CARE OF A BY-2 CULTURE

     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.
 
 

NT KC MEDIA - LIQUID OR PLATES

(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
Autoclave
 

SOLID MEDIA:

For plates only:  Add to flasks  7 g/ L Phytagar before autoclaving

Add kanamycin (100 mg/ml)

Add carbenicillin (250 mg/ml)
 

MEDIA COMPONENTS:

Miller's I:
     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
 
 

TRANSFORMATION PROTOCOL:

Day 1:

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.
 

Day 2:

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.
 

Day 5:

7.    To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml
       carbenicillin (NTC).

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
       rotor.

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 fairly sensitive
       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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