Interactions in bryophytes using a new in vitro culture method reveal negative and positive interspecific effects in the sporelings of two moss species

In vitro culture experiments are crucial for studying chemically-mediated interactions in plants. However, the difficulty in distinguishing spores and sporelings of different bryophyte species hinders research on early developmental stages. Here we propose a modification of the sandwich technique, a standard method for studying plant allelopathic, and present a case study using two common mosses. As in the standard sandwich method, we have created a physical barrier using gelled medium, and inoculated spores of Tortula muralis and Syntrichia ruralis in two layers. To assess their intra- and interspecific interactions, we measured the green coverage of the protonemata using image analysis, and the degree of sporeling development using a categorical index. We successfully obtained physically separated sporelings of target and emitters from spores of these two species. The green coverage analysis showed no differences in any of the comparisons. However, the developmental index showed a negative effect of T. muralis on S. ruralis, whereas S. ruralis appeared to promote the development of T. muralis. The method proposed here is successful for culturing moss spores. The two inocula (the emitter and the receiver) are physically separated but the set-up allows the diffusion of water-soluble and volatile substances. For testing interactions at these early stages of the gametophyte, we recommend measuring the degree of development of moss sporelings rather than their coverage. Our results revealed both positive and negative interspecific interactions between T. muralis and S. ruralis sporelings. This supports that positive interactions in bryophytes may be more common than previously thought.