Agrobacterium tumefaciens transformation

For transformation of Arabidopsis: we use Agrobacterium tumefaciens GV3101 pMP90 . Growth of non-transformed cells should be performed with 50 ug/mL gentamycin sulfate and 25 ug/mL rifampicin added to the media. Gentamycin stock = 50 mg/mL in H2O; rifampicin stock = 50 mg/mL in DMSO. Selection of transformed Agro should be performed with 50 ug/mL kanamycin.

For transformation of Brassica sp: we use Agrobacterium tumefaciens strain LBA4404. Growth of non-transformed cells should be performed with 100 ug/mL streptomycin and 40 ug/mL rifampicin added to the media. Streptomycin stock = 50 mg/mL in H2O; rifampicin stock = 50 mg/mL in DMSO. Selection of transformed Agro should be performed with 50 ug/mL kanamycin.

FREEZE-THAW METHOD

1. Grow Agrobacterium strain with appropriate helper Ti plasmid in 5 ml of YEP medium + antibiotics overnight at 28C with shaking (250 rpm).
2. Add 2 ml of the O/N culture to 50 ml YEP medium with antibiotics + antibiotics in a 250 mL flask and shake at 250rpm and 28C until the culture reaches an OD600 of 0.5-1.0.
3. Chill the culture on ice. Centrifuge the cell suspension at 3000 g for 5 min at 4C.
4. Resuspend the cells in 1 mL of 20 mM CaCl2 solution (ice-cold). Dispense 0.1 mL aliquots into pre-chilled microcentrifuge tubes. OPTIONAL: These cells can be frozen in liquid nitrogen and stored at -80C. Frozen cells can be used for future transformations. Add 1 ug of DNA to the frozen cells and follow steps 7-10.
5. Add about 1 ug of plasmid DNA to the cells.
6. Freeze the cells in liquid nitrogen.
7. Thaw the cells by incubating the test tube in a 37C water bath for 5 min.
8. Add 1 mL of YEP medium (no antibiotics) to the test tube and incubate at 28C for 2-4h with gentle shaking. This period allows the bacteria to express antibiotic resistance genes.
9. Centrifuge the tube for 30 s at ~3,000-4,000 xg (not max speed). Discard supernatant and resuspend in 0.1 ml YEP.
10. Spread the cells on a YEP agar plate containing gent50, rif 25 (for GV3101) or strep100/rif40 (for LBA4404) and appropriate antibiotic carried by binary vector. Use 50 ug/ml for kanamycin. Transformed colonies should appear in 2-3 days.

For alternative protocol see: http://cshprotocols.cshlp.org/cgi/content/full/2006/30/pdb.prot4666?print=true

ELECTROPORATION METHOD, from: http://cshprotocols.cshlp.org/cgi/content/full/2006/30/pdb.prot4665?ijkey=1875311f56f6442472f47615a7b2e7fc2da5ed6c&keytype2=tf_ipsecsha

This protocol was adapted from Shen and Forde (1989) and Mersereau et al. (1990).

Preparation of Competent Cells

1. Inoculate 500 ml of LB (not YEP) with 5 ml of a fresh saturated culture of the appropriate strain of Agrobacterium. Incubate the culture at 28ºC with vigorous agitation. Start the culture in the late afternoon, and harvest it the following morning.

2. When the cells have reached log phase (OD550 0.5-0.8), chill the culture by gently swirling it in an ice-water bath. Keep the cells at 4ºC for all further steps.

3. Pellet the cells by centrifuging at 4000g for 10 minutes at 4ºC in a prechilled rotor.

4. Discard the supernatant, add 5-10 ml of ice-cold H2O, and use a wide-bore pipette to pipette the cells gently up and down until no clumps remain. Adjust the suspension volume to 500 ml with ice-cold H2O.

5. Repeat Steps 3 and 4 twice.

i. After the first repeat, resuspend the cells in a final volume of 250 ml of ice-cold H2O.

ii. After the second repeat, resuspend the cells in a final volume of 50 ml of ice-cold H2O.

