| Summary: | <h4>Background & aims</h4>The bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia-free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia-free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment.<h4>Methods</h4>The Burkholderia-free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia-free plants to the nodulating plants under the same in vitro conditions.<h4>Key results</h4>All the developed plants were Burkholderia-free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development.<h4>Conclusions</h4>Embryo culture is a cultivation technique with a high success rate for the development of Burkholderia-free plants of P. umbellata. The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.
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