I. Surface sterilization of rice seeds
· De-hull rice seeds
· Wash with 70% ethanol for 1 min in a Falcon tube
· Replace 70% ethanol with 20% sodium hypochloride solution and incubate the seeds for 1 hour on a shaker in a slow motion
· In plant-hood, wash the seeds with sterile distilled water for 5-6 times (After the final wash, the seeds can be left over-night in dark in sterile distilled water on a shaker)
II. Callus induction
· Transfer surface sterilized seeds, on by one, to the callus induction medium -ND2 (see Appendix I) in 9cm petri-dishes (30ml of the medium per plate and place 26 seeds per plate). Seal the plates with parafilm. Incubate the plates in the dark at 26 C. Ten days later, transfer the calli induced from the scutellar region to fresh ND2 medium. The calli should be subcultured every two weeks.
III. Pre-culture
· After 2-3 2-week subculture cycles, transfer the looslely attached small globular calli (1-3mm in diameter) to ND2 medium for 4-day-pre-culture. Conditions for culturing the calli are same as above.
IV. Osmotic treatment
· Prepare plates (9cm petri-dishes) containing ND2MS medium (see Appendix II). Arrange the precultured calli in a circle ( the same size as a quarter) on agar surface 4 hours before transformation.
V. Biolistic transformation using the PDS-1000 particle gun
· Microcarrier (gold) preparation
1. Place 30 mg of gold particles (Bio-Rad) in a 1.5 mL microtube
2. Add 0.5 mL 100% ethanol and vortex for 1-2 min
3. Centrifuge the microtube at 10,000 rpm for 1 min. Remove supernatant. Repeat steps 2 and 3 twice
4. Add 0.5 mL sterile distilled water, resuspend, centrifuge, and remove supernatant. Repeat 3 times
5. Resuspend microcarriers in 0.5 ml sterile distilled water.
6. Store at 4 C
· Preparing CaCl2 and Spermidine
1. CaCl2: 2.5 M, autoclaved, store at ¨C20 C
2. Spermidine: 0.1 M, filter, store at ¨C20 C for no more than 1 month
· Sterilization of sonsumables and assemble
1. Soak macrocarrier holders, macrocarries, stopping screen and rupture disks (1100 Psi) in 70% ethanol for at least 15 min
2. Dry in the hood on sterile filter paper
3. Rinse other metallic parts (acceleration tube, rupture sisk retaining cap, and microcarrier launch assembly) in 70% ethanol
4. Dry theses inside the hood
5. Clean the components of PDS eith 70% ethanol. Allow to dry
· DNA precipitation
1. Under continuous vortexing, add to a 50 ul gold aliquot the following in order: 5ul DNA (DNA concentration=1ug mL-1), 50ul 2.5 M CaCl2, 20ul 0.1 M spermidine
2. Continue vortexing for 3 min
3. Incubate the mixture at room temperature for 10 min
4. Spin at 10,000 rpm for 5-10 sec.
5. Remove supernatant
6. Add 250ul 100% ethanol
7. Vortex for 2 min
8. Centrifuge at 13,000 rpm for 1 min
9. Remove supernatant
10. Resuspend microcarriers in 60ul 100% ethanol (for 5 bombardments)
11. Pipet 10ul of the DNA-coated microcarriers onto the center of each macrocarrier
12. Dry for about 1 min
· PDS operation
1. Open the Helium valve, adjust the pressure to 1300 Psi
2. Switch PDS on
3. Switch vacuum pump on
4. Adjust the gap between rupture dish and macrocarier to 9 cm
5. Load the rupture disk
6. Install macrocarriers into macrocarrier holder
7. Load the microcarrier launch assembly
8. Position the sample for bombardment at the desired lever
9. Close the chamber door
10. Ture the VACUUM switch on to the VAC position
11. Press and hold the FIRE switch until the disk ruptures at 26-28 inHg
12. Release the FIRE switch immediately after the disk ruptures
13. Place the VACUUM switch to the VENT position
14. Remove the sample and treat accordingly
15. Discard the macrocarrier, stopping screen, and rupture disk
VI. Selection and regeneration
· The bombarded samples were incubated in the dark for 16-20 h at 26 C. Later, transfer calli in rows in petri-dishes containing ND2H25 medium (for HPT gene) or ND2G25 medium( for NPTII gene). Seal the plates with parafilm. Incubate the plates in dark at 26 C.
· 2-3 weeks later, transfer the small protuberances that grew from the calli onto ND2H50 medium or ND2G50 medium.
· 2 weeks later, transfer the resistant calli onto fresh ND2H50 medium or ND2G50 medium for 2 weeks propagation.
· Then, transfer the resistant calli onto ND2HS medium or ND2GS medium.
· Finally, resistant calli were regenerated on NN.2B2H medium or NN.2B2G medium under continuous light at 26 C.
Appendix
I. N6 medium
|
Bottle |
Component |
Stock solution (g L-1) |
Amount to take per liter of preparation (mL) |
Final concentration (mg L-1) |
|
A |
KNO3 |
141.50 |
20 |
2830.0 |
|
B |
MgSO4 |
18.5 |
10 |
185.0 |
|
|
MnSO4.4H2O |
0.44 |
|
4.4 |
|
|
ZnSO4.7H2O |
0.15 |
|
1.5 |
|
|
(NH4)2SO4 |
46.3 |
|
463.0 |
|
C |
KH2PO4 |
40.0 |
10 |
400.0 |
|
|
KI |
0.08 |
|
0.8 |
|
|
HBO3 |
0.16 |
|
1.6 |
|
D |
CaCl2.2H2O |
16.6 |
10 |
166.0 |
|
E |
FeSO4.7H2O |
2.78 |
10 |
27.8 |
|
|
Na2EDTA |
3.73 |
|
37.7 |
|
|
|
mg 100 mL-1 |
|
|
|
Vatamins |
Nicotinic acid |
50 |
1 |
0.5 |
|
|
Glycine |
200 |
|
2.0 |
|
|
Thiamine HCl |
100 |
|
1.0 |
|
|
Pyridoxine HCl |
50 |
|
0.5 |
|
Sucrose |
|
|
|
30000.0 |
|
Agar |
|
|
|
8000.0 |
|
PH |
5.8 |
|
|
|
II. ND2 medium
N6 + proline 500 mg L-1, casamino acid 300 mg L-1, 2,4-D 2 mg L-1
III. ND2MS medium
ND2 + mannitol 30 g L-1, sorbitol 30 gL-1
IV. ND2H25/ ND2G25/ ND2H50/ ND2G50 media
ND2 + Hygromycin 25 mg L-1/ G 418 25 mg L-1/ Hygromycin 50 mg L-1/ G 418 50 mg L-1
V. NN.2B2H/NN.2B2G media
N6-Agar + casamino acid 300 mg L-1, NAA 0.2 mg L-1, 6-BA 2 mg L-1, phytogel 2.5 g L-1, Hygromycin 50 mg L-1/ G 418 50 mg L–