The potential role of reactive oxygen species (ROS) is studied in the male gametophytes of petunia (Petunia hybrida E. Vilm.) grown in vivo with a focus on its germination, growth support in the progamic stage of fertilization, and the function of the mechanism underlying S-RNase-based self-incompatibility. Exogenous treatment with H₂O₂ influences the in vivo germination and polar growth of pollen tubes (PTs), which manifests as the acceleration or inhibition of these processes depending on its concentration, time interval after pollination, and pollination variant. The H₂O₂ treatment of the stigma somewhat stimulates the PT elongation in the late stages of self-incompatible pollination (4–8 h) versus the strong PT inhibition observed during the first hour of germination. A different pattern is observable in cross-compatible pollination: the H₂O₂ treatment of pistils inhibits PT growth during the overall pollination at all tested concentrations. Treatment of pistils with the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) strongly inhibited the growth of PTs in both pollination variants. In addition, DCF-DA staining confirms that ROS are formed in pollen, PTs, stigma of nonpollinated pistil, and the pistil itself in all pollination variants. The PT growth during the function of the self-incompatibility mechanism is arrested at high ROS concentrations, which is presumably associated with the SI-induced programmed cell death. Our results demonstrate that ROS are a necessary component of pollen, PTs, exudate, and stigma cells and contribute to successful reproduction. This study provides a deeper insight into the ROS functions during the PT growth in an in vivo system.

Distant hybridization is an important breeding strategy that can effectively expand the genetic pool for developing new, high-yielding, and stress-resistant cultivars. However, reproductive isolation often hinders the application of this approach. Intergeneric reproductive barriers (IRBs) manifest during pollen–pistil interactions in plants belonging to different genera, yet the mechanisms underlying these barriers remain largely unknown. In this study, we investigated pollen–pistil interactions during the progamic phase of fertilization in three Solanaceae species using several intergeneric cross combinations. Pollen of all tested species germinated on the stigma surface, grew through the stylar transmitting tissue, and ceased elongation at a distance of 300–920 µm therefrom due to the action of a reproductive barrier. We suggest that programmed cell death (PCD) is a factor contributing to IRBs in Solanaceae. The obtained data indicate that, in all cross combinations, the rejection of incompatible pollen tubes (PTs) is accompanied by increased activity of caspase-like proteases (CLPs), as confirmed by in vivo visualization of active CLPs and spectrophotometric measurement of their activity. Specifically, incompatible PT growth in stylar tissues during the first 4 h after pollination was accompanied by a five- to six-fold increase in caspase-3-like protease activity. Notably, the arrest of PT growth in distant hybridization shows striking similarity to that observed during S-RNase-based self-incompatibility (SI) in Solanaceae. We anticipate that further research will ultimately provide insights into the regulation of reproductive barriers during distant hybridization.