Team:Newcastle/Notebook/Endophyte
Alternative Roots
Root Colonisation
NOTEBOOK
Root Colonisation
Week Beginning 16/07
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 firstly attempted to plant Arabidopsis thaliana seeds in a range of agar concentrations. Groups of 8 replicates were made at concentrations; 0.4 %, 0.6 %, 0.8 % and 1 % in bottomless microcentrifuge tubes and were left to chill over the weekend.
Week Beginning 23/07
The seeds from last week were taken from the fridge and placed in a pipette-tip box filled with water to approximately 1 mm below the microcentrifuge tube bottoms. After 5 days these seedlings were examined and all but the 1 % agar replicates had swollen and fallen through the microcentrifuge tubes into the bottom of the pipette-tip box. This showed that 1 % agar was appropriate for our uses.
Week Beginning 30/07
The team planted a 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 for lab-based plant growth.
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 tryptone-soy agar plates.
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. The wild type Pseudomonas sp. inoculated seedlings from the previous week were taken for microscopy with Dr Vasilios Andriotis where we found DAPI staining seedlings that had been gently washed and mounted on 15 % glycerol was a suitable way to visualise endophytes.
Week Beginning 24/09
A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy with Dr Andriotis, again seedlings were washed and DAPI stained prior to visualisation. An experiment was set up to compare the effects of the wild type and transformed Pseduomonas sp. on germination so a large agar plate containing 70 micrograms per millilitre gentamicin was made [1]. This will show if the transformation has made Pseudomonas sp. detrimental to plant growth.
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. This opportunity was also used to capture more images of the transformed Pseudomonas sp. chassis in Arabidopis thaliana roots.
References & Attributions
Attributions: Frank Eardley, Luke Waller and Lewis Tomlinson
References: Conte S, Stevenson D, Furner I, Lloyd A (2009) Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein. PLANT PHYSIOLOGY 151:559-573.