VACUUM INFILTRATION OF ARABIDOPSIS PLANTS
 
Last Update: December 2006
 
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. III-VIE Paris 316: 1194-1199; Bent A, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, Giraudat J, Leung J, Staskawicz BJ (1994) RPS2 of Arabidopsis thaliana - A leucine-rich repeat class of plant-disease resistance genes. Science 265: 1856-1860; Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383-396; Gamborg OL, Miller RA, Ohyama K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 148-151; Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Based on the protocol for vacuum infiltration developed at Pamela Green's laboratory
 
PREPARE SOLUTIONS
1. Yeast Extract Peptone (YEP) medium (1 L):
Mix 10 g of yeast extract , 10 g of peptone, 5 g of NaCl, in 800 mL of dH2O. adjust to pH 7.0. Add dH2O to 1 L (Autoclave)
2. YEP plates (1 L):
Mx 1 L of YEP media and 15 g of agar (Autoclave). Cool to 50oC (leave in a 50oC water bath until used). Add antibiotics
3. Arabidopsis fertilizer (10 L):
Mix 50 mL of 1M KNO3, 25 mL of 1M KPO4 (pH 5.5), 20 mL of 1M MgSO4, 20 mL of 1M Ca(NO3)2, 5.0 mL of 0.1M Fe.EDTA and 10 mL of micronutrients. Dissolve in ddH2O and store at room temperature
4. Vacuum infiltration suspension solution (2 L):
Mix 1.6 L of dH2O, 4.4 g of MS salts, 6.38 g of Gamborg's B5 basal medium with minimal organics (Zigma, G5893), 100 g of Sucrose, and 1.0 g of Mes. pH to 5.7 w/ KOH. Add 2.0 mL of 10 mg/mL Benzyl adenine and 400 mL of Silwet L-77 (Lehle Seeds, catalog # vis-01). Add dH2O to 2 L
5. Arabidopsis micronutrients (500 mL):
Mix 70 mL of 0.5M boric acid, 14 mL of 0.5M MnCl2, 2.5 mL of 1M CuSO4, 1.0 mL of 0.5M ZnSO4, 1.0 mL of 0.1M NaMoO4, 1.0 mL of 5M NaCl and 0.05 mL of 0.1M CuCl2. Dissolve in ddH2O and store at room temperature
Note: Gamborg's B5 basal medium with minimal organics contains all the micronutrients, macronutrients, and vitamins as described in Gamborg OL et al. (1968)
   
PROCEDURE - Step 1 - Introduction of binary vector into Agrobacterium tumefaciens
There are many ways to introduce a binary vector (a vector used for Agrobacterium-mediated transfer of DNA and lacking the vir genes present in the original Ti-plasmid) into Agrobacteria, such as (1) chemical methods that disrupt the cell wall of the bacteria; (2) tri-parental mating between the bacteria harboring the binary vector and the Agrobacteria with the help of a helper strain, and (3) making Agrobacteria electrocompetent for electroporation
Electroporation is described here as it is one of the simplest and most efficient methods:
1. Start 4x 75 mL YEP overnight cultures of Agrobacteria (strain GV3101) in 250 mL flasks

2. Grow at 28oC, shaking (Agrobacteria will dye at 37oC, and at temperatures of 32-33oC they have a tendency to lose their vector)
3. Prepare: 3.5 L sterile H2O chilled to 4oC, 10 % glycerol, autoclaved chilled to 4oC, Four Centrifuge bottles, autoclaved
4. From each overnight culture, innoculate 1 L of YEP in 2 L flasks (1:200)
5. Grow overnight at 28oC shaking
6. Allow 1 L cultures to grow to OD595 = 1.5

7. Aliquot cultures into centrifuge bottles and centrifuge for 15 mins at 5000 rpm to pellet cells
8. Resuspend cells in cold sterile H2O (2 L total)
9. Pellet cells again for 15 mins at 5000 rpm
10. Resuspend cells in cold sterile H2O (1 L total)
11. Pellet cells a third time for 15 mins at 5000 rpm

12. Resuspend cells in 2x 20 mL aliquots of cold 10% glycerol and transfer to Oakridge tubes
13. Pellet cells one last time for 15 mins at 6000 rpm
14. Resuspend each pellet in 1 mL 10% glycerol (cold)
15. Aliquot into 1.5 mL eppendorfs on ice, 80 µL each and freeze in EtOH/dry ice bath
16. Store at -80°C
   
PROCEDURE - Step 2 - Electroporation
17. Remove from -80oC tubes containing 80 mL of electro-competent Agrobacterium GV3100 cells

18. Thaw cells on ice
19. Add ~1-3 mg of binary vector to the cells. Incubate on ice for up to 3 mins
20. Transfer the mixture of cells and DNA to an ice-cold electroporation cuvette (0.2 cm electrode gap). Make sure the suspension is at the bottom of the cuvette
21. Set a Gene Pulser apparatus (Bio-Rad) at 25 mF and the voltage at 2.5 kV. Set the Pulse resistance controller to 200 W

22. Place the cold cuvette in the chamber slide (Cuvette notch facing away from you)
23. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber
24. Electroporate by pushing both red bottoms, until a beep sound (~4.6-4.8 seconds)
25. Remove the cuvette from the chamber and immediately add 1 mL of YEP/LB medium to the cuvette and quickly resuspend the cells by pipetting
26. Transfer the cell suspension from the cuvette to 1.5 mL tubes and incubate on a shaker (200 rpm) at 28oC for 2 h to allow recovery and expression of the antibiotic resistance markers (Clean cuvettes successively with ddH2O, EtOH, sterile water, then wrap with aluminum foil and finally autoclave)

