Experiment Summary

Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This method allows a first rapid approach, avoiding time consuming stable transformation of maize plants and also allows quantitative promoter expression analysis by a histochemical or fluorometric assay.

Materials and Reagents

1. Plant Material: Maize plants from W64A pure inbred line

2. Ethanol absolute, gradient HPLC grade

3. 1.0 μm Gold Microcarriers

4. Rupture Disks 900 psi

5. Macrocarriers

6. Stopping Screens

7. Murashige and Skoog medium (M&S) with vitamins

8. Spermidine free base

9. Sodium hypochlorite, solution 5-9% Cl active

10. Cell culture dishes 60 mm x 15 mm Style treated polystyrene

11. X-GlcA sodium trihydrate

12. X-glucuronide: 5 bromo-4-chloro-3-indolyl-β-D-glucoronic sodium salt 3H2O

13. N, N-dimethylformamid for spectroscopy

14. MUG: 4 methyl-umbelliferyl-B-D-glucuronide hydrate C

15. Potassium hexacyanoferrate (III)

16. Potassium hexacyanoferrate (II) trihydrate

17. 4-Methylumbelliferone sodium salt (4-MU)

18. Luciferase Assay Reagent

19. CDTA: trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid

20. Protein Assay Dye Reagent Concentrate

21. Histochemical analysis detection buffer

22. Fluorometric lysis buffer

23. Fluorometric analysis reaction buffer

24. Fluorimetric analysis stopping buffer

25. MSO medium

26. 1x Luciferase lysis buffer

Equipment

1. BIORAD PDS-1000/He system

2. Laminar Flow Cabinet AH-100

3. Vacuum pump 50/60 Hz

4. Cylinder compressed helium gas (UN1046) 117 Kg (LYNDE)

5. RAYPA Bathwater sonicator 50 W

6. Mini-shaker

7. Mini centrifuge

8. ND-1000 Spectrophotometer

9. Spectra Max M3 apparatus

10. Olimpus Stereomicroscope SZX16

11. Screw cylinder polypropylene microtube with attached O-Ring cap and conical base

Procedure

1. Embryo excision from the maize kernel

a) Submerge and wash maize ears 16-18 days after manual pollination (dap) successively in different solutions at room temperature.

b) Detach grains with gloved hands from the washed ears and embryos from the grains with the help of a sterile cutter in a sterile Petri glass dish. Place 9 embryos/dish in a 3 x 3 array in the center of cell culture dishes (60 mm diameter) containing MSO medium. The embryo axis side in contact with the medium.

c) Maintain plates at 21-23 °C for 24 h in the dark before bombardment to allow embryos to be recovered of excision treatment.

2. Microcarrier stock preparation (at room temperature)

a) Add 1 ml ethanol (HPLC) to 60 mg of Gold Microcarriers in a cylinder microtube with attached ring cap and a conic base. Vortex at 2,200 rpm with the minishaker for 10 min. Avoid the use of hydrated ethanols.

b) Centrifuge in the Mini Centrifuge for 1 min at 10,000 rpm.

c) Remove the Ethanol with a pipette, without removing microcarriers sediment. Discard ethanol.

d) Add 1 ml sterile water. Vortex 1 min. Sediment by centrifugation as in step 2-b. Discard water (Repeat 3 times).

Suspend microcarriers in 1 ml of water. Vortex 1 min. Make aliquots (30 μl) in conical tubes from the homogeneous suspension.

Store at -20 °C.

3. Microcarriers coating with the plasmid DNA of interest

a) Quantify DNA stocks (prepared by using a commercial plasmid prep kit) of plasmids sharing the studied promoters fused to a gene reporter, usually the beta-glucoronidase enzyme, with a nanodrop Spectrophotometer.

b) Sonicate defreezed microcarrier aliquots for 3 min in a bathwater sonicator.

c) Add succesivelly to the microcarrier aliquots:

12.5 μl DNA in TE buffer (1 μg/μl)

95 μl of water

125 μl CaCl2 2.5 M (while vortexing: Open the tube and decrease speed to avoid sample lost while you add solutions)

25 μl spermidine 0.1 M (while vortexing)

d) Vortex for 3-5 min.

e) Allow sedimentation on ice to minimize ethanol evaporation for 15-20 min.

