However, the lack of such changes does not mean they are not present at the genetic or DNA methylation levels. Īlthough rare, the influence of different factors affecting tissue culture plant regeneration can be seen at the morphological level of regenerants. Apart from DNA mutations, changes in ploidy were observed when tissue cultures kept for a prolonged time. However, with increasing tissue culture time, the transversion rate also rises. Studies devoted to the mutation rate in plant tissue cultures pointed at the prevalence of transition over transversion. In contrast to direct embryogenesis, indirect one may induce somaclonal variation at the DNA sequence level due to callus phase. The other factors that may affect (epi)genetic purity of regenerants are, i.e., time of in vitro induction medium and the presence of callus stage. Silver ions may also influence retrograde (plastid-to-nucleus) signaling, epigenetic processes and activate reparation mechanisms. However, at increased concentration silver ions may be toxic for plant tissues inducing reactive oxygen species followed by oxidative DNA damages. The positive effect of AgNO 3 on somatic embryogenesis, shoot growth, and direct organogenesis is well established. Another ion utilized in tissue culture media is silver. However, overstepping bounds of toxic doses may imbalance the chloroplast redox state altering photosynthesis, and inducing oxidative stress. A somewhat elevated level of copper ions in induction or regeneration media may positively influence somatic embryogenesis or androgenesis. Copper ions at the physiological level are crucial for growth and plant survival. Among media components, copper and silver ions are indispensable for plant growth and development. Many reports pointed out media components as the factors inducing genetic or epigenetic changes shared among in vitro derived plant. The extent of changes may depend on a type of explants, genotype, or time of culture on induction medium. chromosomal changes, point mutations, movement of transposable elements, or changes in methylation status of DNA originating due to numerous stresses (sterilization factors, media components, light conditions or humidity ). Genetic or epigenetic purity of regenerants can be perturbed by e.g. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context. 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 main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful.
JACCARD COEFFICIENT XLSTAT VERIFICATION
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. 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. 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 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. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress.
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