mFs{w_wYwuLabo͔|wz[@Protocols
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Protocols: from in vitro Rice Culture to Genetic Transformation using Helios
Gene Gun
A. Callus Induction and Subculture
Things to do before go through:
a. For callus induction, the following tools/media have to be autoclaved:
Iwaki pyrex bottle, distilled water or Milli-Q water, filter paper, glass Petri
dish, sterile surgical blade, small pinset, and appropriate medium (such as
MS or NB5) with 3 mg 2,4-D, 3 gL-1 Gellan gum and pH 5.8.
b. For callus subculture, the following tools/media have to be autoclaved:
filter papers, glass Petri dish, sterile surgical blade, small pinset, and
appropriate medium (such as NB5 or D1) supplemented with 3 mg 2,4-D,
500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 30gL-1 maltose, 3 gL-1 Gellan
gum and pH 5.8.
Autoclaving can be done for 20 min (1210C). After pouring medium into
Petri dish (around 20-30 ml), do not immediately cover them with the caps,
but let them evaporate for 10 min under Laminar Flow Cabinet (LAF).
Thereafter, cover them with parafilm , then store at refrigerator by
inverting.
1. Mature seeds of rice are dehusk, remove the endosperm by cutting with
sterile surgical blade (remain only embryo with small part of
endosperm), placed into the 100 ml Iwaki pyrex bottle. Cutting the
endosperm part of seed helps to minimize contamination from this part.
2. Around 20-30 ml of seventy percent of ethanol is then added; gently
shake for 2-3 min (manual shaking or placed on the shaker machine,
depend on the number of samples).
3. Remove the excess of ethanol by washing 3 times with distilled
autoclaved water.
4. Surface sterilized in 50% commercial bleach (4.5% sodium hypochlorite)
and supplemented with 2-3 drops of Tween20.
5. Covering the upper part of the bottle with parafilm.
6. Put the bottle on the shaker 50-80 rpm for 45 min.
7. Wash thoroughly with sterile distilled water 3-5 times.
8. Shake on the shaker with sterile distilled water for 30-60 min.
9. Turn on UV light of LAF (Laminar Air Flow) for 20-30 min.
10. Spray fingers, hand and in side of LAF with 70% ethanol.
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11. Remove water; wash again the seeds with sterile water (performed in
LAF).
12. Turn on the burner and blower of LAF.
13. Provide 30-50 ml of 70% ethanol in the Iwaki pyrex for sterilization.
Clean the excess of ethanol by tissue paper.
14. Put the mature embryos on Petri dish covered with filter paper for 10
min air drying.
15. Lay carefully the mature seed embryos on the medium (embryo should
be in contact with the medium).
16. Incubate those calluses in Growth Chamber at 270C for 2-3 weeks.
Calluses formed on scutellar tissues can be sliced, and then subcultured
on new subculture medium. Twenty calluses or more can be cultured per
Petri dish (9 mm in diameter). A three-week subculture will be
appropriate to obtain high number of embryogenic calluses.
B. Plant Regeneration
Things to do before starting
Prepare fresh pre-regeneration medium:
- medium NB5 supplemented with 3 mgL-1 Kinetin, 3 mgL-1 BA, 0.3 mgL-1
IAA, 0.3 mgL-1 NAA, 10 mgL-1 ABA, 500 l-proline, 500 mgL-1 l-glutamine,
800 mgL-1 casein hydrolysate, 30gL-1 maltose, 8 gL-1 Agarose and pH 5.9.
Prepare also regeneration medium:
- medium NB5 supplemented with 3 mgL-1 Kinetin, 3 mgL-1 BA, 0.3 mgL-1
IAA, 0.3 mgL-1 NAA, 500 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1
casein hydrolysate, 30gL-1 maltose, 8 gL-1 Agarose and pH 5.9.
Autoclaving: pre-regeneration and regeneration media, small pinset, 100 ml
Iwaki pyrex or long tube filled with rooting medium.
They can be stored put in refrigerator at 4-100C for 1 month.
1. Subcultured callus is then selected on the basis of its phenotypic
performance. Select cream/ yellow/ white calluses with globular/ nodular
appearance for regeneration.
