Arabidopsis transformation by vacuum infiltration
_____
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].
PLANT GROWTH:
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.
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.
Infiltration Media
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.
5% sucrose
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)
Selection plates:
1x Murashige&Skoog salts
1% sucrose
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).
Top agar:
1x Murashige&Skoog salts.
1% sucrose.
adjust pH 5.7 with 1M KOH.
0.7% Difco agar.
autoclave.
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.
Hypoclorite solution:
for 50 ml:
4ml Na Hypoclorite 15%
255l Tween-20
water to 50ml