Selenium (Se) is an essential micronutrient for animals, including humans (Birringer et al., 2002), although for a long time, it was only known for its toxicity (Schwarz and Foltz, 1957). Recent studies have shown that Se-enriched plants are wanted for both cancer prevention (Finley, 2005) and Se phytoremediation (Berken et al., 2002; Sors et al., 2005). In nature, several Se-hyperaccumulating species of the genus Astragalus originating from seleniferous soils have been characterized (Trelease and Trelease, 1939). They cannot only tolerate high Se in soil, but can also hyperaccumulate Se at concentrations of up to 20 to 40 mg per gram of dry matter in their shoots when they grow under 2 to 10 ppm Se in natural soils (Davis, 1972). However, these Se accumulators are not edible plants for chemopreventive purposes and have very limited applications for Se phytoremediation because of their extremely slow growth and low biomass (Cunningham et al., 1997). In addition, the Se-hyperaccumulating plants cannot cross with any food plants as a result of genetic distance. Molecular cloning and genetic engineering may offer a better way to isolate genes related to the Se-hyperaccumulating property that could be transferred to other crop plants to create Se-enriched transgenic plants.
Currently, the functional analysis of the Astragalus genes related to Se accumulation is limited because there is no transformation and regeneration system available for the Astragalus species. So far, only one Se-accumulating gene, encoding selenocysteine methyltransferase, has been isolated from a Se hyperaccumulator, Astragalus bisulcatus (Neuhierl et al., 1999). Other genes that are also responsible for Se accumulation might exist. The discovery and study of additional Se-accumulating genes will help us to understand the mechanism of Se accumulation in Se hyperaccumulators and ultimately to develop transgenic plants with the Se-hyperaccumulating capacity. Gene transformation, however, needs reliable regeneration systems.
A previous report showed that calli could be induced from the hypocotyls of five Se-hyperaccumulator and three nonaccumulator species of Astragalus by including 13.57 μm 2,4-dichlorophenoxyacetic acid (2,4-D) in the induction medium (Ziebur and Shrift, 1971). Both the A. racemosus and A. canadensis species used in the present study were in their report. When those calli were subcultured, they failed to regenerate plants despite that incompletely developed roots and shoots were observed in some of their early subcultures, although they had been subcultured and maintained for several years (Ziebur and Shrift, 1971). Synthetic auxin, 2,4-D, is a strong hormone that is widely used to induce callus formation (Khanna and Raina, 1998). However, the concentration of 13.57 μm is considerably high for plant tissue culture, in which calli derived from an induction medium with such a high concentration of 2,4-D generally lose further dedifferentiation ability (Xie et al., 1995).
The objective of the current study was to establish a plant regeneration system for both the Se-hyperaccumulator A. racemosus and the nonaccumulator A. canadensis. To induce shoots, another more moderate auxin, a-naphthalene acetic acid (NAA), was chosen to combine with cytokinin 6-benzylaminopurine (BA) and cytokine-like N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU). CPPU has been proven to be able to effectively improve the efficiency of shoot formation and somatic embryogenesis in different plant species (Fiore et al., 2002; Millan-Mendoza, 1998; Murthy and Saxena, 1994; Nakajima et al., 2000; Tsuro et al., 1999; Zhang et al., 2005). We report our newly established plant regeneration system for A. racemosus and A. canadensis. Current results demonstrate that CPPU combined with NAA was good for A. racemosus and A. canadensis tissue cultures. Culture efficiencies of two species and three types of explants are compared.
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