Precise evaluation of tissue culture-induced variation during optimisation of in vitro regeneration regime in barley

Abstract

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.

Exploring the Biochemical Origin of DNA Sequence Variation in Barley Plants Regenerated via in Vitro Anther Culture

“…Genotyping of the isolated DNAs using the metAFLP approach allowed the identification of 407 markers shared by the donor plant and regenerants [25]. Quantitative metAFLP characteristics of the DNAs are listed in Table 1.…”

“…Furthermore, abiotic stresses such as cold and drought are responsible for changes in DNA methylation patterns [22]. Additionally, studies on plant responses to in vitro tissue culture-induced stress show that tissue culture conditions promote changes in stress-induced DNA methylation and/or demethylation patterns [23][24][25][26][27]. Machczyńska and colleagues reported a relatively high level of DNA sequence variation between the donor plant and its regenerants [28].…”

Time of In Vitro Anther Culture May Moderate Action of Copper and Silver Ions that Affect the Relationship between DNA Methylation Change and the Yield of Barley Green Regenerants

“…The approach is being world-wide used, but a growing number of data clearly shows that plant regeneration via anther culture is affected by an in vitro tissue culture-induced variation (TCIV) [ 44 , 45 , 46 , 47 ] that could be transmitted to a progeny [ 48 ]. Among others, the TCIV is due to changes in the DNA methylation patterns that are under either genetic or epigenetic control [ 49 ]. As plant regeneration requires cell reprogramming [ 18 ] involving DNA methylation pattern change, it is of value to understand whether ingredients present in the in vitro medium participating in many cellular biochemical pathways [ 17 , 50 , 51 ] and other factors such as time of in vitro cultures may affect methylation changes and the number of the regenerated green plants that may be limited by the presence of albinos [ 52 ].…”

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

“…The approach is being world-wide used, but a growing number of data clearly shows that plant regeneration via anther culture is affected by an in vitro TCIV [54][55][56][57] that could be transmitted to a progeny [58]. Among others, the TCIV is due to changes in the DNA methylation patterns affecting distinct methylation contexts (symmetric and asymmetric) that are under either genetic or epigenetic control [44]. As plant regeneration requires cell reprogramming [1] involving DNA methylation pattern changes, it is of value to understand whether ingredients present in the in vitro medium and other factors such as the time of in vitro cultures may affect methylation changes and the number of the regenerated green plants that may be limited by the presence of albinos [59].…”

“…Our previous studies have demonstrated that manipulating Cu 2+ , Ag + ion concentration one may optimize in vitro anther cultures of barley towards the increased number of green plants [43]. We have also shown that under varying conditions of Cu 2+ , Ag + , and time (T) differences in DNA methylation of the symmetric and asymmetric context may appear [44]. According to that results, the role of the time seems to be negligible, whereas the others [45] indicated that the longevity of in vitro tissue cultures might influence DNA methylation pattern [46] or even sequence changes [47].…”