New Prospects for Improving Microspore Embryogenesis Induction in Highly Recalcitrant Winter Wheat Lines

Among various methods stimulating biological progress, double haploid (DH) technology, which utilizes the process of microspore embryogenesis (ME), is potentially the most effective. However, the process depends on complex interactions between many genetic, physiological and environmental variables, and in many cases, e.g., winter wheat, does not operate with the efficiency required for commercial use. Stress associated with low-temperature treatment, isolation and transfer to in vitro culture has been shown to disturb redox homeostasis and generate relatively high levels of reactive oxygen species (ROS), affecting microspore vitality. The aim of this study was to investigate whether controlled plant growth, specific tiller pre-treatment and culture conditions could improve the potential of microspores to cope with stress and effectively induce ME. To understand the mechanism of the stress response, hydrogen peroxide levels, total activity and the content of the most important low-molecular-weight antioxidants (glutathione and ascorbate), as well as the content of selected macro- (Mg, Ca, NA, K) and micronutrients (Mn, Zn, Fe, Cu, Mo) were determined. These analyses, combined with the cytological characteristics of the microspore suspensions, allowed us to demonstrate that an increased microspore vitality and stronger response to ME induction were associated with higher stress resistance based on more efficient ROS scavenging and nutrient management. It was shown that a modified procedure, combining a low temperature with mannitol and sodium selenate tiller pre-treatment, reduced oxidative stress and improved the effectiveness of ME in winter wheat lines.