Using seedlings inoculated with wild type Pseudomonas sp. as a positive control,initial microscopy used only water to mount the samples onto the microscope slide. This method was unsuitable for visualising bacteria in the root as bacteria were indistinguishable from the plant. Our Pseudomonas sp. strain produces a fluorescent siderophore causing green fluorescence so we attempted to view the sample through a GFP filter, this was unsuccessful as the auto-fluorecence of plant tissue was too great to see the bacteria. To overcome this issue, DAPI stain was used to stain nucleic acids of the bacteria. DAPI was added to distilled water at a concentration of 1 microlitre per millilitre and this was used to mount the sample and a DAPI filter was used to visualise the sample. At 40x magnification bacteria were visible and this revealed a biofilm was present on the surface of the root, however it was unclear if the bacteria were inside the root so seedlings were washed in distilled water. Examination of the cleaned seedlings was challenged by the fact that root hair cells had wrapped around the main root during the washing process meaning that it was unclear if bacteria were inside the main root or merely on the surface.

To overcome the issue of root hair cells wrapping, the team used 15% glycerol in place of water for mounting samples. This was highly successful as the viscosity of the glycerol caused root hair cells to spread and revealed clear signs of colonisation. Pseudomonas sp. was present in the intercellular spaces along both the root and hypocotyl. These bacteria were still motile and we could see them moving in real time.

Figure 2. Bright field microscopy of a DAPI stained Arabidopsis thaliana. Wild type Pseudomonas sp. is visible in the intercellular spaces.

Figure 3. Bright field microscopy through a DAPI filter, Pseudomonas sp. is visible on the root surface.

We were able to take images of the bright field microscopy with and without a DAPI filter before merging the images to provide a clear visual representation of how the bacteria live within the plant.

Figure 4. A Pseudomonas sp. inoculated Arabidopsis thaliana root without DAPI filter.

Figure 5. A Pseudomonas sp. inoculated Arabidopsis thaliana root with DAPI filter.

Figure 5. An overlay of Figure 4 and Figure 5, clearly showing Pseudomonas sp in the intercellular spaces of plant cells.

Visualising the Transformant

A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed and DAPI stained.Endophytic bacteria were visible inside the root and hypocotyl of both Arabidopsis thaliana and Eruca sativa seedlings showing that transformation had not altered the bacteria’s ability to colonise.

Figure 21. Bright field microscopy of an Arabidopsis thaliana seedling root at x100 magnification,showing transformed Pseudomonas sp. living as an endophyte in the intercellular spaces.

A selection of seedlings were again selected for microscopy, this time negative control E. coli DH5α inoculated seedlings were examined. Microscopy revealed that though E. coli was present on the root surface, there was no sign of endophytic relationship like that of Pseudomonas sp.. Further images were taken of transformant Pseudomonas sp. inoculated seedlings to aid visual demonstration of our transformed endophyte working.

Figure 23.Bright field microscopy showing the root of our negative control Arabidopsis thaliana at100x magnification, where E. coli DH5α was visible on the surface of the root in small numbers but not inside the root as an endophyte.