实验概要

        A breakthrough in Arabidopsis research was the invention ofthe vacuum-infiltration procedure, a simple and reliable methodof obtaining transformants at high efficiency while avoidingthe use of tissue culture. The plant transformation proceduresdescribed here involve floral dip, vacuum infiltration, andspraying. They yield transformants at frequencies ranging upto several percent, with the most common frequency being 0.1%-1%.

主要试剂

Agrobacterium culture carrying a suitable vector

Arabidopsis plants

Infiltration medium for floraldip

LB medium containing antibioticsthat select for both the Ti and the T-DNA plasmids

Silwet L-77 (0.2% [v/v])(optional, for Steps 17-19)

YEP medium (optional; seeStep 4)

主要设备

Centrifuge with GSA rotor (or equivalent)

Cover for plants, transparent

Dessicator with vacuum pump and glass dish (optional, for Steps13-16)

Incubator, preset to 28°C

Spray bottle (optional, for Steps 17-19)

Standard equipment for growing Arabidopsis 

Vessel for dipping plants

Plastic nursery flat

实验步骤

Plant Growth

1. Place the plants in 4-inch pots at a density of 10-15 plantsper pot. Grow the plants in short days (9-12 hours of lightper day) using ample fertilizer, so that large rosettes areobtained. To help hold the soil in the pot, cover the pots withnylon mesh. If short-day conditions are not available, orif time is limited,grow the plants under long days from thebeginning, but increasethe density to 15-20 plants per pot.

2. Remove the first inflorescence shoots as soon as they emerge(to encourage the growth of more inflorescences) and move theplants to long days (16-24 hours of light per day). Plantswill be ready for transformation after about 1 week,when thesecondary inflorescence shoots are ~3 inches tall.Proceed withthe floral dip (Steps 3-12), vacuum infiltration(Steps 13-16),or spraying (Steps 17-19) methods as describedbelow.

Floral Dip of Arabidopsis

3. Three days prior to plant transformation, inoculate a 5-mlliquid culture of Agrobacterium carrying a suitable binary vectorand incubate at 28ºC with  

vigorous agitation. Use LB mediumcontaining antibiotics that select for both the Ti and the T-DNAplasmids.

4. After 2 days, inoculate 200 ml of LB medium with1 ml ofthe preculture and incubate again with vigorous agitationforan additional 24 hours at 28ºC.

Use YEP medium forhigher Agrobacterium density.

5. Stop watering the plantsand allow the soil to dry out alittle, so that it will be lessprone to falling out of thepots during dipping.

6. PelletAgrobacterium by centrifuging at 6000 rpm in a GSArotor (orequivalent) for 10 minutes. If possible, centrifugeat roomtemperature. Note that the cell pellet is pink.

7. Resuspendthe cell pellet in 400 ml of infiltration medium.

8. Transferthe Agrobacterium suspension to a convenient vesselfor dippingplants, e.g., a lid from a box of disposable pipettetips ora 400-ml beaker.

9. Invert a pot of plants and dip the inflorescenceshoots intothe suspension. Rest the pot on the edge of thebeaker and allowthe plants to soak for ~30 seconds. The samesuspension canbe used for 10 or more pots.Try to avoid contaminationof the soil with Agrobacterium, whichproduces a rather unpleasantsmell.

10. After dipping, lay the pots on their sides in aplasticnursery flat and place a transparent cover over themfor thenext 24 hours.

11. After 24 hours, remove the cover,rinse the plants withwater, and return them to their normalgrowing conditions.

12. After about 3 weeks, collect seeds.

Vacuum Infiltration

        Some labs report an increase in transformation efficiency usingvacuum infiltration to draw the Agrobacterium into the planttissue.

13. Follow Steps 3-9 above, and transfer the inverted plantsand the beaker into a desiccator. Under vacuum, the suspensiontends to boil over, so place aglass dish under the vessel inthe desiccator.

14. Connect the desiccator to a vacuum pumpand evacuate untilthe suspension is at a good rolling boil.

If an oil pump is used, the traps must be very good to avoidsaturating the pump oil. Oil-free vacuum pumps, such as recirculating-watervacuum pumps, are less trouble but they take a bit longer todraw the vacuum. House vacuum lines are usually insufficientfor this purpose.

15. Release the vacuum as quickly as possible;the sudden increasein pressure will force the bacterial cellsinto the plant tissue.

16. Proceed through Steps 10-12 above. 

Spraying

        In this procedure, Arabidopsis are transformed by simply sprayingthe plants with an Agrobacterium suspension .

17. Grow Agrobacterium to stationary phase, harvest, and resuspendin two volumes of H₂O with 0.2% (v/v) Silwet L-77.

18. Usea spray bottle to apply the bacterial suspension tofloweringplants at weekly intervals until the first seeds startto dehisce.

19. Place the plants in covered flats for 1 day before returningto normal growing conditions.