f) Discard solution without disturbing microcarrier sediment as in step 2-c.

g) Add 500 μl ethanol (HPLC). Vortex 10-20 sec. Allow sedimentation during 15-20 min on ice.

h) Discard ethanol as in step 2-c.

i) Repeat with 200 μl ethanol (HPLC).

j) Discard ethanol as in step 2-c.

k) Finally add 40 μl ethanol (HPLC).

l) Sonicate in the bathwater sonicator 3 sec (Repeat 3 times).

m) Vortex 3 min.

n) Distribute the 40 μl homogenous solution of DNA coated microcarriers in ethanol (HPLC) (step 3-j) between the surface center of three macrocarriers (≈ 10 μl/macrocarrier) on a flat leveled surface. Macrocarriers have been washed in 70% ethanol and dried, beforehand. Let dry. This will allow the bombardment of three different samples (three cell culture dishes, each with 9 embryos in MSO medium).

o) When microcarriers have dried on macrocarriers surface (5-10 min), they are introduced inside previously autoclaved macrocarriers holders and are ready to be used for maize embryo bombardment.

4. Maize embryos 16-18 dap particle bombardment

a) Wash stopping screens and rupture disks with 70% ethanol. Locate in the adaptor the stopping screen and the macrocarrier holder with microcarriers attached to the macrocarrier. Locate the rupture disk at the end of the acceleration tube.

b) Proceed to the bombardement of the cell culture dishes with the maize embryos in MSO medium with the microcarriers coated with the different studied plasmids following PDS-100/He System instructions (http://www.Bio-Rad.com/biolostics).

c) Bombardement parameters

d) After bombardment and before the quantitative fluorometric or histochemical analysis, Petri dishes are incubated in the dark at 21-23 °C for 24 h to allow expression of the reporter gene.

e) The validity of results is evaluated by the reproducibility of the results. This is achieved by the mean value of different experiments (3 to 4) and their standard deviation.

f) Finally significant differences between constructs or conditions (p-value) are calculated from the means of different experiments in a Student'st test (http://www.physics.csbsju.edu).

g) Different efficiency has been observed in different maize varieties. Efficiency has shown to be in an inverse rapport with the scutellar hydration level degree of the different maize varieties during embryo development.

5. Quantitative histochemical analysis

a) Embryos from each bombarded dish are transferred to 2 ml tubes with 1 ml of histochemical analysis detection buffer and incubated overnight at 37 °C.

b) Blue spots of different intensity appear as result of the precipitation of product derivates reaction.

c) To avoid diffusion of the blue spots on the surface of the embryos, they are transferred to a 50% glycerol solution in water. Blue spots are quantified by observation with an OLYMPUS Stereomicroscope SZX16. Stained embryos are stored at 4 °C.

d) As control one set of embryos can be bombarded with a constitutive promoter for monocots, such as OsActine: GUS-nos ter, to easily evaluate the efficiency of the microcarrier batch and of each specific experiment.

6. Quantitative fluorometric analysis

a) Embryos from each bombarded dish are frozen in liquid N2 and stored at -80 °C until analysis.

b) The nine frozen embryos from a bombarded dish are grinded in 300 μl fluorometric lysis buffer.

c) Centrifuge 15 min 13,000 rpm in the Mini Centrifuge at 4 °C.

d) Freeze the clear extract solution at -80 °C in aliquots of 40 μl, until beta-glucoronidase quantification. Sediment is rejected.

e) For fluorometric analysis 20 μl of clean extract are added to 80 μl fluorometric analysis reaction buffer and warmed at 37 °C.

f) Aliquots of 20 μl are taken at different time courses (10 (zero), 60, 180, 360 min).

g) Reaction is stopped by an addition of 180 μl of fluorimetric analysis stopping buffer to each taken aliquot and stored 4 °C until analysis.

h) Samples are transferred to a microtiter plate to measure fluorescence emission of the beta-glucoronidase enzyme product 4-MU (4-methylumbelliferone) in a plate fluorescence lector, Spectra Max M3 apparatu (excitation 365 nm, emission 455 nm).

i) After analysis defreezed samples cannot be refreezed and are discarded.

j) 4-Methylumbelliferone (4-MU) standards are used to calibrate the system.

k) Protein concentration in the extracts is measured using the Bradford assay in a Spectra Max M3 apparatus.