TRANSFORMATION OF ARABIDOPSIS BY VACUUM INFILTRATION Posted to the Arabidopsis bulletin board; 11 Jan 1994 Andrew Bent e-mail address:

abent@ux1.cso.uiuc.edu This protocol is based on the work of Nicole Bechtold, Jeff Ellis and Georges Pelletier. My modifications were incorporated to streamline their procedure. The most significant changes eliminate the need to uproot and re-plant infiltrated plants. - Andrew Bent Plant Growth: 1. Grow plants of the appropriate genotype to a stage at which bolts are just emerging. I have found that it works well to grow 12-15 plants in a 3.5" pot. If pot is covered with nylon window screen after planting, plants grow through the screen and when pot is inverted for infiltration less dirt falls out. If plants are grown for the first four weeks in short days you will get larger plants and a greater seed yield (transfer plants to long days to induce bolting). Success may also depend on frequent fertilization and strong light intensity. 2. Clip off emerging bolts to encourage growth of multiple secondary bolts. Infiltration will be done four to eight days after clipping. Vacuum Infiltration: 3. Grow a large liquid culture of Agrobacterium carrying the appropriate construct. Start a 25 ml overnight (LB + antibiotics) two to three days ahead of time. Add this culture to 400ml of LB + antibiotic the day before infiltration. My experiments were done using A. tumefaciens GV3101pMP90 (C. Koncz and J. Schell, 1986, Mol. Gen. Genet. 204:383-396). 4. Harvest cells by centrifugation (5K 10min. in GSA rotor, preferably at room temp.) and resuspend in 3 volumes infiltration medium (OD600 approx. 0.8). Harvest cells at an OD600 of >2.0. A 400 ml culture will give enough

cells for infiltration of at least six pots. 5. Add Agrobacterium (in infiltration medium) to a dish or beaker and invert plants (pot, soil, and all) into liquid solution. Be sure bolts and entire rosettes are submerged. A one liter beaker filled with >200 ml of solution fits well with our 3.5" pots. Bacterial solution can be extended by reusing for at least one additional pot. 6. Place beaker into bell jar. Draw a vacuum until bubbles form on leaf and stem surface and solution starts to bubble a bit, then release vacuum very rapidly. The necessary time and vacuum pressure will vary lab-to-lab. Practice on a few dispensable plants first. Good infiltration is visibly apparent as uniformly darkened, water-soaked tissue. Be sure to have good traps in your vacuum system or you will quickly saturate the pump oil. 7. Remove plants from beaker, lay them on their side into a plastic flat and cover with plastic wrap or a dome to maintain humidity. The next day, uncover plants and set upright. 8. Grow approximately four weeks, keeping bolts from each pot together and separated from neighboring pots. 9. When siliques on plants are very dry, harvest seed (all seed from one pot together). Selection of Putative Transformants: Kanamycin selection protocol: (Note that Basta selection is much less labor intensive - but your present binary vector system is more likely to encode antibiotic resistance.) 10. Pour selection plates.

Plastic 150 x 15 mm petri dishes are convenient. 11. Sterilize seed.

A variety of sterilization protocols are appropriate. I place seed in 15 ml plastic orange cap tubes and then treat: 1 minute in ethanol or isopropanol

5 minutes in 50% Bleach/50% water/0.05% Tween. 3 rinses with sterile water. It is advisable to add one or two control seeds from a known transformed plant onto a marked location on at least a few of the selection plates. Sterilize these seed also. 12. Plate seed by resuspending in sterile, room temperature 0.1% agarose and spreading onto selection plates. Dry plates in laminar flow hood until seed no longer flows when plate is tipped. Use one ml agarose for every 500-1000 seed. Plate 2000 to 4000 seed per 150 x 15 mm plate. Higher densities can make antibiotic selection less effective. 13. Vernalize plates for two nights in cold room. growth chamber. Move plates to

14. After about 7 days, transformants should be clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend over and into the selective medium. 15. Transplant plantlets to soil, grow, and collect seed.

Transplanting success is improved by breaking up agar around root prior to pulling, by removing any adhering chunks of agar from root before planting, by saturation of soil with water after transplanting, and by growing plants under a dome (for high humidity) for the first day or two. If you break the root, put plantlet onto a new selection plate for a few days before transplanting. Infiltration Medium: 1/2 X Murashige & Skoog salts 1 X B5 vitamins 5.0% Sucrose .044 uM Benzylamino Purine (10 ul per liter of a 1 mg/ml stock in DMSO) Selection Plates: 1/2 X Murashige & Skoog salts 0.8% Agar Autoclave, cool, then add: 1 X B5 vitamins Antibiotic (such as Km 50 ug/ml)

The citation for the above protocol is: Bent,A.F., Kunkel,B.N., Dahlbeck,D., Brown,K.L., Schmidt,R., Giraudat,J., Leung,J. and Staskawicz,B.J. (1994). RPS2 of Arabidopsis thaliana: A leucine-rich repeat class of plant disease resistance genes. Science 265:1856-1860. It would also be good to cite the inventors of this approach, Bechtold et al.: 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. Good Luck! - Andrew Bent ------------------------------------Andrew Bent Assistant Professor Department of Agronomy University of Illinois Urbana, IL 61801 Phone 217-244-6308 Fax 217-333-4777