Enhancing microspore embryogenesis initiation by reducing ROS, autophagy, and cell death with novel small molecules in rapeseed and barley

When submitted to stress conditions, microspores cultured in vitro can be reprogrammed towards an embryogenic pathway, the so-called microspore embryogenesis, which constitutes a biotechnological tool to rapidly produce double-haploid plants for breeding programs. Nevertheless, not all cells succeed in switching their development and, as a consequence of the stress treatment applied, many of them undergo cell death, which causes a significant reduction of the final yield of the process. In this study, we have analyzed the potential of several novel small molecule antioxidants, never used before in plants, to improve cell viability during microspore embryogenesis induction. The new molecules have been tested in two crop species, Brassica napus and Hordeum vulgare, in which cell reprograming was induced by heat (32 °C) and cold (4 °C) treatments. Using transcriptomic and physiological approaches, we have analyzed changes in oxidative stress and autophagy, and their involvement in cell death during microspore embryogenesis induction. The results provide new evidence of increased ROS production and upregulated oxidative stress and autophagy-related genes during embryogenesis induction, all of which contribute to higher cell death. We identified novel small molecule antioxidants that mitigated these effects, enhancing cell viability and promoting microspore embryogenesis initiation. The findings in two phylogenetically distant crop species suggest a conserved cellular response and highlight the potential of these compounds to improve in vitro protocols in other species where early-stage cell death poses a significant challenge during embryogenesis induction.