An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants.

BACKGROUND: The development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture's induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance-Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed. RESULTS: According to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55. CONCLUSIONS: The amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale's GPRE can be increased.

Corrigendum: Glutathione and copper ions as critical factors of green plant regeneration efficiency of triticale in vitro anther culture.

[This corrects the article DOI: 10.3389/fpls.2022.926305.].

S-Adenosyl-L-Methionine and Cu(II) Impact Green Plant Regeneration Efficiency.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Glutathione and copper ions as critical factors of green plant regeneration efficiency of triticale in vitro anther culture.

Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Length Polymorphism was used to assess tissue culture-induced variation, Fourier Transform Infrared spectroscopy to describe the glutathione spectrum, and a structural equation model to develop the relationship between sequence variation, de novo DNA methylation within asymmetric sequence contexts, and copper ions in the induction medium, as well as, glutathione, and green plant efficiency. An essential aspect of the study is demonstrating the contribution of glutathione to green plant regeneration efficiency and indicating the critical role of copper ions in influencing tissue culture-induced variation, glutathione, and obtaining green regenerants. The model presented here also has practical implications, showing that manipulating the concentration of copper ions in the induction medium may influence cell function and increases green plant regeneration efficiency.

Triticale doubled haploid plant regeneration factors linked by structural equation modeling.

Triticale regeneration via anther culture faces many difficulties, e.g., a low percentage of regenerated plants and the presence of albinos. Plant regeneration may be affected by abiotic stresses and by ingredients added to the induction medium. The latter influences biochemical pathways and plant regeneration efficiency. Among such ingredients, copper and silver ions acting as cofactors for enzymatic reactions are of interest. However, their role in plant tissue cultures and relationships with biochemical pathways has not been studied yet.The study evaluated relationships between DNA methylation, changes in DNA sequence variation, and green plant regeneration efficiency influenced by copper and silver ions during triticale plant regeneration. For this purpose, a biological model based on donor plants and their regenerants, a methylation-sensitive amplified fragment length polymorphism, and structural equation modeling were employed.The green plant regeneration efficiency varied from 0.71 to 6.06 green plants per 100 plated anthers. The values for the components of tissue culture-induced variation related to cytosine methylation in a CHH sequence context (where H is A, C, or T) were 8.65% for sequence variation, 0.76% for DNA demethylation, and 0.58% for de novo methylation. The proposed model states that copper ions affect the regeneration efficiency through cytosine methylation and may induce mutations through, e.g., oxidative processes, which may interfere with the green plant regeneration efficiency. The linear regression confirms that the plant regeneration efficiency rises with increasing copper ion concentration in the absence of Ag ions in the induction medium. The least absolute shrinkage and selection operator regression shows that de novo methylation, demethylation, and copper ions may be involved in the green plant regeneration efficiency. According to structural equation modeling, copper ions play a central role in the model determining the regeneration efficiency.

Effect of copper and silver ions on sequence and DNA methylation changes in triticale regenerants gained via somatic embryogenesis.

Somatic embryogenesis is a plant regeneration method that can be exploited in tissue culture systems for a variety of tasks, such as genetic modification or the selection of somaclones with advantageous characteristics. Therefore, it is crucial to create efficient regeneration procedures and comprehend how medium components affect regeneration effectiveness or the degree of variation created in plant tissue cultures. The level of tissue culture-induced variation in triticale regenerants was examined in the current study in relation to the concentration of copper and silver ions in the induction media as well as the length of time immature zygotic embryo explants were incubated on these media. The high degree of variation (45%) revealed by the methylation-sensitive amplified fragment length polymorphism approach for estimating variation included 38% DNA sequence alterations, 6% DNA demethylation, and 1% de novo DNA methylation. Different levels of variance were found in relation to various DNA sequence settings. The CHG context had the most alterations, whereas CG experienced the fewest; sequence variation predominated in each sequence context. Lower copper ion concentrations showed the most variance. However, it could not be connected to the duration of in vitro culture or the effect of silver ions. Accordingly, we think that altering the concentration of copper ions in the induction medium may throw off the equilibrium of the metabolic processes in which copper is involved, resulting in tissue culture-induced variation.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture.

