From: hauser@mcclb0.med.nyu.edu
Newsgroups: bionet.genome.arabidopsis
Subject: Arabidopsis root culture protocol
Date: 1 Oct 92 01:25:28 GMT
Organization: NYU Medical Center, New York, 10016


Dear Network Readers:

     Having seen repeated questions on the bulletin board
concerning propagation of Arabidopsis roots we feel tempted to
share with the Arabidopsis community our protocol before
publication.

"Protocol for mass clonal propagation of Arabidopsis roots"

This protocol (Czako, M and Marton, L; manuscript in preparation)
has been developed for the 'RLD' ecotype, both wild-type and
transgenic but has also been succesfully used for the Columbia
ecotype and its NR deficient mutant G-5. We have maintained one of
the cultures for 18 months and others for more than a year.

1. Inoculate 25 mL liquid MS or ARC medium with one or more
seedlings or rooted shoots. Incubate at R.T. on an orbital shaker
at approximately 70-80 RPM. They will develop an extensive root
system in about two weeks.
2. The roots are then excised and transferred into fresh ARC liquid
medium and given a two day treatment with 0.05 mg/L IAA.
3. The roots are transferred to fresh ARC medium.
4. The IAA treatment is repeated every 2-4 weeks.
 Comments:
- the sustained root culture has become hormone independent,
because it has been growing without IAA treatment for months now.
- if the roots are chopped into smaller segments at the time of IAA
treatment, they tend to form a tangle of old and fresh roots, from
which the fresh roots are difficult to separate. If a 10-15 mm
diameter size disc of the root mat is excised from the mass and
transferred into fresh medium roots will grow out from the 'disc',
and the fresh roots are easy to separate.
- these roots are suitable for transformation by our root
transformation protocol, though the frequency of shoot regeneration
is not as high as in the cold stored seedling derived roots (see
Marton and Browse, 1991. Plant Cell Reports, 10. 235-239) but still
very good. So far that has been the only way of clonal (mass)
propagation of some important (e.g. heterozygotes in our lab)
material for transformation and biochemical analysis (J. Browse,
WSU) purposes. A further improvement is on the way to restore or
even to surpass the original morphogenic potential and therefore
the transformation efficiency...

     ARABIDOPSIS ROOT CULTURE MEDIUM (ARC)

  For 1000 ml:
             MS salts               4.3  g
             Miller's solution      3   ml
                  [6% (w/v) KH2PO4 stock solution]
             myo-inositol         200   mg
             Vitamix stock          2   ml
             Sucrose               30    g
     Set pH to 5.8 with 1 M NaOH.
     Dispense liquid 25 ml-wise into 125 ml Erlenmeyer flasks.
Vitamix: 500X stock solution:
              100 ml:
     Vitamin B1         500 mg
     Vitamin B6          50 mg
     Glycine            100 mg
     Nicotinic acid      50 mg
     Folic acid          25 mg
     Biotin (Vitamin H)  50 mg

Sincerely,                    Mihaly Czako and Laszlo Marton
                              Department of Biological Sciences
                              University of South Carolina
                              Columbia, SC20208
                              803-777-6676
Newsgroups: bionet.genome.arabidopsis,bionet.molbio.hiv,bionet.molbio.methds-reagnts