Plant transformation using TAC clones

Plant Transformation Using JAtY CLones

-.Arabidopsis transformation with large bacterial artificial chromosomes. Alonso JM, Stepanova AN. Methods Mol Biol. 1062:271-83 (2014)

To obtain uniform and relatively high (0.1-2%) transformation efficiencies, glucose instead of sucrose should be used during the floral dip transformation. The detrimental effect of sucrose on the transformation efficiency is probably the result of a leaky expression of the SacB in some of the clones. The expression of the SacB in Agrobacterium depends on the promoter activity of the Arabidopsis sequences cloned upstream of SacB.

The procedure used in the lab is as follows:

1. Inoculate from a single colony 50 ml of LB supplemented with kanamycin (25 micrograms/ml) in a 250ml Erlenmeyer flask.

2. Grow the cultures at 28ºC for 24-48h (until near saturation) with constant shaking.  [Please, note that some Agro strains do not do well in liquid.  If that is the case for your strain, instead of the initial 50 ml liquid culture, streak the Agro colony on the surface of a 10mm LB/kanamycin plate and grow for 24-48 hours at 28ºC.  Wash off the cells from the surface of the plate with ~20 ml liquid LB/kanamycin directly into 500 ml liquid culture below – see step 3].

3. Use the entire 50 ml of the liquid Agrobacterium culture [or, for some strains, the ~20ml cell suspension from plates] to inoculate 500 ml of LB/kanamycin in a 1 liter Erlenmeyer flask.

4. Grow cells at 28ºC for additional ~24h with constant shaking.

5. Pellet the Agro cells by centrifugation at room temperature.

6. Resuspend Agro in 250 ml of a Dipping Solution (5% glucose in water, plus 200 microliter/liter Silwet-77).

7. Perform floral dip (Clough and Bent. Plant J. 16:735-743) or vacuum infiltration (Alonso and Stepanova. Methods Mol Bio. 236:177-88.). We have not observed any differences in the transformation efficiency between these two procedures.

8. Place the pots with transformed plants horizontally in a clean tray and cover the trays with a transparent plastic dome immediately to maintain high humidity.

9. Move the trays with the transformed plants back to the growth chamber and keep them covered with the transparent plastic domes for ~24h.

10. After ~24h, shift the plastic domes ~2-3 cm sideways to facilitate air exchange (to reduce humidity gradually). Allow the plants to recover for another ~24h.

11. After the second 24h, remove the plastic domes completely and position the pots with the plants right side up, i.e. vertically.

12. Water plants as needed and continue watering until flowering is finished.

13. Allow the plants to fully dry prior to seed collection.

Selection of transformants in BASTA (Phosphinothricin, glufosinate ammonoium) plates.

1.Sterilize seeds for 10-15 minutes in 50 ml tubes using 30-40ml of 50% bleach spiked with 3-4 drops of Triton-100 per liter (to avoid seeds clumping and aggregating together).  Make sure the seeds are fully resuspended.  Invert/shake the tubes with seeds occasionally during the 10-15 minutes of sterilization.

2.Wash the seeds 3-5 times with ~50 ml sterile di H2O.  After each wash, remove as much water as possible.

3.To the seed suspension in di H2O, add Basta and Timentin to 20 microgram/ml and 300 microgram/ml final concentration, respectively.  For example, if the residual volume of seeds in water is 5 ml, add 5 microliters of 10 mg/ml Basta and 300 mg/ml Timentin.  Timentin inhibits Agro growth that survived bleach sterilization under the seed coat.

4.Cold-treat the tubes with seeds at 4ºC for 2-3 days to equalize germination.

5.Equilibrate the tubes with seeds to room temperature for 15-30 minutes and add to the seed suspension melted, pre-cooled 0.7% top agarose in water.  Be careful not to use hot top agarose, as it will kill the seeds.  On the other hand, if the agarose is too cool, it will solidify when mixed with the room temperature seed suspension and make clumps. We typically use 1-3 volumes of top agarose per each seed suspension volume.  For example, add 5-15 ml of 0.7% top agarose to 5 ml seed suspension.

6.Use plastic single use 10ml pipettes to uniformly distribute the seed/agarose suspension on the top of AT plates (1x MS salts, 1% sucrose, pH 6.0; 0.6-0.8% agar) supplemented with 20 micrograms/ml Basta.  Plate up to 8000 seeds (~0.2g dry weight) resuspended in 5-15 ml top agarose per each 150mm Petri plate containing 50-70ml AT/Basta.

7.Incubate plates in the light for 1-2 hours at room temperature to improve germination and place the plates in the dark incubator at 22ºC for 3 days.

8.After 3 days in the dark, move the plates to the light for 2-5 days.  Check the plates periodically.  Basta-resistant plants (transformants) will develop green color upon light exposure.  Sensitive plants (untransformed) will remain bleached or will fail to germinate all together.

9.Transplant Basta-resistant plants to soil and propagate.