like protein; (4) thylakoid membrane protein TERC; (5) xyloglucan endotransglucosylase/hydrolase; and (6) nucleotide-gated ion channel protein ( Table 2). The first of the proteins listed above is associated with a negative regulation of ABA-activated signaling pathway as well as the positive regulation of phosphatase activity. The second is involved in the regulation of short-day photoperiodism and flowering. Another gene potentially related to the studied traits of the mapping population encodes the thylakoid membrane protein TERC, an integral protein that plays a crucial role in thylakoid membrane biogenesis and thylakoid formation in early chloroplast development, essential for the synthesis of photosystem II (PSII) core proteins and is required for the efficient insertion of thylakoid membrane proteins. . Subgenome R identified for the winter triticale DH 'Hewo' × 'Magnat' mapping population, related to seedlings the tolerance to drought, freezing, and pink snow mold caused by M. nivale as well as the tolerance of adult plants to drought, powdery mildew, and yielding capacity in the generative phase. Subgenome regions were determined by bioinformatics using the genetic map published by Tyrka et al. [21] and Windows QTL Cartographer V2.5_011. Figure 3. Subgenome R identified for the winter triticale DH 'Hewo' × 'Magnat' mapping population, related to seedlings the tolerance to drought, freezing, and pink snow mold caused by M. nivale as well as the tolerance of adult plants to drought, powdery mildew, and yielding capacity in the generative phase. Subgenome regions were determined by bioinformatics using the genetic map published by Tyrka et al. [21] and Windows QTL Cartographer V2.5_011 [23,28,31,32,34,38].
Other candidate genes located in a section of chromosome 5R in the rye subgenome database were identified here and include over sixty genes involved mainly in the immune response to abiotic and biotic stimuli, signaling, and oxidoreductase activity (Table 3). At least twenty-four of them are described in the literature as coding proteins associated with the response/tolerance to abiotic and biotic stresses: 2-oxoglutarate-dependent dioxygenase, ankyrin repeat-containing protein, ATP-dependent RNA helicase DeaD, beta-amylase, BTB/POZ and MATH domain-containing protein, chaperone DnaK, cold regulated protein (COR), cysteine proteinase inhibitor, embryogenesis transmembrane protein-like, F-box family protein, flavin-containing monooxygenase, heat shock transcription factor, homeobox leucine-zipper protein, lipid transfer protein, L-type lectin-domain containing receptor kinase VIII, metacaspase-1, NAC domain-containing protein, nascent polypeptideassociated complex subunit beta, peptidoglycan-binding LysM domain protein, peroxidase, photosystem I assembly protein Ycf3, polyubiquitin, rRNA N-glycosidase, and terpene synthase. COR proteins are also known as involved in the vegetative to reproductive phase transition of meristem. Many proteins from those listed in Table 3 such as cyclin delta-3, cysteine proteinase inhibitor, embryogenesis transmembrane protein-like, F-box family protein, folylpolyglutamate synthase, polyamine oxidase, protein FAR1-RELATED SEQUENCE 5, ribosomal RNA small subunit methyltransferase J, senescence regulator (DUF584), WD repeat-containing protein, and xyloglucan 6-xylosyltransferase are reported as involved in seed development. Next, cytochrome P450 identified here is described as associated with the regulation of growth as well as leaf and root development. The other candidate proteins identified here may contribute to transcription regulation: basic helix-loop-helix (bHLH) DNA-binding superfamily protein, Basic-leucine zipper (bZIP) transcription factor family protein, cold regulated protein (COR), histone acetyltransferase of the CBP family 12, homeobox leucine-zipper protein, LURP-one-like protein, NAC domain-containing protein, protein FAR1-RELATED SEQUENCE 5, small nuclear ribonucleoprotein, THO complex subunit 1, transcription elongation factor GreA, and Trihelix transcription factor GT-2. Many other candidate genes may encode proteins involved in response to hormones such as 2-oxoglutarate-dependent dioxygenase, basic helix-loop-helix (bHLH) DNA-binding superfamily protein, ankyrin repeat-containing protein, cold regulated protein (COR), cyclin delta-3, embryogenesis transmembrane protein-like, F-box family protein, homeobox leucine-zipper protein, lipid transfer protein, P1, polyamine oxidase, and polyubiquitin (Table 3).      b. Carotenoid 9,10(9',10')-cleavage dioxygenase-like a. Acts as a processive, ATP-dependent zinc metallopeptidase for both cytoplasmic and membrane proteins. Plays a role in the quality control of integral membrane proteins.
b. Involved in strigolactone biosynthesis, hormones that inhibit tillering and shoot branching, contribute to the regulation of shoot architectural response to phosphate-limiting conditions, and function as a rhizosphere signal that stimulates hyphal branching of arbuscular mycorrhizal fungi and trigger seed germination of root parasitic weeds.  Nucleotide-gated ion channel * Voltage-gated potassium channel activity, cyclic nucleotide gated ion channel, integral transmembrane helical component of membrane *-Positive additive effect from cv. Hewo. Table 3. Other genes located in a section of chromosome 5R in rye database.
7 ATP-dependent RNA helicase DeaD Involved in mRNA turnover, and more specifically in mRNA decapping. Involved in response to cold, salt stress and water deprivation.  Carboxyl-terminal peptidase putative DUF239 (2x) Neprosin, integral component of membrane.

