Nontransformation methods for studying signaling pathways and genes involved in <i>Brassica rapa</i> pollen–stigma interactions

Self-incompatibility (SI) is a mechanism in plants that prevents self-fertilization and promotes outcrossing. SI is also widely utilized in the breeding of Brassicaceae crops. Understanding the regulatory mechanisms of SI is essential but has been greatly restrained in most Brassicaceae crops due to inefficient transformation. In this study, we developed methods for examining signaling pathways and genes of pollen-stigma interactions in Brassicaceae crops lacking an efficient genetic transformation system. We pretreated excised stigmas of Brassica rapa (B. rapa L. ssp. Pekinensis) in vitro with chemicals to modify signaling pathways or with phosphorothioate antisense oligodeoxyribonucleotides (AS-ODNs) to modify the expression of the corresponding genes involved in pollen-stigma interactions. Using this method, we first determined the involvement of reactive oxygen species (ROS) in SI with the understanding that the NADPH oxidase inhibitor diphenyleneiodonium chloride, which inhibits ROS production, eliminated the SI of B. rapa. We further identified the key gene for ROS production in SI and used AS-ODNs targeting BrRBOHF (B. rapa RESPIRATORY-BURST OXIDASE HOMOLOGF), which encodes one of the NADPH oxidases, to effectively suppress its expression, reduce stigmatic ROS, and promote the growth of self-pollen in B. rapa stigmas. Moreover, pistils treated in planta with the ROS scavenger sodium salicylate disrupted SI and resulted in enlarged ovules with inbred embryos 12 d after pollination. This method will enable the functional study of signaling pathways and genes regulating SI and other pollen-stigma interactions in different Brassicaceae plants.