2. Carefully place them on the pre-regeneration medium for 1 week at
270C.
3. Transfer them on regeneration medium for 2-3 weeks.
4. Subsequently transfer green shoot buds or shoots onto rooting medium
(hormone free MS medium).
5. When the leaf height reached 10 cm, the green plants are transferred
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onto the soil and grown in the glass house or SCEPL (27-320C).
Carefully separate them into individual plant.
6. After 3-4 weeks on acclimatization, plants can be transferred into pot or
field for further growth.
C. Transformation of E. coli with Plasmid
This section of protocol provides the amplification of the genes of interest
that have been cloned into plasmid vector. The genes of interest are mostly
cloned into plasmid vector and before they become ereadyf, they have to be
transformed into the competent cells (generally E. coli ) in order to get a
desired amount of DNA concentration for many purposes such as for
manipulation of expression construct or for genetic transformation using
Helios gene gun.
Prepare: LB (Luria-Bertani Broth) plate and SOC medium.
- LB broth plate (1 L): 10g polypepton, 5 g yeast extract, 10 g NaCl and 15
g agar. Add 50 g/mL Ampicilin or Kanamycin (depends on antibiotic
resistant gene in the plasmid for bacterial selection), 2% X-Gal and 100
mM IPTG.
- SOC medium (1 L): 2% bactotryptone, 0.5% yeast extract, 0.4 mL 5M
NaCl, 0.167 mL 3M KCl (autoclaved). Just prior to use add 1% of 1M
MgCl2.6H2O & 1M MgS04.7H2O, and 20 mL of 1M glucose (filter
sterilized).
1. Thaw competent cells E. coli in ice for 10 min. (Styrofoam box).
2. One to fifty nanogram of plasmid DNA is added to competent cells
(usually diluted in 1-5 L TE).
3. Add 100 L E. coli (preferred DH5 strain), mixed well by pipeting, then
immediately incubate in ice for 20-30 min.
4. Prepare the hot water or set the water bath at 420C.
5. Heat shock the mixture for 1 min at temperature stated in step no. 3.
6. Return back immediately on ice for 5 min.
7. Add 500 L SOC medium (in the clean bench).
8. Incubate on shaker machine (200-250 rpm) at 370C for 1 h. At the same
time, pre-warm the LB plate (by putting inverted) in incubator (370C).
9. Centrifuge at 5000-7000 rpm for 1-3 min.
10. Discard 400 L supernatant (performed in LAF from this to step no. 13)
11. Resuspend pellet in remaining 100 L supernatant.
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12. Wash the hockey stick in ethanol and quickly flame it.
13. Grow the bacteria on LB plate with the help of hockey stick.
14. Incubate the plate in incubator at 370C for 13-16 hrs (over night).
15. In day 2, pick a single white colony with toothpaste.
16. Make a master plate by numbering and prepare 1.5 ml sterile micro
tubes (10 tubes, each containing 1 mL LB medium might be sufficient).
17. Scratch them on both the LB plate as well as LB liquid medium (1.5 mL
micro tube). Leave the toothpaste in the micro tubes.
18. Shake the toothpaste in the LB medium, and then discard it.
19. Cover LB plate with parafilm and grow LB plate and LB liquid in
incubator at 370C for 13-15 hrs (over night).
20. Harvest the plasmid with alkaline-SDS lysis method (next protocol).
D. Plasmid Extraction (Alkaline-SDS lysis Method)
Materials to be prepared:
- TE buffer: 10mM Tris-HCl, 1mM EDTA, pH 8. (add 100 g/mL RNase A for
step no. 14 of this section).
- Alkaline buffer: 20 mL of 5% SDS, 4 ml of 5N NaOH, and 76 mL Milli-Q
water.
- Acid buffer: 60 mL of 5M CH3COOK (autoclaved), add 11.5 mL of
CH3COOH and 28.5 mL sterile H2O.
1. After overnight grow at 370C (13-15 hrs), E. coli is then harvested.
2. Centrifuge at 8000 rpm for 2 min (balanced).
3. Remove the supernatant.
4. Add 150 L TE buffer and vortex well.
5. Put in 150 L fresh alkaline buffer and mix gently by inverting. Do not
vortex.