12
Cold regulated protein (COR) 27 Negative regulation of transcription, DNA-templated, regulation of circadian rhythm, regulation of photoperiodism, flowering, response to abscisic acid, response to absence of light, response to blue and red light, response to cold and virus, vegetative to reproductive phase transition of meristem.

13
Cyclin delta-3 G1/S transition of mitotic cell cycle, guard mother cell differentiation, regulation of cyclin-dependent protein serine/threonine kinase activity, response to cytokinin, response to sucrose, seed development.
14 Cysteine protease (4x) Programmed cell death involved in cell development.

15
Cysteine proteinase inhibitor Cellular response to heat, defense response, response to cold, water deprivation and oxidative stress, positive regulation of seed germination. 16 Cytochrome P450 (10x) Leaf and root development, oxidoreductase activity, regulation of growth, response to insect, signalling.

20
Egg cell-secreted protein 1.1 Regulation of double fertilization forming a zygote and endosperm.

21
Embryogenesis transmembrane protein-like (2x) Brassinosteroid mediated signaling pathway, cell death, cellular response to hypoxia, leaf senescence, negative regulation of cell death, protein phosphorylation, regulation of seedling development.

22
F-box family protein (5x) Regulation of cell division, seed development, regulation of stomatal movement, response to abscisic acid, response to water deprivation.

23
Flavin-containing monooxygenase Glucosinolate biosynthetic process from homomethionine, defense response to bacterium, defense response to fungus, L-pipecolic acid biosynthetic process, plant-type hypersensitive response, response to other organism, systemic acquired resistance.

29
Histone acetyltransferase of the CBP family 12 (7x) Histone acetylation, positive regulation of transcription by RNA polymerase II.

30
Homeobox leucine-zipper protein Auxin-activated signaling pathway, negative regulation of transcription DNA-templated, shade avoidance, unidimensional cell growth, reproduction, response to abscisic acid and cytokinin, osmotic stress, virus and water deprivation.

32
L-gulonolactone oxidase (2x) L-gulonolactone oxidase activity, oxidoreductase activity, L-ascorbic acid biosynthetic process.   In summary, the presented results indicate the location of common QTL regions on the 4R, 5R, and 6R rye chromosomes, with particular emphasis on 5R. In silico localized candidate genes potentially play important roles in the trait's expression. Nevertheless, these results should guide further research using molecular gene identification methods and it is worth extending research to other mapping populations.

Drought Tolerance
Soil drought can significantly accelerate the aging of plants by a gradual decrease in metabolic activity, ultimately leading to cell death [35]. Gelang et al. [61] associated the decrease in plant yield with accelerated senescence caused by soil drought and a significant shortening of the grain filling phase. The first visible sign of aging is the yellowing of the leaves as a result of chlorophyll degradation and the appearance of other dominant pigments, mainly carotenoids, xanthophylls, and anthocyanins. Chlorophyll a decays faster than chlorophyll b during aging, leading to a decrease in the chlorophyll a/b ratio [62]. It has also been observed that the degradation of the reaction centers precedes the degradation of the proteins that make up the light-harvesting complex in PSII. Another factor that distinguishes the stay-green genotypes is the increase in the carbohydrate content of the green parts compared to the normal aging genotypes.
Increased accumulation of soluble carbohydrates in long-lasting green plants is often accompanied by increased leaf assimilation area in the grain-filling phase [61]. A higher yield of plants with delayed aging may result, for example, from maintaining a high level of soluble carbohydrates in the leaves. Carbohydrates are used in the synthesis of phenolic compounds involved in plant defense during environmental stresses, and can also serve as indicators of plant aging. In aging plant organs, the level of phenolic compounds increases at the expense of soluble carbohydrates. This indicates the important role of sugars in the integration of environmental signals during the regulation of leaf senescence [63].
Mechanisms related to the triticale aging in conditions of soil drought can be controlled by the genome of wheat and/or rye; however, triticale also does not show the specific drought responses that neither wheat nor rye have [34,64]. The genetic and molecular basis of triticale acclimatization to drought has so far been poorly understood. It is not clear whether triticale responses to drought are specific to the wheat or rye genome or result from the activity of both subgenomes. Cereal aging studies tend to focus on the flag leaf, however, the aging of triticale progresses from its lower parts below the flag leaf, which are the first to show clear signs of drought-induced aging and then run toward the flag leaf [64].

