Monitoring Barley Microspore Embryogenesis: In Vitro Culture and Immunolocalization Analyses.

The agricultural sector has a strong interest in developing new barley varieties to enhance productivity, quality, and resistance to pests and diseases. The production of doubled haploid (DH) plants is crucial in genetic improvement, as it enables the rapid and precise development of homozygous lines, accelerating the creation of enhanced varieties and consolidating new genetic combinations within a single generation. Microspore embryogenesis provides the most efficient tool for generating DH embryos and plants in barley (Hordeum vulgare L.). An appropriate in vitro protocol for inducing microspore embryogenesis must be accompanied by effective methods to monitor the cellular events and molecular determinants underlying the process. This combination is essential to elucidate the regulatory mechanisms involved and enable efficient manipulation. This chapter first presents a detailed, updated protocol for inducing microspore embryogenesis in barley using a 4°C cold stress treatment. It then describes methodologies for sample processing from embryogenic cultures to analyze cellular organization changes and localize key molecular determinants, such as auxin, through immunofluorescence assays. The described in vitro method incorporates several modifications to enhance culture efficiency, including reduced maltose and mannitol volumes during microspore isolation, optimized gradient preparation for clearer separation of viable microspores, improved cleaning to prevent aggregate formation that interferes with reprogramming, lower cell density in culture, or the use of larger Petri dishes to promote embryo differentiation. The cellular analysis protocol encompasses fixation, preembedding in gelatin, acrylic resin embedding, semithin sectioning, cytochemical staining, and immunofluorescence. This approach enables precise observation of cellular structures under light microscopy, offering detailed insights into embryogenic progression and allowing to carry out immunofluorescence studies using a wide range of antibodies. By integrating protocols for in vitro induction and cellular analysis, this chapter provides a comprehensive guide for monitoring barley microspore embryogenesis. It assists researchers in optimizing culture outcomes and advancing cellular studies, including structural analyses and the localization of key molecules like auxin.