6. Again, add 100 L acid buffer and mix gently by inverting. Do not
vortex.
7. Centrifuge 15000 rpm for 5-10 min.
8. Transfer supernatant into new rough tubes. Do not forget to give the
numbers on the cap of the tubes.
9. Add one volume 2-propanol (around 400 L) and mix well.
10. Centrifuge 15000 rpm for 5-10 min.
11. Discard supernatant and rinse with 70% ethanol (2-3x).
12. Centrifuge 15000 rpm for 2 min. Discard supernatant.
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13. Vacuum dry for 4-5 min in vacuum machine.
14. Add 20 L RTE (TE with RNase) to each tube and vortex.
15. Incubate at 370C for 30-60 min.
16. Add 0.6 time volume PEG/NaCl and put on ice for 1 hr.
17. Centrifuge again at 15000 rpm for 10 min.
18. Discard supernatant. Rinse with 70% ethanol (2-3x).
19. Vacuum dry for 4-5 min and resuspend in 10 L TE.
20. Confirm the plasmid by restriction digest using the proper/known
restriction enzyme, then subsequently do electrophoresis.
21. Quantify plasmid DNA concentration by using spectrophotometer if
necessary.
E. Helios Gene Gun Bombardment Procedures
Preparation: put calluses, 3-5 mm in diameter on 0.4 M Mannitol of NB5
medium, 4 hr before bombardment.
Autoclave: glass Petri dish with filter paper, small pinsets, plastic bowl,
10-15 ml tube, 1.5 ml tube, and 2 ml screw-cap tube.
a. Preparation of DNA onto microcarriers
1. Prepare a stock solution of 20 mg/mL PVP in ethanol (100%) in 2 ml
screw-cap tube.
2. Weight out 12.5 mg of 0.6 m gold particles for each 30 inch length of
tubing.
3. Put the gold particles into 1.5 ml sterile micro tube.
4. Add to the gold 40 L of 50 mM spermidine, mix well.
5. Sonicate for 5-8 min.
6. Add 40 L of plasmid-DNA (50-100 g, depends on the desired amount of
DNA delivered per shot) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH
8.0).
7. Turn on the vortexer, add drop by drop 40 ?L of 1M CaCl2, to associate
the DNA with the gold particles.
8. Allow the suspension to settle at room temperature for 10 min.
9. Centrifuge the suspension for 1 min in a microcentrifuge to pellet the
gold-DNA.
10. Wash three times with 100% ethanol.
11. Resuspend the pellet in 3 mL of 0.05 mg/mL PVP-ethanol using 10-15 ml
sterile tube (take 7.5 ?L from the stock prepared in step 1).
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12. Go to Genomic Center for the Gene gun bombardment.
b. Cartridge preparation with the Tubing Prep Station
1. Dry the Gold-Coat tubing by purging with nitrogen (flow 0.3-0.4 liters
per min, LMP) for 10-15 min.
2. Turn off the nitrogen flow.
3. After mixing thoroughly, by using a syringe attached to an adapter,
draw the gold suspension into the Gold-Coat tubing.
4. Slide the loaded tubing into the Tubing Prep Station and allow it to
settle for 3-5 min.
5. The ethanol is then slowly drawn off with a edirty syringef from the
tubing, turn it 1800 and let it settle for a few seconds.
6. Start rotating the Gold-Coat tubing to allow the gold particles to spread
onto its inner surface.
7. After 20-30 sec, open the nitrogen flow (0.3-0.4 LMP) and the residual
ethanol is then removed by passing nitrogen through for 3-5 min.
8. Discard any unevenly coated section and cut the remaining tubing into
0.5 inch pieces with the Tubing Cutter.
c. Particle delivery
1. Connect the Helios gene gun to the helium source, insert an empty
cartridge holder into the gun and pressurize the system by a few
epre-shotsf.
2. Load the cartridge into the cartridge holder and insert it into the Helios
gene gun.
