root culture

[The Fifth Beatle]

> Dear netters,
> Does anyone has experience with root culture conditions?
> Suddendly I'm getting in trouble with my transgenics root cultures obtained
> after A. rhizogenes infection. They are growing very slow and with no
> branching of lateral roots. Furthermore, they start to become dark in the
> region of the central stele after awhile. I tried to change the vitamins
> and MS powder batch with no success. I previously grew my transgenic roots
> in MS/2 medium, + 3% sucrose + vitamins, 1.2% agar and this conditions were
> fine.
> Any advice will be welcome
> Maurizio



Hi Maurizio,

I have similarly-transformed Arabidopsis root cultures growing in 
liquid B5 (Sigma - Gamborg's B-5 basal medium with minimal organics) 
with 3% sucrose (just normal sugar from the supermarket works fine).  
No extra vitamins are necessary.  I do however add Timentin 
(200mg/L) to keep the Agro at bay.  The cultures are maintained in 
250mL glass jars on an orbital shaker (@40-50 rpm) in a 20 degree C 
constant temperature room.

In our lab we mainly use MS medium for growing whole plants or calli 
and only use B-5 for roots (solid or liquid cultures).  

Under these conditions the roots look very healthy and only need sub-
culturing every 4-6 weeks.  Reducing the sucrose levels will allow 
sub-culturing at longer intervals.

Hope you find this helpful,

Eugene


**********************************************************************
Eugene B. Fredericks                     * "And in the end, the love
Dept. Genetics & Developmental Biology   *  you take is equal to the
Monash University                        *  love you make..."
Wellington Rd, Clayton 3168              *    -  The last line of the
Victoria, AUSTRALIA                      *       last song on the last
E-mail: E.B.Fredericks at sci.monash.edu.au *       Beatles album   
**********************************************************************