To readapt a culture on plates, simply transfer
some of the cells back into liquid
media. We usually pipette the cell suspension up and down to break up any clumps. It
may be best to start out with a smaller culture volume when you first go back to liquid;
BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow
until it is the consistency of thin applesauce. At this point, you should be able to go to
a 50 ml culture and start subculturing as described below. In our experience, wild-type
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more
variable, with some producing smooth cultures, some producing clumpy ones, and some going
back and forth between these two states. We've found that clumpy cultures do not interfere
with our half-live measurements, although manipulating them can be a bit more difficult.
We grow our liquid cultures in 50 ml
of media in 250 ml baffle flasks at 28 degrees C
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many
people grow these cells in regular flasks with no problem. We subculture them once a week
by transferring 5% of the culture to fresh media. We generally maintain two flasks
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday)
in case one of the cultures crashes. Also, you can maintain the culture on a plate.
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume,
e.g. 10 ml, for convenience.
(amounts are for 1L of media):
750 ml H2O
4.3 g MS salts (add slowly to liquid)
30 g Sucrose
50 ml 20X MES pH 5.7
10 ml B1 -inositol
3 ml Miller's I
10 ml 2,4-D, 10-4 M
pH to 5.7 with 0.1 N KOH
Bring volume to 1000ml
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving
Add kanamycin (100 mg/ml)
Add carbenicillin (250 mg/ml)
60 g KH2 PO4 per liter
20 X MES:
10 g MES per liter, pH to 5.7 with 1N KOH
B1 - Inositol:
0.1 g Thiamine
10.0 g myo-inositol
H2O to final volume of 1 liter
1. Grow up 1 ml of the Agrobacterium overnight
in LB + all selective drugs.
This culture may be started from frozen glycerol cultures of necessary. We know that
LBA4404 and GV3111 work well for this protocol.
2. BY-2 cells are used 3 days after splitting the
BY-2 cell culture. 4 ml of BY-2
cells are required for each transformation with an additional 4 ml for
the control culture which receives no bacteria.
3. 1 ul Acetosyringone (20 mM in ethanol) is added
per ml of BY-2 cells. I
usually just treat the whole 50 ml culture at this point and discard any
that is left over when I'm finished.
4. Using a 10 ml pipette, the BY-2 cells are pipetted
in and out about 20
times. This helps to induce small lesions in the cells and increases the
efficiency of the transformation.
5. 75 ul of bacteria (dense growth) or 100 ul (moderate
growth) are added to
a petri dish containing 4 ml of BY-2 cells (from step 4) and mixed
thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA.
6. Wrap plates with parafilm and incubate for 3 days
at 28 degrees C.
7. To each plate add 10 ml of NT liquid medium containing
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC.
9. Centrifuge at 1K for 4 minutes at room temperature
in a swinging bucket
10. Aspirate off liquid using pipet capped with a sterile blue
1 ml top.
Change tip for each sample.
11. Resuspend in 50 ml NTC and repeat spin.
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are
to carb require fewer washes. I have found that LBA4404 generally
requires only 2 washes.
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml
on 2 NTKC
(kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no
longer lots of fluid on the plates (leave the plates open in the hood
a few minutes of necessary), wrap them in parafilm.
14. Incubate at 28 degrees C. Transformants should be large
enough to transfer to
fresh NTKC plates in 3-4 weeks.
Supplies for each transformation (Remember the controls):
Day 1 Supplies:
LB + appropriate drugs
Agrobacterium containing plasmid for transformation.
Day 2 Supplies:
1 ml Agrobacterium overnight culture
4 ml BY-2 cells - 3 days post subculture
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer
pipets and pipetman
1 petri plate
Day 5 Supplies:
200 ml NTC liquid
50 ml conical tube
swinging bucket centrifuge at room temp
aspiration setup with 5 ml pipet capped with 1 ml blue tip
2 NTKC plates
pipetmen and tips
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