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Wheat Information
Service
Number 72: 106-108 (1991)
Transient
expression of beta-glucuronidase (GUS) gene in wheat pollen embryos
via microprojectile bombardment
T. Shimada1, M. Seki2, and H.
Morikawa3
1. Ishikawa Agr. College, 2. Kyoto Univ., 3. Hiroshima Univ.
Biolistic gene delivery is a useful method of introducing genes into
cereal crop species, especially wheat in which neither infection of
Agrobacterium
tumefaciens nor
efficient system for regeneration from protoplasts, have been
established (Gasser and Fraley 1989).
Cultured anthers of some wheat cultivars can produce pollen embryos
at high frequencies which grow into green plantlets (Shimada and
Otani, unpublished). These pollen embryos might could serve as
recipients of alien genes to grow transformed wheat. In this work we
demonstrated the transient expression of the beta-glucuronidase (GUS)
gene in wheat embryos derived from anther culture introduced by the
pneumatic particle gun (Iida, et al 1990). Anthers of wheat
(Triticum aestivum cvs.
Haruyutaka, BW2559,
Glenson 81, Seri 82) were cultured on liquid Potato-2 medium. After a
1-month culture, pollen embryos (1 - 2 mm) were transferred onto
regeneration medium in the petri dish (6 mm diameter). The procedure
of wheat anther culture was as previously reported (Shimada and Otani
1988, 1989). The pollen embryos on the regeneration medium were
bombarded with DNA-coated gold particles by a pneumatic particle gun
as reported previously (Iida et al 1990).
Plasmid DNA, pBI221 which has the beta-glucuronidase (GUS) gene under
the control of the cauliflower mosaic virus 35S promoter and nopaline
synthetase (NOS) polyadenylation signal was used.
The embryos and/or germination embryos were assayed for GUS
expression using X-gluc substrate at 48 hr. and 1 week after
bombardment. The expression GUS activity could be observed as
blue-colored spots. GUS activity could be observed in 44 of 56 pollen
embryos from 4 cultivars at 48 hr. after bombardment by DNA-coated
gold particles (Fig.
1,
Table
1). The number of
blue spots varied from one to 15 with an average of 4.3. Two shots of
bombardment increased the number of blue spots. After 1 week the
bombarded embryos on the re eneration medium grew into plantlets.
Young shoots and roots assayed by the X-gluc substrate showed only
pale blue regions. Wheat seedlings might have endogenous GUS
activity. We did. not observe any dark blue spots, which would
indicate a strong GUS positive response.
About 40 - 60% of the pollen embryos bombarded grew into normal green
plants after a month (Table
2) and 10 - 20%,
of the green plants set seeds whose root tips did not express GUS
activity.
Further optimization of the factors that may influence the efficiency
of gene delivery, for example the introduced DNA concentration and
the conformation of plasmid DNA, is required.
References
Iida A, Seki M, Kamada M, Yamada Y and Morikawa H (1990) Gene
delivery into cultured plant cells by DNA-coated gold particles
accelerated by a pneumatic particle gun. Theor Apple Genet:
813-816.
Gasser CS and Fraley RT (1989) Genetically engineering plants for
crop improvement. Science 244: 1293-1299.
Shimada T and Otani M (1988) Efficiency of potato medium on induction
of pollen embryoids in anther culture of Japanese wheat cultivars.
Japan J Breed 38: 212-222 (in Japanese).
Shimada T and Otani M (1989) Varietal differences in green plant
regeneration ability of wheat pollen embryoids. Japan J Breed 39:
187-194 (in Japanese).
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