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.

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.

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.