6. Pellet cells as in Step 3, and resuspend them in 5 ml of 10% (v/v) ice-cold, sterile glycerol.

7. Dispense 50-µl aliquots of cells into microcentrifuge tubes. Snap-freeze them in liquid nitrogen, and store them at -70ºC.

Electroporation and Recovery

8. Thaw competent cells on ice (50 µl per transformation).

9. Add plasmid DNA (1 µl of E. coli miniprep or 1-5 µg of CsCl-purified plasmid DNA) to the cells, and mix them together on ice.

10. Transfer the mixture to a prechilled electroporation cuvette. Carry out electroporation as recommended for E. coli by the manufacturer of the chosen electroporator. For example, when using a Bio-Rad electroporator with a 2-mm cuvette, use the following conditions:

Capacitance: 25 µF

Voltage: 2.4 kV

Resistance: 200 {Omega}

Pulse length: 5 msec

11. Immediately after electroporation, add 1 ml of LB to the cuvette, and transfer the bacterial suspension to a 15-ml culture tube. Incubate for 4 hours at 28ºC with gentle agitation.

12. Collect the cells by centrifuging briefly, and spread them on an LB agar plate containing the appropriate antibiotic (see Table 1). Include the antibiotic for the T-DNA vector.

13. Incubate the cells for 3-4 days at 28ºC.

14. Restreak colonies on a new LB agar plate. Incubate this plate at 28ºC, and, when the colonies have grown, seal the plate with Parafilm. Keep it at 4ºC as a stock plate.

15. Grow small liquid cultures of the restreaked colonies, and carry out minipreps and/or PCR to verify the presence of plasmid DNA (see PCR Analysis of Agrobacterium).

16. Make glycerol stocks of the appropriate clones, and store them at -20ºC.

Arabidopsis growth and seed handling

See The Arabidopsis Biological Resource Center (ABRC) for helpful hints on growing Arabidopsis.

Soil, fertilizer, pots, watering

1. Soil: We use BX soil mix.
2. Fertilizer: Prepare Miracle-Grow Liquid Houseplant Food (8-7-6) by mixing two capfuls of fertilizer per gallon of water.
3. Insecticide: Marathon granular insecticide -- add 5g to the bottom of each flat upon initial sewing.
4. Pots: To start seedlings use 3x5" tray inserts. For transplanting and large-scale growth use 36-pot inserts. Use 3" pots per plant for physiology/phenotyping experiments.

Starting seedlings

1. Sprinkle granular insecticide on the bottom of the plant tray in which the pots will be placed.
2. LOOSELY fill pots or flat inserts with BX soil mix. Lightly wet the top of the soil with tap water and fill bottom of tray with ~1/2 - 1" deep of fertilizer water. Once most of the water is absorbed, seeds can be sewn.
3. To start seedlings, sprinkle ~50-100 seed per 3x5" pot and place in growth chamber or on light racks in lab. Seedlings are usually ready for transplant after 7-10 days.
4. Water plants from the bottom only when the soil is starting to become dry, usually every 3-5 days. DO NOT OVERWATER OR LET PLANTS STAND IN WATER FOR EXTENDED PERIODS! Dump out excess water if it is not soaked up within an hour or so. Approximately 1/2" of water is appropriate for one flat.

Transplanting seedlings

1. Prepare trays and soil as for starting seedlings.
2. Once the soil is thoroughly wet, create a small hole ~1/2" deep with the end of a pen or sharpie. Gently transplant individual seedlings to individual pots and gently pack soil around roots.
3. Fertilize flats during the initial transplanting and every two weeks thereafter with Miracle-Grow Liquid Houseplant Food (8-7-6) by mixing two capfuls of fertilizer per gallon of water. Fertilize as you would for standard watering (see below).
4. Water plants from the bottom only when the soil is starting to become dry, usually every 3-5 days. DO NOT OVERWATER OR LET PLANTS STAND IN WATER FOR EXTENDED PERIODS! Dump out excess water if it is not soaked up within an hour or so. Approximately 1/2" of water is appropriate for one flat.