Effective Microspore Embryogenesis
Another important breeding feature of triticale associated with the effect of low temperature is the ability to undergo a process of somatic microspore embryogenesis (ME)androgenesis, used for the development of new plant lines from single, unfertilized pollen grains or from the anthers. Effective ME requires significant modifications in the pattern of gene expression, followed by changes in the cell's proteome and metabolism. Recent research has also aroused an interest in the role of epigenetic factors in de-differentiation and reprogramming the microspores to develop into an embryo. Therefore, a demethylating agent (2.5-10 µM 5-azacitidine, AC) along with low temperature (3 weeks at 4 • C) for ME induction was used for two doubled haploid triticale lines selected from the 'Saka 3006' × 'Modus' population, and their effect was analyzed in relation to the protein profiles of pollen grains as well as the expression of selected genes (TAPETUM DETERMINANT1 (similar to TaTPD1), SOMATIC RECEPTOR KINASES EMBRYOGENESIS 2 (SERK2), and GLUTATHIONE S-TRANSFERASE (GSTF2) as well as the efficiency of ME [65]. The use of a concentration of 5.0 µM AC was the most effective for ME induction; this was associated with the inhibition of intensive anabolic processes, mainly photosynthesis and light-dependent reactions, transition for effective catabolism and mobilization of carbohydrate reserves to satisfy the high energy demand of cells during microspore reprogramming and effective defense against stress-inducing effects (i.e. protection of the correct one folding during protein biosynthesis and efficient degradation of dysfunctional or damaged proteins). Additionally, the use of a demethylating agent at a concentration of 5.0 µM AC enhanced the expression of all genes previously identified as related to the embryogenic potential of microspores (i.e. similar to TaTPD1, SERK, and GSTF2) [65].
The effectiveness of ME is determined by a complex network of internal and environmental factors. In the presented tests of triticale, a strong positive correlation between the generation of hydrogen peroxide and ME efficiency confirmed the important role of reactive oxygen species in microspore reprogramming toward somatic embryogenesis [66]. However, for high efficiency ME induction, intensive hydrogen peroxide production must be associated with high activity antioxidant enzymes, superoxide dismutase and catalase. As revealed in a study, a strong seasonal influence on the physiological state of microspores suggests a kind of "biological clock" controlling plant reproduction, crucial for the viability of microspores and embryogenic potential. Although the impact of various modifications of plant material pre-treatment causing stress inducing ME was determined mainly by condition microspores, but with higher stress intensity (3 weeks at 4 • C), positive effects induced by antioxidant molecules were observed for reduced glutathione and its precursor, 1-2-oxothiazolidine-4-carboxylic acid. A high level of variability in response to the inducing initial ME was also demonstrated in the processing of the material between two DH lines of triticale and among microspores isolated from later-developing spikes [66].

Conclusions
The biggest challenges in modern triticale breeding are abiotic stresses such as drought and freezing as well as biotic stresses caused by fungal pathogens. In response to this demand, several genetic maps have been developed and described so far, together with the quantitative trait loci and candidate genes linked to important triticale traits. For many years, studies conducted on a winter triticale mapping population based on doubled haploid lines obtained from a cross of cultivars 'Hewo' and 'Magnat' focused on the analysis of quantitative trait loci regions associated with abiotic and biotic stress tolerance as well as related to other important breeding traits. A comparison analysis of those results revealed the location of common quantitative trait loci regions on the rye chromosomes 4R, 5R, and 6R, with particular emphasis on chromosome 5R. The most valuable are the QTL regions that have been repeated in different experiments as well as in different locations and years. Less reliable are the QTL regions obtained in a single experiment. As described in this paper, the candidate genes identified in the above genome regions may potentially play an important role in analyzing trait expression. Nevertheless, these results should guide further research using molecular methods of gene identification and it is worth extending the research to other mapping populations.