Non-transformation methods for studying signaling pathways and genes involved in Brassica rapa pollen-stigma interactions.

Self-incompatibility (SI) is a mechanism in plants that prevents self-fertilization and promotes out-crossing. 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. Here, we developed methods for studying signaling pathways and genes of pollen-stigma interactions in Brassicaceae crops lacking an efficient genetic transformation system. We pretreated excised stigmas of Brassica rapa (Brassica 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 firstly determined the involvement of reactive oxygen species (ROS) in SI with the understanding that the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), which inhibits ROS production, eliminated SI of B. rapa. We further identified the key gene for ROS production in SI and used AS-ODNs targeting BrRBOHF (Brassica 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 (Na-SA) disrupted SI and resulted in enlarged ovules with inbred embryos 12 days 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.