Arabidopsis seed sterilization

For small amounts of seed (~10-500), work in 1.5 mL microfuge tubes. Note: 100 microliters of dry seed = ~2,500 seed

1. To each tube, add 1 mL of 50% bleach/0.02% Tween 20, incubate with shaking/inversion for 7 minutes.
2. Briefly centrifuge tubes to pellet seeds, remove bleach.
3. Add 1 mL of sterile water and resuspend seeds to rinse off bleach. Spin briefly and remove wash.
4. Repeat step 3 three more times (total of 4 rinses).
5. Rususpend seed in ~100-200 uL of sterile 0.1% agarose and spread on growth media plates.

Suggestions: After the bleach is removed, you must work in a sterile hood. Work with sterile P1000 filter tips, freshly sterilized water and 0.1% agarose in wide-mouth bottles. BE CAREFUL not to touch the inner lip of microcentrifuge tube lids with anything nonsterile. For large amounts of seed (>2,500), work in 15 mL conical tubes.

Arabidopsis transformation

Transformation of Arabidopsis by the floral dip method Most plant transformation experiments are performed in vitro, meaning they are not performed on intact plants. However, in planta transformation with Agrobacterium is possible, and was first described in Arabidopsis. Briefly, whole plants with young developing flowers are dipped into a culture of Agrobacterium, which in turn partially infect the growing flowers, and transfer the T-DNA to developing ova (future seeds). The plant is then allowed to self-fertilize and set seed. Seed are then collected and plated on selection media. The most common Agrobacterium tumefaciens strain used for Arabidopsis transformation is GV3101.

Experimental “This protocol (Clough and Bent, 1998; modified from Bechtold et al. 1993) is extremely simple. We have found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1% (one transformant for every 100 seed harvested from Agrobacterium-treated plants).”

Steve Clough and Andrew Bent, University of Illinois at Ubana-Champaign

1. Grow healthy Arabidopsis plants until they are flowering. Grow under long days in pots in soil covered with bridal veil, window screen or cheesecloth.

2. (optional) Clip first bolts to encourage proliferation of many secondary bolts. Plants will be ready roughly 4-6 days after clipping. Clipping can be repeated to delay plants. Optimal plants have many immature flower clusters and not many fertilized siliques, although a range of plant stages can be successfully transformed.

3. Prepare Agrobacterium tumefaciens strain (GV3101) carrying gene of interest on a binary vector (pBI121). Grow a large liquid culture @ 28oC in LB with antibiotics to select for the binary plasmid, or grow in other media. You can use mid-log cells or a recently stationary culture.

4. Spin down 500 mL of Agrobacterium, resuspend in an equal volume of in 5% Sucrose solution (if made fresh, no need to autoclave sucrose; an OD600 = 0.8 is ideal, but can be higher or lower). You will need 100-200 ml for each two or three small pots to be dipped, or 400-500 ml for each two or three 3.5" (9cm) pots.

5. Before dipping, add Silwet L-77 to a concentration of 0.05% (500 ul/L) and mix well. If there are problems with L-77 toxicity, use 0.02% or as low as 0.005%.

6. Dip above-ground parts of plant in Agrobacterium solution for 10 seconds, with gentle agitation. You should then see a film of liquid coating plant. Some investigators dip inflorescence only, while others also dip rosette to hit the shorter axillary inflorescences.

7. Place dipped plants under a dome or cover for 16 to 24 hours to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight (air under dome can get hot).

8. Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes, twist-ties, or other means. Stop watering as seeds become mature.

9. Harvest dry seed. Transformants are usually all independent, but are guaranteed to be independent if they come off of separate plants.

10. Select for transformants using antibiotic or herbicide selectable marker. For example, vapor-phase sterilize and plate 40 mg = 2000 seed (resuspended in 4 ml 0.1% agarose) on 0.5X MS/0.8% tissue culture Agar plates with 50 ug/ml Kanamycin, cold treat for 2 days, and grow under continuous light (50-100 microEinsteins) for 7-10 days.

11. Transplant putative transformants to soil. Grow, test, and use! For higher rates of transformation, plants may be dipped two or three times at seven day intervals. We suggest one dip two days after clipping, and a second dip one week later. Do not dip less than 6 days apart.

Experiments adapted from: Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.