Microspore embryogenesis (ME) relies on the cellular reprogramming of the default gametophytic developmental pathway, which normally directs microspores toward pollen formation, into an embryogenic pathway that leads to the development of embryo-like structures (ELS) and, subsequently, haploid or doubled haploid (DH) plants. To test how redox control underpins this switch, we have carried out an extended in silico analysis of previously published RNA-seq data from two barley cultivars differing in ME competence (Igri, responsive; Golden Promise, recalcitrant) across four early induction stages (0-III). A curated set of 472 antioxidant/redox genes-core detoxification enzymes, the ASC-GSH cycle, TRX/GRX/PRX systems and GSTs-was examined. The analysis revealed that the expression of antioxidative defense genes is dynamically modulated during ME induction, underscoring the importance of redox homeostasis in successful microspore reprogramming. Both cultivars shared a late (stages II-III) program with increased SODs, selected CAT/GPX genes, rising MDHARs, deployment of specific TRX/GRX/PRX members and broad GSTs upregulation. Divergence emerged during progression: Igri showed a pronounced stage-III rise of GRs and targeted TRX/GRX/PRX transcripts, together with stronger activation of multiple GSTs. When considered alongside diverse experimental data, these stage-restricted, cultivar-biased signatures support a hypothetical model in which strengthened ASC-GSH recycling and thiol-redox hubs sustain H2O2 signaling while limiting oxidative damage. Targeting MDHARs, GRs, selected TRX/GRX/PRX genes, and GST subsets could improve ME efficiency and accelerate the integration of DH technology into modern crop breeding programs.

Sex determination in Cannabis sativa L. has major implications for breeding, biotechnology, and crop management. In this species, sex is primarily governed by an XX/XY sex chromosome system, with genetic regulation constituting the principal level of control. Variation in sex expression, encompassing dioecious, monoecious, or hermaphroditic forms, may arise from interactions between sex chromosomes and autosomal factors X-to-autosome (X: A) chromosome balance. Secondary modulation of sex phenotype involves environmental and physiological influences, with phytohormonal regulation representing an important layer of sex plasticity. Epigenetic mechanisms may further contribute to the downstream fine-tuning of phenotypic sex expression. Importantly, sex expression in Cannabis can also be manipulated chemically. Several silver-based compounds, such as silver thiosulfate, are widely used to induce staminate flower formation in genetically female plants, whereas application of ethephon promotes female flower formation in genetically male individuals. Although both agents act through ethylene signalling pathway, their effects likely involve broader and more complex interactions within the molecular network, including cross-talk with other hormonal pathways. Within this context, transcriptional regulators associated with auxin signalling, including Aux/IAA - TOPLESS-RELATED co-repressors such as TPR1, may contribute to the activation of male developmental programmes by modulating hormonal response pathways. However, their precise role and position within the sex-regulatory hierarchy are yet to be clarified. The multi-layered regulation of sex determination in Cannabis sativa L. provides a valuable framework for investigating the molecular basis of this process through the integration of genetic, environmental, and hormonal factors. A more comprehensive understanding of these interacting regulatory layers may facilitate the development of strategies for early sex identification, improved control over flower sex conversion, and the optimisation of cannabinoid-rich female inflorescence production. Such advances would support both fundamental and applied approaches in Cannabis biotechnology and cultivation.

In theory, microspore embryogenesis (ME) is the most efficient method for producing doubled haploids, however the actual effectiveness of this process is strongly genotype dependent. Here, we analysed how treatment with 5 µM 5-azacytidine (AC) is associated with early physiological and molecular events in triticale anthers and microspores redirected towards embryogenic development. The effectiveness of ME was evaluated in anther cultures of two DH lines with contrasting ME responsiveness and was related to differences in reactive oxygen species (ROS) generation, cysteine protease activity profiling, and the frequency of programmed cell death (PCD). AC increased microspores viability, doubled the number of embryo-like structures (ELS), and improved green plant regeneration in the responsive DH28 line, but did not efficiently overcome the recalcitrancy of the DH19 line. AC treatment elevated total ROS level and reduced DNA fragmentation in DH28. However, the accumulation of one of the ROS, hydrogen peroxide (H2O2), was not affected by AC treatment but significantly higher compared with the recalcitrant DH19. Activity-based protease profiling using specific inhibitors revealed that AC treatment strongly suppress of PCD-associated cysteine proteases, particularly papain-like cysteine proteases (PLCPs) and vacuolar processing enzymes (VPEs-like) in DH28. On the contrary, caspase-3-like activity was consistently present and appeared to play a regulatory or signalling role rather than functioning as a primary executor of PCD. In contrast, DH19 maintained higher overall protease activity and showed no AC-related changes in ROS generation. AC enhances the effectiveness of triticale ME in a genotype-dependent manner, and its positive effect is accompanied with reduced PCD-associated proteolysis during early cellular reprogramming. Genotypic differences in the ability to suppress PCD-associated protease activity may therefore contribute to variation in ME efficiency. The observed relationships between AC treatment, ROS levels and protease activity remain correlative and warrant future mechanistic investigation to clarify the causal links among these processes.

