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Results and discussion
Previously, we found that the rice Act1 promoter showed
the highest activity of transient gusA expression in three
non-embryogenic cultured cell lines of T. monococcum, T. durum
and T. aestivum (Takumi et al. 1994). To estimate
the efficiency in other cultured cell lines, transient gusA
expression was confirmed by using three embryogenic cell lines
(Table 1). Two of them were cultured cells
derived from immature embryos of nullitetrasomic and ditelosomic
lines of T. aestivum cv. Chinese Spring (Sears 1966;
Sears and Sears 1978). The other callus line was Aegilops
cylindrica, a wild wheat species. These calli were cultured for
ten days and then bombarded with the particles coated with a plasmids
pAct1-F. Transient expression of the gusA gene
was observed in two days after bombardment by using a histochemical
staining. pAct1-F yielded a high activity of transient
gusA expression in all three cell lines (data not shown). This
suggests that the rice Act1 promoter efficiently induces
expression of a marker gene in cultured cells of various wheat
species and accessions including aneuploid lines. Of the cultured
cells of Ae. cylindrica bombarded with pAct1-F after
2-14 days of incubation, the highest expression was obtained in the
cells bombarded after 14 days incubation (Fig.
1). This supported our previous findings using the cells of T.
monococcum (Takumi et al. 1994), that the efficiency of transient
expression was severely influenced by culture duration of the target
tissues before bombardment.
To examine the transient expression of the gusA gene connected to the
rice Act1 promoter in wheat pollen embryoids, pAct1-F
was introduced by particle bombardment into pollen embryos of four
wheat cultivars after two or five days incubation. The number of blue
spots in pollen embryoids was only few compared to that observed in
immature embryos. Fig. 2 shows the number of
blue spots per pollen embryoid incubated for two or five days before
bombardment. The number in the four wheat cultivars was 7.1 to 11.7
and 4.6 to 8.8 per pollen embryoid incubated for two and five days,
respectively. The activity of transient gusA expression was
similar among the four wheat cultivars, but the activity expressed in
two-days old embryoids was slightly higher than that observed in the
five-days old embryoids in all cultivars but Gernard 81. Loeb and
Reynolds (1994) demonstrated that the CaMV 35S promoter is not
effective in wheat pollen embryoids and that the maize Ubi1
promoter controls a high level of gusA expression in the
pollen embryoids. It was not known whether the rice Act1 promoter is
as efficient in the pollen embryoids as the maize Ubi1
promoter, but the activity of the Act1 promoter in the
pollen embryoids was not so high as that in immature embryos. This
fact suggests that production of transgenic plants from bombarded
pollen embryoids with a selectable marker gene under control of the
Act1 promoter has no promise at present, considering the lower
regeneration rate from pollen embryoids than from immature embryos.
It is essential for production of transgenic wheats from pollen
embryoids to increase drastically their regeneration rate by
improving the anther culture.
To examine transient expression of gusA gene under control of
the rice Act1 promoter in immature embryos of some tetraploid
and hexaploid wheats, the scutellar tissues cultured for one to nine
days were bombarded with the particles coated with a plasmid
pAct1-F. Immature embryos at stage III (14 days after
anthesis) were isolated because the developmental stage of immature
embryos is important for embryogenesis and immature embryos at stages
II and III are most suitable for induction of scutellum callus (Scott
et al. 1990). Transient expression of the gusA gene was
observed in two days after bombardment by using a histochemical
staining. Fig. 3 shows the relationship
between transient gusA expression and culture duration prior
to bombardment. No clear correlation between the culture duration and
transient gusA expression was recognized in all four wheats,
as other wheat cultivar Akadaruma (Takumi and Shimada 1996). However,
transient expression of gusA gene in immature embryos of CS
was higher than that in two emmer wheats. The rice Act1
promoter provided a high level of the gusA expression in
scutellar tissues of both tetraploid and hexaploid wheats. Transient
gusA expression was gradually decreased during 1- 31 days
after bombardment with pAct1-F by using immature embryos of
T. aestivam cv. Akadaruma (Fig.
4). The number of blue spots in immature embryos decreased with
the increasing culture duration after bombardment. A sharp decrease
was observed in the first ten days. This indicated that most blue
spots are due to transient expression of the reporter gene and stable
transformation is rather difficult.
The rice Act1 promoter which is known to cause a high level of
gusA expression in transformed rice and maize cells (McElroy,
Zang et al. 1990; McElroy et al 1991) controlled the transient
gusA expression efficiently in cultured cells, pollen
embryoids and scutellar tissues of common wheat and its relatives.
The high levels of transient expression in these materials seem to be
caused by the constitutive expression of the rice Act1
(McElroy, Rothernberg et al. 1990; Zang et al. 1991).
Moreover, we previously demonstrated that the rice Act1
promoter showed higher activity of transient expression in
cultured cells of common wheat than the maize Ubi1 promoter,
although these two promoters showed similar activity in einkorn wheat
cells (Takumi and Shimada 1995). These findings suggest that the rice
Act1 promoter is an efficient and useful promoter in
transformation of monocotyledonous crops.
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