Team:Newcastle/Notebook/Endophyte
Alternative Roots
Root Colonisation
NOTEBOOK
Characterising plant colonisation by Pseudomonas sp.
Week Beginning 20/07
Preliminary work began with the team developing agar-based germination methods, this was mainly due to the lack of plant research experience in the team. The team conceptualised growing Arabidopsis in microcentrifuge tubes within pipette-tip boxes.
The team planted their first set of 16 Arabidopsis seeds in 1 % agar in a pipette-tip box placed on the lab windowsill. This would give an indication as to if these conditions were suitable for growth. After 7 days 12/16 seeds had germinated showing this method was appropriate.
Week Beginning 27/08
Following engagement with GrowUp Urban Farms, it was suggested that we use a seed coating inoculation method, rather than wounding as we intended, this makes our engineered microbe more accessible for commercial use. As a preliminary experiment to test this, Arabidopsis seeds were sterilised before being coated in Pseudomonas sp. liquid culture. Seeds were then planted in 1 % agar and allowed to germinate. After 1 week, 7 of these seedlings were surface sterilised, cut and plated on nutrient agar plates. On all 7 of these plates Pseudomonas sp. was re-isolated in pure culture.
Figure 1. Pseudomonas sp. on a TSA plate that has been re-isolated from Arabidopsis thaliana seedlings which have been inoculated by the seed-coating method suggested by GrowUp Urban Farms.
Week Beginning 03/09
The team decided that the most valuable way to assess endophytic relationship would be to use microscopy to visualise Pseudomonas sp. inside the plant. As a positive control a set of 96 Arabidopsis seeds were surface sterilised and coated in wild type Pseudomonas sp. liquid culture. These seeds were planted in 1 % agar and allowed to germinate on the laboratory windowsill.
Week Beginning 10/09
Following successful transformation of Pseudomonas sp. a smorgasbord of Arabidopsis thaliana and Eruca sativa seeds were sterilised and coated in either transformed Pseudomonas sp. liquid culture or E. coli DH5α liquid culture (as a negative control) allowed to germinate on the windowsill ready for microscopy.
Figure 3. Eruca sativa seedlings growing in a contained environment on the laboratory windowsill with a nice view of sunny Newcastle.
Week Beginning 24/09
A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed and DAPI stained prior to visualisation.
Week Beginning 01/10
A selection of seedlings were again selected for microscopy, this time negative control E. coli DH5α inoculated seedlings were examined with bright field microscopy and DAPI staining.
REFERENCES & Attributions
Attributions: Frank Eardley and Lewis Tomlinson