Transient Transformation of Ipomoea purpurea flower petals via particle bombardment
In the absence of a reliable, efficient and timely stable genetic transformation system, transient transformation allows researchers to directly test genetic hypotheses in vivo. We developed the following protocol to assay promoter function though, in principle, the protocol should be effective for functionally evaluating protein coding sequences as well. This protocol makes use of two independent reporter genes: in the presence of the substrate 4-methylumbelliferae beta-D-glucuronide trihydrate, beta-galacturonidase (GUS) emits visible light when stimulated by nm wavelengths. We used GUS as the reporter for our experimental promoter sequences. The luciferase gene was isolated from jellyfish; when presented with its substrate luciferin, luciferase emits detectable light. We used luciferase as the reporter gene for a constitutively active promoter, CaMV 35S, which provided an estimate of transformation efficiency in each particle bombardment replicate. Both of these reporter constructs were delivered to the host tissue in derivatives of the widely available pUC18 plasmid which is easily stored and isolated from E. coli.
The following protocol is the amalgamation of several pre-existing protocols, reported previously. In practice, nearly all host species and tissue types will have their own optimal experimental parameters.
Preparing the gold for shooting leaf or flower tissue
1. Weigh 60 mg of gold, pour into a 1.5 mL eppie.
2. Add 1 mL of 70% EtOH to the tube.
3. Vortex 5 min, let stand for 15 min.
4. Spin 5 sec, remove the supernatant.
5. Add 1 mL of dH20 to the tube, vortex 10 sec, let stand 1 min.
6. Spin 5 sec, remove supernatant.
7. Repeat step 5-6 three more times.
8. Add 1 mL 50% glycerol to the eppendorf tube, store at -20ºC.
Preparing DNA and Gold mixture
1. Allow gold mixture to warm slightly, then vortex. Add 50 µL to a 1.5 mL eppie.
2. Sequentially add, while vortexing at medium speed
10 µL of DNA mixture (1 µg/µL) (5 µL each of control and experimental vector)
50 µL of 2.5 M CaCl2
20 µL of 0.1M spermidine (free base; comes as 6.3M)
3. Vortex an additional 3 min.
4. Let sit on the benchtop for 1 min.
5. Spin down at high speed for 2 sec.
6. Discard supernatant.
7. Add 140 µL 70% EtOH, flick gently (don't disturb the pellet), remove supernatant.
8. Add 140 µL 100% EtOH, flick gently (don't disturb the pellet), remove supernatant.
9. Add 50 µL 100% EtOH. This ethanol needs to be very pure, as any water will cause clumping during the next step.
Coating the Microcarrier
1. Submerge microcarriers in a small dish of 100% EtOH (I typically do this before starting the gold preparation and let it sit).
2. Using forceps, remove microcarriers from EtOH and let dry on a Kimwipe.
3. Load microcarriers into the disks.
4. Vortex gold mixture and quickly pipette off 10 ¿L.
5. Add 10 ¿l gold-particle mix to center of microcarrier.
6. Repeat for as many microcarriers as needed (NOTE: re-vortex after every two).
7. Let particle mix dry on microcarrier.
1. Turn on gas cylinder. Set regulator to 1500 psi.
2. Turn on vacuum pump; turn on power to chamber (left red switch).
3. Place rupture disk into center of screw-on cylinder; screw cylinder on; tighten it with torque tool (until the bar just touches the black sleeve).
4. Place stopping screen and microcarrier into holder and screw on lid.
5. Place tissue on shelf (may adjust shelf height) and close door.
6. Turn on vacuum switch (middle red switch, up position); make sure vacuum takes and door is held shut.
7. When vacuum guage reads 25, it's ready to fire.
8. Turn vacuum switch to "hold" (down position).
9. Press "fire" switch (right red switch) and hold until it fires.
10. Turn vacuum switch to vacuum to vent.
11. Repeat steps 3 - 10 for as many shots as you want to make.
12. When done, turn off everything: turn off gas tank, hold the fire switch on until pressure drops to 0, turn off chamber, turn off vacuum pump.
14. Incubate bombarded tissue overnight at room temperature.
Quantitative assay (luciferase)
1. Prepare grinding buffer (keep at -20ºC):
100 mM Na Phosphate pH 7.0 1ml of 1M/10 ml
10 mM DTT 100 µL of 1M/10 ml
20 µg/ml Leupeptin 20 µL 10mg/ml stock/10 ml
20% (v/v) glycerol 4 ml 50% stock/10 ml
H20 4.88 ml/ 10 ml
2. Prepare 2X Luciferase Assay Buffer (LAB) (keep at -20ºC)
60 mM Tris-SO4 pH 7.7* 600¿L 1M/ 10 mL
20 mM MgCl2 200¿L 1M/ 10 mL
20 mM DTT 200 ¿L 1M/ 10 mL
2 mM EDTA 40¿L 0.5M/ 10 mL
H20 8.95mL/ 10 mL
- Tris adjusted to pH w/ H2S04
3. Prepare 100 ¿l/sample of 1X LAB + 1 mM luciferin (Luciferin stock- dissolve 3.18mg in 100¿L H20 for a 100 mM solution. Store at -20C). Load these into white-bottom 96-well plate.
4. Prepare 100 ¿l/sample of 2X LAB + 2¿l of 0.1 M ATP (Assay Buffer).
5. Grind tissue in 300 ¿L grinding buffer with mortar and pestle.
6. Spin extract in microfuge for 10 min. at 4ºC, and use it for assays (the extracts can be kept at -20ºC).
7. Remove 75 ¿L supernatant to a tube containing 100 ¿l of 2X LAB+ATP.
8. Add complete sample to 100 ¿l of 1X LAB with Luciferin (in 96-well plates) as prepared above. Pipette up and down.
9. Visualize samples by measuring the luminescence of each for 5 seconds in a spectrophotometer.
Quantitative assay (GUS)
1. Prepare GUS assay buffer (keep at -20ºC)
2.5 mM MUG 250 ¿L 0.1M/ 10 mL
50 mM NaPO4, pH 7.0 500 ¿L 1M/ 10 mL
10 mM EDTA 200 ¿L 0.5M/ 10 mL
10mM DTT 100 ¿l 1M/ 10 mL
20 µg/ml Leupeptin 20 µL stock (10 mg/mL)/ 10 mL
20% (v/v) MeOH 2 mL/ 10 mL
H20 6.93 mL/ 10 mL
2. Add 200 ¿L/ sample to clear-bottom 96-well plate
3. Add 50 ¿L of sample to well
4. Seal plate with parafilm and incubate overnight (~20 hr) at 37oC
5. (The next day) add 50 ¿L of overnight reaction to 200¿L 0.2M Na2CO3
6. Visualize samples by exciting samples at 365 nm and measuring the light emitted at 455 nm.