Efficient Protocol for Doubled Haploid Production in Rye (Secale cereale L.)

The cultivation of rye (Secale cereale L.) for functional foods is economically important due to its high-dietary fiber content and health-related bioactive compounds. Breeders are seeking modern biotechnology techniques to accelerate traditional breeding in this predominantly cross-pollinated species. The development of efficient protocols for double haploid (DH) production in rye breeding faces challenges. Despite five decades of research, no universally efficient DH production protocol has emerged, largely due to genotypic variability and recalcitrance to in vitro regeneration. The protocols presented here incorporate antioxidants, such as reduced glutathione (GSH) and selenium (SS), into the tiller low temperature pretreatment procedure, as well as arabinogalactan proteins (AGPs) and phytagel into the induction medium. All these modifications aim to reduce the generation of reactive oxygen species (ROS). As a result, microspores are more likely to survive stress pretreatment, the isolation process and transfer to in vitro culture better. A higher population of viable microspores could result in an increase in the number of reprogrammed, embryogenic microspores and microspore-derived embryo-like structures (ELS). The protocols presented have been tested on 15 F1 rye breeding lines, with the greatest effectiveness in lines with moderate and high responsiveness to androgenesis induction, also known as microspore embryogenesis (ME) induction.