Abstract
Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5–10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).
Highlights
The application of AC at 5–10 μM concentration range during ME-inducing low temperature tillers treatment decreased the expression of TaMET2B gene what was associated with increased efficiency of ELS formation (AC5.0) or green plant regeneration capacity (AC10) in anther cultures of highly responsive triticale line DH28
Stimulation of ELS development induced by AC5.0, enhanced the expression of GSTF2, TaTPD1-like and SERK2 genes involved in ME regulation
The AC2.5 and AC5.0 treatments resulted in some green plant regeneration in recalcitrant line DH19, the efficiency of the process was so low that, none of applied treatments could be consider as having significant positive influence on ME effectiveness in this recalcitrant line
Summary
Two DH lines (DH19, DH28) of winter hexaploid triticale with different ME effectiveness were chosen for the study. Both lines were selected from the mapping population of 90 DH lines ‘Saka 3006’ × ‘Modus’[57] obtained from the State Plant Breeding Institute at the Hohenheim University (Stuttgart, German), complies with relevant institutional, national, and international guidelines and legislation. Based on the data collected from three separate phenotyping, DH28 was identified as highly responsive to ME induction, while DH19 was identified as a recalcitrant model, which was confirmed later in several experiments[29,30,32,33]. The procedures of seeds germination, vernalisation and plant growth conditions were described by Żur et al.[29]
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