Microspore embryogenesis (ME) is the shortest route to total homozygosity, as it results in the production of doubled haploid (DH) plants. However, the huge theoretical potential of this method is only rarely realized in practice, as there are no effective protocols available for a large number of crop species. In addition, the effectiveness of DH plant production is genotype dependent and highly variable not only between different species, but also between different cultivars, lines, and even individuals. The majority of modern triticale breeding materials are moderately responsive to ME induction. Therefore, procedures are still being developed and optimized for the requirements of specific genotypes. Based on the hypothesis of the important role of redox homeostasis in effective ME induction, we describe here a protocol modified by the application of reduced glutathione (GSH) as a factor that maintains microspore viability and promotes proper embryo-like structure (ELS) development in triticale.

Winter survival of crops is influenced by multiple environmental stress factors, including low temperature, excessive soil moisture, and pathogen pressure. With climate change, the role of flooding during cold acclimation has become increasingly important. This study examined the effect of cold flooding on frost tolerance of two winter rye (Secale cereale L.) lines differing in resistance to snow mould. Plants were subjected to cold acclimation for three weeks at 4 °C, followed by ten days of flooding at the same cold temperature. Cold flooding improved frost tolerance only in the snow mould-resistant line (343), which maintained the presence of a 50 kDa β-1,3-glucanase isoform and exhibited a flooding-induced decrease in cytosolic sucrose synthase (SuS) activity in leaves, while maintaining higher SuS activity in roots. In contrast, the less resistant line (620) lacked the 50 kDa isoform in roots and showed substantially lower SuS activity than line 343; however, leaf SuS activity increased during prolonged flooding to levels comparable with those of line 343. The 35 kDa isoform was no longer detectable in the leaves of either line after one or ten days of flooding, whereas in the roots of both lines, its accumulation was maintained after one day but was abolished after ten days of flooding. Additionally, line 620 exhibited prolonged flooding-induced upregulation of glu-8 and TLP3 genes, which, in the absence of protein accumulation, suggests post-transcriptional or translational constraints associated with prolonged stress. These findings demonstrate that higher frost tolerance under cold flooding involves the accumulation of specific pathogenesis-related (PR) proteins, maintaining a high reduced and oxidised glutathione ratio and metabolic adjustment of sucrose synthesis. Furthermore, cold acclimation alone did not differentiate frost tolerance between rye genotypes differing in snow mould resistance, whereas flooding under low-temperature conditions enhanced freezing tolerance only in the resistant line, indicating that flooding may function as a positive signal involved in frost tolerance induction.

The cultivation of rye (Secale cereale L.) for functional foods is economically important due to its high-dietary fiber content and health-related bioactive compounds. Breeders are seeking modern biotechnology techniques to accelerate traditional breeding in this predominantly cross-pollinated species. The development of efficient protocols for double haploid (DH) production in rye breeding faces challenges. Despite five decades of research, no universally efficient DH production protocol has emerged, largely due to genotypic variability and recalcitrance to in vitro regeneration. The protocols presented here incorporate antioxidants, such as reduced glutathione (GSH) and selenium (SS), into the tiller low temperature pretreatment procedure, as well as arabinogalactan proteins (AGPs) and phytagel into the induction medium. All these modifications aim to reduce the generation of reactive oxygen species (ROS). As a result, microspores are more likely to survive stress pretreatment, the isolation process and transfer to in vitro culture better. A higher population of viable microspores could result in an increase in the number of reprogrammed, embryogenic microspores and microspore-derived embryo-like structures (ELS). The protocols presented have been tested on 15 F1 rye breeding lines, with the greatest effectiveness in lines with moderate and high responsiveness to androgenesis induction, also known as microspore embryogenesis (ME) induction.

