Abstract
BackgroundSomatic 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.ResultsThis 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.ConclusionsThe 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.
Highlights
Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress
The methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) analyses of donor and regenerants with eight primer pairs (Additional file 1: Table S1) resulted in 192 markers evaluated for all trials (M1–M9) and shared between KpnI/MseI and Acc65I/MseI platforms
Presented research showed that by manipulating the concentration of the in vitro tissue culture medium ingredients such as copper and silver ions and the time of tissue cultures, it is possible to regenerate plants that are either close to the source of explant or vice versa at the level of the tissue culture-induced variation
Summary
Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Genetic or epigenetic purity of regenerants can be perturbed by e.g. chromosomal changes [1], point mutations [2], movement of transposable elements [3], or changes in methylation status of DNA [4] originating due to numerous stresses (sterilization factors [5], media components [6], light conditions [7] or humidity [8]). Many reports pointed out media components as the factors inducing genetic [13] or epigenetic [14] changes shared among in vitro derived plant. A somewhat elevated level of copper ions in induction or regeneration media may positively influence somatic embryogenesis [16, 17] or androgenesis [18].
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