This protocol is modified from Bechtold, Ellis and Pelletier (1993). “In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants”. [C.R. Acad. Sci. Paris, Life Sciences 316: 1194-1199].
1. Take seeds with a brush and place them into 8cm square pots filled with soil. Don’t compress the soil too much and water the pots thoroughly with 2-3 pot-vol to remove excess nutrients. Place 12-16 seeds in each pot.
Place the pots in the cold room for two days before transfering them to the growth chamber. Grow the plants for three weeks in short days (10 hr or less) to get large plants and a greater seed yield. Transfer the pots to long days to induce bolting. Grow plants to a stage at which bolts are around 10 cm tall.
2. Clip off emerging bolts close to rosette leaves to encourage growth of multiple secondary bolts. Infiltration will be done 7 to 9 days after clipping (plants will be 10-15 cm high and the biggest of the inflorescens will have made the first tiny silique. Do not water the plants the day before vacuum infiltration.
3. Start a 4ml agrobacterium culture (YEP+antibiotics) inoculated from a -800C stock or from a plate. Grow cells O/N to 48h depending on the strain. Add this culture to 250 ml of YEP+antibiotics (A 250ml culture will give enough cells for infiltration of 6 pots). Grow the culture between O/N and 2 days (depending on the strain) to OD600 = 1.2-1.8. The culture will have a mother of pearl appearance (not lumpy or black).
4. Spin down agros at 5000rpm for 10 min in 250ml centrifuge bottles, resuspend in infiltration media to an OD600 = 0.8 in a minimum volume of 300ml.
5. Poor the agro suspension into a beaker of an appropiate size (400ml is ok). Place the beaker into the vacuum jar. Degass the solution by drawing vacuum until bubles form. Place a paper towel under the beaker to avoid that the beaker gets stuck in the bottom of the vacuum jar.
6. Sprinkle the plants with water 5 min prior to infiltration (optional) and then invert plants into the culture solution. Make sure that all the flowers are submerged and leave 2cm between the rosettes leaves and the agro suspension. Don’t let the culture contact the rosette or soil as this could kill the plants. Avoid that the solution boils over when you pull the vacuum. Make sure that the soil is only moist, so that the water from the pots does not enter into the culture suspension (therefore we recommend not to water the plants the day before infiltation). Draw vacuum for 15-20 min for WS and 30 min for Col-0 at a pressure close to 0.05 Bar (we are using an oil pump).
7. Before removing the plants from the vacuum jar place a plastic bag over the pot and beaker. Pull out and remove plants from the beaker, lay pots on their side (to avoid that excess infiltration media runs down into the soil). Fold over the top of the plastic bag and staple them twice. The other possibility is to place the pots laying on their side into a tray and cover the whole box with saranwrap. Put them in a growth chamber for one night. Next day move them to the green house. Put the plants in vertical position and open the bags. Next day get rid off the bags. In case you have the plants in trays: put also the plants in vertical position and use sticks and saranwrap to make a kind of a tend around the plants. Next day remove the plastic. In hot summers, we recommend to give plants a shower after we have placed them in vertical position (the purpose of this is to remove the sugars from the infiltration media which decrease fungal infection).
8. Grow plants for approx. four weeks, keeping bolts from each pot together but separated from neighbouring pots
9. When the siliques begin to turn yellow, place the pot on its side with the plants inside a big envelope. Leave them for one week to dry out and cut off the plants. Let the seeds dry in the envelope and clean them 10 days later (keep all the seeds from one pot together). Store the seeds in the cold room for one week before plating them.
KANAMYCIN SELECTION PROTOCOL
1. Sterilisation of seeds:
aliquot seeds in 15ml falcon tubes (approx 700 seeds/tube, you can estimate the ammount of seeds by first drawing a square plate of 9cmx9cm on a paper and spreading the seeds on it). Add 10 ml of hypoclorite solution. Shake tubes for 10 min. Remove the solution and add 10ml of 70% ethanol. Wait 2 minutes. Discard EtOH and wash seeds 2-3 times with 10ml of sterile water.
Resuspend seeds with 8ml 0.7% top agar (no warmer than 55oC ).
2. Spread seeds onto selection plates (MS+Kan). Dry plates in laminar flow hood until the top agar has solidified.
3. Vernalize plates for two nights in the cold room at 4oC. Transfer the plates to the growth chamber (21oC with continous light).
4. After aprox 7 days transformants should be clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend into the selective medium. Root growth is the most clear maker to identify transformants at an early stages.
To make sure that the transformants are positive transfer them to a new MS+Kan plate and leave them there for a few days (if they turn yellow is because they are faulse positives). Transfer the seedlings to soil.
If you have contamination on your plates at this step, transfer the transformants as early as possible to soil.
5. Grow the plants and collect the seeds.
1/2 x Murashige&Skoog salts (SIGMA #5524)
1X B5 vitamines (1ml of 1000x stock) (SIGMA; #G-2519) Gamborg’s vitamine powder, to prepare the 1000x stock disolve 11.2g in 100ml water.
adjust to pH 5.7 before autoclaving
after autoclaving add:
– Benzylamino Purine (BAP), 10 µl per liter of a 1 mg/ml stock in DMSO. By adding the hormone just before use, you can keep infiltration media as a stock for at least one week prior to infiltration.
– we recommend to add 0.01% silwet to the infiltration media to increase transformation efficiency especially for Landsberg and colombia ecotypes. (silwet is from LEHLE SEEDS, cat no VIS-01 VAC-IN-STUFF (silwet L-77)
1x Murashige&Skoog salts
adjust pH 5.7 with 1M KOH.
0.7% Difco agar.
autoclave, cool, and add:
1x MS vitamines (SIGMA #M-7150). Take 1ml of 1000x stock prepared by disolving 10.3gr in 100ml of water.
antibiotic (kanamycin 50mg/l).
1x Murashige&Skoog salts.
adjust pH 5.7 with 1M KOH.
0.7% Difco agar.
before use: boil in the microwave and keep in water bath at 50-55C.
YEP media (liquid):
10 g /l Bacto peptone (Difco)
10 g/l Yeast extract (Difco)
5 g /l NaCl
For YEP plates add 15gr/l Difco bacto agar.
for 50 ml:
4ml Na Hypoclorite 15%
water to 50ml