Plants are constantly subjected to stresses such as herbivore attacks, which continuously activate the jasmonic acid (JA) signaling pathway in nature. However, knowledge about the effect of long-term activation of the JA signaling pathway on plant defense response remains limited. THIOGLUCOSIDE GLUCOHYDROLASE 1 (TGG1) and TGG2 are enzymes that activate defensive metabolites, namely glucosinolates, which are involved in defense against herbivores and pathogens. Here, we show that prolonged exposure to the wounding hormone methyl jasmonate (MeJA) enhances TGG1 and TGG2 expression in the rosette leaves of 12-d-old Arabidopsis thaliana, and this response does not appear to be strictly dependent on the canonical JA signaling pathway. Airborne MeJA treatment for up to 5 d enhanced both TGG1 and TGG2 gene expression and their protein levels in Arabidopsis leaves. Notably, TGG1 and TGG2 gene expression was also significantly upregulated in two JA signaling pathway mutants, namely coi1-16 and myc2,3,4, following 5 d of MeJA treatment. TGG1 and TGG2 proteins accumulate in specialized myrosin cells of rosette leaves. Myrosin cell area expanded in response to MeJA treatment in a leaf-age-dependent manner. Consistent with this observation, the expression of FAMA, a transcription factor known to regulate TGG1 and TGG2 expression, was also increased in a leaf-age-dependent manner after 5 d of MeJA treatment. Taken together, our results suggest the existence of additional regulatory mechanisms beyond canonical JA signaling pathway that is activated by long-term exposure to MeJA and regulates the expression of defense-related genes TGG1 and TGG2.

Hormonal homeostasis plays a critical role in the regulation of microspore embryogenesis (ME). The balance between endogenous phytohormones must be altered to induce microspore reprogramming from the classical pollen-formation pathway to embryogenic development, but too extensive changes may be detrimental. In the present study, the levels of auxins, cytokinins and abscisic acid were monitored in the anthers of two Polish winter wheat F1 lines and the spring cultivar Pavon highly differentiated in terms of ME effectiveness. Analyses were carried out at subsequent steps of the ME induction procedure that combined low temperature, sodium selenate and mannitol tiller pre-treatment. Of all the factors tested, mannitol induced the most profound effect on phytohormones and their homeostasis in wheat anthers. It significantly increased the accumulation of all auxins and decreased the levels of most cytokinins, while the change in ABA content was limited to cv. Pavon. In an attempt to alleviate this hormonal shock, we tested several modifications of the induction medium hormonal composition and found thidiazuron to be the most promising in stimulating the embryogenic development of wheat microspores. The lack of ABA-driven stress defence responses may be one of the reasons for the low effectiveness of ME induction in winter wheat microspore cultures. Low cytokinin level and a disturbed auxin/cytokinin balance may then be responsible for the morphological abnormalities observed during the next phases of embryogenic microspore development. One possible solution is to modify the hormonal composition of the induction medium with thidiazuron identified as the most promising component. Mannitol, used to induce microspore embryogenesis in wheat, stimulates intense auxin accumulation, which disrupts hormonal homeostasis and can lead to disturbances in the next stages of embryogenic development.

Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors. Haploids/doubled haploids (DHs) derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering. However, the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined. Here, RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis (ME) in barley cultivars Golden Promise and Igri, which exhibit contrasting responsiveness to microspore-derived plant formation. Our experiments revealed fundamental regulatory networks, specific groups of genes, and transcription factor (TF) families potentially regulating the developmental switch. We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME. Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.

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

Myrosinase is an enzyme that activates a group of metabolites, namely glucosinolates, which are involved in the defense against herbivores and pathogens. In Arabidopsis thaliana , THIOGLUCOSIDE GLUCOHYDROLASE 1 (TGG1) and TGG2 are two myrosinases that accumulate in specialized myrosin cells of rosette leaves. Here, we show that prolonged exposure to the wounding hormone methyl jasmonate (MeJA) enhances TGG1 and TGG2 expression independent of the canonical jasmonic acid (JA) signaling pathway. We found that airborne MeJA treatment for up to 5-days enhanced both TGG1 and TGG2 gene expression and their protein levels in Arabidopsis leaves. TGG1 and TGG2 gene expression did not stop in two JA signaling pathway mutants, namely coi1-16 and myc2,3,4 , after 5-days of MeJA treatment, although other typical JA responses were completely stopped in these two mutants. FAMA is a transcription factor that participates in the specification of myrosin cell development, but it does not appear to be involved in TGG1 and TGG2 gene expression regulation in the 5-day MeJA treatment, as its expression did not increase. Taken together, our results suggest the existence of an alternative JA signaling pathway that is activated by long-term exposure to MeJA.

