in planta transformation

HAUGHN at SASK.USASK.CA HAUGHN at SASK.USASK.CA
Mon Sep 2 11:20:00 EST 1991


Dear networkers:

  This is in response to the requests of Tom Moran and Bob Whittier
(Aug 9) for information concerning the in 'planta' transformation
method of Chang et al.  We have preliminary results which suggest
that the method works.  
  In our experiments we used the Columbia ecotype grown in
continuous light at 21 oC and Agrobacterium tumefaciens strain
GV3101 (pMP90) (Koncz and Schell,1986, MGG 204:383-396) transformed
with a binary plasmid constructed by Dr. Ragu Datla: binsyn
backbone with a GUS-NPT fusion gene behind a CaMV 35S promoter. 
Otherwise, we followed the protocol as described at the Vienna
meeting last summer. 
  Our first attempt was last summer immediately following the
Vienna meeting.  We treated 15-20 plants and got nothing.  However,
encouraged by the E-mail report last Fall that a group at Dupont
had had success,  we tried again with more plants (80 treated). 

All of the seed obtained from those plants (I estimate over 100,
000 separated into four batches) were tested for resistance to
Kanamycin.  Seven plants were clearly resistant and all of these
tested positive for GUS activity.  We know that the seven resistant
plants represent at least four independent events since at least
one resistant plant was obtained from each of the four batches. 
Both resistance to kanamycin and the GUS activity cosegregated 3:1
(KanR/+GUS: KanS/-GUS) among the progeny of one of those lines. 
The other lines have not been analyzed.  We have not yet tested
for the presence of the T-DNA in any of the lines.  We have
initiated a third and fourth transformation experiment but have no
results as yet so it is too early to tell how reliable the method
will be but we feel that the preliminary results are quite
promising.
  
George Haughn and Marilyn Martin
Haughn at sask.usask.ca



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