1.      Excise root and leaf segments, wash with 100mM potassium
phosphate buffer (pH7.0) for 3 times.

2.      Immerse in the Gus substrate solution, 37C, dark, 12-24h

3.      Rinse in phosphate buffer

4.      Fix for 4h or longer

5.      Rinse in the same buffer

6.      Observe as whole specimens or as sections.

1M potassium phosphate buffer (pH7.0)

K2HPO4: 34.8g —>200ml

KH2PO4: 27.2g—->200ml

184.5ml K2HPO4+115.5ml KH2PO4—-> 300ml 1M potassium phosphate buffer
(pH7.0)

Gus substrate solution

Volume(500ml)

Final concentration

Potassium ferricyanide

82mg

0.5mM

Potassium ferrocyanide

105.6mg

0.5mM

Potassium phosphate buffer (pH7.0, 1M)

50ml

100mM

Store at -20C. Add 1mM x-gulc (MW521.8~~0.5mg/ml) freshly when use.

Fix solution

2.5% glutaraldehyde

200mM sodium cacodylate, pH7.2

1. Harvest tissue (can be stored at -70C)

2. Grind tissue in 100ul extraction buffer.

3. Spin 1min. Take supernatant, put on ice.

4A. 20ul for BCA assay (add 500ul assay reagent—-> 37C 30min
—–>562nm

4B. Add 0.05ul mercaptoethanol to the remnant 80ul, spin 10min.

5. Set up GUS assay:

In borosilicate tube (VWR 60825-550) combine:

Plant extract: 20ul

Assay mix: 80ul

Mix well. Assay mix is 70 ul extraction buffer and 10ul substrate
solution.

6. Incubate at 37C covered with parafilm for 1 hour exactly.

1.Stop FUS assay by adding 900ul 0.2M sodium carbonate solution.

2.Measure fluorescence in Sequoia-Turner fluorimeter using a MU standard
as a control.

Filters: 365nm/455nm

Fluorimeter must warm up for 15 minutes before use.

Gain 1 or Gain 10 should be sufficient.

Substrate solution: 1mM MUG in extraction buffer (22mg per 50ml)

Dissolve MUG in DMF first before adding to extraction buffer.

Store at 4C, but it is not stable for more than a few weeks.

Extraction buffer

50mM sodium phosphate pH7.0

10mM EDTA

0.1% laurylsarcosine

0.1% Triton-X-100

10mM beta-mecaptoethanol (can also try 1mM fresh DTT)

Stop solution

2.12g sodium carbonate in 100ml water.

BCA assay

Mix reagents A and B as indicated 50:1 just before use. Make a standard
of 0, 5, 10, 20ug BSA (in 1ml). Use 5, 10, 20ul protein solution per
assay and add 500ul assay reagent. Incubate for 30min at 37C, then read
Absorbance at 562nm.

MU standard

MU is not stable in solution and has to be prepared fresh, dissolved in
DMF and diluted to 1mM in extraction buffer, every time the standard is
measured.

9nmole MU in 1ml stop buffer will give a reading of 1559 at gain 1.

X=8.56 X 10(exp-3) X f/A

With

X=GUS activity in [pmole/min/ug protein]

F=fluorescence units

A=absorbance of BCA assay assuming that 20ul protein was assayed.