Diverse processes leading to doubled haploid (DH) plant production, such as microspore embryogenesis, gynogenesis, and distant hybridization followed by genome elimination, are based on the unique ability of plant cells to form haploid embryos without fertilization. All of these are possible because of various in vitro culture systems that enable the growth and development of tissues or single cells outside of the parental organism. The possibility of re-directing cell development from its original pathway to embryogenesis brings several benefits to many research areas, but the most important is the possibility of its implementation in breeding programs. This review summarizes the achievements of Polish research groups in studies of the mechanisms of haploid/DH embryo development and demonstrates the practical applications of these systems in basic studies and plant breeding. It shows the results of studies on economically important crops including barley (Hordeum vulgare L.), oilseed rape (Brassica napus L.), triticale (×Triticosecale Wittm.), oat (Avena sativa L.), rye (Secale cereale L.), sugar beet (Beta vulgaris ssp. vulgaris L.), and some vegetable species, including carrot (Daucus carota L.), onion (Allium cepa L.), red beet (Beta vulgaris L.), and members of the Brassicaceae.

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 (H 2 O 2 ) and ME effectiveness confirmed the important role of reactive oxygen species in microspore reprogramming. However, for high effectiveness of ME induction, intensive H 2 O 2 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.

BACKGROUND:A mannitol stress treatment and a subsequent application of n-butanol, known as a microtubule-disrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. The degree of MT fragmentation was examined by the relative fluorescence intensity quantification.RESULTS:In uni-nucleated mannitol-treated microspores, severe CMT and EMT fragmentation occurs, although a complex network of short EMT bundles protected the nucleus. Additional treatment with n-butanol resulted in further depolymerization of both CMT and EMT, simultaneously with the formation of MT aggregates in the perinuclear region. Some aggregates resembled a preprophase band. In addition, a portion of the microspores progressed to the first mitotic division during the treatments. Bi-nucleate pollen-like structures showed a high MT depolymerization after mannitol treatment and numerous EMT bundles around the vegetative and generative nuclei after n-butanol. Interestingly, bi-nucleate symmetric structures showed prominent stabilization of EMT.CONCLUSIONS:Fragmentation and stabilization of microtubules induced by mannitol- and n-butanol lead to new configurations essential for the induction of microspore embryogenesis in bread wheat. These results provide robust insight into MT dynamics during EM induction and open avenues to address newly targeted treatments to induce ME in recalcitrant species.

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5–10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes ( TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE ( GSTF2 )) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores ( TaTPD1-like, SERK and GSTF2 ).

In this review, we describe and integrate the latest knowledge on the signaling role of proteins and peptides in the stress-induced microspore embryogenesis (ME) in some crop plants with agricultural importance (i.e., oilseed rape, tobacco, barley, wheat, rice, triticale, rye). Based on the results received from the most advanced omix analyses, we have selected some inconspicuous but possibly important players in microspores reprogramming toward embryogenic development. We provide an overview of the roles and downstream effect of stress-related proteins (e.g., β-1,3-glucanases, chitinases) and small signaling peptides, especially cysteine—(e.g., glutathione, γ-thionins, rapid alkalinization factor, lipid transfer, phytosulfokine) and glycine-rich peptides and other proteins (e.g., fasciclin-like arabinogalactan protein) on acclimation ability of microspores and the cell wall reconstruction in a context of ME induction and haploids/doubled haploids (DHs) production. Application of these molecules, stimulating the induction and proper development of embryo-like structures and green plant regeneration, brings significant improvement of the effectiveness of DHs procedures and could result in its wider incorporation on a commercial scale. Recent advances in the design and construction of synthetic peptides–mainly cysteine-rich peptides and their derivatives–have accelerated the development of new DNA-free genome-editing techniques. These new systems are evolving incredibly fast and soon will find application in many areas of plant science and breeding.

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.

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.