Particle Bombardment Protocol

Particle Bombardment Protocol
Preparing Gold
1. 1 µm gold particle 60 mg + 1 ml 100% EtoH
2. Vortex for 2 minutes
3. Spin down
4. Remove EtOH
5. Wash two times with SDW then dissolve in 1 ml SDW
6. Vortex and leave on ice until used, or can store at -80 o C as gold stock
a. It is possible to store stock solution at 4 o C, or even at room temperature if
necessary
Spermidine
1. Melt spermidine free base at room temperature (37 o C)
2. 14 µl spermidine + 986 µl SDW = 0.1 M Spermidine
2.5 M CaCl2
1. 18.375 g CaCl2 / 50 ml SDW
2. Autoclave
Coat Gold with DNA (for 1 shot)
1. To 10 µl of gold stock (votex well), add…
2µg DNA (should be in high concentration, recommend 1 µg/µl)
16 µl 2.5 M CaCl2 (final 1M)
6.4 µl 0.1 M spermidine (final 0.016 M)
Total volume: 40 ul
2. Vortex for 10 minutes
a. Place on ice for ten seconds, every minute
3. Spin down briefly (~10 seconds) to settle loose gold
4. Wash two times with 100 µl 70% EtOH
5. Spin down briefly
6. Wash once with 100 µl of 100% EtOH
7. Resuspend gold pellet in 10 µl 100% EtOH
8. Vortex well before spreading the coated gold out on each flyer
Cleaning
1. Soak macrocarrier, flyer, and stopper in 70% EtOH
2. Let air dry completely before use
Plant material preparation
Coat DNA on gold and spread 10 ul of each on flyer using micropipette before
preparing plant materials
A. Tobacco pollen
1. Collect pollen from 10-15 flowers per membrane
2. Suspend pollen in 300 µl PGM w/o PEG

3. Pre-wet nylon membrane with 50 µl PGM w/o PEG and spread all pollen
suspension onto the membrane
4. Let membrane dry out slightly and add PGM w/o PEG onto filter paper
around membrane
5. Bombard immediately
a. 1100 psi rupture disc, 2-3 shots per membrane
6. Rinse pollen off the membrane with 2 ml PGM w/ PEG
7. Pool pollen from the two membranes onto one small plate (~ 4 ml/plate)
8. Incubate at room temperature with gentle agitation (60 RPM) for 2 hours to
overnight
9. Check the result under a fluorescent microscope
B. Onion epidermal cells
10. Peel a thin layer of onion skin from the inner side of onion bulb
11. Lay flat on top of wet filer paper
12. Use 900 psi rupture disc
13. Transfer to a MS plate after the shot, incubate at least 8 hr before examining
using a fluorescent microscope.
Bombardment
Prepare plate as designed below. Wet filter paper in Petri dish with ddH2O.
Plate preparation diagram
Filter Paper
Nylon
Membrane
1. Turn on vacuum pump
2. Turn on valve on helium tank (pressure ~ 1500 – 2000 psi)
3. Turn on the He-1000 system
4. Set 1100 psi rupture disc in place
5. Set stopper in place
6. Set flyer (dried) in place
7. Place sample on level 4 or 5
8. Recheck settings to ensure proper placing

9. Press Vacuum on the He-1000 system until the vacuum in the chamber reachs ~
25 – 28 in. Hg
10. Switch Vacuum to Hold
11. Press on Fire button until rupture disc bursts
12. Release vacuum and remove sample from chamber
13. Remove rupture disc, flyer, and stopper
14. Finish
a. Close valve on helium tank
b. Vacuum and fire to release pressure
c. Release vacuum
d. Turn off gene gun and vacuum pump
Recipes
Pollen germination medium w/o PEG
Ingredients
50% Sucrose 9 ml 1.8 ml
0.5M MES pH 6.0 1 ml 200 µl
2% MgSO4 0.25 ml 50 µl
1% KNO3 0.25 ml 50 µl
1% Boric Acid 0.25 ml 50 µl
1M Ca(NO3) · 4H2O 75 µl 15 µl
H2O 14.178 ml 2.835 ml
Totals 25ml 5ml
Pollen germination medium w PEG
Ingredients PGM w/ PEG
40% PEG · 4000 15 ml 30 ml
50% Sucrose 1.25 ml 2.5 ml
0.5M MES pH 6.0 1 ml 2 ml
2% MgSO4 0.25 ml 0.5 ml
1% KNO3 0.25 ml 0.5 ml
1% Boric Acid 0.25 ml 0.5 ml
1M Ca(NO3) · 4H2O 75 ul 150 ul
H2O 6.925 ml 13.085 ml
Totals 25 ml 50 ml