Preparation of Cells

1.      Inoculate 1l of L-broth with 1/100 volume of a fresh overnight culture

2.      37C, shaking, to ABS600 = 0.5-0.8

3.      Chill the flask on ice for 15-30min, centrifuge in a cold rotor 6000G, 10min

4.      Remove as much of the supernatant as possible, resuspend pellets in 1l 10% glycerol, repeat step 3

5.      Resuspend in 0.5 l 10% glycerol, repeat step 3

6.      Resuspend in 20ml ice cold 10% glycerol, repeat step 3

7.      Resuspend in 2-3ml 10% glycerol

8.      Frozen in aliquots in liquid N2, store at -80C.

Electroporation

1.      Chill electroporation cuvettes (use sterile ones, can be reused for 2-3 times) and white chamber slide

2.      Add 1-2ul plasmid/ligation product

3.      Add 40ul electroporation competent cells on the same spot where the plasmid was placed

4.      Agitate to mix, incubate for 1-2min

5.      Pulse (Setting: 1.8KV/200Ù/25uFD)

6.      Add 1ml LB in cuvette, transfer to eppendorf tube

7.      Shake for 1 hour

8.      Plate

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^–

1.        Grow Bradyrhizobium strain in HM medium (Nieuwkoop et al., 1987) containing only arabinose as carbon source to an OD600nm= 0.4 to 0.8. Grow E. coli strain(s) in LB to approximately the same OD.
2.        Pellet 1 ml of both strains in a sterile microcentrifuge tube at 10,000 rpm for 3 min. If either stain was grown with antibiotics, then wash twice with 1 ml of HM medium and pellet again. For triparental mating, do the same to the strain with the helper plasmid.
3.        Decant the supernatant and resuspend the cell pellet in 50-100 µl of HM broth by pipetting (no vortexing).
4.        Mix the strain suspensions together and transfer the mixture onto a sterile 0. 45µM filter and place the filter (cell side up) onto a HM agar plate. Let the dry to remove any excess moisture and then incubate at 30oC for two days. Using a day-old HM plate helps to reduce the moisture. Set up controls with filters containing both donor and recipient alone.
5.        After incubation, lift the filter from the plate and place into a 1.5 ml microcentrifuge tube. Add 0.5-1.0 ml HM medium and vortex to suspend cells.
6.        Spread 100-200 µl of the cell suspension onto RDY agar plates (So et al., 1987) containing the appropriate antibiotics. The same is done for controls. Incubate plates at 30oC. Colonies usually appear in approximately 6-10 days.

References:

Nieuwkoop, A.J., Z. Banfalvi, N. Deshman, D. Gerhold, M.G. Schell, K. Sirotkin, and G. Stacey. 1987. A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum. J. Bacteriol. 169: 2631-2638.

So, J.-S., A.L.M. Hodgson, R. Haugland, M. Leavitt, Z. Banfalvi, A.J. Nieuwkoop, and G. Stacey. 1987. Mol. Gen. Genet. 207: 15-23.

This protocol is modified from Bechtold, Ellis and Pelletier (1993). “In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants”. [C.R. Acad. Sci. Paris, Life Sciences 316: 1194-1199].

PLANT GROWTH:

1. Take seeds with a brush and place them into 8cm square pots filled with soil. Don’t compress the soil too much and water the pots thoroughly with 2-3 pot-vol to remove excess nutrients. Place 12-16 seeds in each pot.

Place the pots in the cold room for two days before transfering them to the growth chamber. Grow the plants for three weeks in short days (10 hr or less) to get large plants and a greater seed yield. Transfer the pots to long days to induce bolting. Grow plants to a stage at which bolts are around 10 cm tall.

2. Clip off emerging bolts close to rosette leaves to encourage growth of multiple secondary bolts. Infiltration will be done 7 to 9 days after clipping (plants will be 10-15 cm high and the biggest of the inflorescens will have made the first tiny silique. Do not water the plants the day before vacuum infiltration.

VACUUM INFILTRATION:

3. Start a 4ml agrobacterium culture (YEP+antibiotics) inoculated from a -800C stock or from a plate. Grow cells O/N to 48h depending on the strain. Add this culture to 250 ml of YEP+antibiotics (A 250ml culture will give enough cells for infiltration of 6 pots). Grow the culture between O/N and 2 days (depending on the strain) to OD600 = 1.2-1.8. The culture will have a mother of pearl appearance (not lumpy or black).

4. Spin down agros at 5000rpm for 10 min in 250ml centrifuge bottles, resuspend in infiltration media to an OD600 = 0.8 in a minimum volume of 300ml.

5. Poor the agro suspension into a beaker of an appropiate size (400ml is ok). Place the beaker into the vacuum jar. Degass the solution by drawing vacuum until bubles form. Place a paper towel under the beaker to avoid that the beaker gets stuck in the bottom of the vacuum jar.

6. Sprinkle the plants with water 5 min prior to infiltration (optional) and then invert plants into the culture solution. Make sure that all the flowers are submerged and leave 2cm between the rosettes leaves and the agro suspension. Don’t let the culture contact the rosette or soil as this could kill the plants. Avoid that the solution boils over when you pull the vacuum. Make sure that the soil is only moist, so that the water from the pots does not enter into the culture suspension (therefore we recommend not to water the plants the day before infiltation). Draw vacuum for 15-20 min for WS and 30 min for Col-0 at a pressure close to 0.05 Bar (we are using an oil pump).