27. Pipette 200 mL of each transformation on YEP plates containing selection (for Agrobacterium strain GV3101 add 50 mg/mL gentamycin)

28. Spread the cells with a bent glass rod. Place plates inverted at 28oC for 2-3 days in the dark
29. Check for proper transformation by performing a plasmid mini-prep and checking by restriction digest
   
PROCEDURE - Step 3 - Preparation of Arabidopsis plants
1. Arabidopsis seed are sown onto mesh-covered pots containing enough fertilizer -soaked-soil to form a mound that rises ~1 inch above the pot. Hold the mesh firmly against the pot with a rubber band. Plants are grown under 16 hours of light and at ~22oC (see Figure 1)
Figure 1
Figure 2
Figure 3

2.Since a large amount of seeds were planted to insure germination, as plants germinate and grow they need to be thinned to only a few per pot. Make sure plants are constantly humid by irrigating with fertilizer. Drying will result in early bolting but also in small weak plants (see Figure 2)

3.Once plants start to bolt, they are ready to be transformed. Clipping of the primary bolts can be performed to induce lateral bolt formation (more flowers) and if the plants are not needed just yet. Otherwise, this is not necessary (see Figure 3)
   
PROCEDURE - Step 4 - Growth of Agrobacterium containing clone of interest for transformation
1. Streak Agrobacteria containing binary vector onto a YEP/LB plate containing the appropiate antibiotics
2. Incubate at 28oC for 1-3 days

3. Inoculate a large amounts of Agrobacteria from the plate into 5 mL YEP/LB containing the appropiate selection. Incubate at 28oC while shaking (150 rpm) for 1-2 days. It is normal to see a clumpy culture
   
PROCEDURE - Step 5 - Vacuum infiltration of Arabidopsis plants
1. Start Agrobacterium cultures (two per construct) by inoculating the 5 mL from step 4 (3.) onto 500 mL of LB with the appropiate antibiotics in a 2.5 L flask
2. Grow for 1-2 days (ON) at 28oC in the dark at 250 rpms. An OD600 of about 1-2 is good (clowdy culture)
3. Transfer Agrobacterium cells to centrifuge tubes and centrifuge at 6,000 g for 5 mins at 4oC
4. Discard supernatant and resuspend cells in a total of 300 mL of Vacuum infiltration suspension solution (add less at the beginning in order to make the resuspension easier -use a pipette if necessary)

5. Bring the total volume to 1 L for each 500 mL culture (2 L per construct) with Vacuum infiltration suspension solution
6. Add ~1 L of Agro suspension to a 4 L vacuum jar (12 cm diameter). The idea is to cover enough at the bottom so that the plants will be completely submerged. Place 3 plastic pots containing plants on the jar grooves (flowers facing down)
Figure 4
Figure 5
6.1 Transfer Arabidopsis plants to a clean area covered with some bench top paper. Surface sterilize a vacuum jar and fill the bottom with the Agrobacterium solution (see Figure 4)
6.2 Transfer the pots upside-down into the jar so that ALL the plant material is in contact with the Agrobacterium solution. Try to minimize the amount of soil that is in contact with the solution (see Figure 5)
7. Cover the vacuum jar and apply vacuum (400 mm Hg or about 17 inches). Bubbles should appear as the pressure drops. Once the desired level of vacuum is reached, close suction. Keep the vacuum for 2-5 mins and then release immediately
Figure 6
Figure 7
Figure 8
8. Let the plants sit for 2-5 mins in the solution. At this point the plants will look droopy and saturated (the Agrobacterium solution that was introduced will maketheleaves look somewhat translucent and a darker shade of green) (see Figure 6)
9. Transfer the pots to a big tray and place them horizontally (this allows drainage of the Agrobacterium solution). Cover tray with plastic, transfer to a growth chamber and leave for 24 hours (see Figure 7)
9.1 Wash vacuum jar with chlorox and then with water, followed at the end with ethanol
10. After two days, remove plastic, upright plants by supporting them with sticks and irrigate them

11. Irrigate plants for 2-4 more weeks or until they are fully mature (finished flowering and setting seed)

12. After less than 4 weeks, stop irrigation and leave plants to dry (see Figure 8)
   
PROCEDURE - Step 6 - Selection of Transformants
1. Once plants are dry collect seed. The siliques/pods should be dry enough by this point that seeds should fall just by touching the pods. Separate the seeds from the pods using a mesh. Seeds may be stired at 4oC (this aids in germination) (see Figure 9)
Figure 9
Figure 10
Figure 11
2. Sterilize seeds (100 mL/2500 seeds per plant) using a 30-50% bleach/0.1-0.02 % Triton X-100 solution (6-10 minutes rocking). Wash away any trace of bleach by washing at least 4x with ddH2O (see Figure 10)
3. Resuspend seeds in 8.0 mL sterile 0.1% agarose and pour onto 150x15 mm GM plates containing the appropiate selection. Spread seeds evenly over the entire area and let dry for 10-30 minutes. Seal plates with surgical tape and place in a growth chamber (see Figure 11)
4. After about a week will start developing their first true leaves. At this point it should be possible to select putative transgenics (green plants) from untransformed plants (bleached plants)
Figure 12
Figure 13
Figure 14
5. Transfer putative transgenics (green plants) onto a second selection plate. This will reduce the chance of selecting false positives
6. Transfer 2-3 week old plants to soil
Note: when plating seed, antibiotics that kill Agrobacterium: Carbenecillin 500 mg/mL, Vancomycin 500 mg/mL; Ampicillin 500 mg/mL; Cloxacillin 500 mg/mL