Plant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870, Błonie, Poland.
Institute of Biology and Biotechnology, University of Rzeszow, Al. Rejtana 16C, Rzeszow, 35-959, Poland.
BMC Plant Biol. 2024 Sep 12;24(1):853. doi: 10.1186/s12870-024-05572-w.
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).
The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO: 0.1; 5; 10 µM) and silver salts (AgNO: 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).
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.
小孢子胚胎发生是一种在组织培养环境中产生双倍单倍体的过程，广泛应用于谷类作物。绿色再生体的高效生产需要能够在糖酵解水平上感知到的应激，随后是三羧酸循环和电子传递链。后者可以受到诱导培养基中 Cu(II)离子浓度的影响，Cu(II)离子作为生化反应的辅因子，间接影响涉及 DNA 甲基化（去甲基化-DM、从头甲基化-DNM）的表观遗传机制。这些结论是通过对黑小麦再生体的研究得出的，但对大麦没有类似的研究。通过这种方式，本研究探讨了 DNM、DM、Cu(II)、SAM、GSH 和β-葡聚糖如何影响绿色植物再生效率（GPRE）。
该实验涉及通过花药培养获得的春大麦再生体。通过添加铜（CuSO：0.1；5；10µM）和银盐（AgNO：0；10；60µM），创建了 9 种（试验）诱导培养基变体，并对花药的孵育时间（21、28 和 35 天）进行了不同的处理。使用 DArTseqMet 分子标记方法估计 DNA 甲基化的变化，该方法还检测胞嘧啶甲基化。使用基于衰减全反射-傅里叶变换红外光谱（ATR-FTIR）的暂定分配来评估营养处理诱导的β-葡聚糖、SAM 和 GSH 的表型变异。绿色植物再生的有效性范围为每 100 个接种的花药 0.1 到 2.91 株。去甲基化水平范围为 7.61 到 32.29，而从头甲基化达到 6.83 到 32.27 的范围。本文表明，来自特定体外条件（试验）的样本形成了紧密的组群，与导致两个主要成分的因素相关，这两个主要成分负责 55.05%的方差（第一成分 DNM、DM，第二成分 GSH、β-葡聚糖、Cu(II)、GPRE）。
我们可以得出结论，体外组织培养条件会影响生化水平、DNA 甲基化变化和 GPRE。在 IM 中增加 Cu(II)浓度会影响代谢和 DNA 甲基化，从而提高 GPRE。因此，改变 IM 中的 Cu(II)浓度有望提高 GPRE。