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

Structural Equation Modeling (SEM) Analysis of Sequence Variation and Green Plant Regeneration via Anther Culture in Barley.

The process of anther culture involves numerous abiotic stresses required for cellular reprogramming, microspore developmental switch, and plant regeneration. These stresses affect DNA methylation patterns, sequence variation, and the number of green plants regenerated. Recently, in barley (Hordeum vulgare L.), mediation analysis linked DNA methylation changes, copper (Cu2+) and silver (Ag+) ion concentrations, sequence variation, ß-glucans, green plants, and duration of anther culture (Time). Although several models were used to explain particular aspects of the relationships between these factors, a generalized complex model employing all these types of data was not established. In this study, we combined the previously described partial models into a single complex model using the structural equation modeling approach. Based on the evaluated model, we demonstrated that stress conditions (such as starvation and darkness) influence ß-glucans employed by cells for glycolysis and the tricarboxylic acid cycle. Additionally, Cu2+ and Ag+ ions affect DNA methylation and induce sequence variation. Moreover, these ions link DNA methylation with green plants. The structural equation model also showed the role of time in relationships between parameters included in the model and influencing plant regeneration via anther culture. Utilization of structural equation modeling may have both scientific and practical implications, as it demonstrates links between biological phenomena (e.g., culture-induced variation, green plant regeneration and biochemical pathways), and provides opportunities for regulating these phenomena for particular biotechnological purposes.

Understanding In Vitro Tissue Culture-Induced Variation Phenomenon in Microspore System.

In vitro tissue culture plant regeneration is a complicated process that requires stressful conditions affecting the cell functioning at multiple levels, including signaling pathways, transcriptome functioning, the interaction between cellular organelles (retro-, anterograde), compounds methylation, biochemical cycles, and DNA mutations. Unfortunately, the network linking all these aspects is not well understood, and the available knowledge is not systemized. Moreover, some aspects of the phenomenon are poorly studied. The present review attempts to present a broad range of aspects involved in the tissue culture-induced variation and hopefully would stimulate further investigations allowing a better understanding of the phenomenon and the cell functioning.

Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley.

A plant genome usually encompasses different families of transposable elements (TEs) that may constitute up to 85% of nuclear DNA. Under stressful conditions, some of them may activate, leading to sequence variation. In vitro plant regeneration may induce either phenotypic or genetic and epigenetic changes. While DNA methylation alternations might be related, i.e., to the Yang cycle problems, DNA pattern changes, especially DNA demethylation, may activate TEs that could result in point mutations in DNA sequence changes. Thus, TEs have the highest input into sequence variation (SV). A set of barley regenerants were derived via in vitro anther culture. High Performance Liquid Chromatography (RP-HPLC), used to study the global DNA methylation of donor plants and their regenerants, showed that the level of DNA methylation increased in regenerants by 1.45% compared to the donors. The Methyl-Sensitive Transposon Display (MSTD) based on methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach demonstrated that, depending on the selected elements belonging to the TEs family analyzed, varying levels of sequence variation were evaluated. DNA sequence contexts may have a different impact on SV generated by distinct mobile elements belonged to various TE families. Based on the presented study, some of the selected mobile elements contribute differently to TE-related SV. The surrounding context of the TEs DNA sequence is possibly important here, and the study explained some part of SV related to those contexts.

Comparative Study of the Genetic and Biochemical Variability of Polyscias filicifolia (Araliaceae) Regenerants Obtained by Indirect and Direct Somatic Embryogenesis as a Source of Triterpenes.

