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

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.

Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley

“…Then, it is not evident whether such results should be interpreted concerning methylation of the mobile elements or their mobility. On the other hand, the level of methylation (and sequence) changes evaluated in the study is only slightly higher than that for the same species using metAFLP alone [76,119,120]. Assuming MSTD amplifies short fragments (the range is 45-500 bp [121]), our results may, at least partly, reflect mobile elements' movement due to methylation changes due to microspore reprogramming.…”

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

“…However, the TCIV and SV terms are often used mutually [7]. TCIV may be pronounced at the morphological [8], biochemical [9], genetic [10] and epigenetic [11] levels, which do not necessarily overlap [12]. Possibly the most investigated level is based on DNA markers.…”

“…Consequently, another AFLP based technique, the so-called methylation sensitive AFLP (metAFLP) approach, was developed [ 1 , 39 ], allowing quantification of sequence and DNA methylation changes during a single experiment. Further advances in the technique showed that one might study not only CG and CHG but also the CHH methylation context, an essential tool in studies on cereals [ 1 , 12 ].…”

“…The role of copper and silver ions in sequence variation [ 63 ] and green plant development [ 40 ] was also documented in barley regenerants derived via anther culture. Similarly, in embryo-derived regenerants of barley copper ions participated in sequence variation, possibly via modification of methylated sequences [ 12 ]. The respective relationships could be evaluated using mediation and moderation analyses [ 64 ] applied to DNA methylation changes and the ATR-FTIR spectroscopy data.…”

“…As such ingredients may be toxic, they may lead to genomic DNA degradation as indicated by comet analysis [ 101 ]. It was also demonstrated that copper and silver ions containing media might also affect DNA methylation patterns [ 12 ], resulting in DNA sequence changes [ 62 ]. Moreover, the two ions most probably change respiratory chain balance affecting the Yang cycle [ 62 ] and inducing sequence changes [ 63 ].…”