3. Turn the helium regulator to the selected pressure.
4. Spray the Gene gun with ethanol 70%. Clean with tissue paper.
5. Place the calluses on the center of Petri dish with filter papers, then
covered by plastic bowl with a hole that suites the spacer of the Gene
gun (perform inside the clean bench).
6. Point the target tissues with the spacer, activate the safety interlock
switch and press the trigger simultaneously to deliver the DNA/gold to
the target tissues.
7. Immediately transfer calluses into the NB5 medium with 0.4 M
mannitol.
8. After all the cartridges have been discharged, remove them carefully
with the Cartridge Extraction Tool.
9. Depressurize and disconnect the gun from the helium source.
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10. Place the bombarded calluses (on osmotic medium) in Growth Chamber
at 270C.
11. Sixteen hours after bombardment, rice calluses then transferred onto
new subculture medium (NB5) without selective agent (hygromycin,
bialaphos or other selectable marker) for one week.
12. Thereafter, calluses are subcultured on the subculture medium (NB5)
with appropriate selective agent for 2-3 weeks.
13. The survived calluses are then selected on the basis of their phenotypic
appearance. Transformant callus will be cream, yellow, or white which
can be distinguished from non-transformant. For plant regeneration,
please follow the above mentioned procedures.
F. PCR Detection of the Transgene in Putative Transgenic Plants
Material to be prepared:
- Prepare the template genomic DNA of transformants and
non-transformants by isolating their DNA and check the concentration
using spectrophotometer. Adjust their concentration to 10-50 ng per
micro liter.
- Primers that designed specific to the target gene/DNA sequence. It can
be designed by manually or by the help of software such as FastPCR. To
get a working concentration for PCR, the easy way is that primer is
diluted in one-tenth volume with TE buffer as recommended by company.
From this primer stock, we just diluted in 1:19 with TE buffer.
1. Prepare ice in Styrofoam box.
2. Thaw all PCR components at room temperature (it takes 10-20 min,
depend on room temperature condition).
3. Prepare PCR tubes and one 1.5 mL sterile micro tube.
4. Gently vortex and briefly centrifuge all solutions after thawing.
5. Put in 1.5 mL sterile tube, a mixture of 36.5 L Milli-Q water, 5 L PCR
buffer, 5 L dNTPs, 1 L forward primer and 1 L reverse primer and
0.5 L polymerase enzyme (prefer to use KOD Dash polymerase from
Toyobo, Jpn). This mixture is only for one sample, however if the
samples are more, just simple multiply it.
6. Gently vortex the mixture.
7. Distribute the mixture, 49 L, to each PCR tube.
8. Add 1 L template, gently mix and briefly spin down.
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9. Overlay the sample with half volume of mineral oil. This step may be
omitted if the thermal cycler is equipped with a heated lid.
10. Start PCR with the designed program. For the wheat Glu-1Dx5 gene:
i. Denature 940C, 2 min.
ii. Denaature 940C, 30 sec.
iii. Annealing 590C, 4 sec.
iv. Extension 740C, 1 min 36 sec.
v. Go to cycle ii) to iv), 30 cycles.
vi. Final extension 740C, 8 min.
vii. End 40C, 0 min.
11. Put samples immediately on thermal cycler if the temperature reaches
940C.
12. Most programs take 1-2 hrs. Wait until finish the program.
13. After the PCR is complete, run out samples on a mini-gel electrophoresis
or store in the freezer.
14. The presence of the band can be visualized by staining with ethidium
bromide under UV light.
To know further? Wish to help me in improving this protocol, please do not
hesitate to contact me at ncarsono(at)yahoo.com. Your generous assistance
would be grateful for further research.
Thanks.
Nono Carsono
Lab. of Plant Breeding,
Padjadjaran University, Indonesia
Jl. Raya Jatinangor Ujungberung Bandung 40600
Phone: +62-22-779320
Literature cited
Bio-Rad (1997) Helios Gene Gun System. Instruction Manual. Bio-Rad
Laboratories. USA. 1-49.
Helenius E, Boije M, Niklander-Teeri V, Palva ET and Teeri TH (2000) Gene
delivery into intact plants using the Heliostm gene gun. Plant Mol Biol
Reporter 18: 287a-287l.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: a
Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor
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Laboratory.

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