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The GUS activity was observed as bluecoloured spots at 48 h after
bombardment. The control explants were bombarded with tungsten
particles lacking DNA and blue spots were not observed, The number of
blue spots per embryo varied between 0.9 + or - 0.3 and 4.2 + or -
1.0 (Table 1). All spots were dark
blue-coloured, which indicate a strong GUS positive response
(Fig. 1). Increasing the distance to 15 cm
from 6 cm and to 15 cm from 12 cm resulted in a slight increase of
mean number of blue spots per embryo for 1550-psi and 1800-psi
pressure, respectively. But changing the target distance between 6 cm
and 9 cm does not have effect on transformation efficiency at
1100-psi pressure. At 1800 psi, the greatest pressure tested, there
was a significant reduction in the number of blue spots. This could
be due to the higher degree of aggregation of the particles when
rupture disk pressure is high. The highest number of blue spots was
observed when the targets were 15 cm away from the stopping plate
under the pressure of 1550 psi.
The data showed that plasmid DNA was efficiently introduced into the
mature embryos of winter durum wheat 'Berkmen 469' and the embryos
are suitable recipients of foreign DNA introduced via particle
bombardment. Therefore, mature embryos can be used as an effective
explant source in direct gene transfer studies of durum wheat.
Recently, mature embryos of oat (Torbert et al. 1998) and rice
(Valdez et al. 1998) have successfully been used as an alternative
source of totipotent target cells for microprojectile
bombardment-mediated transformation. This study is the first
demonstration of the transient GUS expression in mature embryos of
winter durum wheat. Several factors might affect the efficiency of
foreign gene delivery by particle bombardment. In this study, the
effect of distance and pressure combination was evaluated. Further
optimization of the other factors that may influence the efficiency
of gene delivery, such as DNA concentration and the confirmation of
plasmid DNA, is required.
In conclusion, mature embryos might become a useful target tissue in
the transient transformation of durum, wheat, when all bombardment
parmeters are optimized further and a reliable selection technique of
bombarded tissue is developed. Stable transformant was not obtained
but this transient assay could be used to optimize some conditions
for stable transformation of durum wheat. The next step of this study
is to establish a stable transformation system of durum wheat.
Acknowledgments
This work was supported by a grant from the Scientific and
Technical Council of Turkey and Ankara University (contract no:
TOGTAG/1285). Dr. A.C. Cuming kindly provides the pBI221.23 DNA
construct.
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