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One of the most important factors affecting the transformation efficiency is the activity of the promoter which should drive the introduced gene. Three promoters, i.e., the promoters of maize alcohol dehydrogenase gene (Adh1 ), maize ubiquitin gene (Ubi1 ) and rice actin gene (Act1 ), are widely used in wheat transformation. The cauliflower mosaic virus (CaMV) 35S promoter which is often used in dicot transformation systems shows low activity in transient gusA expression in pollen embryoids of wheat (Shimada et al. 1991), immature embryos (Chibbar et al. 1991) and cultured cells (Wang et al. 1988; Takumi et al. 1994). However, the promoter and the first intron of the maize Adh1 that were placed after the CaMV 35S promoter greatly stimulate expression of a foreign gene in callus (Wang et al. 1988), leaf base and apical tissue (Oard et al. 1989) and immature embryos (Chibbar et al. 1991) of wheat. Taylor et al. (1993) have demonstrated that the maize Ubi1 showed higher activity of transient expression in cells of cereals including wheat than the maize Adh1 promoter plus its first intron. The efficiency of the Ubi1 promoter has also been confirmed in immature embryos (Vasil et al. 1993) and pollen embryoids (Loeb and Reynolds 1994). On the other hand, Nehra et al. (1994) have demonstrated that the rice Act1 promoter showed higher transient activity than the maize Adh1 promoter plus its first intron in wheat immature embryos. In our previous work, the rice Act1 promoter was also confirmed to show higher activity than any other examined promoters including the Adh1 promoter plus its first intron in cultured cells of three Triticum species (Takomi et al. 1994). These results have indicated that the rice Act1 promoter was efficient and useful in wheat cells.

In this study, we evaluated the efficiency of the rice Act1 promoter in various cell types including embryogenic callus, immature embryos and pollen embryoids, as revealed by transient expression of the gusA gene encoding beta-glucuronidase (GUS).


Materials and methods

Plant materials
Calli derived from immature embryos of two aneuploid lines of Chinese Spring (CS) and Aegilops cylindrica, immature embryos of four common wheat cultivars and two emmer wheats, and pollen embryoids derived from anther culture of four common wheat cultivars were used, as shown in Table 1. Three callus lines were induced from immature embryos, which grew vigorously on Linsmaier-Skoog (LS) medium (Linsmaier and Skoog 1965) containing 2 mg A 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.25% (w/v) Gelrite (Merck). These calli were subcultured using fresh medium every three weeks. Approximately 1 ml (fresh packed cell volume) of callus was spread onto 90 mm x 15 mm Petri-dishes containing solid LS medium supplemented with 2 mg /1 2,4-D. Immature seeds were sterilized with 70% ethanol and immature embryos isolated were placed with scutellar tissues exposed on LS medium containing 2 mg /l 2,4-D. Pollen embryoids (1 to 2 mm) were obtained from anther cultures after the methods of Otani and Shimada (1993) and were also placed on LS medium containing 2 mg /l 2,4-D.

Plasmids, particle bombardment and enzyme assay
The pAct1-F (McElroy, Sang et al. 1990) including the gusA coding region under the control of the 1.3 kb 5' region of the rice actin 1 gene (Act1) was used as the reporter gene. The plastids were amplified in liquid cultures of Escherichia coli, isolated by alkaline Iysis, and purified twice by CsCl/ethidium bromide density centrifugation (Maniatis et al. 1982). The BiolisticR PDS1000/He Particle Delivery System (Bio-Rad) was used as a particle accelerator. Plasmid DNAs were adsorbed to gold particles (1.6 micrometer diameter) according to the protocol described for the BiolisticR PDS-1000/He Particle Delivery System (Klein et al. 1988). The call), pollen embryoids and immature embryos were bombarded after incubation for several days on the LS medium containing 2 mg /1 2,4- D. GUS activity in cultured cells, immature embryos and pollen embryoids was assessed histochemically by the directed addition of the substrate of glucuronidase enzyme as described previously (Takumi et al. 1994) and the average numbers of blue spots, showing transient expression of the gusA gene, per embryo in two separate experiments were counted. In each experiment, more than 20 immature embryos and pollen embryoids were used.

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