In Planta Transformation of Arabidopsis

In Planta Transformation of ArabidopsisDetlef Weigel and Jane Glazebrook

This protocol was adapted from "How to Transform Arabidopsis ," Chapter 5, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

A breakthrough in Arabidopsis research was the invention of the vacuum-infiltration procedure, a simple and reliable method of obtaining transformants at high efficiency while avoiding the use of tissue culture. The plant transformation procedures described here involve floral dip, vacuum infiltration, and spraying. They yield transformants at frequencies ranging up to several percent, with the most common frequency being 0.1%-1%.

RELATED INFORMATION

Transgenic plants can also be generated via root transformation in tissue culture (see Root Transformation of Arabidopsis ). Although it has largely been superseded by the vacuum-infiltration method described here, the root transformation method can be useful for transforming sterile mutants.

The most commonly used markers for selection of transgenic Arabidopsis are resistance to the antibiotic kanamycin (see Kanamycin Selection of Transformed Arabidopsis ) and to the herbicide glufosinate ammonium (see Glufosinate Ammonium Selection of Transformed Arabidopsis ).

MATERIALS

Reagents

Agrobacterium culture carrying a suitable vector (see Transformation of Agrobacterium Using Electroporation or Transformation of Agrobacterium Using the Freeze-Thaw Method )

See Vectors and Agrobacterium Hosts for Arabidopsis Transformation for considerations regarding Agrobacterium strains and T-DNA vectors.

Arabidopsis plants

Infiltration medium for floral dip

LB medium containing antibiotics that 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; see Step 4)

Equipment

Centrifuge with GSA rotor (or equivalent)

Cover for plants, transparent

Dessicator with vacuum pump and glass dish (optional, for Steps 13-16)

Incubator, preset to 28°C

Spray bottle (optional, for Steps 17-19)

Standard equipment for growing Arabidopsis (see Step 1 and Cultivation of Arabidopsis )

Vessel for dipping plants (see Step 8)

Plastic nursery flat

METHOD

These methods are modified versions of those described by Bechtold et al. (1993) .

Plant Growth

1. Place the plants in 4-inch pots at a density of 10-15 plants per pot. Grow the plants in short days (9-12 hours of light per day) using ample fertilizer, so that large rosettes are obtained. To help hold the soil in the pot, cover the pots with nylon mesh. If short-day conditions are not available, or if time is limited, grow the plants under long days from the beginning, but increase the 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 the plants to long days (16-24 hours of light per day). Plants will be ready for transformation after about 1 week, when the secondary inflorescence shoots are ~3 inches tall. Proceed with the floral dip (Steps 3-12), vacuum infiltration (Steps 13-16), or spraying (Steps 17-19) methods as described below.

Floral Dip of Arabidopsis

This method was adapted from Clough and Bent (1998) .

3. Three days prior to plant transformation, inoculate a 5-ml liquid culture of Agrobacterium carrying a suitable binary vector and incubate at 28ºC with vigorous agitation. Use LB medium containing antibiotics that ｓｅｌｅｃｔ for both the Ti and the T-DNA plasmids. 4. After 2 days, inoculate 200 ml of LB medium with 1 ml of the preculture and incubate again with vigorous agitation for an additional 24 hours at 28ºC. Use YEP medium for higher Agrobacterium density. 5. Stop watering the plants and allow the soil to dry out a little, so that it will be less prone to falling out of the pots during dipping. 6. Pellet Agrobacterium by centrifuging at 6000 rpm in a GSA rotor (or equivalent) for 10 minutes. If possible, centrifuge at room temperature. Note that the cell pellet is pink. 7. Resuspend the cell pellet in 400 ml of infiltration medium. 8. Transfer the Agrobacterium suspension to a convenient vessel for dipping plants, e.g., a lid from a box of disposable pipette tips or a 400-ml beaker. 9. Invert a pot of plants and dip the inflorescence shoots into the suspension. Rest the pot on the edge of the beaker and allow the plants to soak for ~30 seconds. The same suspension can be used for 10 or more pots. Try to avoid contamination of the soil with Agrobacterium, which produces a rather unpleasant smell . 10. After dipping, lay the pots on their sides in a plastic nursery flat and place a transparent cover over them for the next 24 hours. 11. After 24 hours, remove the cover, rinse the plants with water, and return them to their normal growing conditions. 12. After about 3 weeks, collect seeds.

Vacuum Infiltration

Some labs report an increase in transformation efficiency using vacuum infiltration to draw the Agrobacterium into the plant tissue.

13. Follow Steps 3-9 above, and transfer the inverted plants and the beaker into a desiccator. Under vacuum, the suspension tends to boil over, so place a glass dish under the vessel in the desiccator. 14. Connect the desiccator to a vacuum pump and evacuate until the suspension is at a good rolling boil. If an oil pump is used, the traps must be very good to avoid saturating the pump oil. Oil-free vacuum pumps, such as recirculating-water vacuum pumps, are less trouble but they take a bit longer to draw the vacuum. House vacuum lines are usually insufficient for this purpose. 15. Release the vacuum as quickly as possible; the sudden increase in pressure will force the bacterial cells into the plant tissue. 16. Proceed through Steps 10-12 above.

Spraying

In this procedure, Arabidopsis are transformed by simply spraying the plants with an Agrobacterium suspension (Chung et al. 2000 ).

17. Grow Agrobacterium to stationary phase, harvest, and resuspend in two volumes of H2 O with 0.2% (v/v) Silwet L-77. 18. Use a spray bottle to apply the bacterial suspension to flowering plants at weekly intervals until the first seeds start to dehisce. 19. Place the plants in covered flats for 1 day before returning to normal growing conditions.

DISCUSSION

Factors that determine experiment-to-experiment variability in transformation efficiency are poorly understood. However, there are marked differences between the various laboratory strains. For example, transformation of Columbia by vacuum infiltration is about 10-fold more efficient than transformation of Landsberg erecta by the same means. This may be due in part to different floral morphologies, since the main target of transformation in this method is the ovule. A mutation in CRABS CLAW , which affects carpel development and accessibility of ovules, increases transformation efficiency of Landsberg erecta (Desfeux et al. 2000 ).

REFERENCES

Bechtold N., Ellis J., Pelletier G. 1993. In planta Agrobacterium -mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. 316: 1194�1199. Chung M.H., Chen M.K., Pan S.M. 2000. Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res. 9: 471�476.[Medline] Clough S.J., Bent A. 1998. Floral dip: A simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J. 16: 735�743.[Medline] Desfeux C., Clough S.J., Bent A.F. 2000. Female reproductive tissues are the primary target of Agrobacterium -mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123: 895�904.[Abstract/Free  Full Text]