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Summary: rooting Arabidopsis regenerates in soil

Kathy Wilhelm wilhelmk at studentw.msu.edu
Tue Sep 20 10:38:00 EST 1994


Many thanks to those who replied to my question about rooting
Arabidopsis regenerates in soil!  Here are the replies:
From: "Michael J. Ronemus" <ronmicj at minerva.cis.yale.edu>
I've found that tranferring Arabidopsis regenerants to soil directly from 
shoot induction medium is possible; however, what works best for me is 
taking the regenerants from SIM plates, placing them in magenta 
boxes/culture tubes in GB-5 (no hormones) with selection for approx. 2 
weeks, and then transferring to soil.  The advantages are not having to worry 
about fungal contamination and VASTLY greater seed set.  After two weeks 
on GB-5 media without hormones, the regenerants have typically developed 
more leaves and have begun to initiate roots.  The disadvantage of 
transferring to soil is that many will not survive, although the two week 
intermediate culturing step increases the survival rate markedly.  I have 
also found that the type of potting soil used makes a big difference -- 
we use Pro Mix, which works quite well; Metro Mix (what we previously 
used) is not so good.
  After transfer to soil, I keep the plants under very humid conditions 
(under cover, top-watering daily) for a week or so until the regenerants 
are established and then treat them like any other Arabidopsis plants.  
Most (>80%) survive; some will not produce substantial roots but will still 
set seed. 
     Hope this helps!
     Michael J. Ronemus  
_____________________________________________________________________________
Kathy, We used to do this all the time and it works quite well.  It helps
to have a little bit of callus still attached to the shoot.  This way you
don't need to get a real great root and it will still set seed.  We used
rootene F--with Fungicide from the local discount store.  Dip the callus or
end of the shoot in the  power, shake off the excess and put in the soil.
When you put it in the soil, place a small hole in the soil, place the
plant in and push the soil carefully around it.  Then put the shoot in a
plastic bag (Consolidated Plastics, Ohio).  Fold the bag over at the top
and staple it.  Be careful to keep the time the plant is out in the
non-humid air to a min.  After 3-4 days or when growth is obvious, open the
stapled bag.  Then slowly over approx. 5-7 days ease the plant out by
progressively cutting the bag.  We have switched to more advanced methods
(e.g., in planta and whole plant) nowadays.  GOOD LUCK!
Dr. June I. Medford
Department of Biology
506 Wartik Laboratory
Pennsylvania State University
University Park, PA 16802 USA
Telephone:  (814)-863-6092
FAX: (814)-865-9131
EMAIL:  jim2 at psu.edu
_____________________________________________________________________________
                                    
                                Your fellow servant,
                                        Kathy_|_
                                              |



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