Renata Orłowska

Plant Breeding and Acclimatization Institute - National Research Institute

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Piotr Tomasz Bednarek

Plant Breeding and Acclimatization Institute - National Research Institute

J. Zimny

Plant Breeding and Acclimatization Institute - National Research Institute

Agnieszka Niedziela

Plant Breeding and Acclimatization Institute - National Research Institute

Joanna Machczyńska

Plant Breeding and Acclimatization Institute - National Research Institute

Katarzyna A. Pachota

Plant Breeding and Acclimatization Institute - National Research Institute

JŻ

Jacek Żebrowski

Rzeszów University

Dariusz R. Mańkowski

Plant Breeding and Acclimatization Institute - National Research Institute

Piotr Androsiuk

University of Warmia and Mazury in Olsztyn

Wioletta M. Dynkowska

Plant Breeding and Acclimatization Institute - National Research Institute

Sylwia Oleszczuk

Plant Breeding and Acclimatization Institute - National Research Institute

Cooperative Institutions

Plant Breeding and Acclimatization Institute - National Research Institute

Polish Academy of Sciences

National Research Institute

Warsaw University of Life Sciences

Medical University of Warsaw

Leibniz Institute of Plant Genetics and Crop Plant Research

University of Warsaw

University of Nottingham

John Innes Centre

University of Life Sciences in Lublin

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

Journal of Biological Research - Thessaloniki (2021)

Renata Orłowska

Abstract 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.

Somaclonal variation

DNA demethylation

Plant tissue culture

10.1186/s40709-021-00138-5

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Citations (14)

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

Research Square (Research Square) (2020)

Piotr Tomasz Bednarek Renata Orłowska

Abstract BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu 2+ and Ag + ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu 2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu 2+ and Ag + ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu 2+ and Ag + ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu 2+ and Ag + play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

Demethylation

DNA demethylation

Somaclonal variation

10.21203/rs.3.rs-21697/v1

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Citations (3)

Molekularna charakterystyka wpływu elementów mobilnych na zmienność genetyczną w zbożowych kulturach in vitro

Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin (2019)

Renata Orłowska Piotr Tomasz Bednarek Sławomir Bany

10.37317/biul-2019-0024

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Extended metAFLP approach in studies of tissue culture induced variation (TCIV) in triticale

Molecular Breeding (2014)

Joanna Machczyńska Renata Orłowska J. Zimny Piotr Tomasz Bednarek

We present the development of the theoretical background of the metAFLP approach which allows for partition of complex variation into sequence changes, de novo methylation and demethylation of the regenerants derived via in vitro tissue culture methods in the case of triticale. It was demonstrated that, independent of whether andro- or embryogenesis was used for plant regeneration, the level of sequence changes identified between regenerants is about 10 %. Moreover, DNA demethylation prevails over de novo methylation of the regenerants compared to the donor plant. The metAFLP approach allows for the evaluation of numerous quantitative characteristics. For instance, one may quantify the number of sites unaffected by tissue culture approaches, global site DNA methylation etc. It is suggested that the approach could be useful for breeders in order to control plant material uniformity or for the evaluation of modified in vitro tissue culture approaches allowing for control of the (epi)mutation level. The extended metAFLP approach presented here delivers sufficient background for the evaluation of software that could facilitate analyses of the tissue culture induced variation.

Somaclonal variation

Demethylation

Triticale

DNA demethylation

10.1007/s11032-014-0079-2

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Citations (36)

Medium composition affects the tissue culture-induced variation in triticale regenerants

Plant Cell Tissue and Organ Culture (PCTOC) (2022)

Katarzyna A. Pachota Renata Orłowska Piotr Tomasz Bednarek

Triticale

Somaclonal variation

10.1007/s11240-022-02327-z

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Citations (9)

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

BMC Plant Biology (2024)

Renata Orłowska J. Zimny Jacek Żebrowski Piotr Androsiuk Piotr Tomasz Bednarek

Abstract 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.

Triticale

Somaclonal variation

Avena

Attenuated total reflection

Protoplast

10.1186/s12870-023-04679-w

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Citations (2)

Triticale doubled haploid plant regeneration factors linked by structural equation modeling

Journal of Applied Genetics (2022)

Renata Orłowska

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.

Triticale

Doubled haploidy

10.1007/s13353-022-00719-7

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Citations (5)

Plant tissue culture environment as a switch-key of (epi)genetic changes

Plant Cell Tissue and Organ Culture (PCTOC) (2019)

Piotr Tomasz Bednarek Renata Orłowska

Abstract The in vitro tissue cultures are, beyond all difficulties, an essential tool in basic research as well as in commercial applications. Numerous works devoted to plant tissue cultures proved how important this part of the plant science is. Despite half a century of research on the issue of obtaining plants in in vitro cultures, many aspects remain unknown. The path associated with the reprogramming of explants in the fully functioning regenerants includes a series of processes that may result in the appearance of morphological, physiological, biochemical or, finally, genetic and epigenetic changes. All these changes occurring at the tissue culture stage and appearing in regenerants as tissue culture-induced variation and then inherited by generative progeny as somaclonal variation may be the result of oxidative stress, which works at the step of explant preparation, and in tissue culture as a result of nutrient components and environmental factors. In this review, we describe the current status of understanding the genetic and epigenetic changes that occur during tissue culture.

Somaclonal variation

Plant tissue culture

Reprogramming

Plant tissue

10.1007/s11240-019-01724-1

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Citations (110)

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

Frontiers in Plant Science (2021)

Renata Orłowska J. Zimny Piotr Tomasz Bednarek

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.

Somaclonal variation

DNA demethylation

10.3389/fpls.2020.614837

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Citations (12)

β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants’ DNA methylation and GPRE

BMC Plant Biology (2024)

Renata Orłowska Wioletta M. Dynkowska Agnieszka Niedziela Jacek Żebrowski J. Zimny

DNA demethylation

Avena

Somaclonal variation

10.1186/s12870-024-05572-w

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Citations (0)