Author:

Abstract:

Interactions between plants and symbiotic microorganisms are one of the most widespread and prevalent mutualisms on earth. Most research focuses on plant-bacteria interactions with immediate applications. Due to this narrow focus, however, there is an inherent risk of ignoring associations that might prove beneficial in the future. In Rubiaceae, several plant species closely interact with endophytic bacteria. These endophytes can either be found in specialized leaf nodules or are freely distributed among the mesophyll cells. The nodulated type can be found in the genera Pavetta, Psychotria, and Sericanthe, whereas the second interaction type can be found in the tribe Vangueria but also in the genus Psychotria. In this study, we investigate the nodulated Psychotria species in which the endophytes are found in specialised nodules and are host specific. Due to this specificity, vertical transmission of bacteria to the other plant generations is hypothesized. However, when this transmission is impeded, the resulting plants have distorted leaves and plant growth is stunted. Our aim is to cultivate bacteria-free plants and assess whether it is indeed the absence of bacteria that results in the aforementioned phenotypical changes. Furthermore, we will determine the exact location of the endophytes in reproductive parts of the host plants. We hypothesize that bacteria are present in the endosperm. To reach our goals, two techniques are applied: in vitro culture, a method in which sterilized seeds are germinated, and embryo rescue, a method that allows us to extract embryos from mature seeds. We assume that the embryo rescue method will result in bacteria free plants, while bacteria will still be present when using the in vitro seed culture method. Using morphological and genetic analyses we want to investigate whether the bacteria are present. The results of the first genetic analysis show that bacterial DNA is lacking when applying both methods, nevertheless some plants appear to have nodules. The possible presence of nodules and the absence of bacterial DNA is rather contradictory, as nodules are considered to be intrinsically associated with bacteria. When bacteria are absent in both methods, it is possible that the bacteria use the endocarp/testa to infect to the next generation. It is shown that this structure is permeable for alcohol, which was used in the sterilisation process and thus could have killed the bacteria. More research is needed to clarify this intriguing query.