7. Before removing the plants from the vacuum jar place a plastic bag over the pot and beaker. Pull out and remove plants from the beaker, lay pots on their side (to avoid that excess infiltration media runs down into the soil). Fold over the top of the plastic bag and staple them twice. The other possibility is to place the pots laying on their side into a tray and cover the whole box with saranwrap. Put them in a growth chamber for one night. Next day move them to the green house. Put the plants in vertical position and open the bags. Next day get rid off the bags. In case you have the plants in trays: put also the plants in vertical position and use sticks and saranwrap to make a kind of a tend around the plants. Next day remove the plastic. In hot summers, we recommend to give plants a shower after we have placed them in vertical position (the purpose of this is to remove the sugars from the infiltration media which decrease fungal infection).

8. Grow plants for approx. four weeks, keeping bolts from each pot together but separated from neighbouring pots

9. When the siliques begin to turn yellow, place the pot on its side with the plants inside a big envelope. Leave them for one week to dry out and cut off the plants. Let the seeds dry in the envelope and clean them 10 days later (keep all the seeds from one pot together). Store the seeds in the cold room for one week before plating them.

KANAMYCIN SELECTION PROTOCOL

1. Sterilisation of seeds:

aliquot seeds in 15ml falcon tubes (approx 700 seeds/tube, you can estimate the ammount of seeds by first drawing a square plate of 9cmx9cm on a paper and spreading the seeds on it). Add 10 ml of hypoclorite solution. Shake tubes for 10 min. Remove the solution and add 10ml of 70% ethanol. Wait 2 minutes. Discard EtOH and wash seeds 2-3 times with 10ml of sterile water.

Resuspend seeds with 8ml 0.7% top agar (no warmer than 55oC ).

2. Spread seeds onto selection plates (MS+Kan). Dry plates in laminar flow hood until the top agar has solidified.

3. Vernalize plates for two nights in the cold room at 4oC. Transfer the plates to the growth chamber (21oC with continous light).

4. After aprox 7 days transformants should be clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend into the selective medium. Root growth is the most clear maker to identify transformants at an early stages.

To make sure that the transformants are positive transfer them to a new MS+Kan plate and leave them there for a few days (if they turn yellow is because they are faulse positives). Transfer the seedlings to soil.

If you have contamination on your plates at this step, transfer the transformants as early as possible to soil.

5. Grow the plants and collect the seeds.

Infiltration Media

1/2 x Murashige&Skoog salts (SIGMA #5524)

1X B5 vitamines (1ml of 1000x stock) (SIGMA; #G-2519) Gamborg’s vitamine powder, to prepare the 1000x stock disolve 11.2g in 100ml water.

5% sucrose

adjust to pH 5.7 before autoclaving

after autoclaving add:

– Benzylamino Purine (BAP), 10 µl per liter of a 1 mg/ml stock in DMSO. By adding the hormone just before use, you can keep infiltration media as a stock for at least one week prior to infiltration.

– we recommend to add 0.01% silwet to the infiltration media to increase transformation efficiency especially for Landsberg and colombia ecotypes. (silwet is from LEHLE SEEDS, cat no VIS-01 VAC-IN-STUFF (silwet L-77)

Selection plates:

1x Murashige&Skoog salts

1% sucrose

adjust pH 5.7 with 1M KOH.

0.7% Difco agar.

autoclave, cool, and add:

1x MS vitamines (SIGMA #M-7150). Take 1ml of 1000x stock prepared by disolving 10.3gr in 100ml of water.

antibiotic (kanamycin 50mg/l).

Top agar:

1x Murashige&Skoog salts.

1% sucrose.

adjust pH 5.7 with 1M KOH.

0.7% Difco agar.

autoclave.

before use: boil in the microwave and keep in water bath at 50-55C.

YEP media (liquid):

10 g /l Bacto peptone (Difco)

10 g/l Yeast extract (Difco)

5 g /l NaCl

For YEP plates add 15gr/l Difco bacto agar.

Hypoclorite solution:

for 50 ml:

4ml Na Hypoclorite 15%

255l Tween-20

water to 50ml

Immature embryo (12-15 days after pollination ) —->

Precultured on ND2 medium for 4 days at 26C in darkness —->

Immersed in the bacterial suspension for 20 minutes —->

Incubated on ND2-As medium for 3 days at 26C in darkness —->

Cultured on ND2CCH25 medium for 2 weeks for selection —->

Subcultured on ND2CCH50 medium for 4 weeks ( 2 generations ) for
selection —->

Resistant calli were put on 3M filter paper overnight for osmotic
treatment —->

Resistant calli were transferred to NN.1B2H medium for regeneration
—->

Regenerated seedlings were transferred to MS0 medium for further growth
—->

Plantlets about 10cm high were grown to maturity in greenhouse in soil