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Wheat Information Service
Number 89: 30-32 (1999)
Research information
Transient expression of beta-glucuronidase (GUS)
gene in mature embryos of winter durum wheat via microprojectile
bombardment
M. Ozgen1, S. Onde2, M.
Birsin1 and C. Sancak1
1Department of Field Crops, Faculty of Agriculture,
University of Ankara, 06110 Diskapi, Ankara, Turkey
2 Department of Biological Sciences, Faculty of Science
and Art, Middle East Technical University, 06531 Ankara, Turkey
Key words: Particle bombardment, Transformation, Transient
expression, Triticum durum, Wheat
In durum wheat (Triticum turgidum L.), a biolistic
transformation method has been developed, for the first time, by
using scutella isolated from immature embryos (Bommineni et al. 1997;
Takumi and Shimada 1997). Immature embryos are commonly used as
targets for particle bombardment because the highest frequencies of
callus induction and plant regeneration have been obtained from the
immature embryos in both common and durum wheat. However, another
explant is required. because it is usually difficult to get immature
embryos throughout the year and their suitable stage for bombardment
is also strictly limited. Mature embryos are easily available without
limit any time, but they are not used as targets for bombardment
because of the low frequency of callus induction. However, some new
techniques have successfully been developed in callus induction and
plant regeneration from mature embryos of common and durum. wheat
(Ahmed et al. 1992; Ozgen et al. 1996, 1998).
In this study, the winter durum wheat (Triticum durum Desf.
cv. 'Berkmen 469') was used in transformation because of its high
frequency of regeneration from mature embryo culture to fertile
plants (Ozgen et al. 1996). The objective of the present study was to
investigate distance between stopping plate and target tissue, and
rupture disk pressure affecting transient expression of
beta-glucuronidase (GUS) gene in winter durum wheat mature embryos
introduced by the particle delivery system.
Mature seeds were sterilized and imbibed according to Ozgen et al.
(1998). For bombardment, mature embryos were aseptically excised with
a scalpel from the imbibed seeds and placed with the scutellum
upwards on a solid agar medium in the centre of sterile petri plates.
The GUS gene was used as a reporter gene in order to calibrate and
optimize physical conditions of the bombardment. All bombardments
were carried out with the Bio-Rad Biolistic (R), PDS
1000/He particle delivery system according to the manufacturer's
protocol. Bombardment was performed under partial vacuum (25" Hg).
Tungsten particles, 0.7 micro-m mean diameter, were coated with 0.6
micro-gram per shoot of pBI221.23 DNA which has the GUS gene and the
hpt gene (Lonsdale et al. 1990) as described in manufacturer's
protocol. Both genes are under the control of the cauliflower mosaic
virus (CaMV) '35 S' promoter. Four different distances (6, 9, 12 and
15 cm) from stopping plate to the target mature embryos and three
strengths (1100, 1550 and 1800 psi) of rupture disk were examined as
physical parameters. Each plate, containing 60 mature embryos, was
bombarded once. The mature embryos were subjected to histochemical
GUS assay according to Lonsdale et al. (1990).
1E-mail: mozgen@dialup.ankara.edu.tr
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