Abstract
In vitro culture experiments are crucial for the studies of chemical-mediated interactions in plants. However, distinguishing spores and sporelings of different species of bryophytes in mixed cultures poses a serious drawback for research on early developmental stages. Here we propose a modification of the sandwich technique, a standard method to explore allelopathic effect of plants, 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 protonemata green coverage using image analysis, and 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 shows a negative effect of T. muralis on S. ruralis, while S. ruralis apparently promotes the development of T. muralis. The method here proposed is successful for culturing moss spores, so that the different inocula are physically separated while allowing diffusion of water-soluble and volatile substances. For testing interactions in these early stages of the gametophyte, we recommend measuring the degree of development of moss sporelings rather than their coverage. Our results have revealed the existence of both positive and negative interspecific relationships between T. muralis and S. ruralis sporelings, supporting that positive interactions in bryophytes might be more common than previously thought.