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Plant Physiol, July 2000, Vol. 123, pp. 895-904
Female Reproductive Tissues Are the Primary Target of
Agrobacterium-Mediated Transformation by the
Arabidopsis Floral-Dip Method1
Christine
Desfeux,
Steven J.
Clough, and
Andrew F.
Bent2*
Department of Crop Sciences, University of Illinois, Urbana,
Illinois 61801
The floral-dip method for Agrobacterium-mediated
transformation of Arabidopsis allows efficient plant transformation
without need for tissue culture. To facilitate use with other plant
species, we investigated the mechanisms that underlie this method. In
manual outcrossing experiments, application of Agrobacterium
tumefaciens to pollen donor plants did not produce any
transformed progeny, whereas application of
Agrobacterium to pollen recipient plants yielded
transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding  -glucuronidase
[GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed
delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the
same seed pod contained independent T-DNA integration events. In
Arabidopsis flowers, the gynoecium develops as an open, vase-like
structure that fuses to form closed locules roughly 3 d prior to
anthesis. In correlation with this fact, we found that the timing of
Agrobacterium infection was critical. Transformants were
obtained and GUS staining of ovules and embryo sacs was observed only
if the Agrobacterium were applied 5 d or more prior
to anthesis. A 6-fold higher rate of transformation was obtained with a
CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest
that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be
delivered to the interior of the developing gynoecium prior to locule
closure if efficient transformation is to be achieved.
1
This research was supported by the North Central
Soybean Research Program.
2
Present Address: Department of Plant Pathology,
University of Wisconsin, Madison, WI 53706.
*
Corresponding author; e-mail afb{at}plantpath.wisc.edu; fax
608-263-2626.
© 2000 American Society of Plant Physiologists
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