Barley Anther Culture

2020

Plant Mol Biol

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.

Section: Discussionmentioning

confidence: 99%

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

2020

Plant Mol Biol

“…In vitro anther tissue cultures is an essential approach for the evaluation of uniform plant materials useful for breeding programs [ 40 , 41 , 42 , 43 ]. 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 ].…”

Section: Discussionmentioning

confidence: 99%

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

2020

Plants

Plant anther culture allows for the regeneration of uniform and homozygous double haploids. However, off-type regenerants may appear as a result of so-called tissue culture-induced variation (TCIV). In addition, the presence of Cu2+ and Ag+ ions in the culture medium might influence the number of green plants. The regenerants were obtained via anther cultures of barley under varying Cu2+ and Ag+ ion concentrations in the induction medium during distinct time conditions. DArTseqMet markers were evaluated based on regenerants and donor plants and delivering data on DNA demethylation (DM) and de novo methylation (DNM) and changes in methylation (Delta). The number of green regenerated plants per 100 anthers (GPs) was evaluated. The Cu2+ and Ag+ ion concentrations moderated relationships between Delta and the number of green plants conditional on time of tissue cultures. Depending on the ions, moderated moderation is valid within the different time of anther culture. When the highest concentration of copper is analyzed, plant regeneration is possible under short ‘Time’ (21 days) of anther culture wherein Delta is negative or under elongated Time when Delta is positive. Under 21 days of culture, the highest concentration of silver ions and when Delta is negative, some regenerants could be evaluated. However, under high Ag+ concentration when Time of culture is long and Delta positive, the highest number of green plants could be obtained.

“…In vitro anther tissue cultures is an essential approach for the evaluation of uniform plant materials useful for breeding programs [50][51][52][53]. 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].…”

Section: Discussionmentioning

confidence: 99%

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

2020

Preprint

BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.