Editing Agrobacterium tumefaciens transformation (section)

==  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.


[[Category:Protocols]]