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Acquiring transgenic lines is fundamental strategy for plant gene/protein function study. How to successfully obtain the transgenic lines is a big matter with transformation methods. Now Arabidopsis floral dip transformation method is more routinely used in hundreds of laboratories because of easily operating and high successful rate. Here simplified Arabidopsis transformation protocol was described, which was commonly used in our lab and developed from several protocols.

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[Bio101] Simplified Agrobacterium-mediated Transformation of Arabidopsis thaliana

Molecular Biology > DNA > Transformation

[Abstract] Acquiring transgenic lines is fundamental strategy for plant gene/protein function study. How to successfully obtain the transgenic lines is a big matter with transformation methods. Now Arabidopsis floral dip transformation method is more routinely used in hundreds of laboratories because of easily operating and high successful rate. Here simplified Arabidopsis transformation protocol was described, which was commonly used in our lab and developed from several protocols.

Materials and Reagents

 

1.         Agrobacterium strain(s): ABI (refs. 16, 17), GV3101 (ref. 16), EHA105 (ref. 18), LBA 4404 (ref. 19) or others

2.         1/2MS medium (2.15g Murshige&Skoog salts  (Phytotechnology laboratoriesTM), 10g sucrose, 0.8% agar per liter, pH 5.7~5.8)  autoclaved  Liquid LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter;

3.         Bio 101)

4.         5% (wt/vol) sucrose, prepared fresh (Sigma)

5.         Silwet L-77 (Lehle Seeds)

6.         Bleach (VWR Scientific )

7.         Concentrated hydrochloric acid 

8.         Appropriate antibiotics (Sigma/Fisher)

 

Equipments

 

1.         Growth chambers or light room or greenhouse, adjustable to long-day condition of 16 h light/8 h dark

2.         Plant pots: e.g., 3.5 in× 3.5 in.

3.         Laminar-flow hood

4.         Petri dishes: (100×15cm VWR)

5.         Whatwan filter paper

6.         Desiccator  jar

 

Procedure

 

1.         Grow healthy Arabidopsis plants until they are flowering. Grow under long days in individual pots in soil (optional: soil can be covered with bridal veil, window screen or cheesecloth). 

2.         When constructs are not ready, the transformation experiment can be delayed by clipping the first bolts to proliferate secondary bolts. Plants will be ready roughly 4-7 days after clipping.  Clipping is another option to get more immature flower cluster.

3.         (optional) If  you want to increase the transformation efficiency, to relatively supply more nutrient  to new buds as well,  try to remove the silliques on plants before transformation.

4.         Prepare Agrobacterium tumefaciens strain harboring gene of interest on a binary vector. Inoculate one big colony into 5-10ml liquid LB medium with the appropriate antibiotics for vector selection till saturation at 28°C, or grow in other media. Normally it takes 1.5-2 days

5.          Subculture cells to a 300ml liquid LB with the appropriate antibiotics until cells grow to stationary phase (OD600:1.5~2.0) at 28°C

6.         Collect Agrobacterium cells by centrifugation at 4000g for 15min at room temperature, and resuspend  cells to OD600 =0.6~0.8 in 5% sucrose solution ( made freshly, no need to autoclave). 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. 

7.         Sliwet L-77 need to be added to a concentration of 0.02~0.05% (vol/vol) and mix well before dipping. If there are problems with L-77 toxicity, use 0.02% or as low as 0.005%. 

8.         Pour the transformation solution with cells into proper size beaker. Then invert plants and dip above-ground parts of plant in Agrobacterium solution for 2 to 3 seconds with gentle agitation. You have to use fingers or sticks to prevent loss of soil if the soil is not coved by bridal veil, window screen or cheesecloth. You can use pipette to drop some solution to some axillary floral buds which are too short to submerge into solution. A film of liquid coating the plants should then be visible. After two spots are done, solution need to be stirred using one stick to avoid cells falling down.

9.         Place dipped plants in the tray under a dome or covered by a big plastic trash bag for 16~24 hours to maintain high humidity. Lay down the treated plants on their side. Do not expose to excessive sunlight (air under dome can get hot). 

10.     Remove the cover next day. Send the treated plants back to the greenhouse or the growth chamber. 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. 

11.     Harvest dry seed.

12.     For screening of primary transformants.  Pour 1/2MS plates containing carbenicillin to prevent bacterial contamination and the appropriate antibiotic or herbicide for positive trnasformants selection.

13.     Sterilize seeds by vapor-phase sterilization method or other methods. Place a 250 ml beaker containing 50 ml bleach into the desiccator jar, carefully add 2.5ml concentrated hydrochloric acid  to the bleach prior to seeling the jar. Keep this overnight.

14.     Place seeds on the medium evenly by gently shaking the hands holding a autoclaved whatman filter paper with seeds on.

15.     Vernalize seeds at 4°C for 3 days, then move to normal growth chamber till transformants can be readily distinguished as seedlings with healthy green cotyledons and true leaves and roots that grow into the selective medium.

16.     Transplant putative transformants to soil.

For higher rates of transformation, plants may be dipped two or three times at seven day intervals. Do not dip less than 6 days apart.  For the many different background materials transformed with the same construct, spraying transformation method is more efficient.

 

References

 

1.         Bechtold N., Ellis J., and Pelletier  G. (1993). In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199. 

2.         Clough S.J., Bent A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16(6): 735-43. 

3.         Additional commentary can be found by searching the Arabidopsis newsgroup archives.

4.         Zhang X., Henriques R., Lin S.S., Niu Q.W., Chua N.H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-6. 

5.         Clough S and Bent A.  Simplified Arabidopsis Transformation Protocol (Brief version for those who are familiar with the method) 

 



How to cite: Chen, L. (2011). Simplified Agrobacterium-mediated Transformation of Arabidopsis thaliana. Bio-protocol Bio101: e41. DOI: 10.21769/BioProtoc.41; Full Text



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