β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants' DNA methylation and GPRE

Abstract

Background: Microspore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation-DM, de novo methylation-DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE).

Results: The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO₄: 0.1; 5; 10 µM) and silver salts (AgNO₃: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation. Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE).

Conclusions: We can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

Keywords: β-glucans; Anther culture; Barley; DNA methylation; DNA sequence; GSH; Green plant regeneration efficiency; SAM.

Fig. 1

The mean (central line) and the standard deviation (ribbon) of the spectra absorbance (bottom) and the absolute values of calculated the 1^st derivative (top) collected from the barley leaves of young regenerants derived via androgenesis. The inset presents the spectra and the 1^st derivative for region around 2500 cm⁻¹, which can be tentatively assigned to GSH (shaded). The wavenumber ranges corresponding to SAM (1630–1620 cm⁻¹) and β-glucans (990–950 cm^-1), are marked grey shaded

Fig. 2

Principal component analysis biplot. Active variables (for explanation see Table 1) are given in red whereas trials are indicated in colored dots

Fig. 3

Classification and regression tree analysis illustrating trials classification

Fig. 4

Standardized characteristics of DM, DNM, SAM, GSH, β-glucans, and GPRE for trials