The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A

Abstract

Genotype-limited plant regeneration is one of the main obstacles to the broader use of genetic transformation in barley breeding. Thus, developing new approaches that might improve responses of in vitro recalcitrant genotypes remains at the center of barley biotechnology. Here, we analyzed different barley genotypes, including "Golden Promise," a genotype commonly used in the genetic transformation, and four malting barley cultivars of poor regenerative potential. The expression of hormone-related transcription factor (TF) genes with documented roles in plant regeneration was analyzed in genotypes with various plant-regenerating capacities. The results indicated differential expression of auxin-related TF genes between the barley genotypes in both the explants and the derived cultures. In support of the role of auxin in barley regeneration, distinct differences in the accumulation of free and oxidized auxin were observed in explants and explant-derived callus cultures of barley genotypes. Following the assumption that modifying gene expression might improve plant regeneration in barley, we treated the barley explants with trichostatin A (TSA), which affects histone acetylation. The effects of TSA were genotype-dependent as TSA treatment improved plant regeneration in two barley cultivars. TSA-induced changes in plant regeneration were associated with the increased expression of auxin biosynthesis-involved TFs. The study demonstrated that explant treatment with chromatin modifiers such as TSA might provide a new and effective epigenetic approach to improving plant regeneration in recalcitrant barley genotypes.

Keywords: Barley; Hormones level; Plant regeneration; Transcription factors; Trichostatin A.

Fig. 1.

Plant regeneration in barley following the protocol of Hensel et al. (2009) in which scutella of immature zygotic embryos were used as explants. The freshly isolated 0 day explants (A) and explants cultured for 14 days (B), 28 days (C) on the CIM medium, and 56 days (D) on the PRM medium. The timeline represents the culture time points selected for gene expression and hormone concentration analysis. The induction of callus tissue on CIM medium (E, F) and plant regeneration on PRM medium (G, H) in the culture of different barley cultivars: “Golden Promise”, “Dema”, “Scarlett”, “Krona”, and “Morex”. The cultures at 14 (E), 28 (F), 35 (G), 42nd (H), and analysis day — 56 days (I) were shown

Fig. 2.

Plant regeneration potential of different barley cultivars: “Golden Promise”, “Scarlett”, “Krona”, “Dema”, and “Morex”. Plant regeneration efficiency (A) and productivity (B) of culture. * — values significantly different from “Golden Promise” (p < 0.05; n = 3; means ± SD are given). Histological analysis of the “Golden Promise” culture: somatic embryos at 35 days of culture (C) and regeneration of shoots (D) and roots (E) at 42 days of culture. Different types of structures were marked: SAM (shoot apical meristem), LB (leaf bud), v (vascular bundle), and RAM (root apical meristem). The sections were stained with methyl and toluidine blue (C, D) and PAS (E)

Fig. 3.

Expression pattern of plant regeneration-related TF genes, including LEC1 (A), FUS3 (B), BBM (C), PHB (D), and ERF022 (E), in the barley cultures of different plant regeneration potential, including “Golden Promise” of high regeneration potential and two genotypes poorly responding in vitro, “Scarlett” and “Dema”. * — values significantly different from 0 day of GP (p < 0.05; n = 3; means ± SD are given); ** — values significantly different from 0 day of “Scarlett” (p < 0.05; n = 3; means ± SD are given); # — values significantly different from 0 day of “Dema” (p < 0.05; n = 3; means ± SD are given)

Fig. 4.

The expression level of TF genes LEC1 (A), FUS3 (B), BBM (C), PHB (D), and ERF022 (E) in poorly responding in vitro genotypes of “Scarlett” and “Dema” in relevance to “Golden Promise” of high regeneration potential. The expression level of genes in “Scarlett” and “Dema” cultures was calibrated to expression in GP culture at the same age. * — values significantly different from “Golden Promise” at the same time point (p < 0.05; n = 3; means ± SD are given)

Fig. 5.

Accumulation of different hormones (ng/gDW), including free-IAA (A), IAAox (B), ABA (C), JA (D), and SA (E) in freshly isolated (0 day) in vitro and cultured barley explants on CIM medium (14 days, 28 days) and PRM medium (35 days) of different genotypes: “Golden Promise”, “Scarlett”, and “Dema”. DW, dry weight. * — values significantly different from 0 day explants of the same genotype (p < 0.05; n = 3; means ± SD are given); # — values significantly different from the “Golden Promise” at the same culture time point (p < 0.05; n = 3; means ± SD are given)

Fig. 6.

Effect of TSA treatment on plant regeneration in different barley cultivars: “Golden Promise,” “Krona,” “Scarlett,” “Dema,” and “Morex.” Plant regeneration efficiency and productivity evaluated in barley cultures treated with 0.0, 1.0, 2.5, 5.0, and 7.5 μM of TSA for different times (1, 2, and 4 weeks) in different barley genotypes: “Golden Promise” (A), “Krona” (B), “Scarlett” (C), “Dema” (D), “Morex” (E). W, weeks. * — values significantly different from the control (untreated with TSA) culture (p < 0.05; n = 3; means ± SD are given)

Fig. 7.

TSA-induced increased expression level of the plant regeneration-related TF genes: LEC1 (A), FUS3 (B), BBM (C), PHB (D), and ERF022 (E) in the culture of barley cultivars: “Golden Promise”, “Scarlett”, and “Dema”. Explants were treated with 7.5 μM of TSA for 4 weeks. The expression level of genes in the TSA-treated culture was calibrated to expression in the untreated (control) culture of the same genotype. * — values significantly different from the control culture (untreated with TSA) of the same genotype at the same time point (p < 0.05; n = 3; means ± SD are given)