2.1. Plasmid DNA extraction (mini- prep)
A culture of E. coli (DH5-a) was initiated the day before the extraction. 1,5 mL of that culture was transferred to a sterile microtube tube and centrifuged at maximum speed for 30 seconds. The supernatant was discarded and the pellet was ressuspended in 200 μL of Sucrose-Tris-EDTA-Triton (STET) buffer [8% (w/v) sucrose; 0.1% (v/v) Triton X-100; 50 mM EDTA; 50 mM Tris-HCl, pH 8.0] and 5 μL of lysozyme (50 mg/ml). The samples were incubated for 5 minutes at room temperature. The samples were boiled for 45 seconds for inactivation of DNases and lysozyme and centrifuged for 5 minutes at maximum speed. The pellet was removed with a sterile toothpick and 200μL of 2- propanol was added to the tube for DNA precipitation. The samples were homogenized by vortexing, followed by a 10 minutes spin at maximum speed. The supernatant was removed and the pellet was washed with 70% (v/v) ethanol. The pellet was air-dried, at room temperature. The DNA was ressuspended in 20 μL of sterile water with RNase (10 μg/ml) and stored at - 20 ºC.
2.2. Transformation of A. tumefaciens by Electroporation
Electrocompetent Agrobacterium GV3101 were thawed on ice. 10 μL of pure plasmid DNA were added to the cells and the mixture was carefully transferred to the bottom of the cuvette. The Biorad Micropulser was set to “Agr” mode. The cuvette was placed in the
chamber slide. After the pulse, 1mL of LB medium was immediately added to the cuvette. This mixture was incubated for 4h without shaking at 28ºC for recovery of the cells. The cells were centrifuged for 4 minutes at 1300 x g. 900 μL supernatant were discarded and the pellet was ressuspended and plated in LB-agar supplemented with antibiotics. The plates were incubated for 48h at 28ºC.
2.3. Transient transformation of Arabi- dopsis thaliana
Figure 1. Arabidopsis seedlings infiltration. a. Vacuum infiltration apparatus, b. detail of Arabidopsis in 6-well plate during infiltration
process
Arabidopsis thaliana seedlings were transiently transformed by vaccum infiltration based on the work of Marion et al, 2008 [5].
Agrobacterium tumefaciens cells trans-
formed with the various constructs were grown overnight in 5 ml pre-culture and used to inoculate a 30 ml culture (LB liquid medium). After overnight growth at 28°C, A.
tumefacienscells were centrifuged at 1537 x
g and resuspended at the appropriate OD600 in 2 ml of MS liquid medium.. Infiltration was performed by submerging the seedlings in the Agrobacterium solution and by applying vacuum (-70 KPa) twice for 1 min (Fig. 1). The remaining infiltration medium was subsequently removed and the
plates were transferred to a culture room for 3 days.
2.3. Stable expression of Arabidopsis
(Floral-dip method)
Figure 2. Floral dip method proceedings. a. Flowering Arabidopsis plants used for floral
dip transformation, b. Arabidopsis flower detail, c. Arabidopsis dipping with gentle agitation and d. covered plants for humidity
maintenance after floral dip
Stable transgenic lines of Arabidopsis
thaliana plants were produced by floral dip
method [7]. Agrobacterium culture was centrifuged for 15 minutes at 1537 xg at room temperature. The supernatant was removed and the cells were resuspended in
floral dip inoculation medium. The floral dip medium was added to a beaker and the plants were inverted into this suspension (Figure 2.8 c). A minimum of three robust plants without siliques (Fig. 2 a and b) were used per transformation. The plants were submerged two times for 2 minutes each with gentle agitation (Fig. 2c). Plants were removed from the beaker and placed in a plastic tray and covered with a clear-plastic to maintain humidity (Fig. 2d)
2.4. Agrobacterium infiltration of N. tabacumLeaves
Figure 3. Tobacco plants and leaf infiltration with a needleless syringe
Agrobacterium-mediated transient transfor- mation of tobacco leaves was performed according to [6]. One mL of a fresh culture of A. tumefaciens transformed with the cons- truct of interest was centrifuged at 16000 xg for 1 minute. The pellet obtained was resuspended in 1 mL of infiltration buffer (10 mM MgCl2 and 10 mM MES) supplemented with 100 mM of acetosyringone, which contributes to increase the virulence of A. tumefaciens. Using a 1 mL capacity syringe, without needle, a tobacco leaf was infiltrated, controlling the pressure applied with the syringe on the lower epidermis (Fig.
3) until the liquid entered through the stomata and infiltrates in the intercellular spaces.
Figure 4. Schematic representation and characteristics of the fluorescent markers/constructs (pre-existent in the laboratory) used for the transformation. A.
Endoplasmic reticulum marker. B. Golgi apparatus fluorescent marker. SP, Signal peptide for targeting to the ER; GFP, Green Fluorescent Protein; HDEL, aminoacidic motif
in the C-terminal end for ER-retention; ST - Sialyl-transferase, a Golgi-membrane protein 2.5. Constructs for Transient Expre-
ssion
Binary vectors containing fluorescent protein fusion constructs were prepared using standard molecular biological techniques. The pVKH18En6 binary vector used in this work is available from John Runions, Oxford Brookes University. Fluorescent fusions pre- existent in the laboratory used were 35s-SP- GFP-HDEL and 35s-SP-ST-GFP (Fig. 4). 2.6. Isolation of protoplasts from Ara-
bidopsis thaliana leaves
Leaves of were placed in Petri dishes with the lower epidermis facing down, floating in
digestion medium, containing 1% (w/v) cellulase and 0.25% (w/v) macerozyme. Petri dishes were placed in vacuum for 15 min and incubated in the dark, without shaking for 2-3 h, at 25 ºC followed by incubation with shaking, in the dark for 15 min at room temperature. Subsequently to the digestion, the protoplasts were gently released from the leaf portions with a plastic pipette and recovered into a new Petri dish WKURXJKDȝPQ\ORQPHVK )LJ
Figure 5. Isolation of protoplasts from Arabidopsis thaliana leaves 2.7. Fluorescence microscopy
The monitoring of the cells’ transformation was performed through fluorescence microscopy. Small portions (about 1 cm2) of the tobacco leaf infiltrated area were cut and placed in a glass slide with abaxial face upwards. A drop of water and a coverslip were placed on top of the leaf. Three days after infiltration the Arabidopsis cotyledons were excised from the seedlings and placed on the top of a drop of water in a slide with the abaxial face upwards. The fresh material was covered with a cover slide. Cells were imaged using Fluorescence microscope OPTIPHOT-2 (Nikon).