in planta transformation
HAUGHN at SASK.USASK.CA
HAUGHN at SASK.USASK.CA
Fri Sep 6 15:10:00 EST 1991
Since my E-mail message concerning 'in planta' transformation (see
below) several people have asked for a detailed protocol. The
protocol we used is outlined below. I would like to stress three
points concerning this protocol. First, it is based on the
technique described by Chang, Park and Nam at the Arabidopsis
meeting in Vienna (June, 1990). We have not modified the technique
significantly. Second, we have not done controlled experiments to
test the relative importance of the different parameters to the
efficiency of transformation. Third, our analysis of the
transformants is not complete so our report of success should be
IN PLANTA TRANSFORMATION
An overnight culture of an appropriate Agrobacterium strain
(see last E-mail message) was prepared by inoculating 25 ml of LB
+ antibiotics in a 250 ml flask with a single colony in the
previous E-mail message and growing for 16 to 20 hrs at 28oC (225
Plants were grown at 22oC under continuous light. 25 seeds
were planted (evenly spaced) in each of 4 (4"). Plants germinating
late were rogued such that all remaining plants (80) were at the
same stage of maturity. At the time of bolting, when the primary
shoot was 2 to 5 cm in height, the shoot and 1 to 3 of the upper
rosette leaves were removed with fine scissors. Using a pasteur
pipette, one drop of culture (35 ul) was applied to the wound.
After all the plants in the pot were innoculated once, a second
drop of inoculum was applied. Plants were returned to growth
chamber for approx. 7 days. When the largest lateral shoots were
5 cm high, all visible lateral shoots were removed regardless of
size and the wounds inoculated with a drop of overnight culture as
before. Plants were then allowed to set seed. Putative
transformed seedlings were selected by sowing sterilized seed on
minimal agar medium containing 50 mg/L Kanamycin.
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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