Polyscias filicifolia (Araliaceae) is broadly used in traditional medicine in Southeast Asia due to its antimicrobial, immunomodulating and cytotoxic activities. The main groups of compounds responsible for pharmacological effects are believed to be oleanolic triterpene saponins. However, Polyscias plants demonstrate relatively slow growth in natural conditions, which led to applying a developing sustainable source of plant material via primary (PSE), secondary (DSE) and direct somatic embryogenesis from DSE (TSE). The AFLP and metAFLP genotyping resulted in 1277 markers, amplified by a total of 24 pairs of selective primers. Only 3.13% of the markers were polymorphic. The analysis of variance showed that the PSE and TSE regenerants differed only in terms of root number, while the DSE plantlets differed for all studied morphological characteristics. Further, the chemical analysis revealed that oleanolic acid (439.72 µg/g DW), ursolic acid (111.85 µg/g DW) and hederagenin (19.07 µg/g DW) were determined in TSE regenerants. Our results indicate that direct somatic embryogenesis ensures the production of homogeneous plant material, which can serve as a potential source of triterpene compounds. Plants obtained via somatic embryogenesis could also be reintroduced into the natural environment to protect and preserve its biodiversity.

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture.

BACKGROUND: Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. RESULTS: This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. CONCLUSIONS: The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium.

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Time of In Vitro Anther Culture May Moderate Action of Copper and Silver Ions that Affect the Relationship between DNA Methylation Change and the Yield of Barley Green Regenerants.

Plant anther culture allows for the regeneration of uniform and homozygous double haploids. However, off-type regenerants may appear as a result of so-called tissue culture-induced variation (TCIV). In addition, the presence of Cu2+ and Ag+ ions in the culture medium might influence the number of green plants. The regenerants were obtained via anther cultures of barley under varying Cu2+ and Ag+ ion concentrations in the induction medium during distinct time conditions. DArTseqMet markers were evaluated based on regenerants and donor plants and delivering data on DNA demethylation (DM) and de novo methylation (DNM) and changes in methylation (Delta). The number of green regenerated plants per 100 anthers (GPs) was evaluated. The Cu2+ and Ag+ ion concentrations moderated relationships between Delta and the number of green plants conditional on time of tissue cultures. Depending on the ions, moderated moderation is valid within the different time of anther culture. When the highest concentration of copper is analyzed, plant regeneration is possible under short 'Time' (21 days) of anther culture wherein Delta is negative or under elongated Time when Delta is positive. Under 21 days of culture, the highest concentration of silver ions and when Delta is negative, some regenerants could be evaluated. However, under high Ag+ concentration when Time of culture is long and Delta positive, the highest number of green plants could be obtained.

Exploring the Biochemical Origin of DNA Sequence Variation in Barley Plants Regenerated via in Vitro Anther Culture.

Tissue culture is an essential tool for the regeneration of uniform plant material. However, tissue culture conditions can be a source of abiotic stress for plants, leading to changes in the DNA sequence and methylation patterns. Despite the growing evidence on biochemical processes affected by abiotic stresses, how these altered biochemical processes affect DNA sequence and methylation patterns remains largely unknown. In this study, the methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach was used to investigate de novo methylation, demethylation, and sequence variation in barley regenerants derived by anther culture. Additionally, we used Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to identify the spectral features of regenerants, which were then analyzed by mediation analysis. The infrared spectrum ranges (710-690 and 1010-940 cm-1) identified as significant in the mediation analysis were most likely related to ß-glucans, cellulose, and S-adenosyl-L-methionine (SAM). Additionally, the identified compounds participated as predictors in moderated mediation analysis, explaining the role of demethylation of CHG sites (CHG_DMV) in in vitro tissue culture-induced sequence variation, depending on the duration of tissue culture. The data demonstrate that ATR-FTIR spectroscopy is a useful tool for studying the biochemical compounds that may affect DNA methylation patterns and sequence variation, if combined with quantitative characteristics determined using metAFLP molecular markers and mediation analysis. The role of ß-glucans, cellulose, and SAM in DNA methylation, and in cell wall, mitochondria, and signaling, are discussed to highlight the putative cellular mechanisms involved in sequence variation.

CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.

Precise evaluation of tissue culture-induced variation during optimisation of in vitro regeneration regime in barley.

KEY MESSAGE: The Taguchi method and metAFLP analysis were used to optimise barley regenerants towards maximum and minimum levels of tissue culture-induced variation. The subtle effects of symmetric and asymmetric methylation changes in regenerants were identified. Plant tissue cultures (PTCs) provide researchers with unique materials that accelerate the development of new breeding cultivars and facilitate studies on off-type regenerants. The emerging variability of regenerants derived from PTCs may have both genetic and epigenetic origins, and may be desirable or degrade the value of regenerated plants. Thus, it is crucial to determine how the PTC variation level can be controlled. The easiest way to manipulate total tissue culture-induced variation (TTCIV) is to utilise appropriate stress factors and suitable medium components. This study describes the optimisation of in vitro tissue culture-induced variation in plant regenerants derived from barley anther culture, and maximizes and minimizes regenerant variation compared with the source explants. The approach relied on methylation amplified fragment length polymorphism (metAFLP)-derived TTCIV characteristics, which were evaluated in regenerants derived under distinct tissue culture conditions and analysed via Taguchi statistics. The factors that may trigger TTCIV included CuSO4, AgNO3 and the total time spent on the induction medium. The donor plants prepared for regeneration purposes had 5.75% and 2.01% polymorphic metAFLP loci with methylation and sequence changes, respectively. The level of TTCIV (as the sum of all metAFLP characteristics analyzed) identified in optimisation and verification experiments reached 7.51 and 10.46%, respectively. In the trial designed to produce a minimum number of differences between donor and regenerant plants, CuSO4 and AgNO3 were more crucial than time, which was not a significant factor. In the trial designed to produce a maximum number of differences between donor and regenerant plants, all factors had comparable impact on variation. The Taguchi method reduced the time required for experimental trials compared with a grid method and suggested that medium modifications were required to control regenerant variation. Finally, the effects of symmetric and asymmetric methylation changes on regenerants were identified using novel aspects of the metAFLP method developed for this analysis.

Improvement of anther cultures conditions using the Taguchi method in three cereal crops

Background: Plant tissue cultures have the potential to reprogram the development of microspores from normal gametophytic to sporophytic pathway resulting in the formation of androgenic embryos. The efficiency of this process depends on the genotype, media composition and external conditions. However, this process frequently results in the regeneration of albino instead of green plants. Successful regeneration of green plants is affected by the concentration of copper sulfate (CuSO4) and silver nitrate (AgNO3) and the length of induction step. In this study, we aimed at concurrent optimization of these three factors in barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and triticale (x Triticosecale spp. Wittmack ex A. Camus 1927) using the Taguchi method. We evaluated uniform donor plants under varying experimental conditions of in vitro anther culture using the Taguchi approach, and verified the optimized conditions. Results: Optimization of the regeneration conditions resulted in an increase in the number of green regenerants compared with the control. Statistic Taguchi method for optimization of the in vitro tissue culture plant regeneration via anther cultures allowed reduction of the number of experimental designs from 27 needed if full factorial analysis is used to 9. With the increase in the number of green regenerants, the number of spontaneous doubled haploids decreased. Moreover, in barley and triticale, the number of albino regenerants was reduced. Conclusion: The statistic Taguchi approach could be successfully used for various factors (here components of induction media, time of incubation on induction media) at a one time, that may impact on cereals anther cultures to improve the regeneration efficiency

Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants.

In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo-derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Amplified Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modification of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a significant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress.

BACKGROUND: We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms. METHODS: Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward's Agglomeration method whereas MSAP characteristics for ANOVA. RESULTS: Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines. CONCLUSIONS: Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII-MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.