The Arabidopsis thaliana ecotype Columbia (Col-0, used in the Arabidopsis Genome
Initiative, 2000) was used exclusively in this work. Col-0 ecotype plants with a gnl1 knockout background, making them susceptible to brefeldin A treatment, were transformed with selected vectors for experiments described in section 5.2.6.
2.5.2 Arabidopsis thaliana seed sterilisation and sowing
Seeds were sterilised using either sodium hypochlorite (bleach) and sterile dH2O or chlorine gas. To use bleach, seeds were shaken for 5 minutes in 1ml of 30% bleach and tween in a 1.5ml Eppendorf. They were then pelleted at 16,000 x g for 20 seconds and rinsed in sterile dH2O. This step was repeated twice more before 900µl sterile dH2O was removed after the final spin, and seeds were plated directly onto petri dishes containing ½
43
MS-agar (Murashige and Skoog (MS) basal medium (4.4g/l), adjusted to pH5.8 with 1M KOH, and containing 0.8% (w/v) bacto agar and a selective antibiotic if desired). To use chlorine gas, less than 100 seeds were placed into an open 1.5ml Eppendorf which was then placed into a rack inside a large plastic lidded box within the fume hood. 100ml of sodium hypochlorite was added to a glass beaker within this box, and 3ml of 35% hydrochloric acid was added slowly. The lid was immediately placed over the box and seeds were incubated overnight. Once sterile, seeds were aerated for 10 minutes in a flow hood. Seeds were then sterile and ready to be sown at any time.
2.5.3 Maintenance of Arabidopsis thaliana
½ MS-agar plates (containing appropriate antibiotics for selection as necessary) were sealed with micropore tape and typically subjected to 24-48 hours of stratification at 4°C before being transferred to a 24 hour light chamber at 25°C. Seedlings were transferred to Arabidopsis-mix soil (University of Warwick) 14 D.A.G and cultivated in a designated transgenic glasshouse facility with conditions at 16 hours light / 8 hours dark and 18°C.
2.5.4 Transformation of Arabidopsis thaliana
The primary stem of Arabidopsis plants was removed once the plants had bolted in order to encourage further stems and increased number of flowers. Plants were deemed ready for transformation once 20-50% of the flowers were open. At this point, a 10ml culture of LB supplemented with the appropriate antibiotic was inoculated with Agrobacterium
tumefaciens containing the plasmid of interest, and was incubated at 28°C, shaking at
200rpm for 18-24 hours. This was decanted into 100ml of LB and incubated for a further 6 hours before pelleting cells at 3,000rpm for 15 minutes (Beckman J2-21MIE). The supernatant was discarded and the pellet re-suspended in sterile dH2O containing 5% (w/v) sucrose. Silwet L-77 was added to a final concentration of 0.01%. Flowers of the plants were submerged in the suspension for 30 seconds. The suspension was then shaken and foam was pipetted onto the flowers. Plants were incubated in sealed plastic sleeves for 24 hours and left to dry. Dry seeds were collected, sterilised and plated on ½ MS-agar plates containing appropriate antibiotics for selection.
2.5.5 Arabidopsis thaliana seed collection
Plants were left to dry once lowest siliques began dehiscing. Once dry, stems were cut from the base of the stem and left for a further 1 week before these were placed into
44
paper bags. Bags were folded over and left for at least 1 week for seeds to completely dry before collection.
2.5.6 BASTA selection of transgenic Arabidopsis thaliana seedlings
T1 and T2 seeds were sown directly onto Arabidopsis-mix soil (2:1 potting mix to perlite) soaked in BASTA (Kasper, 150g/L glufosinate ammonium, Bayer, Germany) diluted 1/1000 with H2O, and subjected to 2 days in the dark at 4˚C. Trays of seedlings were given a second BASTA treatment two weeks after this, with regular watering in between. Positive transformants were transferred to larger pots three weeks after sowing and brought to seed.
2.5.7 Seed preparation for germination and seedling growth assays
Prior to these assays, all seeds were grown in a Panasonic MLR-352-PE cabinet at the same time in the same conditions. They were first subjected to a 2 day 4˚C dark treatment, then grown at 16hr 22˚C days and 18˚C night. This was reduced after flowering to 18˚C day and 10˚C night for enhanced dormancy. Seeds were collected at silique dehiscing and left to dry for 1 week before being desiccated in calcium nitrate for 7 days to bring seeds to 8-10% moisture. They were then stored at -20˚C.
2.5.8 Germination experiment set up and data analysis
Sterilised seeds were evenly spaced on a layer of fine mesh which lay on filter paper dampened with 10ml sterile dH2O. This was contained within lidded plastic boxes, and subjected to a 3 day cold treatment at 4°C for 24 hours in the dark. After this, the mesh with seeds on was transferred to a box of the same set up but containing 25ml of polyethylene glycol (PEG) solution at the desired drought condition (MPa), as described in table 2.5 and based on Michel et al. 1973. Boxes were kept at 15°C with 24 hour light for 14 days. Seeds were checked for germination using a stereomicroscope at the same time each day. The point of germination was defined as emergence of radicle. Results were analysed using a Two-Way ANOVA in GenStat, and calculating confidence intervals in Microsoft Excel.
2.5.9 Seedling establishment experiment set up and data analysis
Sterilised seeds were plated on ½ MS-agar for a 3 day cold treatment at 4°C in 24 hours dark. Plates were then transferred to 15°C 24 hours light for 2 days to germinate. Germinated seeds were transferred to ½ MS-agar plates containing varying amounts of
45
mannitol to create different drought conditions, as described in table 2.4. Seedlings were grown at 20°C for 14 days in 24 hour light. Seedling length (radicle, hypocotyl and lateral roots) was measured using electronic callipers for 35S promoter lines, and in Image J for native promoter lines. Results were analysed using a Two-Way ANOVA in GenStat.
Table 2.7 Amount of PEG or mannitol added to create levels of drought (Bill Finch- Savage, University of Warwick)
Nominal Water Potential (MPa) -0.4 -0.6 -0.8
Polyethylene Glycol 8000 (g) 66.5 82.8 96.6
Mannitol (g) 29.1 43.8 58.3
2.5.10 Growth and maintenance of Nicotiana benthamiana
Seeds were sown onto a mix of 50% soil and 50% vermiculite. These were germinated and grown in conditions of 16 hours light / 8 hours dark and 18°C. After 5-6 weeks of growth plants were typically ready for infiltrations.
2.5.11 Transformation of Nicotiana benthamiana leaves by infiltration with Agrobacterium tumefaciens
A 5 ml culture of A. tumefaciens containing the plasmid of interest was grown overnight to an OD of 0.5–1.5 at 28°C, shaking at 200 rpm. 1.5ml of this culture was centrifuged at
room temperature in a 2ml Eppendorf tube at 1000×g for 10 minutes (Eppendorf bench
microfuge). The supernatant was discarded and pellet re-suspended in 1ml of infiltration media (50 mM MES, pH5.6; 0.5% glucose (w/v); 2mM Na3PO4; 100µM acetosyringone (1M in DMSO stock)). This was repeated to wash cells. Further infiltration buffer was added as necessary to reach OD600 0.05-0.5. A wound was made with a small needle on the lower epidermis of a younger leaf and the cell suspension was injected using a 1 ml disposable syringe. Plants were left at room temperature for 3 days until tissue collection.