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

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.

Comparison of methods for obtaining doubled haploids of carrot

Doubled haploid lines of carrot can be obtained through androgenesis in anther cultures and in isolated microspore cultures. The two methods were compared using three carrot cultivars (‘Kazan F1’, ‘Feria F1’, and ‘Narbonne F1’) at the androgenesis induction stage, during plant regeneration from embryos, and during acclimatization of androgenetic plants as well as their characterization. It was found that cultivar was the main factor affecting the efficiency at each stage of plant production in both anther and isolated microspore cultures. The efficiency of androgenesis in anther cultures of ‘Feria F1’ was considerably higher in comparison with isolated microspore cultures, and more plants were obtained from the embryos of androgenesis-cultured plants. In ‘Kazan F1’ and ‘Narbonne F1’, more acclimatized androgenetic plants were produced from anther cultures. Ploidy assessment of acclimatized plants of ‘Narbonne F1’ showed that the majority of the plants in the population derived from anther cultures had a doubled chromosome (DH) set. On the other hand, the majority of plants obtained from isolated microspore cultures were haploids. When assessing homozygosity, it was found among plants obtained in anther cultures that the percentage of homozygotes for phosphoglucose isomerase (PGI) and aspartate aminotransferase (AAT) depended on the cultivar. In contrast, the majority of plants derived from isolated microspore cultures were homozygous regardless of cultivar.