Background: When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.

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. Key message Variation appeared in regenerated plants as well as variation inherited by generative progeny of regenerants can may many, positive or negative impact, of gained plant materials. This review focused on factors that triggered this phenomenon with underlying oxidative stress.

Key message The Taguchi method and metAFLP analysis were used to optimise barley regenerants towards maximum andminimum levels of tissue culture-induced variation. The subtle effects of symmetric and asymmetric methylationchanges in regenerants were identified. Abstract Plant tissue cultures (PTCs) provide researchers with unique materials that accelerate the development of new breeding cultivars and facilitate studies on off-type regenerants. The emerging variability of regenerants derived from PTCs may have both genetic and epigenetic origins, and may be desirable or degrade the value of regenerated plants. Thus, it is crucial to determine how the PTC variation level can be controlled. The easiest way to manipulate total tissue cultureinduced variation (TTCIV) is to utilise appropriate stress factors and suitable medium components. This study describes the optimisation of in vitro tissue culture-induced variation in plant regenerants derived from barley anther culture, and maximizes and minimizes regenerant variation compared with the source explants. The approach relied on methylation amplified fragment length polymorphism (metAFLP)-derived TTCIV characteristics, which were evaluated in regenerants derived under distinct tissue culture conditions and analysed via Taguchi statistics. The factors that may trigger TTCIV included CuSO 4 , AgNO 3 and the total time spent on the induction medium. The donor plants prepared for regeneration purposes had 5.75% and 2.01% polymorphic metAFLP loci with methylation and sequence changes, respectively. The level of TTCIV (as the sum of all metAFLP characteristics analyzed) identified in optimisation and verification experiments reached 7.51 and 10.46%, respectively. In the trial designed to produce a minimum number of differences between donor and regenerant plants, CuSO 4 and AgNO 3 were more crucial than time, which was not a significant factor. In the trial designed to produce a maximum number of differences between donor and regenerant plants, all factors had comparable impact on variation. The Taguchi method reduced the time required for experimental trials compared with a grid method and suggested that medium modifications were required to control regenerant variation. Finally, the effects of symmetric and asymmetric methylation changes on regenerants were identified using novel aspects of the metAFLP method developed for this analysis.

Doubled haploids of triticale are of interest for plant breeders due to hybrid breeding programs based on cytoplasmic male sterility Tt phenomenon. However, (epi)mutations appearing during in vitro culture regeneration may lead to a phenotypic variation that makes the uniformity of plant materials questionable. Using RP-HPLC genomic DNA methylation of donor doubled haploid plants utilized as a source of tissues for the in vitro regeneration (via androgenesis and somatic embryogenesis) of triticale cv. Bogo and their consecutive generative progeny was evaluated. It was demonstrated that in vitro cultures induced a decrease of the DNA methylation of the regenerants independently of the approach used for plant regeneration. The decrease in DNA methylation of genomic DNA proceeded up to the first/second successive generations followed by the beginning of its reestablishment. Moreover, somatic embryogenesis resulted in a higher level of genomic DNA demethylation in regenerants than androgenesis and the process of methylation seems to be affected by donor plant. It is being speculated that long term changes in genomic DNA methylation may be a source of off-type individuals that may spontaneously arise during plant breeding.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-014-0079-2) contains supplementary material, which is available to authorized users.